InteractiveFly: GeneBrief

little imaginal discs: Biological Overview | References

Gene name - little imaginal discs

Synonyms -

Cytological map position-26B2-26B2

Function - enzyme, transcription factor

Keywords - trithorax group, cell growth, myc pathway

Symbol - lid

FlyBase ID: FBgn0031759

Genetic map position - 2L: 5,990,441..5,999,483 [-]

Classification - JmjC domain trimethyl H3K4 demethylase with C5HC2 zinc finger motif

Cellular location - nuclear

NCBI links: EntrezGene
lid orthologs: Biolitmine

Recent literature
Morán, T., Bernués, J. and Azorín, F. (2015). The Drosophila histone demethylase dKDM5/LID regulates hematopoietic development. Dev Biol [Epub ahead of print]. PubMed ID: 26183107
dKDM5/LID regulates transcription of essential developmental genes and, thus, is required for different developmental processes. This study reports the essential contribution of dKDM5/LID to hematopoiesis in Drosophila. The study shows that dKDM5/LID is abundant in hemocytes and that its depletion induces over-proliferation and differentiation defects of larval hemocytes and disrupts organization of the actin cytoskeleton. It was also shown that dKDM5/LID regulates expression of key factors of hematopoietic development. In particular, dKDM5/LID depletion up-regulates expression of several transcription factors involved in hemocytes proliferation and differentiation as well as of several small-GTPases that link signaling effectors to actin cytoskeleton formation and dynamics.

Tarayrah, L., Li, Y., Gan, Q. and Chen, X. (2015). Epigenetic regulator Lid maintains germline stem cells through regulating JAK-STAT signaling pathway activity. Biol Open. PubMed ID: 26490676
Signaling pathways and epigenetic mechanisms have both been shown to play essential roles in regulating stem cell activity. While the role of either mechanism in this regulation is well established in multiple stem cell lineages, how the two mechanisms interact to regulate stem cell activity is not as well understood. This study reportd that in the Drosophila testis, an H3K4me3-specific histone demethylase encoded by little imaginal discs (lid) maintains germline stem cell (GSC) mitotic index and prevents GSC premature differentiation. Lid is required in germ cells for proper expression of the Stat92E transcription factor, the downstream effector of the JAK-STAT signaling pathway. These findings support a germ cell autonomous role for the JAK-STAT pathway in maintaining GSCs and place Lid as an upstream regulator of this pathway. This study provides new insights into the biological functions of a histone demethylase in vivo and sheds light on the interaction between epigenetic mechanisms and signaling pathways in regulating stem cell activities.

Liu, X. and Secombe, J. (2015). The Histone demethylase KDM5 activates gene expression by recognizing chromatin context through its PHD reader motif. Cell Rep 13: 2219-2231. PubMed ID: 26673323
KDM5 family proteins are critically important transcriptional regulators whose physiological functions in the context of a whole animal remain largely unknown. Using genome-wide gene expression and binding analyses in Drosophila adults, this study demonstrates that KDM5 (Lid) is a direct regulator of genes required for mitochondrial structure and function. Significantly, this occurs independently of KDM5's well-described JmjC domain-encoded histone demethylase activity. Instead, it requires the PHD motif of KDM5 that binds to histone H3 that is di- or trimethylated on lysine 4 (H3K4me2/3). Genome-wide, KDM5 binding overlaps with the active chromatin mark H3K4me3, and a fly strain specifically lacking H3K4me2/3 binding shows defective KDM5 promoter recruitment and gene activation. KDM5 therefore plays a central role in regulating mitochondrial function by utilizing its ability to recognize specific chromatin contexts. Importantly, KDM5-mediated regulation of mitochondrial activity is likely to be key in human diseases caused by dysfunction of this family of proteins.

Gajan, A., Barnes, V. L., Liu, M., Saha, N. and Pile, L. A. (2016). The histone demethylase dKDM5/LID interacts with the SIN3 histone deacetylase complex and shares functional similarities with SIN3. Epigenetics Chromatin 9: 4. PubMed ID: 26848313
Two histone-modifying enzymes, RPD3, a deacetylase, and dKDM5/LID, a demethylase, are present in a single complex, coordinated through the SIN3 scaffold protein. This study analyzed the developmental and transcriptional activities of dKDM5/LID in relation to SIN3. Knockdown of either Sin3A or lid resulted in decreased cell proliferation in S2 cells and wing imaginal discs. Conditional knockdown of either Sin3A or lid resulted in flies that displayed wing developmental defects. Interestingly, overexpression of dKDM5/LID rescued the wing developmental defect due to reduced levels of SIN3 in female flies, indicating a major role for dKDM5/LID in cooperation with SIN3 during development. Together, these observed phenotypes strongly suggest that dKDM5/LID as part of the SIN3 complex can impact previously uncharacterized transcriptional networks. Transcriptome analysis revealed that a significant affect was observed on genes required to mount an effective stress response. Together, the data provide a solid framework for analyzing the gene regulatory pathways through which SIN3 and dKDM5/LID control diverse biological processes in the organism.

Liu, X., Greer, C. and Secombe, J. (2014). KDM5 interacts with Foxo to modulate cellular levels of oxidative stress. PLoS Genet 10: e1004676. PubMed ID: 25329053
Increased cellular levels of oxidative stress are implicated in a large number of human diseases. This study describes the transcription co-factor KDM5 (also known as Lid) as a new critical regulator of cellular redox state. Moreover, this occurs through a novel KDM5 activity whereby it alters the ability of the transcription factor Foxo to bind to DNA. Microarray analyses of kdm5 mutants revealed a striking enrichment for genes required to regulate cellular levels of oxidative stress. Consistent with this, loss of kdm5 results in increased sensitivity to treatment with oxidizers, elevated levels of oxidized proteins, and increased mutation load. KDM5 activates oxidative stress resistance genes by interacting with Foxo to facilitate its recruitment to KDM5-Foxo co-regulated genes. Significantly, this occurs independently of KDM5's well-characterized demethylase activity. Instead, KDM5 interacts with the lysine deacetylase HDAC4 to promote Foxo deacetylation, which affects Foxo DNA binding.
Navarro-Costa, P., McCarthy, A., Prudencio, P., Greer, C., Guilgur, L. G., Becker, J. D., Secombe, J., Rangan, P. and Martinho, R. G. (2016). Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling. Nat Commun 7: 12331. PubMed ID: 27507044
Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. It was hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. This study comprehensively defines the Drosophila female germ line epigenome throughout oogenesis and shows that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. The perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, these results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I.
Zhaunova, L., Ohkura, H. and Breuer, M. (2016). Kdm5/Lid regulates chromosome architecture in meiotic prophase I independently of its histone demethylase activity. PLoS Genet 12: e1006241. PubMed ID: 27494704
During prophase of the first meiotic division (prophase I), chromatin dynamically reorganises to recombine and prepare for chromosome segregation. Histone modifying enzymes are major regulators of chromatin structure, but knowledge of their roles in prophase I is still limited. This study reports on crucial roles of Kdm5/Lid, one of two histone demethylases in Drosophila that remove one of the trimethyl groups at Lys4 of Histone 3 (H3K4me3). In the absence of Kdm5/Lid, the synaptonemal complex was only partially formed and failed to be maintained along chromosome arms, while localisation of its components at centromeres was unaffected. Kdm5/Lid was also required for karyosome formation and homologous centromere pairing in prophase I. Although loss of Kdm5/Lid dramatically increased the level of H3K4me3 in oocytes, catalytically inactive Kdm5/Lid can rescue the above cytological defects. Therefore Kdm5/Lid controls chromatin architecture in meiotic prophase I oocytes independently of its demethylase activity.
Zamurrad, S., Hatch, H. A. M., Drelon, C., Belalcazar, H. M. and Secombe, J. (2018). A Drosophila model of intellectual disability caused by mutations in the histone demethylase KDM5. Cell Rep 22(9): 2359-2369. PubMed ID: 29490272
Mutations in KDM5 family histone demethylases cause intellectual disability in humans. However, the molecular mechanisms linking KDM5-regulated transcription and cognition remain unknown. This study establish Drosophila as a model to understand this connection by generating a fly strain harboring an allele in the fly KDM5C homolog Little imaginal discs, analogous to a disease-causing missense mutation in human KDM5C (kdm5A512P). Transcriptome analysis of kdm5A512P flies revealed a striking downregulation of genes required for ribosomal assembly and function and a concomitant reduction in translation. kdm5A512P flies also showed impaired learning and/or memory. Significantly, the behavioral and transcriptional changes in kdm5(A512P) flies were similar to those specifically lacking demethylase activity. These data suggest that the primary defect of the kdm5A512P mutation is a loss of histone demethylase activity and reveal an unexpected role for this enzymatic function in gene activation. Because translation is critical for neuronal function, it is proposed that this defect contributes to the cognitive defects of kdm5A512P flies.
Drelon, C., Belalcazar, H. M. and Secombe, J. (2018). The histone demethylase KDM5 is essential for larval growth in Drosophila. Genetics. PubMed ID: 29764901
Regulated gene expression is necessary for developmental and homeostatic processes. The KDM5 family of transcriptional regulators are histone H3 lysine 4 demethylases that can function through both demethylase-dependent and independent mechanisms. While loss and overexpression of KDM5 proteins are linked to intellectual disability and cancer, respectively, their normal developmental functions remain less characterized. Drosophila melanogaster provides an ideal system to investigate KDM5 function, as it encodes a single ortholog in contrast to the four paralogs found in mammalian cells. To examine the consequences of complete loss of KDM5, a null allele of Drosophila kdm5, also known as little imaginal discs (lid), was generated, and it was shown to be essential for viability. Animals lacking KDM5 show a dramatically delayed larval development that coincides with decreased proliferation and increased cell death in wing imaginal discs. Interestingly, this developmental delay is independent of the well-characterized Jumonji C (JmjC) domain-encoded histone demethylase activity of KDM5, suggesting key functions for less characterized domains. Consistent with the phenotypes observed, transcriptome analyses of kdm5 null mutant wing imaginal discs revealed the dysregulation of genes involved in several cellular processes, including cell cycle progression and DNA repair. Together, these analyses reveal KDM5 as a key regulator of larval growth and offer an invaluable tool for defining the biological activities of KDM5 family proteins.
Varma, P. and Mishra, R. K. (2018). Little imaginal discs, a Trithorax group member, is a constituent of nuclear matrix of Drosophila melanogaster embryos. J Biosci 43(4): 621-633. PubMed ID: 30207309
Attachment of chromatin to the Nuclear Matrix (NuMat) brings significant regulation of the transcriptional activity of particular genes. This study has identified Lid (Little imaginal discs) as one of the components of NuMat. It belongs to the TrxG group of proteins involved in activation of important developmental genes. However, unlike other activator proteins of TrxG, Lid is a Jumonji protein involved in H3K4me3 demethylation. This study reports the association of Lid and its various domains with NuMat which implicates its structural role in chromatin organization and epigenetic basis of cellular memory. Both N and C terminal regions of this protein were found to be capable of associating with NuMat. The association of individual domains was further mapped and it was found that, PHD, ARID and JmjC domains can associate with NuMat individually. Based on these findings, it is hypothesized that C terminal region of Lid which contains PHD fingers might be responsible for its NuMat association via protein-DNA interactions. However, for the N terminal region harboring both a PHD and an ARID finger, Lid anchors to the NuMat via both protein-protein and protein-DNA interactions. It is suggested that Lid, a demethylase, being part of NuMat might be involved in regulating the chromatin dynamics via its NuMat association.
Chen, K., Luan, X., Liu, Q., Wang, J., Chang, X., Snijders, A. M., Mao, J. H., Secombe, J., Dan, Z., Chen, J. H., Wang, Z., Dong, X., Qiu, C., Chang, X., Zhang, D., Celniker, S. E. and Liu, X. (2019). Drosophila histone demethylase KDM5 regulates social behavior through immune control and gut microbiota maintenance. Cell Host Microbe 25(4): 537-552.e538. PubMed ID: 30902578
Loss-of-function mutations in the histone demethylases KDM5A, KDM5B, or KDM5C are found in intellectual disability (ID) and autism spectrum disorders (ASD) patients. This study used the model organism Drosophila melanogaster to delineate how KDM5 contributes to ID and ASD. Reducing KDM5 causes intestinal barrier dysfunction and changes in social behavior that correlates with compositional changes in the gut microbiota. Therapeutic alteration of the dysbiotic microbiota through antibiotic administration or feeding with a probiotic Lactobacillus strain partially rescues the behavioral, lifespan, and cellular phenotypes observed in kdm5-deficient flies. Mechanistically, KDM5 was found to transcriptionally regulate component genes of the immune deficiency (IMD) signaling pathway and subsequent maintenance of host-commensal bacteria homeostasis in a demethylase-dependent manner. Together, this study uses a genetic approach to dissect the role of KDM5 in the gut-microbiome-brain axis and suggests that modifying the gut microbiome may provide therapeutic benefits for ID and ASD patients.
Torres-Campana, D., Kimura, S., Orsi, G. A., Horard, B., Benoit, G. and Loppin, B. (2020). The Lid/KDM5 histone demethylase complex activates a critical effector of the oocyte-to-zygote transition. PLoS Genet 16(3): e1008543. PubMed ID: 32134927
Following fertilization of a mature oocyte, the formation of a diploid zygote involves a series of coordinated cellular events that ends with the first embryonic mitosis. In animals, this complex developmental transition is almost entirely controlled by maternal gene products. How such a crucial transcriptional program is established during oogenesis remains poorly understood. This study performed an shRNA-based genetic screen in Drosophila to identify genes required to form a diploid zygote. The Lid/KDM5 histone demethylase and its partner, the Sin3A-HDAC1 deacetylase complex, are necessary for sperm nuclear decompaction and karyogamy. Surprisingly, transcriptomic analyses revealed that these histone modifiers are required for the massive transcriptional activation of deadhead (dhd), which encodes a maternal thioredoxin involved in sperm chromatin remodeling. Unexpectedly, while lid knock-down tends to slightly favor the accumulation of its target, H3K4me3, on the genome, this mark was lost at the dhd locus. It is proposed that Lid/KDM5 and Sin3A cooperate to establish a local chromatin environment facilitating the unusually high expression of dhd, a key effector of the oocyte-to-zygote transition.
Belalcazar, H. M., Hendricks, E. L., Zamurrad, S., Liebl, F. L. W. and Secombe, J. (2021). The histone demethylase KDM5 is required for synaptic structure and function at the Drosophila neuromuscular junction. Cell Rep 34(7): 108753. PubMed ID: 33596422
Mutations in the genes encoding the lysine demethylase 5 (KDM5) family of histone demethylases are observed in individuals with intellectual disability (ID). Despite clear evidence linking KDM5 function to neurodevelopmental pathways, how this family of proteins impacts transcriptional programs to mediate synaptic structure and activity remains unclear. Using the Drosophila larval neuromuscular junction (NMJ), this study shows that KDM5 is required presynaptically for neuroanatomical development and synaptic function. The Jumonji C (JmjC) domain-encoded histone demethylase activity of KDM5, which is expected to be diminished by many ID-associated alleles, is required for appropriate synaptic morphology and neurotransmission. The activity of the C5HC2 zinc finger is also required, as an ID-associated mutation in this motif reduces NMJ bouton number, increases bouton size, and alters microtubule dynamics. KDM5 therefore uses demethylase-dependent and independent mechanisms to regulate NMJ structure and activity, highlighting the complex nature by which this chromatin modifier carries out its neuronal gene-regulatory programs.
Hatch, H. A. M., Belalcazar, H. M., Marshall, O. J. and Secombe, J. (2021). A KDM5-Prospero transcriptional axis functions during early neurodevelopment to regulate mushroom body formation. Elife 10. PubMed ID: 33729157
Mutations in the lysine demethylase 5 (KDM5) family of transcriptional regulators are associated with intellectual disability, yet little is known regarding their spatiotemporal requirements or neurodevelopmental contributions. Utilizing the mushroom body (MB), a major learning and memory center within the Drosophila brain, this study demonstrates that KDM5 is required within ganglion mother cells and immature neurons for proper axogenesis. Moreover, the mechanism by which KDM5 functions in this context is independent of its canonical histone demethylase activity. Using in vivo transcriptional and binding analyses, a network of genes directly regulated by KDM5 was identified that are critical modulators of neurodevelopment. KDM5 directly regulates the expression of prospero, a transcription factor that this study demonstrates to be essential for MB morphogenesis. Prospero functions downstream of KDM5 and binds to approximately half of KDM5-regulated genes. Together, these data provide evidence for a KDM5-Prospero transcriptional axis that is essential for proper MB development.
Torres-Campana, D., Horard, B., Denaud, S., Benoit, G., Loppin, B. and Orsi, G. A. (2022). Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain. PLoS Genet 18(1): e1009615. PubMed ID: 34982772
The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. This study identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, it was found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, it was shown that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, this study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.


The Myc oncoprotein is a potent inducer of cell growth, cell cycle progression, and apoptosis. While many direct Myc target genes have been identified, the molecular determinants of Myc's transcriptional specificity remain elusive. A genetic screen was carried out in Drosophila and the Trithorax group protein Little imaginal discs (Lid) was identified as a regulator of dMyc-induced cell growth. Lid was originally identified in intergenic noncomplementation with a mutation in ash1, a trithorax group gene (Gildea, 2000; full text of article). Lid binds to dMyc and is required for dMyc-induced expression of the growth regulatory gene Nop60B. The mammalian Lid orthologs, Rbp-2 (JARID1A) and Plu-1 (JARID1B), also bind to c-Myc, indicating that Lid-Myc function is conserved. Lid is a JmjC-dependent trimethyl H3K4 demethylase in vivo, and this enzymatic activity is negatively regulated by dMyc, which binds to Lid's JmjC domain. Because Myc binding is associated with high levels of trimethylated H3K4, it is proposed that the Lid-dMyc complex facilitates Myc binding to, or maintenance of, this chromatin context (Secombe, 2007). Identication of Lid as a histone H3 trimethyl-Lys4 demethylase has also been reported by Lee (2007) and Eissenberg (2007).

Lid is a 1838-amino-acid protein possessing numerous conserved motifs including an ARID (A/T-rich interaction domain), implicated in binding A/T-rich DNA; a single C5HC2 zinc finger; three PHD fingers (plant homeobox domain), implicated in forming protein-protein interactions; and Jumonji N and C (JmjN and JmjC) domains. JmjC-containing proteins have recently been shown to act as histone demethylase enzymes in a Fe2+ and -ketoglutarate-dependent manner (Klose, 2006). To test whether Lid can demethylate histones in vivo, Lid was overexpressed in fat body and in wing disc cells and the levels of mono-, di-, and tri-methylated histone H3K4 and H3K27 were examined. Di- and tri-methylated histone H4K20 and trimethylated histone H3K9 and H3K36 were also examined. Overexpression of Lid specifically decreased the levels of the tri-methylated form of H3K4 but had no effect on the other methylated histones examined in either GFP-marked fat body or wing disc cells. Significantly, expression of Lid in the wing disc reduced trimethyl H3K4 in a dose-dependent manner, with two copies of the UAS-Lid transgene reducing trimethyl H3K4 more efficiently than one copy. Moreover, levels of trimethylated H3K4 are increased in wing discs from lid homozygous mutant animals, consistent with the model that Lid regulates the levels of this histone modification during normal development. To determine whether the JmjC domain of Lid is required for the observed H3K4 demethylation, transgenic flies were generated carrying a Gal4-inducible form of full-length Lid containing Ala substitutions at His637 and Glu639 (Lid-JmjC*) that abolishes the protein's ability to bind the Fe2+ cofactor required for demethylase activity. Similar mutations have been shown to disrupt the demethylase function of the JmjC domains of JHDM2A, JHDM3A, JHDM1, and JMJD2A. Unlike wild-type Lid, expression of full-length Lid-JmjC* did not decrease levels of trimethylated H3K4 in fat body or in wing disc cells, demonstrating that an active JmjC domain is required for Lid-mediated H3K4 demethylation. Interestingly, expression of Lid-JmjC* resulted in increased levels of trimethyl H3K4 in the fat body, perhaps due to a dominant interfering effect on wild-type Lid function in these cells. Taken together, these results demonstrate that Lid is a trimethyl H3K4 demethylase that modifies nucleosomal histone H3 in vivo. The global regulation of H3K4 trimethylation status by Lid is not, however, likely to be mediated by recruitment by dMyc, since no effect was observed of reduced or increased dMyc expression on trimethyl H3K4 levels in either fat body or wing disc cells (Secombe, 2007).

Forty other genomic regions were identified that enhanced or suppressed the GMR-Gal4, UAS-dMyc (GMM) phenotype when heterozygous. Two of these regions delete genes encoding known regulators of dMyc stability, such as ago, or are involved in Myc transactivation, such as Pcaf. Specific mutations in both of these genes have been shown to enhance or suppress the GMM rough eye phenotype, respectively. Interestingly, none of the known direct transcriptional targets of dMyc were identified as genetic modifiers of the GMM phenotype, suggesting that the GMM phenotype arises from modulation of multiple genes and provides a powerful tool to identify proteins directly required for dMyc function in vivo (Secombe, 2007).

TrxG proteins are renowned for their essential role in maintaining homeotic (hox) gene expression during development, with mutations in many TrxG genes resulting in lethality due to homeotic transformations. Six TrxG protein complexes have been identified to date. While one function of these complexes is to antagonize Polycomb group (PcG) repression to maintain active hox gene expression, TrxG proteins are also recruited to other developmentally important genes to either activate or repress their transcription in a context-dependent manner. Based on the suppression of the GMM phenotype, the physical interaction between Lid and dMyc, and the requirement of Lid for dMyc-dependent activation of Nop60B, it is predicted that Lid acts as a dMyc coactivator involved in cell growth. The interaction between endogenous Lid and dMyc proteins is also likely to be essential for normal larval development since reducing the gene dose of lid is lethal in combination with the dmyc hypomorphic allele dmP0. In addition, genetically reducing lid enhances a small bristle phenotype induced by expression of a dMyc RNAi transgene. The original small discs phenotype described for lid mutants also suggests a role for Lid in the regulation of cell growth or proliferation during larval development. Unfortunately, this phenotype occurs at a frequency far too low (<1% of lid mutant larvae) to allow characterization (Secombe, 2007).

It is expected that the function of the Lid-Myc complex is evolutionarily conserved, since the human orthologs of Lid, Rbp-2 (JARID1A) and Plu-1 (JARID1B), bind strongly to c-Myc and dMyc in vitro, and both have been implicated in transcriptional regulation. Originally described as a binding partner for the tumor suppressor protein Retinoblastoma (RB), Rpb-2 has been shown to behave as a coactivator for RB at some promoters while antagonizing RB function at others (Benevolenskaya, 2005). Rbp-2 has also been identified as a transcriptional coactivator for nuclear hormone receptors (NRs) (Chan, 2001) and for the LIM domain transcription factor Rhombotin-2 (Mao, 1997). In addition, Plu-1 acts as a transcriptional corepressor for BF1 and PAX9 (Lu, 1999; Tan, 2003). While the transcriptional repression activities of Rbp-2 and Plu-1 are likely to be linked to a conserved trimethyl H3K4 demethylase activity, the molecular mechanism by which they activate transcription remains unclear. The mechanism by which Lid functions is currently being addressed by carrying out genetic screens using phenotypes generated by gain or loss of lid function (Secombe, 2007).

Coimmunoprecipitation analyses revealed that dMyc is likely to form multiple distinct complexes comprising TrxG proteins: One includes the Brm (SWI/SNF) nucleosome remodeling complex, and another contains Lid and Ash2. Consistent with the physical interaction observed between dMyc and Brm, components of the Brm complex suppress the GMM phenotype when genetically reduced, indicating that they are required for dMyc-induced cell growth. An interaction between Myc and the Brm complex has been observed in mammalian cells, where c-Myc interacts with the Brm (Brg1) subunit Ini1, and expression of a dominant-negative Brg1 allele inhibits c-Myc-dependent activation of a synthetic E-box reporter. However, the interaction between dMyc and the Brm complex described in this study, using Drosophila, provides the first demonstration of a biological significance for this complex (Secombe, 2007).

The second dMyc-TrxG complex identified includes Lid and Ash2, with Ash2 being immunoprecipitated with both anti-dMyc and anti-Lid antisera. In addition, decreased levels of Ash2 suppress, and increased levels of Ash2 levels enhance, the GMM phenotype, suggesting that Lid and Ash2 are limiting for dMyc-induced cell growth. In Schizosaccharomyces pombe, the orthologs of Ash2 and Lid (Ash2p and Lid2p) interact in vivo. While Ash2 has no known enzymatic activity, it is an integral component of several conserved complexes, including the SET1 histone methyltransferase complex (TAC1 in Drosophila; MLL in mammals) that is essential for methylation of histone H3K4. Biochemical purification of SET1, Lid2p, and Ash2p complexes from S. Pombe has demonstrated that the Lid2p-Ash2p complex is distinct from the SET1-Ash2 complex. Reducing the gene dose of the SET1 ortholog trx does not affect the GMM phenotype, consistent with the Drosophila Lid-Ash2-dMyc complex also being independent of TAC1 methyltransferase complex. The observation that Ash2 is a component of both H3K4 methylating (MLL) and demethylating (Lid) complexes is intriguing and suggests that it may be a crucial modulator of H3K4 methylation status. Whether Ash2 is required for Lid-mediated H3K4 demethylation is currently being tested (Secombe, 2007).

Lid is the first enzyme characterized that specifically demethylates trimethylated histone H3K4 in vivo. Based on the similarity between Lid and its mammalian orthologs Rbp-2 and Plu-1, it is expected that this demethylase activity to be conserved. The enzymatic activity of Lid requires a functional JmjC domain; however, Lid's specificity for a trimethylated lysine target is likely to be determined by the presence of a conserved N-terminal JmjN domain. Evidence to date suggests that proteins that possess both a JmjN and a JmjC domain prefer di- or trimethylated lysine substrates, whereas JmjC proteins that lack a JmjN domain demethylate mono- or dimethylated lysines. Indeed, analysis of the crystal structure of JMJD2A, which targets trimethylated H3K9 and K36, has revealed that the JmjN domain makes extensive contacts within the catalytic core of the JmjC domain, presumably accounting for the differences in target specificity between JmjC and JmjN/JmjC proteins (Secombe, 2007).

Trimethylated H3K4 is often found surrounding the transcriptional start site of active genes and is strongly correlated with binding by c-Myc. The trimethyl H3K4 demethylase activity of Lid would predict that Lid/Rbp-2 proteins may act as transcriptional repressors in a similar manner to LSD1, which demethylates mono- and di-methylated H3K4. Consistent with this hypothesis, it is observed that a large number of genes are derepressed in microarrays of homozygous lid mutant wing discs. However, expression of dMyc abrogates Lid's enzymatic activity, indicating that Lid is not acting as a demethylase when bound to dMyc. This is consistent with the observation that expression of Lid-JmjC* (a Gal4-inducible form of full-length Lid containing Ala substitutions at His637 and Glu639 that abolishes the protein's ability to bind the Fe2+ cofactor required for demethylase activity) enhances the GMM eye phenotype. Indeed, Lid behaves as a dMyc coactivator based on the requirement for Lid in dMyc-induced expression of the growth regulator Nop60B. Both activation and repression functions have been previously suggested for Rbp2. Interestingly, LSD1's demethylase activity is also negatively regulated by an associated protein, BHC80, in a similar manner to the inhibition of Lid's enzymatic activity by dMyc. Dynamic regulation of histone demethylase activity is therefore likely to be a common feature of regulated gene expression in vivo (Secombe, 2007).

Recently, analysis of c-Myc target gene promoters revealed a strong dependence on trimethylated H3K4 for E-box-dependent c-Myc binding. Based on this observation, it is tempting to speculate that although Lid is likely to be enzymatically inactive when complexed with dMyc, Lid may retain its ability to recognize trimethylated H3K4 (perhaps through its JmjN domain) and thereby facilitate appropriate E-box selection. The inhibition of Lid demethylase activity by dMyc may also result in maintenance of local H3K4 trimethylation to permit binding of additional dMyc molecules or other transcription factors. The maintenance of trimethylated H3K4 by dMyc may allow binding of the NURF chromatin remodeling complex that specifically recognizes trimethylated H3K4. NURF binding, through its large BPTF subunit, has been correlated with spatial control of Hox gene expression and is thought to link H3K4 methylation to ATP-dependent chromatin remodeling. Finally, considering the fact that Lid contains multiple domains potentially involved in DNA binding and protein interaction, it is likely that interaction of Lid/Rbp-2 with Myc in Drosophila and mammalian cells will promote association of other proteins with the Myc-Lid complex, allowing further diversification of Myc function (Secombe, 2007).

Functional antagonism between histone H3K4 demethylases in vivo

Dynamic regulation of histone modifications is critical during development, and aberrant activity of chromatin-modifying enzymes has been associated with diseases such as cancer. Histone demethylases have been shown to play a key role in eukaryotic gene transcription; however, little is known about how their activities are coordinated in vivo to regulate specific biological processes. In Drosophila, two enzymes, dLsd1 [(Suppressor of variegation 3-3), Drosophila ortholog of lysine-specific demethylase 1)] and Lid (little imaginal discs), demethylate histone H3 at Lys 4 (H3K4), a residue whose methylation is associated with actively transcribed genes. These studies show that compound mutation of Lid and dLsd1 results in increased H3K4 methylation levels. However, unexpectedly, Lid mutations strongly suppress dLsd1 mutant phenotypes. Investigation of the basis for this antagonism revealed that Lid opposes the functions of dLsd1 and the histone methyltransferase Su(var)3-9 in promoting heterochromatin spreading at heterochromatin-euchromatin boundaries. Moreover, the data reveal a novel role for dLsd1 in Notch signaling in Drosophila, and a complex network of interactions between dLsd1, Lid, and Notch signaling at euchromatic genes. These findings illustrate the complexity of functional interplay between histone demethylases in vivo, providing insights into the epigenetic regulation of heterochromatin/euchromatin boundaries by Lid and dLsd1 and showing their involvement in Notch pathway-specific control of gene expression in euchromatin (Di Stefano, 2011).

Molecular studies have identified an increasingly large number of histone-modifying enzymes, and biochemical assays readily allow these proteins to be classified, but the more difficult and more important challenge is to understand how these various enzymatic activities are integrated, in vivo, to control biological processes. This study examined the effects of combining mutations in the two H3K4 demethylases Lid and dLsd1 in Drosophila. Thise studies, performed in vivo, show that the interplay between Lid and dLsd1 is dependent on the chromatin context and active signaling pathways. The results show a consistent pattern of genetic interactions between Lid and dLsd1 that is evident in multiple tissues and phenotypes. Unexpectedly, despite their activity as histone H3K4 demethylases, these proteins function antagonistically in a number of functional and developmental contexts. For example, dLsd1 and Lid have opposing functions in the establishment of euchromatin and heterochromatin boundaries. At these locations, the antagonism does not seem to stem from the effects of Lid on H3K4 methylation, but rather from its indirect effects on the spreading of H3K9me2. In addition, while the data show that both Lid and dLsd1 can repress Notch targets within euchromatin when Notch signaling is not active, and that Notch signaling is an important component of the dLsd1 mutant phenotype, genetic evidence supports the hypothesis that Lid and dLsd1 have antagonistic functions in the context of activated Notch signaling. This complex pattern of interactions illustrates that the functional interplay between demethylases, and most likely between other types of chromatin-associated proteins, cannot be rationalized into a single generic model. The evidence that dLsd1 can switch from being a negative regulator of Notch target genes to a positive regulator adds an extra layer of complexity to the interplay between Lid and dLsd1, and strongly supports the concept that the activity of histone demethylases is highly regulated and context-dependent (Di Stefano, 2011).

Genetic and biochemical data support a model for the creation and maintenance of heterochromatin boundaries, proposed by (Rudolph, 2007), in which dLsd1 promotes deacetylation of H3K9 by RPD3 and subsequent methylation of H3K9 by Su(var)3-9, thereby facilitating spreading of heterochromatin. In addition, this study shows an increase in H3K4me1 at the white-rough-est locus in dLsd1 mutant flies, suggesting that active demethylation of H3K4me1 by dLsd1 is an important step in the establishment of heterochromatin. Furthermore, it was found that Lid antagonizes dLsd1 function by promoting euchromatin formation, and that the spreading of heterochromatin seen in Lid mutants is dependent on dLsd1 and Su(var)3-9 activities. Consistent with this notion, H3K9 methylation levels are increased in Lid mutant flies compared with control at the white-rough-est locus and in pericentric heterochromatin. Interestingly, the levels of H3K4me2 and H3K4me3 at the white-rough-est locus are very low and increase only marginally upon Lid mutation, suggesting that Lid function in this context is independent of its histone H3K4 demethylase activity. Previously, Lid had been reported to facilitate activation of Myc target genes in a demethylase-independent manner, and to antagonize Rpd3 histone deacetylase function; moreover, mutation of Lid has been shown to cause a decrease of H3K9 acetylation levels. It is therefore tempting to speculate that Lid opposes the spreading of heterochromatin, independent of its function as a histone H3K4 demethylase, by antagonizing the activity of the dLsd1/Su(var)3-9/Rpd3 complex. This antagonism would explain why, in double mutants for dLsd1 and Lid, the balance between euchromatin and heterochromatin is artificially reset to wild-type levels. Consistently, reorganization of chromatin domains observed in dLsd1 mutant flies affects the expression of genes located at the 2R euchromatin-heterochromatin boundary, an effect that is reversed by mutation of Lid (Di Stefano, 2011).

Given the predominant presence of H3K4 methylation in euchromatin and its important role in determining the transcription status of a gene, it was of interest to establishing the nature of the interplay between Lid and dLsd1 in a euchromatic context. Previous studies had implicated Lid as a crucial factor in the silencing of Notch target genes. The current study shows a cooperative role for Lid and dLsd1 in repressing Notch target gene expression, and suggests that they contribute to repression by maintaining low levels of H3K4 methylation. Repression of Notch target genes is essential for the establishment of Notch-inhibited cell fates, suggesting that Lid and dLsd1 could play a role in proper cell fate specification during Drosophila development. Interestingly, the role of dLsd1 does not seem to be limited to repression of Notch target genes. Indeed, genetic analysis suggests that, in a context in which the Notch signaling pathway is active, dLsd1 switches from a repressor to an activator role. Such a dual role had already been described for Su(H), whose switch from a repressor to an activator has been suggested to be mediated through an exchange of associated proteins. Similarly, in mammalian cells, studies have shown that LSD1 activity can be modulated by changes in composition of the complexes present at the Gh promoter, and, depending on the cell type (somatotroph or lactotroph), LSD1 can act as either an activator or a repressor. Therefore, a possible explanation for the current data is that, depending on the complexes available, dLsd1 can switch from being a repressor to acting as an activator of Notch target genes. Alternatively, dLsd1 mutation could promote derepression of negative regulators of Notch activity, or could directly modulate Notch activity by demethylating crucial components of the Notch-activating complex. Further studies are required to distinguish between these possibilities (Di Stefano, 2011).

These results provide the basis for future studies aimed at investigating whether the dual role of dLsd1 in modulating Notch signaling is conserved in mammals. In mice, LSD1 has been shown to repress the Notch target Hey1 in late stages of pituitary development, suggesting that its ability to regulate Notch target genes is conserved. This pathway-specific function of LSD1 could potentially be exploited to create novel strategies to manipulate Notch-mediated carcinogenesis (Di Stefano, 2011).

Collectively, these results reveal an intricate interplay between the histone demethylases Lid and dLsd1 in the control of higher-order chromatin structure at euchromatin and heterochromatin boundaries affecting developmental gene silencing. They also demonstrate an involvement of dLsd1 and Lid in Notch pathway-specific control of gene expression in euchromatin, and support the idea that, depending on the context, Lid and dLsd1 can favor either transcriptional activation or transcriptional repression (Di Stefano, 2011).

dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes

H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3 was analyzed. ChIP-seq (Little imaginal discs) results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling shows that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol lloser5) form. The results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription (Lloret-Llinares, 2012).

This study reports that dKDM5/LID localizes at TSS of developmental genes and regulates H3K4me3. dKDM5/LID target genes are actively transcribed and, though weakly, they are significantly downregulated in lidRNAi mutant flies. Previous reports already suggested a positive contribution of dKDM5/LID to transcriptio. The current results also show that dKDM5/LID target genes are bound by ASH2, an evolutionarily conserved component of H3K4-KMT2 complexes that localizes at TSS and is required for H3K4me3. In addition, dKDM5/LID target genes are strongly downregulated in ash2 mutant flies. These observations indicate that dKDM5/LID and ASH2 act coordinately to regulate H3K4me3 at TSS of developmental genes for their efficient transcription. dKDM5/LID and ASH2, however, do not bind chromatin simultaneously, indicating that they act at different moments during transcription. These observations strongly favor a model by which ASH2 and dKDM5/LID act sequentially during transcription to facilitate its progression. On this regard, work performed in budding yeast links chromatin modification events to sequential RNApol II activation. At a first step, TFIIH-mediated phosphorylation of CTDSer5 recruits scKMT2/SET1 to methylate H3K4, and induces promoter escape. Later, the onset of productive transcription involves phosphorylation of CTDSer2, which results in recruitment of H3K36 KMT3/SET2 both in budding yeast and mammals. dKDM5/LID recruitment might also be regulated during transcription cycle progression. In this context, it is possible that, after RNApol II activation and subsequent H3K4-methylation, dKDM5/LID is recruited and transient demethylation resets chromatin to the original 'unmethylated' state, facilitating the next RNApol II molecule to initiate progression through the transcription cycle. Consistent with this model, it was shown that the C-terminal PHD-finger of dKDM5/LID, or the mammalian homolog KDM5A/JARID1A, specifically binds H3K4me2,3 (Wang, 2009) and, furthermore, this study has shown that dKDM5/LID binds chromatin enriched in H3K4me3, whereas chromatin bound by ASH2 is poor in H3K4me3. Finally, the results also show that dKDM5/LID depletion significantly reduces RNApol ll occupancy, in particular by the promoter-proximal Pol IIoser5 active form, providing a basis for the positive contribution of dKDM5/LID to transcription. In contrast, occupancy by the elongating Pol IIoser2 form is not similarly affected, showing a tendency to be slightly increased. It is possible that, in the absence of dKDM5/LID, constitutive/increased H3K4me3 at TSS affects RNApol II pausing and, hence, transcription efficiency. Actually, it has been shown that depletion of NELF, a factor required for RNApol II pausing, results in a general downregulation of its target genes both in Drosophila and human cells (Lloret-Llinares, 2012).

Several reasons could account for the weakness of the observed effect of dKDM5/LID depletion on gene expression. On one hand, though dKDM5/LID content is strongly reduced in lidRNAi, depletion is not complete. Note that null lid mutations could not be used, as they are lethal during late embryo/early larvae development. Second, although dKDM5/LID is the only enzyme known to specifically demethylate H3K4me3 in Drosophila, additional KDMs might exist capable of playing a similar function. At this respect, it was reported that dKDM2, which was originally found to demethylate H3K36me2, might also be capable of demethylating H3K4me3. Thus, it is possible that loss of dKDM5/LID is partially compensated by dKDM2. As a matter of fact, a genetic interaction was recently reported between dKDM5/lid and dKDM2 (Lloret-Llinares, 2012).

The proposed function of dKDM5/LID in the regulation of transcription is likely conserved, as it was recently reported that mammalian KDM5B/JARID1B preferentially localizes at TSS of developmental genes and regulates H3K4me3. Mammalian KDM5C/JARID1C has also been shown to bind at TSS. Interestingly, although KDM5B/JARID1B is required to efficiently silence stem and germ cell specific genes during neuronal differentiation, its depletion in ESCs shows also a weak downregulation of target genes. In Drosophila, dKDM5/LID has also been shown to be involved in repression of some developmental genes. In fact, in the wing imaginal disc, ∼20% of dKDM5/LID target genes show no detectable H3K4me3. Altogether, these observations suggest that dKDM5/LID might play a dual function; repressing specific genes during development and, in differentiated cells, regulating H3K4me3 dynamics at TSS during transcription (Lloret-Llinares, 2012).

Histone lysine demethylases function as co-repressors of SWI/SNF remodeling activities during Drosophila wing development

The conserved SWI/SNF chromatin remodeling complex uses the energy from ATP hydrolysis to alter local chromatin environments through disrupting DNA-histone contacts. These alterations influence transcription activation, as well as repression. The Drosophila SWI/SNF counterpart, known as the Brahma or Brm complex, has been shown to have an essential role in regulating the proper expression of many developmentally important genes, including those required for eye and wing tissue morphogenesis. A temperature sensitive mutation in one of the core complex subunits, SNR1 (SNF5/INI1/SMARCB1), results in reproducible wing patterning phenotypes that can be dominantly enhanced and suppressed by extragenic mutations. SNR1 functions as a regulatory subunit to modulate chromatin remodeling activities of the Brahma complex on target genes, including both activation and repression. To help identify gene targets and cofactors of the Brahma complex, advantage was taken of the weak dominant nature of the snr1E1 mutation to carry out an unbiased genetic modifier screen. Using a set of overlapping chromosomal deficiencies that removed the majority of the Drosophila genome, genes were sought that when heterozygous would function to either enhance or suppress the snr1E1 wing pattern phenotype. Among potential targets of the Brahma complex, components were identified of the Notch, EGFR and DPP signaling pathways important for wing development. Mutations in genes encoding histone demethylase enzymes were identified as cofactors of Brahma complex function. In addition, it was found that the Lysine Specific Demethylase 1 gene (lsd1) was important for the proper cell type-specific development of wing patterning (Curtis, 2011).

Although chromatin remodeling is an important component of gene activation, its role in gene repression is not as well understood. The unbiased genetic screen using a weak dominant temperature sensitive mutant allele of a key Brm complex regulatory subunit has provided new insights into the involvement of chromatin remodeling complexes in developmental tissue patterning. Mutations in components of several signaling pathways, including Notch, EGFR and DPP/TGFβ, genetically interacted in these assay. These results, combined with candidate gene genetic analyses, have confirmed a previous hypotheses that the Brm complex participates in both gene activation and gene repression to help coordinate several key signaling pathways that lead to proper animal patterning. The results are largely concordant with the results of previous limited screens that identified a set of dominant modifiers of brmK804R mutant phenotypes. Among 14 chromosomal deficiencies that enhanced the brmK804R rough eye phenotype, this study found that 6/14 were also dominant enhancers of the snr1E1 wing phenotype and 3/14 were suppressors, suggesting that dominant modifier screens are effective tools for identifying unknown loci important for Brm complex regulatory functions. Consistent with this view, the Brm complex has been shown to interact the Notch ligand, Delta, in the developing fly eye. The genetic modifier screen results presented in this study indicate that Notch signaling functions may also be mediated through the Brm complex in the developing fly wing. Given the strong evolutionary conservation of these pathways, it is anticipated that the vertebrate SWI/SNF orthologs will play a similarly important role in patterning the tissues of vertebrate animals (Curtis, 2011).

What are the target genes regulated by the Brm complex in the developing wing? Previous studies have found that loss of snr1 function results in ectopic dpp and rhomboid expression in intervein cells. These data are consistent with the genetic interactions shown in this report that were observed using mutants affecting both the DPP and EGFR pathways. These studies have additionally provided an important insight into gene regulatory factors beyond signaling pathways that contribute to transcription repression in collaboration with chromatin remodeling complexes at key points in the development and differentiation of tissues. In the present analyses, several lines of evidence are provided suggesting that the mechanism of Brm complex-mediated gene repression is not only dependent upon a tight, physical and genetic relationship between two core subunits, SNR1 and MOR, but also on histone lysine demethylase enzymes (Curtis, 2011).

It has been reported that the full in vitro chromatin remodeling activity of the mammalian BRM/BRG1 complex on reconstituted nucleosomes can be accomplished with a subset of three or four core components, including the SNF5 (SNR1), BAF155/BAF170 (MOR) and BRM/BRG1 ATPase subunits that are highly conserved from yeast to vertebrates. Each of these subunits is required for complex stability in vivo as RNAi depletion of the individual components in cultured Drosophila cells leads to reduced stability of the other subunits with corresponding changes in target gene expression. Loss of BRM function in vivo, using either a dominant negative ATPase deficient mutant (brmK804R) or an amorphic allele (brm2), can suppress the snr1E1 wing phenotype revealing an important role for SNR1 in restraining Brm complex transcription activation functions. In contrast, mor mutants enhance mutant phenotypes associated with reduced brm function and show allele-specific interaction with snr1E1, suggesting an important functional relationship between the MOR, BRM and SNR1 subunits. MOR likely serves as a scaffolding protein, since physical associations were observed between SNR1-MOR and MOR-BRM. Two independent domains of MOR, the SWIRM and SANT, domains respectively, are critical for the binding interaction. Therefore, the contribution of SNR1 regulatory function on Brm complex chromatin remodeling activities may depend on crosstalk through MOR since no direct physical contacts between SNR1 and the BRM subunit have been observed (Curtis, 2011).

An unbiased dominant modifier genetic screen allowed identification of histone lysine demethylase enzymes as novel coregulators of the Brm complex in controlling gene expression. Previous screens looking for modifiers of a brm dominant negative allele (brmK804R) did not uncover mutations in histone-modifying families, such as acetyltransferases, deacetylases, and methyltransferases. However, the wing patterning defect associated with snr1E1 is highly sensitive, allowing observation of subtle changes in remodeling activities, and identification a family of epigenetic modifiers as potential Brm regulators. Previous studies have found that histone deacetylases (HDACs) were important corepressors that worked in direct collaboration with the Brm complex. In the present study, mutations in predicted demethylase genes genetically interacted with snr1E1 and LSD1 was shown to associate with the Brm complex in vivo, suggesting demethylases are also potential cofactors. While a functional cooperation between histone deacetylation and demethylation activities has been suggested previously, the current data implicates at least three chromatin modifying activities—ATP-dependent chromatin remodeling, histone deacetylation and demethylation—cooperating to regulate tissue-specific gene repression through multiple bridging interactions. In this scenario, the commitment of a gene promoter to be repressed in a cell type-specific manner would depend on the collateral influence of several chromatin modifying activities that would serve to help establish a repressed transcriptional environment, refractory to the influence of signaling pathways operational in adjacent cells (Curtis, 2011).

There appears to be no correlation between the predicted demethylase lysine substrate and enhancement/suppression of the snr1E1 phenotype. This is not surprising, since a high degree of functional redundancy exists amongst demethylase enzymes. It is likely that multiple demethylase enzymes cooperate to regulate a variety of target genes. This is supported by experimental evidence showing that knockdown experiments of individual demethylases, for example lsd1, in cell culture often showed little or no change in global methylation status, though significant changes were observed on a gene-specific level in vivo. Independent loss of function mutations in two JARID family members, lid and Jarid2/CG3654, resulted in an opposite genetic interaction with snr1E1. This study observed that a loss of function mutation in lid, (lid2) dominantly suppressed, whereas a loss of function mutation in Jarid2 (CG3654EY02717) enhanced the ectopic vein phenotype associated with snr1E1. LID is an H3K4me3/me2 specific demethylase. JARID2 is predicted to have the same substrate specificity, though overexpression analyses in cell culture experiments showed no global increase in H3K4me3/2. The observed opposite genetic interaction with snr1E1 may reflect differences in target gene regulation by LID and JARID2, either as a consequence of different target genes controlled in the developing wing or through opposite mechanisms in controlling gene transcription. Importantly, JARID2 homologs in Xenopus and mammalian model systems physically associate with the Polycomb Repressor Complex-2 (PRC2) and directly contribute to transcriptional repression by preventing the methylation of the histone lysine residues correlated with transcriptional activation. Therefore, mutation of JARID2 (CG3654EY02717) may enhance the snr1E1 phenotype if the normal role of CG3654 is to suppress transcription of a particular gene involved in wing vein development (Curtis, 2011).

The cell-fate decision to become vein or intervein is largely based on cell-type specific expression of transcription factors. In vein cells, transcription factors with gene targets that promote vein development are highly expressed, whereas those with gene targets that block vein fate are repressed. In intervein cells, the opposite is observed, with heightened expression intervein-promoting factors and decreased expression of vein promoting factors. The Brm complex has an important role in development of both cell fates, serving a positive role to promote vein development in vein cells, and repress vein development in intervein cells. The opposite genetic interaction phenotypes observed with lid and Jarid2 could be partially explained if the Brm complex is coordinating with the each specific demethylase to regulate different target genes. This study found that loss of function mutations in vein promoting genes, such as Egfr, suppressed the snr1E1 phenotype. The results suggest that LID and EGFR may regulate the expression of similar target genes and indeed EGFR (as well as other signaling pathways) may function in wing vein development through LID. In this scenario, a loss of function mutation in lid would result in a decrease in the expression of vein promoting genes, thereby suppressing the snr1E1 ectopic vein phenotype. Enhancement of the snr1E1 phenotype by Jarid2/CG3654EY02717 can be explained if JARID2 promotes activation of genes required to block vein differentiation, just as loss of function mutations in vein-inhibiting factors, such as net, enhanced the snr1E1 phenotype (Curtis, 2011).

The candidate genetic screen results suggest that histone lysine demethylase enzymes are likely cofactors of Brm chromatin remodeling activity. However, it is highly unlikely that stable physical associations are made between the complex and all six demethylases. The possibility cannot be eliminated that the Brm complex and demethylase enzymes are independently regulating genes involved in wing patterning or eliciting their functions on different targets at different times during development to contribute to the final read-out of vein/intervein patterning in the adult wing. However, a direct physical association was detected between the Brm complex and LSD1 in coimmunoprecipitation and GST-pulldown experiments, implying that LSD1 is a potential cofactor of Brm complex remodeling activities (Curtis, 2011).

The genetic epistasis experiments demonstrated an important in vivo functional relationship between LSD1 and the core subunits of the Brm complex, SNR1, MOR, and BRM. Brm complexes can be subdivided into two groups: PBAP complexes contain BAP170, POLYBROMO/BAP180, and SAYP, whereas BAP complexes contain OSA. These complexes can regulate target genes in a synergistic, antagonistic, or independent manner. BAP and PBAP complexes likely have differential regulatory functions, since they have distinct, but overlapping, localization patterns on larval salivary gland polytene chromosomes and targeted knockdown of OSA, POLYBROMO, or BAP180 using RNAi in cultured Schneider cells, leads to differential expression profiles on whole genome arrays. OSA, BAP170, BAP180, and SAYP likely have different roles in development, as mutation of each leads to different abnormalities. For example, BAP180 is required for proper egg shell development, whereas BAP170 is necessary to stabilize BAP180, important for adult viability, and vein cell differentiation. OSA is necessary for photoreceptor development, normal embryonic segmentation, and wing patterning. BAP, but not PBAP complexes have an important role in regulating cell cycle progression through mitosis (Curtis, 2011).

In mice, knockout of Baf180 causes misregulation of retinoic acid receptor target genes and heart developmental defects, indicating that PBAP complexes may have a role in nuclear receptor transcriptional regulation. The LSD1 corepressor complex, including the cofactor proteins, CoREST (see Drosophila CoREST), and histone deacetylase, HDAC1/2, have also been indicated in nuclear receptor transcriptional regulation. LSD1 association in complexes containing the Estrogen Receptor (ER) or Androgen Receptor (AR) leads to a switch in methylated lysine specificity, and results in demethylation of mono- and dimethylated H3K9 and gene activation (Curtis, 2011).

It is not known how BAP vs. PBAP complexes are differentially recruited to target genes. Recruitment of BAP complexes to specific target genes may depend on the physical associations made by OSA and sequence-specific transcription factors. For example, OSA is required for expression of target genes associated with the transcription factors Pannier and Apterous and can promote transcriptional repression of genes regulated by Wnt/Wingless signaling. Genetic epistasis experiments reveal that LSD1 cooperates with PBAP, but not BAP containing complexes in the Drosophila wing, suggesting that the physical association observed between LSD1 and Brm complex may be limited to PBAP complexes and provide a mechanism for selective target gene recruitment and regulation by Brm remodeling complexes. Further analyses, such as GST-pulldown and coimmunoprecipitation experiments using PBAP specific components need to be performed to address this possibility (Curtis, 2011).

Ectopic vein development within intervein tissue can result from two different possibilities: 1) the loss of a factor necessary to block vein cell development, or 2) the gain of a factor that promotes vein cell differentiation. Knockdown experiments suggest LSD1/dCoREST functions through the first mechanism. Loss of LSD1/dCoREST throughout the entire developing wing imaginal disc resulted in the development of vein material in intervein tissue, but no changes in vein morphology were observed. If LSD1/dCoREST normally functioned to promote vein development, then loss throughout the entire wing should have led to a loss of vein phenotype (Curtis, 2011).

Several lines of evidence suggest that LSD1 may be capable of regulating gene transcription in a cell-type or stage dependent manner. The affect of homozygous loss of lsd1 on transcriptional regulation of known target genes, including the Sodium Channel and NicotinicAcetylcholine Receptor-β is minimal in embryos and larvae, but significant in pupae. This implies that LSD1 has an important role in regulating gene transcription during later developmental stages. Moreover, LSD1 negative regulation of the homeobox genes, Ultrabithorax (Ubx) and abdominal-B (abd-B) continues into adulthood, as lsd1 null animals display significantly increased expression of these genes as the animals continue to age. This stage-dependent requirement appears to be conserved, as the conditional knock-out of LSD1 in the developing mouse pituitary gland causes little or no morphological defects early in pituitary development (E9-9.5), but significantly alters cell-fate determination choices during later stages (E17.5). Furthermore, LSD1 mediates both gene activation and gene repression of different target genes by associating with several multisubunit complexe (Curtis, 2011).

Knockdown and genetic epistasis experiments further support the idea that LSD1 is important for regulating terminal differentiation, since patterning phenotypes are similar to those observed with defects in DPP and EGFR signaling, the pathways active during pupal development, rather than observed with defects in HH signaling, an early pathway component. Previous work has demonstrated an important role in Brm complex involvement in EGFR, DPP, and Delta/N signaling. More recently, it has been demonstrated that OSA, the defining subunit of the BAP complex, is required to activate EGFR targets in the developing wing. In this regard, the Brm complex may be cooperating with LSD1 to regulate several conserved signaling pathways, but this cooperation may be tissue and developmental time-point dependent (Curtis, 2011).

Alcohol-induced behaviors require a subset of Drosophila JmjC-domain histone demethylases in the nervous system

Long-lasting transcriptional changes underlie a number of adaptations that contribute to alcohol use disorders (AUD). Chromatin remodeling, including histone methylation, can confer distinct, long-lasting transcriptional changes, and histone methylases are known to play a role in the development of addiction. Conversely, little is known about the relevance of Jumonji (JmjC) domain-containing demethylases in AUDs. This study systematically surveyed the alcohol-induced phenotypes of null mutations in all 13 Drosophila JmjC genes. A collection of JmjC mutants, the majority of which were generated by homologous recombination, and assayed in the Booze-o-mat to determine their naive sensitivity to sedation and their tolerance (change in sensitivity upon repeat exposure). Mutants with reproducible phenotypes had their phenotypes rescued with tagged genomic transgenes, and/or phenocopied by nervous system-specific knock down using RNA interference (RNAi). Four of the 13 JmjC genes (KDM3, lid, NO66 and HSPBAP1) showed reproducible ethanol-sensitivity phenotypes. Some of the phenotypes were observed across doses, e.g. the enhanced ethanol-sensitivity of KDM3KO and NO66KO, but others were dose-dependent, such as the reduced ethanol sensitivity of HSPBAP1KO, or the enhanced ethanol tolerance of NO66KO. These phenotypes were rescued by their respective genomic transgenes in KDM3KO and NO66KO mutants. While this study could not rescue lidk mutants, knock down of lid in the nervous system recapitulated the lidk phenotype, as was observed for KDM3KO and NO66KO RNAi-mediated knock down. This study reveals that the Drosophila JmjC-domain histone demethylases Lid, KDM3, NO66, and HSPBAP1 are required for normal ethanol-induced sedation and tolerance. Three of three tested of those four JmjC genes are required in the nervous system for normal alcohol-induced behavioral responses, suggesting that this gene family is an intriguing avenue for future research (Pinzon, 2017).

The histone demethylase KDM5 is essential for larval growth in Drosophila

In Drosophila, the larval prothoracic gland integrates nutritional status with developmental signals to regulate growth and maturation through the secretion of the steroid hormone ecdysone. While the nutritional signals and cellular pathways that regulate prothoracic gland function are relatively well studied, the transcriptional regulators that orchestrate the activity of this tissue remain less characterized. This study shows that lysine demethylase 5 (KDM5) is essential for prothoracic gland function. Indeed, restoring kdm5 expression only in the prothoracic gland in an otherwise kdm5 null mutant animal is sufficient to rescue both the larval developmental delay and the pupal lethality caused by loss of KDM5. These studies show that KDM5 functions by promoting the endoreplication of prothoracic gland cells, a process that increases ploidy and is rate limiting for the expression of ecdysone biosynthetic genes. Molecularly, it was shown that KDM5 activates the expression of the receptor tyrosine kinase torso, which then promotes polyploidization and growth through activation of the MAPK signaling pathway. Taken together, these studies provide key insights into the biological processes regulated by KDM5 and expand understanding of the transcriptional regulators that coordinate animal development (Drelon, 2019).

This study demonstrates that KDM5 is essential for the function of the ecdysone-producing prothoracic gland during Drosophila larval development. Crucial to this conclusion was the finding that expressing kdm5 in the prothoracic gland was sufficient to rescue the lethality and developmental delay phenotypes of kdm5140> null allele homozygous mutant animals. Consistent with this observation, prothoracic gland function was defective in kdm5 mutants, with mutant larvae having low levels of ecdysone and reduced expression of downstream hormone-responsive target genes. Demonstrating the importance of KDM5-mediated regulation of ecdysone production, dietary supplementation of 20E restored normal developmental timing to kdm5 mutant larvae. At the cellular level, loss of KDM5 slowed the prothoracic gland endoreplicative cycles that increase the ploidy of these cells and are important for ecdysone biosynthesis. Restoring these endocycles by expressing cyclin E re-established normal developmental timing to kdm5140 mutants but was not able to rescue their lethality. In contrast, ectopic activation of the Torso/MAPK pathway that functions upstream of cyclin E was able to restore both developmental timing and rescue the lethality caused by loss of KDM5. It is therefore proposed that KDM5-mediated activation of the Torso/MAPK pathway in the prothoracic gland is important for larval growth regulation through its role in promoting polyploidization, and for adult eclosion by mechanisms that remain to be determined (Drelon, 2019).

Consistent with an important developmental role for KDM5-mediated regulation of Torso/MAPK, mutations in torso or ablation of the neurons that produce its ligand PTTH cause a 5-day delay to larval development similar to that observed for kdm5140. Because loss of KDM5 reduces, but does not eliminate, torso expression, the kdm5140 phenotype cannot be accounted for solely by the twofold change to Torso/MAPK signaling observed. The dysregulation of additional KDM5-regulated genes is therefore likely to contribute to the prothoracic gland phenotypes of kdm5 mutant larvae. Little is known about the transcriptional regulation of torso and other genes that make up the upstream pathways that regulate ecdysone production. One possibility is that KDM5 functions as a direct transcriptional activator of the torso gene: thus, decreased expression of this receptor would be expected in kdm5 mutant animals. Because of the small size of the prothoracic gland, it is not currently feasible to carry out ChIP experiments to examine KDM5 promoter binding in this tissue. It is, however, notable that the torso promoter was not bound by KDM5 in existing ChIP-seq datasets from larval wing imaginal discs or from whole adult flies. This could be because torso expression is largely restricted to the prothoracic gland during larval development and so may not be expected to have promoter-bound KDM5 in the tissues examined to date. Alternatively, KDM5 might regulate torso indirectly. In the silkworm Bombyx mori, expression of the torso gene is repressed in response to starvation conditions. Although the mechanism by which this occurs is unknown, it does indicate that other cellular defects caused by loss of KDM5 could lead to changes to torso transcription and subsequent decrease in MAPK activity and ecdysone production. Whether the regulation of torso by KDM5 is direct or indirect, it occurs in a demethylase-independent manner, as larvae lacking enzymatic activity show a normal developmental profile (Drelon, 2018). Consistent with this observation, components of KDM5 complexes that regulate gene expression through demethylase-independent mechanisms also affect developmental timing. For example, a development delay similar to that of kdm5140 is caused by RNAi-mediated knockdown of the histone deacetylase HDAC1 or the NuRD complex components asf1 and Mi-2. KDM5 could therefore interact with these proteins to regulate the expression of genes that are crucial to the regulation of larval development (Drelon, 2019).

Increased ploidy of prothoracic gland cells is important for optimal expression of steroidogenic genes and can be induced by activation of the Torso/MAPK pathway. Because the genes required for ecdysone biosynthesis are among the most abundantly expressed in the prothoracic gland, their mRNA levels may be entirely limited by gene copy number. A similar requirement for copy number amplification to produce peak gene expression levels has been observed in other cell types in Drosophila, including chorion gene expression in ovarian follicle cells. However, it is not known how Torso/MAPK activation promotes prothoracic gland cell cycle progression. One mechanism might be by affecting levels of cell cycle regulators such as the transcription factor E2f1, which is essential for both mitotic and endoreplicative cell cycles. This model is based on studies of the polyploid enterocytes of the adult midgut, in which activation of the MAPK pathway via the EGF receptor stabilizes E2f1 protein, leading to transcription activation of cyclin E. Although restoring correct endocycling in kdm5 mutant prothoracic glands was able to rescue their developmental timing, it did not impact their eclosion defect. This could be because loss of KDM5 leads to additional defects within the prothoracic gland that are ultimately detrimental to the function of this endocrine tissue, such as increased oxidative stress, which this study has shown to be affected in kdm5 hypomorphic mutant wing discs. Alternatively, KDM5 could have a cell cycle independent role in maintaining ecdysone levels during pupal development. kdm5140 mutant animals die as pharate adults that have no obvious morphological abnormalities but fail to eclose (Drelon, 2018). Nevertheless, these animals could have significant defects in, for example, nervous system development, which requires ecdysone and is important for eclosion (Drelon, 2019).

The observed role of KDM5 in the growth and polyploidization of larval prothoracic gland raises the possibility that it might play key roles in other cell types that use endoreplicative cycles. This could have broad consequences for understanding of KDM5 biology, as polyploidization is observed in many plant and animal cell types and is widely used during Drosophila larval development. In addition, while the role of polyploid cells in the etiology or maintenance of cancers remains a topic of ongoing research, KDM5-regulated endocycling could contribute to its tumorigenic activities in humans. Regulation of polyploidization in cells of the nervous system could also contribute to the link between KDM5 protein dysregulation and intellectual disability. This could, for example, be mediated by KDM5 function in glial cells, as polyploidization of superineurial glial cells in Drosophila is required for normal brain development. Although it is not clear the extent to which a similar phenomenon occurs during human brain development, it is interesting to note that glial cell types contribute to the severity of intellectual disability disorders such as Rett syndrome. Thus, although there is still much to be learned regarding the contribution of polyploid cells to normal development and to clinically relevant disorders, KDM5-regulated transcriptional programs could be key to the function of cells that use this variant cell cycle (Drelon, 2019).

The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing

Epigenetic chromatin marks restrict the ability of differentiated cells to change gene expression programs in response to environmental cues and to transdifferentiate. Polycomb group (PcG) proteins mediate gene silencing and repress transdifferentiation in a manner dependent on histone H3 lysine 27 trimethylation (H3K27me3). However, macrophages migrated into inflamed tissues can transdifferentiate, but it is unknown whether inflammation alters PcG-dependent silencing. This study shows that the JmjC-domain protein Jmjd3 is a H3K27me demethylase expressed in macrophages in response to bacterial products and inflammatory cytokines. Jmjd3 binds PcG target genes and regulates their H3K27me3 levels and transcriptional activity. The discovery of an inducible enzyme that erases a histone mark controlling differentiation and cell identity provides a link between inflammation and reprogramming of the epigenome, which could be the basis for macrophage plasticity and might explain the differentiation abnormalities in chronic inflammation (De Santa, 2007).

Since maintenance of cell identity and differentiation depends on H3K27me3 and Polycomb-enforced gene silencing, the existence of enzymes that can erase the methyl mark on which cellular memory relies is counterintuitive at first sight. It is even more counterintuitive the idea that the expression of such an enzyme can be induced by stimuli as common as bacterial components and inflammatory cytokines. In this regard it is important to notice the different properties of Utx, which is a constitutively expressed H3K27me3 histone demethylase (HDM), and Jmjd3, which is endowed with a potent HDM activity but is expressed mainly in an inducible and cell-type restricted fashion. Moreover, Utx escapes X inactivation, and the requirement for a biallelic expression is suggested by the presence in males of a highly related gene (Uty) encoded by the Y chromosome. The properties of the two H3K4me3 HDMs Smcx and Smcy are remarkably similar to those of Utx and Uty: they are both encoded by sex chromosomes, and Smcx escapes X inactivation. Overall, it is likely that Utx/Uty (and Smcx/Smcy) act as 'housekeeping' HDMs whose constant availability allows for continuous surveillance over global and gene-specific H3 Lys 27 (or H3K4) methylation. Conversely, tissue-specific and inducible expression of Jmjd3 restricts its field of competence to specific and well-defined developmental stages or functional states, thus limiting the intrinsic dangerousness of the increased availability of a potent H3K27me3 demethylase (De Santa, 2007).

Considering the risks, what is the advantage of the potential increase in epigenomic plasticity afforded by the elevation in Jmjd3 levels? A limited level of plasticity may allow cells to reprogram their properties according to environmental cues, which is a well-known event in flies (Lee, 2005). Tissue regeneration that follows injury requires an efficient reconstitution of the damaged parts, and in some cases the ability of resident stem cells to reconstitute the tissue may be exceeded. When such a deficiency occurs, restoration of tissue integrity may require the recruitment of circulating cells whose plasticity enables them to transdifferentiate and generate a variety of tissue-specific cell types. However, plasticity of recruited cells may itself represent a risk, as it may result in the generation of abnormally differentiated cells and even tumorigenesis. A striking example of the progression through chronic inflammation > recruitment of circulating cells > transdifferentiation and tumor development, is provided by a mouse model of chronic gastritis caused by Helicobacter Felis, a relative of the major cause of gastric cancer in humans, H. Pilori. In this model, chronic inflammation associated with bacterial infection causes extensive apoptosis of the gastric epithelium, followed by the invasion of the stomach by circulating bone marrow-derived cells (BMDCs). BMDCs proliferate in the inflamed stomach and undergo a transdifferentiation-like event known as metaplasia: although metaplasia is supposed to be an entirely epigenetic event not associated with mutations, it leads to displasia and eventually to cancer (Houghton, 2004 ) (De Santa, 2007).

Macrophages are among the most abundant cells in an inflamed tissue, and they may help tissue repair also by transdifferentiation. Although several claims of macrophage transdifferentiation in the past could be explained by cell fusion, the case of lymphoendothelial transdifferentiation in inflamed tissues is supported by strong experimental evidence. In a mouse model of corneal transplantation macrophages infiltrating the transplanted cornea start expressing lymphoendothelial markers and the fate-determining transcription factor Prox1, which is essential for lymphatic vessel development. Later on, macrophage markers are lost and infiltrating cells retaining only lymphoendothelial markers generate a new network of lymphatic vessels. Similarly, neo-lymphoangiogenesis in transplanted and rejected kidneys in humans depends on recipient-derived myeloid cells. Interestingly, the Prox1 promoter in macrophages bears both high H3K4me3 and H3K27me3, and transcription is undetectable. However, it was not possible to observe H3K27me3 demethylation and Prox1 gene reactivation in the conditions used, which suggests the requirement for additional stimuli present in inflamed tissues. Lymphoendothelial trans-differentiation of macrophages during inflammation may help removal of tissue debris and enhance transport of microbial components to lymph nodes for presentation to lymphocytes. Therefore, it may not represent an accidental event but a controlled and desirable outcome (De Santa, 2007).

The direct targets of Jmjd3 include late HoxA cluster genes in differentiating bone marrow cells and Bmp-2 in activated macrophages. While regulation of Hox genes tentatively identifies Jmjd3 as a component of the MLL system (an assumption supported by the inclusion of Jmjd3 in MLL complexes), regulation of Bmp-2 provides interesting mechanistic and biological clues. In macrophages the Bmp-2 gene promoter contains a bivalent chromatin domain with high H3K4me3 and H3K27me3 levels. It has been proposed that in ES cells genes in this configuration are silenced but poised for rapid activation when differentiation is triggered, which implies a functional dominance of the inhibitory mark (H3K27me3) over the active one (H3K4me3). In differentiated cells bivalent domains may have a more complex function in fine-tuning gene expression, as suggested by a genome-wide analysis in T lymphocytes: genes bearing H3K4me3 could be clustered according to their H3K27me3 levels in genes with low transcriptional activity (high H3K27me3), genes with high transcriptional activity (low H3K27me3), and finally genes with intermediate levels of H3K27me3 and intermediate degrees of activity. Therefore, not only the presence or absence of the H3K27me3 mark is relevant, but also its abundance relative to H3K4me3. The data on Bmp-2 are consistent with this model. Bmp-2 gene transcription is rapidly activated in both WT and Jmjd3-depleted cells, although initial activation is rather low. Between 8 and 24 hr poststimulation a sharp increase in Bmp-2 mRNA is observed, but only in WT cells, in which H3K27me3 levels at the Bmp-2 promoter are reduced in a Jmjd3-dependent manner (De Santa, 2007).

The observation that newly synthesized Jmjd3 is incorporated in RbBP5-containing complexes suggests a possible model for the control of histone marks at genes bearing a bivalent domain. An attractive possibility is that a constitutively bound MLL complex devoid of a H3K27me3 HDM component is exchanged for a Jmjd3-containing MLL complex generated after LPS stimulation, thus allowing H3K27me3 demethylation while keeping constant the levels of H3K4me3 (De Santa, 2007).

Bmp-2 is a morphogen essential for embryonic development. It controls cell growth and promotes tumorigenesis, and it induces the differentiation of mesenchymal cells into osteoblasts. Bmp-2 production by activated macrophages participates in healing of bone fractures but also underlies heterotopic bone formation in inflamed joints and other tissues. Therefore, control of macrophage production of Bmp-2 by Jmjd3 is an indirect mechanism by which H3K27me3 demethylation in activated macrophages can impinge on the physiology of inflamed tissues (De Santa, 2007).

In conclusion, these data support the conceptually challenging idea that a histone mark involved in the control of differentiation and in the maintenance of cellular memory can be erased by a rapidly inducible enzyme whose expression is regulated by common environmental stimuli (De Santa, 2007).

Coordinated regulation of transcriptional repression by the RBP2 H3K4 demethylase and Polycomb-Repressive Complex 2

Polycomb group (PcG) proteins regulate important cellular processes such as embryogenesis, cell proliferation, and stem cell self-renewal through the transcriptional repression of genes determining cell fate decisions. The Polycomb-Repressive Complex 2 (PRC2) is highly conserved during evolution, and its intrinsic histone H3 Lys 27 (K27) trimethylation (me3) activity is essential for PcG-mediated transcriptional repression. This study shows a functional interplay between the PRC2 complex and the H3K4me3 demethylase Rbp2 (Jarid1a) in mouse embryonic stem (ES) cells. By genome-wide location analysis it was found that Rbp2 is associated with a large number of PcG target genes in mouse ES cells. PRC2 complex recruits Rbp2 to its target genes, and this interaction is required for PRC2-mediated repressive activity during ES cell differentiation. Taken together, these results demonstrate an elegant mechanism for repression of developmental genes by the coordinated regulation of epigenetic marks involved in repression and activation of transcription (Pasini, 2008).

The best-described members of the JARID proteins are RBP2 and PLU1. PLU1 expression is progressively decreased in advanced and metastatic melanomas. Likewise melanoma cell lines in general exhibit low expression levels of RBP2 suggesting a possible role in tumorigenesis. To date no cellular function has been ascribed for SMCX and SMCY; however, studies have shown that mutations in SMCX are frequently found in patients with X-linked mental retardation, suggesting a role for these proteins in development (Pasini, 2008).

RBP2 was originally identified by virtue of its ability to bind the retinoblastoma protein (pRB). RBP2 has been suggested to function as a transcriptional repressor and to play a role in differentiation. Consistent with this, interference of RBP2 expression using siRNA results in the transcriptional upregulation of two homeotic genes BRD2 and BRD8 in U937 cells. However, in the presence of pRB, RBP2 appears to function as a coactivator of transcription, probably by either modulating or dissociating RBP2 from the target-gene promoter. The demonstration that RBP2 is an H3K4me3 demethylase taken together with the fact that it is dissociated from genes activated during differentiation strongly suggests that RBP2 is a transcriptional repressor. This suggestion is further supported by the observation that another JARID1 family member, PLU1, works as transcriptional repressor when fused to the DNA binding domain of GAL4 (Pasini, 2008).

Interestingly, the S. pombe RBP2 homolog, Lid2, copurifies with Ash2 and Sdc1 the yeast orthologs of human ASH2L and DPY30. Ash2 and Sdc1 are found in the yeast COMPASS H3K4 methyltransferase complex, thus providing the possibility that histone methyltransferases and demethylases with specificity for the same histone mark have common complex partners. Drosophila lid was initially identified in a genetic screen for identification of new members of the trithorax group. Interestingly, the screen was performed in a genetic background having a Trithorax gene mutation in Ash1, a histone methyltransferase with specificity for H3K4. Of further interest, Ash1 has been shown to be necessary for Hox gene expression. Taken together with the fact that Trithorax is required for the maintenance of Hox expression, these data suggest a role for RBP2 in the regulation of Hox genes. In agreement with this, it was found that RBP2 is specifically associated with several genes of the Hoxa cluster in murine ES cells (Pasini, 2008).

In this study, advantage was taken of the fact that C. elegans encodes only a single member of the JARID1 family. Interestingly, the results also point toward an important role for the C. elegans RBP2 homolog, rbr-2, in development and differentiation. The demonstration that deletion of the JmjC domain of RBR-2 or RNAi-mediated knockdown of rbr-2 results in a significant increase of H3K4me3 levels strongly suggests that RBR-2 is an important regulator of H3K4 trimethylation in vivo. Moreover, the fact that the functional inactivation of rbr-2 results in specific defects in vulva development suggests that the regulation of H3K4me3 levels is essential for normal differentiation. The stronger vulva phenotype observed in the rbr-2(tm1231) mutant may suggest that the residual protein levels in the RNAi-treated worms are sufficient for some stages of vulva development. In support of this suggestion, the increase in H3K4me3 levels in the RNAi-treated worms was not as pronounced as in the mutant animal (Pasini, 2008).

Modulation of H3K4 methylation has also been observed in other biological systems. One example is the circadian variation of the transcription of the albumin D-element binding protein gene (Dbp) in the mouse liver. Here, during transcriptional repressive phases, H3K4 trimethylation at the Dbp gene is markedly reduced in the matter of hours. X-chromosome inactivation (XCI) represents another example of a biological process featuring a rapid loss of H3K4 trimethylation. XCI occurs in the early embryonic development of female mammals, where epigenetic silencing of one X chromosome takes place to attain dosage parity between XX females and XY males. One of the earliest and most characteristic epigenetic features of XIC is loss of tri- and dimethylation of H3K4, concomitant with transcriptional silencing of the affected X-linked genes. The mechanism causing the loss of the activating H3K4me3 mark is presently unknown but has been speculated to involve demethylating enzymes (Pasini, 2008).

In mice, only a very limited number of genes have been reported to evade X-chromosome inactivation. Strikingly, two of the seven genes known to escape silencing are JmjC-domain proteins: the ubiquitously transcribed tetratricopeptide repeat gene on X chromosome (Utx) and Smcx. The human orthologs of these two genes likewise evade epigenetic silencing, and given the putative demethylating activity of these proteins it is tempting to speculate on their involvement in establishing or maintaining inactivation, perhaps through removal of the activating H3K4me3/me2 marks (Pasini, 2008).

A model is proposed for how RBP2 could be involved in regulating the transcription of genes during cellular differentiation. It is suggested that RBP2 is implicated in this modulation in two ways, either (1) by being released from genes that have been kept silenced, such as the Hox genes, which require activation during differentiation, or (2) by being recruited to genes that require silencing after cellular differentiation. Such genes may also include silent developmental genes carrying a bivalent H3K4me3/H3K27me3 epigenetic signature and that are 'poised for transcription'. In this way, RBP2 may contribute to transcriptional silencing by keeping the levels of H3K4me3 low. Thus, RBP2 could constitute one of several mechanisms by which chromatin-modifying enzymes, including histone acetylase/deacetylase complexes and the Polycomb group proteins, orchestrate the fine-tuned epigenetic regulation of genes during cellular differentiation (Pasini, 2008).

An open question is how RBP2 and other JARID1 proteins are recruited to certain chromatin areas. For PLU1 two interacting proteins, brain factor 1 (BF1) and paired box 9 (PAX9), both of which are developmental transcription factors, have been identified. BF1 and PAX proteins interact with members of the Groucho corepressor family, suggesting that PLU1 has a role in Groucho-mediated transcriptional repression. Thus, most likely, JARID1 proteins take part of specific chromatin-remodeling complexes homing to certain genomic areas. Alternatively, JARID1 proteins may bind directly to chromatin; in this context, it is pertinent to note that all JARID1 proteins contain an ARID/BRIGHT domain, which can bind to DNA. In addition, the JARID proteins feature PHD and C5HC2 zinc fingers, which may mediate interactions with specific histone marks. Further studies are required to unravel the link between JARID1 proteins and transcriptional regulation of developmental genes. It would also be of great interest to study the role of these proteins in stem cell differentiation, where epigenetic changes are important players (Pasini, 2008).

Jarid2/Jumonji coordinates control of PRC2 enzymatic activity and target gene occupancy in pluripotent cells

Polycomb Repressive Complex 2 (PRC2) regulates key developmental genes in embryonic stem (ES) cells and during development. Jarid2/Jumonji, a protein enriched in pluripotent cells and a founding member of the Jumonji C (JmjC) domain protein family, is a PRC2 subunit in ES cells. Genome-wide ChIP-seq analyses of Jarid2, Ezh2, and Suz12 binding reveal that Jarid2 and PRC2 occupy the same genomic regions. Jarid2 promotes PRC2 recruitment to the target genes while inhibiting PRC2 histone methyltransferase activity, suggesting that it acts as a 'molecular rheostat' that finely calibrates PRC2 functions at developmental genes. Using Xenopus laevis as a model, Jarid2 knockdown was shown to impair the induction of gastrulation genes in blastula embryos and results in failure of differentiation. These findings illuminate a mechanism of histone methylation regulation in pluripotent cells and during early cell-fate transitions (Peng, 2009).

Jarid2 and Jarid1a regions responsible for Suz12 binding do not overlap with any discernible structural domains and display low similarity, with the exception of a highly homologous short sequence 'GSGFP.' It is hypothesized that this motif may play a role in Suz12 recognition. Indeed, mutations of GSGFP to GAGAA diminished binding of Jarid2 and Jarid1a fragments to full-length Suz12. This motif is conserved in all vertebrate Jarid2 proteins, as well as in C. elegans Jarid2, whereas D. melanogaster and other Drosophila species contain a non-conservative substitution within the motif (GYGFP). The GSGFP motif is also conserved in all four Jarid1 family proteins: Jarid1a/RBP2, Jarid1b/PLU-1, Jarid1c/SMCX and Jarid1d/SMCY, as well as in the single Jarid1 homolog in Drosophila, Lid. The presence of the GSGFP motif in metazoan Jarid proteins suggests that the association with Suz12 may be a common feature of Jarid family members. However, the possibility that additional molecular interactions control Jarid-PRC2 complex formation in vivo cannot be excluded (Peng, 2009).

Jumonji modulates Polycomb activity and self-renewal versus differentiation of stem cells

Trimethylation on histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) regulates the balance between self-renewal and differentiation of embryonic stem cells (ESCs). The mechanisms controlling the activity and recruitment of PRC2 are largely unknown. This study demonstrates that the founding member of the Jumonji family, JMJ (JUMONJI or JARID2), is associated with PRC2, colocalizes with PRC2 and H3K27me3 on chromatin, and modulates PRC2 function. In vitro JMJ inhibits PRC2 methyltransferase activity, consistent with increased H3K27me3 marks at PRC2 targets in Jmj-/- ESCs. Paradoxically, JMJ is required for efficient binding of PRC2, indicating that the interplay of PRC2 and JMJ fine-tunes deposition of the H3K27me3 mark. During differentiation, activation of genes marked by H3K27me3 and lineage commitments are delayed in Jmj-/- ESCs. These results demonstrate that dynamic regulation of Polycomb complex activity orchestrated by JMJ balances self-renewal and differentiation, highlighting the involvement of chromatin dynamics in cell-fate transitions (Shen, 2009).

Histone chaperones ASF1 and NAP1 differentially modulate removal of active histone marks by LID-RPD3 complexes during NOTCH silencing

Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, the interaction networks of histone chaperones ASF1 (Anti-silencing factor 1), CAF1, HIRA, and NAP1 were identified. This study analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) Notch-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. It is concluded that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing (Moshkin, 2009).

Regulated modulation of the chromatin structure is essential for the transmission, maintenance, and expression of the eukaryotic genome. The combined actions of ATP-dependent chromatin-remodeling factors (remodelers), histone chaperones, and histone-modifying enzymes drive chromatin dynamics. Histones are subjected to a wide range of reversible posttranslational modifications, including acetylation, phosphorylation, methylation, and ubiquitylation. Histone modifications, in turn, can promote the recruitment of selective regulatory factors and modulate chromatin accessibility. Chromatin remodelers control DNA accessibility by mediating nucleosome mobilization either through sliding or by nucleosome (dis)assembly (Moshkin, 2009).

Whereas originally considered mainly as mere chaperones, it has become clear that histone chaperones play diverse roles during chromatin transactions. Histone chaperones bind selective histones and include the highly conserved H3/H4 chaperones ASF1, CAF1, HIRA, and Spt6 and the H2A/H2B chaperones NAP1, Nucleoplasmin, and FACT. Although their biochemical activity, binding and release of histones, appears rather mundane, in conjunction with other factors, histone chaperones participate in a variety of chromatin transactions and other cellular tasks. For example, yeast NAP1 participates in an extensive interaction network including a diverse set of transcription initiation/elongation factors, chromatin remodelers, RNA-processing factors, cell-cycle regulators, and other proteins (Moshkin, 2009).

ASF1 is one of the major H3/H4 chaperones, and through association with other proteins, it contributes to diverse chromatin transactions. (1) In conjunction with CAF1 and the MCM2-7 DNA helicase, ASF1 participates in replication-coupled chromatin assembly. (2) When associated with HIRA, ASF1 participates in replication-independent chromatin assembly and histone replacement. (3) DNA-repair-associated chromatin assembly requires the cooperation between ASF1 and the H3K56 acetyltransferase Rtt109. (4) ASF1 functionally cooperates with the Drosophila BRM chromatin remodeler, and (5) interaction of ASF1 with transcription activators stimulates histone eviction from promoter areas and facilitates recruitment of chromatin-specific coactivator complexes. (6) ASF1 itself is one of the targets of Tousled-like kinase (TLK), which controls cell-cycle progression and chromatin dynamics. (7) Finally, ASF1 is involved in developmental gene expression control by mediating transcriptional repression of Notch target genes. ASF1 is recruited to E(spl) genes by the sequence-specific DNA-binding protein Su(H) and its associated corepressor complex, harboring Hairless (H) and SKIP (Moshkin, 2009).

Notch is the central component of a highly conserved developmental signaling pathway that is present in all metazoans. Notch is a single-pass transmembrane protein that is activated through ligand binding, resulting in the release of the Notch intracellular domain (Nicd), which is targeted to the nucleus to activate gene expression. The CSL (CBF1, Su(H), and Lag1) family of sequence-specific DNA-binding proteins is the key targeting factor of Nicd and coactivators and, in the absence of Nicd, corepressors. The repression of Notch target genes involves multiple chromatin-modifying activities including histone deacetylases, H3K9 methyltransferases, CtBP, NcoR/SMRT, and Goucho (GRO). In the absence of the Nicd, loss of ASF1 leads to derepression of the E(spl) genes, revealing its essential role in silencing (Moshkin, 2009).

The molecular mechanism by which ASF1 achieves gene-specific transcription repression and the potential roles of other histone chaperones in developmental gene regulation remains largely unknown. To address these issues, a proteomics survey was performed of the protein interaction networks of ASF1, CAF1, HIRA, and NAP1 in Drosophila embryos. This analysis revealed that ASF1 and NAP1 interact with two related but distinct corepressor complexes: LAF and RLAF. LAF, comprising LID/KDM5 SIN3A, PF1, EMSY, and MRG15, associates with ASF1 (forming LAF-A). RLAF, comprising LAF plus RPD3, interacts with NAP1 (forming RLAF-N). Through a combination of biochemistry and developmental genetics, it was established that LAF-A and RLAF-N are tethered to Notch target genes by the Su(H)/H complex and mediate gene-selective silencing. Both ASF1 and NAP1 are required for the targeted removal of the positive H3K4me3 mark by facilitating LID/KDM5 recruitment to chromatin. Furthermore, NAP1 mediates nucleosome assembly at regulatory elements of Notch target genes and histone deacetylation by RLAF. These results uncover extensive crosstalk between distinct histone chaperones and histone-modifying enzymes in developmental gene regulation (Moshkin, 2009).

These results emphasize that, rather than generic, redundant factors, histone chaperones play highly specialized roles in gene-specific regulation. This study has dissected the molecular mechanism underpinning coordinate silencing of Notch target genes by the histone H3/H4 chaperone ASF1 and the H2A/H2B chaperone NAP1. ASF1 interacts with LAF, comprising SIN3A, PF1, EMSY, MRG15, and the histone H3K4me2/3 demethylase LID/KDM5, forming LAF-A. A closely related complex, RLAF that includes the deacetylase RPD3, does not bind ASF1. Instead, RLAF associates with NAP1, forming RLAF-N. The chaperones ASF1 and NAP1 link, respectively, LAF and RLAF to the Su(H)/H DNA-binding complex, tethering them to the E(spl) genes. Both ASF1 and NAP1 bind the SKIP subunit of the Su(H)/H complex (Goodfellow, 2007). Thus, at least in part, ASF1 and NAP1 facilitate H3K4me3 demethylation activity at the E(spl) genes through LID recruitment. Other LAFs might provide additional links to the Su(H)/H complex by contacting GRO and CtBP, which themselves associate with the Su(H)/H complex. For example, mammalian PF1, MRG15, and SIN3A have been reported to bind GRO. This study identified CtBP in LID, PF1, and NAP1 immunopurifications, providing an additional contact between the Su(H)/H complex and (R)LAF (Moshkin, 2009).

ASF1 does not bind RLAF and has no effect on histone H3 deacetylation by RPD3. In contrast, NAP1 does associate with RLAF and stimulates both H3K4 demethylation by LID and H3 deacetylation by RPD3. SIN3A had a mild effect, but the other LAF subunits played no apparent role in deacetylation. Finally, NAP1 depletion caused a dramatic loss of histones at the E(spl) regulatory elements, whereas ASF1 depletion had no effect on local histone density (Moshkin, 2009).

ASF1 has been proposed to function in chromatin assembly by acting as a donor that hands off the H3/H4 tetramer to either CAF1 or HIRA (De Koning, 2007). Because LAF-A does not associate with either CAF1 or HIRA, this might explain that ASF1 does not modulate nucleosome density at the E(spl) genes. In conclusion, the H3/H4 chaperone ASF1 mediates silencing of Notch target genes by (1) providing a connection between LAF and the Su(H)/H tether and (2) facilitating H3K4 demethylation by LID. The H2A/H2B chaperone NAP1 participates in E(spl) silencing by (1) linking RLAF to Su(H)/H, (2) facilitating H3K4 demethylation by LID, (3) facilitating H3 deacetylation by RPD3, and (4) directing high nucleosome density at repressed loci. The functioning of the H2A/H2B chaperone NAP1 in demethylation and deacetylation of histone H3 provides an example of trans-histone regulation (Moshkin, 2009).

LID and its interacting factors appear to work in a context-dependent manner. For example, LID facilitates activation of dMYC target genes in a manner independent of its demethylase activity. Suggestively, this study observed a genetic interaction between ASF1 and dMYC, indicating a potential role for LAF-A. Recently, it has been suggested that selective RLAF subunits could interact with a homolog of GATA zinc-finger domain-containing protein 1 to facilitate expression of targets by inhibition of RPD3 activity. In mammalian cells, LID homolog RBP2 and MRG15 have been implicated in transcription elongation by restricting H3K4me3 levels within transcribed regions. Identification of SIN3A as a LAF and RLAF subunit provides a molecular explanation for the recent observation that SIN3A is involved in genome-wide removal of both H3K4 methyl and acetyl marks. Collectively, these findings suggest that LID and RPD3 enzymatic activities can be modulated through association with specific partners. The proteomics analysis of the LID, PF1, and EMSY interaction networks further emphasizes the diverse involvement of LAFs in regulation of chromatin dynamics (Moshkin, 2009).

In conclusion, these results emphasize the close interconnectivity between distinct chromatin transactions and reveal cooperation between histone chaperones and targeted histone modifications during developmental gene control. The proteomic survey of ASF1, CAF1, HIRA, and NAP1 provides a starting point for the functional analysis of the regulatory networks in which these chaperones participate. As illustrated by the analysis of LAF-A and RLAF-N, specific protein-protein associations and gene targeting provide an intricate network of combinatorial gene expression control (Moshkin, 2009).

The H3K4 demethylase lid associates with and inhibits histone deacetylase Rpd3

JmjC domain-containing proteins have been shown to possess histone demethylase activity. One of these proteins is the Drosophila histone H3 lysine 4 demethylase Little imaginal discs (Lid), which has been genetically classified as a Trithorax group protein. However, contrary to the supposed function of Lid in gene activation, the biochemical activity of this protein entails the removal of a histone mark that is correlated with active transcription. To understand the molecular mechanism behind the function of Lid, a Lid-containing protein complex was purified from Drosophila embryo nuclear extracts. In addition to Lid, the complex contains Rpd3, CG3815/Drosophila Pf1, CG13367, and Mrg15. Rpd3 is a histone deacetylase, and along with Polycomb group proteins, which antagonize the function of Trithorax group proteins, it negatively regulates transcription. By reconstituting the Lid complex, it was demonstrated that the demethylase activity of Lid is not affected by its association with other proteins. However, the deacetylase activity of Rpd3 is greatly diminished upon incorporation into the Lid complex. Thus, these finding that Lid antagonizes Rpd3 function provides an explanation for the genetic classification of Lid as a positive transcription regulator (Lee, 2009).

To shed light on the molecular mechanism of how the histone demethylase Lid regulates transcription, a Lid-containing protein complex, which includes dPf1, Rpd3, CG13367, and Mrg15, was purified from Drosophila embryonic NE. Previous studies have shown that the activities of chromatin-modifying enzymes can be modulated through association with other proteins in a complex. Although no alteration was observed in histone demethylase activity upon the formation of the Lid complex compared to the activity of recombinant Lid alone, the possibility cannot be ruled out that additional factors are required to mediate this stimulatory effect. Since nucleosomes could not be used as a substrate for reasons of sensitivity, it is possible that the Lid complex is irresponsive to enhanced demethylase activity on methylated histones. In contrast, a different Lid-containing complex that is primarily responsible for histone demethylation may exist. Previously, Lid has been reported to interact with dMyc and another TrxG protein, Ash2, in larval eye imaginal discs, implying that other, tissue- and developmental stage-specific Lid-containing complexes may exist (Lee, 2009).

Intriguingly, inhibition of the HDAC activity of Rpd3 was observed in the Lid complex. In this respect, the major function of Lid in this particular complex may be to counteract the transcriptional repression mediated by the deacetylase activity of Rpd3. Notably, Rpd3 has been shown previously to interact with the PRC2 (Polycomb repressive complex 2) complex and to enhance PcG-mediated gene silencing through histone deacetylation. As the H3K4 demethylase activity of Lid is not required for odd gene activation, it is tempting to speculate that the genetic characterization of lid as a TrxG gene is due in part to its inhibitory effect on Rpd3. By inhibiting the HDAC activity of Rpd3, Lid may counteract the full extent of PcG-mediated suppression of gene expression, providing an explanation for the contradictory genetic classification of lid as a TrxG gene and the enzymatic activity of Lid to remove an active histone mark. From this point of view, it appears possible that the histone demethylase activity of Lid is developmentally dispensable. However, it was observed that lid homozygous mutant flies can be rescued only by a transgene encoding wild-type Lid and not by a transgene encoding a catalytically inactive mutant form of Lid, indicating that H3K4 demethylation is developmentally important. Thus, Lid appears to fulfill two possibly distinct functions during development, and these functions may act independently of each other. One function is to demethylate H3K4, whereas the other is to antagonize HDAC activity to promote transcription (Lee, 2009).

The findings of a recent study substantiate the antagonistic behavior of Lid toward Rpd3. Lloret-Llinares (2008) reported that lid mutant alleles act as an enhancer of position effect variegation, whereas some mutations in Rpd3 have been found to confer suppressor-of-variegation phenotypes. Moreover, polytene chromosomes of lid mutants have been shown to have reduced levels of AcH3 (Lloret-Llinares, 2008), which is consistent with the finding that the overexpression of Lid is able to reduce the binding of Rpd3 to polytene chromosomes. Thus, in the absence of Lid, the balance between Lid and Rpd3 would be tilted toward Rpd3, resulting in reduced levels of AcH3 (Lee, 2009).

A similar HDAC complex containing Pf1 and Mrg15 in mammals has been described previously. It is envisaged that Lid is recruited to a core HDAC complex consisting of Rpd3, dPf1, and Mrg15 (and possibly including additional factors that are part of the HDAC complex) and thereby inhibits the HDAC activity. The recruitment of Lid to the sites of the HDAC complex may act as a switch to turn on the expression of target genes during development. It was shown on a gene-specific level for the odd gene in S2 cells by ChIP analysis and on a global level by the immunostaining of polytene chromosomes that the overexpression of Lid results in a marked decrease in Rpd3 binding, suggesting that excessive Lid is able to interact with and displace Rpd3 from its target sites. It has to be pointed out, however, that these findings are based on conditions of robust overexpression of Lid and that the observations need to be confirmed for target genes of the Lid complex in the context of development (Lee, 2009).

It is surprising that Mrg15 was found to negatively regulate the HDAC activity of Rpd3 in vitro, because Mrg15 has been shown previously to contribute to transcription repression. In this regard, it is possible that the interaction solely between Rpd3 and Mrg15 results in enzymatic inhibition and that interaction with additional factors, such as Sin3, may be required to restore the HDAC activity. Provided that the Lid complex identified in this study does play a role in regulating dynamic histone methylation, another role for Mrg15 is conceivable. The chromodomain of Mrg15 may potentially be involved in recruiting the Lid complex to target genes. The trimethylation of H3K4 peaks in the promoter region, whereas the trimethylation of H3K36 is enriched in the 3′ region of genes. During the process of transcription, the chromodomain of Mrg15 may target the Lid complex to the bodies of genes through its interaction with H3K36me3 and induce the removal of H3K4 trimethylation, resulting in the enrichment of the 5′ region of genes with this modification. In the absence of Lid, this distinct border of the different methyl marks would not be sustained and transcription efficiency would deteriorate, thus offering an explanation for the function of Lid in active transcription (Lee, 2009).

Future genome-wide location studies of Lid and the other components of the complex will reveal which target genes are controlled by this complex. Furthermore, it will be interesting to find out where within target genes the complex is located. Does the complex bind to the bodies of genes to demethylate H3K4, or does the binding take place at promoter regions to regulate dynamic histone deacetylation? The identification of Lid-associated proteins has set the stage for these detailed studies, which will reveal insight into the mechanism underlying transcription regulation by Lid (Lee, 2009).

Histone demethylase KDM5A is an integral part of the core Notch-RBP-J repressor complex

Timely acquisition of cell fates and the elaborate control of growth in numerous organs depend on Notch signaling. Upon ligand binding, the core transcription factor RBP-J activates transcription of Notch target genes. In the absence of signaling, RBP-J switches off target gene expression, assuring the tight spatiotemporal control of the response by a mechanism incompletely understood. This study shows that the histone demethylase KDM5A (Little imaginal discs in Drosophila) is an integral, conserved component of Notch/RBP-J gene silencing. Methylation of histone H3 Lys 4 is dynamically erased and re-established at RBP-J sites upon inhibition and reactivation of Notch signaling. KDM5A interacts physically with RBP-J; this interaction is conserved in Drosophila and is crucial for Notch-induced growth and tumorigenesis responses (Liefke, 2010).

Histone lysine demethylases reversibly remove methyl marks, thus facilitating changes in chromatin formation and transcriptional regulation. Histone demethylases have therefore been proposed as promising therapeutic targets of human diseases, including cancer, that are often associated with aberrant histone methylation. This study identified KDM5A as an enzyme responsible for the removal of H3K4me3 at Notch target genes; KDM5A interacts directly with RBP-J via a domain located between PHD2 and PHD3 domain and its C-terminal PHD3 domain. Interestingly, the PHD3 domain was shown recently to bind to H3K4me3 (Wang, 2009). Although it has been suggested that the Arid domain of KDM5A can bind to a short DNA sequence, CCGCCC, the importance of this finding is challenged by the fact that this sequence is very common in CGIs, yet KDM5A is found only at a small number of genes in ChIP-on-chip experiments. Morover, 11 of these putative KDM5A DNA-binding sites can be found at the CGI of Deltex-1, but only one is present in the Deltex-1 enhancer. Therefore, no correlation exists between the position with a high density of putative KDM5A-binding sites and the dynamically regulated H3K4 trimethylation site at the Deltex-1 gene. Furthermore, in EMSA assays no corroborate binding of KDM5A was seen at the proposed sites. Thus, it is hypothesized that the PHD3 domain of KDM5A binds to H3K4m3 at active promoters, and once the H3K4me3 substrate is demethylated, KDM5A is released (Liefke, 2010). The Polycomb group (PcG) proteins play important roles in maintaining gene silencing during development and adult tissue homeostasis, and recent studies have shown that histone demethylase KDM5A is part of a Polycomb complex. Thus, a KDM5A/Polycomb complex, recruited to Notch target genes, could facilitate the removal of active mark H3K4me3 and the subsequent addition of the repressive H3K27me3 marks. Paradoxically, original genetic analysis of Drosophila lid mutations classified Lid as a member of the Trithorax group of genes. Biochemical experiments suggest that Drosophila Lid affects the HDAC activity of Rpd3 (Lee, 2009), and molecular and genetic data show that Lid facilitates activation of dMYC target genes in a demethylase-independent manner, explaining in part the original classification of lid as a positive transcriptional regulator. However, more recent data and this study support a key role for KDM5A demethylase in the dynamics of gene silencing (Liefke, 2010).

Human KDM5A-containing and Drosophila KDM5A/Lid-containing complexes have been analyzed by several groups. KDM5A is found to be part of an MRG15-containing complex comprising multiple subunits, including Sin3B, HDAC1/2, and RbAp46, and two histone acetyltrasferases, TRRAP and Tip60 (Hayakawa, 2007). Interestingly, similar to the current data, these studies unveiled effects of KDM5A/RBP2 on H3K4 trimethylation away from the TSSs in intergenic regions. Moreover, the current findings on dynamic removal of H3K4me3 associated with changes in acetylated H3K9 also point to a combined action of KDM5A and histone deacetylases. Importantly, Drosophila KDM5A/Lid complexes also contain histone deacetylase activity along with histone chaperones ASF1 and NAP1. In agreement with with the current data, several of these Drosophila corepressors affect Notch target gene expression (Moshkin, 2009). However, although all of these data clearly point to a repressive role of KDM5A/Lid, how these enzymes silence specific genes was unknown (Liefke, 2010).

KDM5A is a member of the KDM5 family, which consists of four proteins (KDM5A-D) in mammals. Particularly, KDM5A is highly expressed in the hematopoietic system. KDM5A (RBP2)-deficient mice appear grossly normal but display a mild hematopoietic phenotype, especially in the myeloid compartment. The relatively mild phenotype of KDM5A mouse knockout might be explained in part by some redundancy between the KDM5 paralogs. Yet, one of the up-regulated genes in the KDM5A knockout microarray is Ifi2004, a Notch target gene, indicating that KDM5 paralogs might play redundant as well as specific roles. Loss of KDM5 orthologs in organisms that encode a single KDM5 gene show more severe phenotypes, underscoring the important role of KDM5 demethylases in development. Thus, mutations in Drosophila KDM5A homolog lid often result in lethality before hatching, some animals show a small optic brain lobe and small imaginal discs, and functional inactivation of the C. elegans KDM5A ortholog, Rbr-2, results in undeveloped vulvas or a multivulval phenotype (Liefke, 2010).

Although the discovery of histone demethylases implicates a reversible state of epigenetic gene silencing, it was unanticipated that these chromatin-modifying enzymes exert pathway-specific effects on gene regulation. The dynamic switch-off (and back on) system for Notch target gene expression used in this study allowed revealing of dynamic changes of histone modifications at Notch target genes. It was found that the H3K4me3 is removed with a half-time of about 4 h after inhibition of the Notch pathway by the γ-secretase inhibito GSI. These 4 h cannot be explained by out-dilution through cell cycling; the cell division time here is 24 h. Modulation of H3K4 methylation has also been observed in other biological systems, such as the circadian variation of the transcription of the albumin D-element-binding protein gene in the mouse liver or the X inactivation in early embryonic development, where loss of H3K4me3 is one of the earliest and most characteristic features of chromosome-wide silencing (Liefke, 2010).

Disruption of Notch signaling results in a reduction of H3K4me3 at RBP-J sites, while reactivation re-establishes H3K4me3 levels. This suggests that switch-off and switch-on of Notch target genes depends on a tightly controlled balance of histone H3 methylation and demethylation. This study further identifies the histone demethylase KDM5A as a fundamental element in the switch-off process. For re-establishing H3K4me3 levels, Notch-IC could recruit an H3K4me3 methyltransferase to RBP-J sites of Notch target genes. In contrast to Drosophila genes, ~70% of mammalian genes possess CpG islands (CGIs), including many Notch target genes like Deltex-1, Hes-1, Hes-5, Nrarp, and Hey-1. Genome-wide studies proposed that H3K4 trimethylation remains very constant at CGI-containing promoters in different cell types. This study showed that H3K4me3 stays stable at the CGIs of Deltex-1 and Hes-1 after switching off Notch, but is regulated at the RBP-J-binding site. This finding shows for the first time that H3K4me3 does not necessarily have to be removed from the entire promoter to facilitate gene silencing. Instead, modulation of H3K4me3 at specific regulator elements could be sufficient to regulate gene expression. It will be of interest if the dynamic versus constant H3K4me3 is a more common feature of CGI-containing promoters. Recent technical advances in analyzing histone modifications genome-wide might help to address this question (Liefke, 2010).

In summary, this study unveils that histone methylation is dynamically regulated by Notch signaling: Inhibition of Notch leads to a reduction of H3K4me3 levels at regulatory RBP-J sites, while reactivation of signaling re-establishes high levels of H3K4me3. These biochemical and in vivo data support a role for the histone H3K4me3 demethylase KDM5A/Lid in facilitating the switch from activation to repression state via Su(H)/RBP-J in both Drosophila and mammals. Thus, the histone lysine demethylase KDM5A/Lid is a crucial factor in the silencing process. With the in vivo evidence of Drosophila lid/KDM5A in Notch-induced tumorigenesis, this study suggests a pathway-specific tumor suppressor role of KDM5A in cancer, and provides the basis for studies in novel strategies to manipulate Notch-mediated carcinogenesis (Liefke, 2010).

Jarid2 and PRC2, partners in regulating gene expression

The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. This study found Jarid2 (see Drosophila Jarid2) associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing (Li, 2010).

Since the first characterization of the PRC2 core complex, the subsequent, persuasive evidence supports that PRC2 is actually a family of complexes whose composition varies during development, as a function of cell type, or even from one promoter to another. This study identified two new components that interact with PRC2: MTF2 and Jarid2. These analyses of the proteins that interact with the PRC2 complex initiated with transformed cells. Yet it has become clear that interactions observed using transformed cells might be specific to such cells, and not a determinant to the integrity of a normal organism. Thus, studies of a developmentally relevant process was incorporated and it was confirmed that the interactions observed between PRC2 and Jarid2 were of consequence to the developmental program (Li, 2010).

MTF2 is a paralog of Drosophila Pcl. PHF1, another mammalian paralog of Pcl, is required for efficient H3K27me3 and gene silencing in HeLa cells. Although PHF1 appears dispensable for PRC2 recruitment in HeLa cells, work in Drosophila has suggested that the absence of Pcl could impair PRC2 gene targeting. It is possible that the other paralogs of Pcl (MTF2 and PHF19) exhibit a role that is partially redundant with PHF1 function and thereby maintain PRC2 recruitment upon its knockdown. Pcl and its mammalian paralogs contain two PHD domains and a tudor domain, domains reported to potentially recognize methylated histones. Although the ability of Pcl to specifically bind modified histone has not been elucidated to date, it is tempting to speculate that the PHD and tudor domains could target Pcl to specific chromatin regions. Its presence would then stabilize PRC2 recruitment and promote its enzymatic activity. In support of this hypothesis, it was observed that, whereas Ezh2 targeting is severely impaired in Eed-/- ES cells, MTF2 recruitment is affected in a promoter-dependent manner and to a lesser extent than that of Ezh2. This observation suggests that MTF2 gene targeting could be partially independent of PRC2 (Li, 2010).

The exact function of Jarid2 is more enigmatic. Indeed, Jarid2 is a member of a family of enzymes capable of demethylating histones. However, Jarid2 is devoid of the amino acids required for iron and αKG binding, and consequently is unable to catalyze this reaction. It is considered that Jarid2 could act as a dominant negative and inhibit the activity of other histone demethylases; however, coexpression of Jarid2 with, for instance, SMCX did not affect H3K4me3 demethylation. Jarid2 has two domains that could potentially bind DNA: the ARID domain and a zinc finger. Although the ARID domain of Jarid2 was reported to bind DNA, band shift assay suggests that other parts of the Jarid2 C terminus (potentially a zinc finger) are also important for binding to DNA. The SELEX experiment performed with the full-length Jarid2 did not allow identification of any sequence-specific DNA binding, but did result in a slight enrichment of GC-rich DNA sequences. Importantly, it was found that the N-terminal part of Jarid2 could robustly stimulate PRC2-Ezh2 enzymatic activity on nucleosomes. A knockdown of Jarid2 decreased the enrichment of PRC2 at its target genes. Conversely, overexpression of a Gal4-Jarid2 chimera recruited PRC2 at a stably integrated reporter and increased PRC2 enrichment at its target genes, supporting the hypothesis that Jarid2 contributes to PRC2 recruitment (Li, 2010).

In the case of Drosophila, PRE (Polycomb group response element) sequences have been described, and PRC2 access to chromatin is expected to involve the concerted action of several distinct and specific DNA-binding proteins that interact directly or indirectly with PRC2. However, these same DNA-binding factors, or even a combination thereof, are also found at active genes devoid of PRC2. What distinguishes PRE sequences harboring PRC2 from active genes is still not clear. During the evolution from Drosophila to mammals, only a few of the DNA-binding factors that bind PREs (Dsp1 and Pho) are conserved. Either PRC2 recruitment in mammals involves other mechanisms, or distinct transcription factors have emerged to stabilize PRC2 at its target genes. A recent study has identified a presumed mammalian PRE; however, the role of this putative PRE at the endogenous locus that is enriched for PRC2 is not reproduced when the element is integrated upstream of a transgene, as PRC2 is absent. Of note, whereas DNA-binding proteins are likely to play an important role for PRC2 recruitment in mammals, some studies have now suggested that long noncoding RNA could also be involved in this process. These observations together suggest that the recruitment of PRC2 to target genes is complex and requires more than one factor. These findings suggest that the DNA-binding activity of Jarid2 is one such factor, but its affinity for DNA is low and likely requires the help of other factors (Li, 2010).

A critical issue at this juncture is whether or not the composition of PRC2 changes during development. This study reports that Jarid2 interacts with PRC2, but its expression, unlike the PRC2 core components, seems to be restricted to some cell lines. In agreement with previous gene expression profiles that monitored mRNA levels during the reprogramming of mouse embryonic fibroblast cells into ES cells, it is observed that Jarid2 expression is higher in undifferentiated ES cells and decreases upon differentiation. Polycomb target genes are enriched with the H2A variant H2A.Z in undifferentiated ES cells; furthermore, H2A.Z and PRC2 targeting are interdependent in these cells. This result suggests that PRC2 recruitment might involve distinct mechanisms in ES cells and differentiated cells. It is possible that Jarid2 somehow contributes to this specificity (Li, 2010).

Knockdown of Jarid2 in undifferentiated ES cells does not give rise to an obvious phenotype; gene expression patterns appear to be only moderately affected, and cell proliferation is unchanged. In contrast, when cells are induced to differentiate, a process that entails dramatic changes in gene expression, impairments were observed as a function of Jarid2 knockdown. Interference with Jarid2 resulted in a failure to accurately coordinate the expression of genes required for the differentiation process, consistent with the previous report on Suz12 knockout cells. Instead of the requisite silencing of OCT4 and Nanog loci that occurs upon normal differentiation, each of which become enriched in H3K27me3, Jarid2 knockdown prevented such H3K27 methylation at these genes, and this correlated with their delayed repression. Thus, the Jumonji family of proteins that usually exhibits demethylase activity that might function in opposition to the role mediated by PRC2 contains the member Jarid2 that is devoid of such activity and instead facilitates the action of PRC2 through enabling its access to chromatin (Li, 2010).

Transcriptional regulation of rod photoreceptor homeostasis revealed by in vivo NRL targetome analysis

A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL (Drosophila homolog: Traffic Jam ) determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. This study reports an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) data with global expression profiling and in vivo knockdown studies. Approximately 300 direct NRL target genes were identified. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP-Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX, an Orthodenticle homolog. Evaluation of 26 ChIP-Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. Histone demethylase Kdm5b (Drosophila homolog: Little imaginal discs) was identified as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. These studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis (Hao, 2012).


Search PubMed for articles about Drosophila Little imaginal discs

Benevolenskaya, E. V., Murray, H. L., Branton, P., Young, R. A., and Kaelin, W. G. (2005). Binding of pRB to the PHD protein RBP2 promotes cellular differentiation. Mol. Cell 18: 623-635. PubMed ID: 15949438

Chan, S. W. and Hong, W. J. (2001). Retinoblastoma-binding protein 2 (Rbp2) potentiates nuclear hormone receptor-mediated transcription. J. Biol. Chem. 276: 28402-28412. PubMed ID: 11358960

Curtis, B. J., Zraly, C. B., Marenda, D. R. and Dingwall, A. K. (2011). Histone lysine demethylases function as co-repressors of SWI/SNF remodeling activities during Drosophila wing development. Dev Biol 350: 534-547. PubMed ID:21146519

De Koning, L., Corpet, A. Haber, J. E. and Almouzni, G. (2007). Histone chaperones: an escort network regulating histone traffic. Nat. Struct. Mol. Biol. 14: 997-1007. PubMed ID: 17984962

De Santa, F., et al. (2007). The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing. Cell 130(6): 1083-94. PubMed ID: 17825402

Di Stefano, L., et al. (2011). Functional antagonism between histone H3K4 demethylases in vivo. Genes Dev. 25(1): 17-28. PubMed ID: 21205864

Drelon C., Belalcazar H. M. and Secombe J. (2018). The histone demethylase KDM5 is essential for larval growth in Drosophila. Genetics 209: 773-787. PubMed ID: 31862793

Drelon, C., Rogers, M. F., Belalcazar, H. M. and Secombe, J. (2019). The histone demethylase KDM5 controls developmental timing in Drosophila by promoting prothoracic gland endocycles. Development 146(24). PubMed ID: 31862793

Eissenberg, J. C., Lee, M. G., Schneider, J., Ilvarsonn, A., Shiekhattar, R., Shilatifard, A. (2007). The trithorax-group gene in Drosophila little imaginal discs encodes a trimethylated histone H3 Lys4 demethylase. Nat. Struct. Mol. Biol. 14(4): 344-6. PubMed ID: 17351630

Gildea, J. J., Lopez, R. and Shearn, A. (2000). A screen for new trithorax group genes identified little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2. Genetics 156(2): 645-63. PubMed ID: 11014813

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Biological Overview

date revised: 22 February 2022

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