The recent discovery of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. Histone H3K27 trimethylation (H3K27me3) has been linked to polycomb-group-protein-mediated suppression of Hox genes and animal body patterning, X-chromosome inactivation and possibly maintenance of embryonic stem cell (ESC) identity. An imbalance of H3K27 methylation owing to overexpression of the methylase EZH2 has been implicated in metastatic prostate and aggressive breast cancers. This study show that the JmjC-domain-containing related proteins UTX and JMJD3 catalyse demethylation of H3K27me3/2. UTX is enriched around the transcription start sites of many HOX genes in primary human fibroblasts, in which HOX genes are differentially expressed, but is selectively excluded from the HOX loci in ESCs, in which HOX genes are largely silent. Consistently, RNA interference inhibition of UTX led to increased H3K27me3 levels at some HOX gene promoters. Importantly, morpholino oligonucleotide inhibition of a zebrafish UTX homologue resulted in mis-regulation of hox genes and a striking posterior developmental defect, which was partially rescued by wild-type, but not by catalytically inactive, human UTX. Taken together, these findings identify a small family of H3K27 demethylases with important, evolutionarily conserved roles in H3K27 methylation regulation and in animal anterior-posterior development (Lan, 2007).
Histone methylation regulates chromatin structure and transcription. The recently identified histone demethylase lysine-specific demethylase 1 (LSD1) is chemically restricted to demethylation of only mono- and di- but not trimethylated histone H3 lysine 4 (H3K4me3). The X-linked mental retardation (XLMR) gene SMCX (JARID1C), which encodes a JmjC-domain protein, reversed H3K4me3 to di- and mono- but not unmethylated products. Other SMCX family members, including SMCY, RBP2, and PLU-1, also demethylated H3K4me3. SMCX bound H3K9me3 via its N-terminal PHD (plant homeodomain) finger, which may help coordinate H3K4 demethylation and H3K9 methylation in transcriptional repression. Significantly, several XLMR-patient point mutations reduced SMCX demethylase activity and binding to H3K9me3 peptides, respectively. Importantly, studies in zebrafish and primary mammalian neurons demonstrated a role for SMCX in neuronal survival and dendritic development and a link to the demethylase activity. These findings thus identify a family of H3K4me3 demethylases and uncover a critical link between histone modifications and XLMR (Iwase, 2007).
How epigenetic information is transmitted from generation to generation remains largely unknown. Deletion of the C. elegans histone H3 lysine 4 dimethyl (H3K4me2) demethylase spr-5 leads to inherited accumulation of the euchromatic H3K4me2 mark and progressive decline in fertility. This study identified multiple chromatin-modifying factors, including H3K4me1/me2 and H3K9me3 methyltransferases, an H3K9me3 demethylase, and an H3K9me reader, which either suppress or accelerate the progressive transgenerational phenotypes of spr-5 mutant worms. These findings uncover a network of chromatin regulators that control the transgenerational flow of epigenetic information and suggest that the balance between euchromatic H3K4 and heterochromatic H3K9 methylation regulates transgenerational effects on fertility (Greer, 2014).
A series of transcription factors critical for maintenance of the neural stem cell state have been identified, but the role of functionally important corepressors in maintenance of the neural stem cell state and early neurogenesis remains unclear. Previous studies have characterized the expression of both SMRT (also known as NCoR2, nuclear receptor co-repressor 2) and NCoR in a variety of developmental systems; however, the specific role of the SMRT corepressor in neurogenesis is still to be determined. This study reports a critical role for SMRT in forebrain development and in maintenance of the neural stem cell state. Analysis of a series of markers in SMRT-gene-deleted mice revealed the functional requirement of SMRT in the actions of both retinoic-acid-dependent and Notch-dependent forebrain development. In isolated cortical progenitor cells, SMRT is critical for preventing retinoic-acid-receptor-dependent induction of differentiation along a neuronal pathway in the absence of any ligand. These data reveal that SMRT represses expression of the jumonji-domain containing gene JMJD3, a direct retinoic-acid-receptor target that functions as a histone H3 trimethyl K27 demethylase and which is capable of activating specific components of the neurogenic program (Jepsen, 2007).
Many jumonji-C-domain containing proteins catalyse removal of methylated histone tails but nevertheless show distinct substrate specificity among various protein families. In vitro histone demethylase assays revealed selective removal of H3K27me3 and expression of full-length JMJD3, but not of jumonji-C-domain deleted protein, results in a dramatic decrease of global trimethyl histone H3K27 level, indicating that JMJD3 demethylates H3K27me3. Other histone modifications evaluated, including H3K27me2, were not diminished by overexpression of JMJD3. These data demonstrate that JMJD3 is a functional demethylase that specifically targets histone H3K27me3 and this function has also been reported for both JMJD3 and the highly homologous UTX protein (Jepsen, 2007).
Trimethyl K27 modification is important in transmitting epigenetic information during development, imprinting and X-chromosome inactivation and has limited reversibility. A global 'passive' decrease of K27me3 has been observed in embryonic stem cell differentiation, accompanied by concurrent loss of the Polycomb group protein Ezh2, the only known histone methyltransferase that targets K27. It was observed, however, that Ezh2 levels remain constant after RA-stimulated differentiation of neural stem cells, prompting the suggestion that epigenetic regulation via histone H3K27me3 modification by the histone demethylase JMJD3 is a contributory early event in neural differentiation. Indeed, RA-induced neuronal differentiation caused a decrease in histone H3K27me3 and recruitment of JMJD3 to the Dlx5 promoter. These results are consistent with several recent genome-wide location analyses that have suggested histone H3K27me3 to be a dynamic marker reflecting developmental potential (Jepsen, 2007).
The results suggest that expression of JMJD3, a novel histone H3K27 demethylase, is regulated in neural stem cell differentiation in response to RA by SMRT-dependent, RA-receptor-mediated programs, and serves as a mechanistic component of this neuronal fate program. Together, these studies have uncovered a specific role of SMRT in maintaining the neural stem cell state, defending against an ability of unliganded RA receptor to initiate a differentiation program along a neuronal pathway based, at least in part, on the RA-dependent regulation of a component of the histone methyl ation/demethylation machinery (Jepsen, 2007).
The trithorax and the polycomb group proteins are chromatin modifiers, which play a key role in the epigenetic regulation of development, differentiation and maintenance of cell fates. The polycomb repressive complex 2 (PRC2) mediates transcriptional repression by catalysing the di- and tri-methylation of Lys 27 on histone H3 (H3K27me2/me3). Owing to the essential role of the PRC2 complex in repressing a large number of genes involved in somatic processes, the H3K27me3 mark is associated with the unique epigenetic state of stem cells. The rapid decrease of the H3K27me3 mark during specific stages of embryogenesis and stem-cell differentiation indicates that histone demethylases specific for H3K27me3 may exist. This study shows that the human JmjC-domain-containing proteins UTX and JMJD3 demethylate tri-methylated Lys 27 on histone H3. Furthermore, it was demonstrated that ectopic expression of JMJD3 leads to a strong decrease of H3K27me3 levels and causes delocalization of polycomb proteins in vivo. Consistent with the strong decrease in H3K27me3 levels associated with HOX genes during differentiation, it was shown that UTX directly binds to the HOXB1 locus and is required for its activation. Finally mutation of F18E9.5, a Caenorhabditis elegans JMJD3 orthologue, or inhibition of its expression, results in abnormal gonad development. Taken together, these results suggest that H3K27me3 demethylation regulated by UTX/JMJD3 proteins is essential for proper development. Moreover, the recent demonstration that UTX associates with the H3K4me3 histone methyltransferase MLL2 supports a model in which the coordinated removal of repressive marks, polycomb group displacement, and deposition of activating marks are important for the stringent regulation of transcription during cellular differentiation (Agger, 2007).
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).
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).
DNA methylation is a fundamental epigenetic modification in vertebrate genomes and a small fraction of genomic regions is hypomethylated. Previous studies have implicated hypomethylated regions in gene regulation, but their functions in vertebrate development remain elusive. To address this issue, epigenomic profiles were generated that include base-resolution DNA methylomes and histone modification maps from both pluripotent cells and mature organs of medaka fish, and the profiles were compared with those of human ES cells. It was found that a subset of hypomethylated domains harbor H3K27me3 (K27HMDs) and their size positively correlates with the accumulation of H3K27me3. Large K27HMDs are conserved between medaka and human pluripotent cells and predominantly contain promoters of developmental transcription factor genes. These key genes were found to be under strong transcriptional repression, when compared with other developmental genes with smaller K27HMDs. Furthermore, human-specific K27HMDs show an enrichment of neuronal activity-related genes, which suggests a distinct regulation of these genes in medaka and human. In mature organs, some of the large HMDs become shortened by elevated DNA methylation and associate with sustained gene expression. This study highlights the significance of domain size in epigenetic gene regulation. It is proposed that large K27HMDs play a crucial role in pluripotent cells by strictly repressing key developmental genes, whereas their shortening consolidates long-term gene expression in adult differentiated cells (Nakamura, 2014).
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).
Changes in histone methylation status regulate chromatin structure and DNA-dependent processes such as transcription. Recent studies indicate that, analogous to other histone modifications, histone methylation is reversible. Retinoblastoma binding protein 2 (RBP2), a nuclear protein implicated in the regulation of transcription and differentiation by the retinoblastoma tumor suppressor protein, contains a JmjC domain recently defined as a histone demethylase signature motif. This study reports that RBP2 is a demethylase that specifically catalyzes demethylation on H3K4, whose methylation is normally associated with transcriptionally active genes. RBP2−/− mouse cells displayed enhanced transcription of certain cytokine genes, which, in the case of SDF1, was associated with increased H3K4 trimethylation. Furthermore, RBP2 specifically demethylates H3K4 in biochemical and cell-based assays. These studies provide mechanistic insights into transcriptional regulation by RBP2 and provide the first example of a mammalian enzyme capable of erasing trimethylated H3K4 (Klose, 2007).
Histone methylation is a posttranslational modification regulating chromatin structure and gene regulation. BHC110/LSD1 has been described as a histone demethylase that reverses dimethyl histone H3 lysine 4 (H3K4). This study shows that JARID1d, a JmjC-domain-containing protein, specifically demethylates trimethyl H3K4. Detailed mapping analysis revealed that besides the JmjC domain, the BRIGHT and zinc-finger-like C5HC2 domains are required for maximum catalytic activity. Importantly, isolation of native JARID1d complexes from human cells revealed the association of the demethylase with a polycomb-like protein Ring6a/MBLR. Ring6a/MBLR not only directly interacts with JARID1d but also regulates its enzymatic activity. JARID1d and Ring6a occupy human Engrailed 2 gene and regulate its expression and H3K4 methylation levels. Depletion of JARID1d enhances recruitment of the chromatin remodeling complex, NURF, and the basal transcription machinery near the transcriptional start site, revealing a role for JARID1d in regulation of transcriptional initiation through H3K4 demethylation (Lee, 2007a).
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).
During cellular differentiation, both permissive and repressive epigenetic modifications must be negotiated to create cell-type-specific gene expression patterns. The T-box transcription factor family is important in numerous developmental systems ranging from embryogenesis to the differentiation of adult tissues. By analyzing point mutations in conserved sequences in the T-box DNA-binding domain, it was found that two overlapping, but physically separable regions are required for the physical and functional interaction with H3K27-demethylase and H3K4-methyltransferase activities. Importantly, the ability to associate with these histone-modifying complexes is a conserved function for the T-box family. These novel mechanisms for T-box-mediated epigenetic regulation are essential, because point mutations that disrupt these interactions are found in a diverse array of human developmental genetic diseases (Miller, 2009).
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