Transcriptional Regulation

The genes fushi tarazu, and especially engrailed, appear to act as transcriptional activating factors of abdominal-A. abd-A is normally expressed in parasegments 7 to 13. The initial distribution of the product is approximately uniform within this domain, but the subsequent elaboration of the expression pattern results in differences between, as well as within, parasegments. The establishment of the original abd-A expression domain is independent of any of these genes, but most of them are required for the subsequent elaboration of abd-A expression within the domain (Macias, 1994).

trithorax encodes a positive regulatory factor required throughout development for normal expression of multiple homeotic genes of the bithorax and Antennapedia complexes (BX-C and ANTP-C). To determine how trx influences homeotic gene expression, the expression of the BX-C genes Ultrabithorax, abdominal-A, Abdominal-B and the ANTP-C genes Antennapedia, Sex combs reduced and Deformed were examined in trx embryos. Each of the genes examined exhibits different tissue-specific, parasegment-specific and promoter-specific reductions in their expression in response to trx. This implies that each of these genes have different requirements for trx in different spatial contexts in order to achieve normal expression levels, presumably depending on the promoters involved and the other regulatory factors bound at each of their multiple tissue- and parasegment-specific cis-regulatory sites in different regions of the embryo (Breen, 1993).

The Fab-7 chromatin domain boundary ensures functional autonomy of the iab-6 and iab-7 cis-regulatory domains in the bithorax complex (BX-C). Chromatin insulators such as gypsy or scsmin are potent insulators that cannot substitute for Fab-7 function within the BX-C. During the early stages of these swapping experiments, a fragment of scs was initially used that was slightly larger than a minimal scs element (scsmin). This scs fragment, unlike scsmin, interferes in an orientation-dependent manner with the output of a regulatory region covering 80 kb of DNA (from iab-4 to iab-8). At the core of this orientation-dependent phenotype is a promoter located immediately adjacent to the scs insulator. In one orientation, the promoter traps the activity of the iab-3 through iab-5 cis-regulatory domains, diverting them from the abd-A gene. In the opposite orientation, the promoter is transcribing the iab-7 cis-regulatory domain, resulting in ectopic activation of the latter. These data suggest that transcription through a Polycomb-Response Element (PRE) interferes with the maintenance of a Polycomb repression complex. Since the large cis-regulatory regions of the bithorax complex are known to be transcribed, transcriptional activity probably reflects a fundamental mechanism to protect an actively transcribed gene from being inactivated by the Pc-G proteins that are present in all cells (Hogga, 2002).

There are precedents where transcription has been suggested to play a role in chromatin remodeling. For example, the human ß-globin locus is subdivided into three chromatin domains, each of which become more accessible to nuclease digestions upon gene activation. Interestingly, large intergenic transcripts delineate each of these domains and chromatin remodeling of each domain is preceded by its transcription. Another example has been reported in which it was found that transcription across a PRE could interfere with silencing. Evidence has been provided that transcription across the iab-2 cis-regulatory domains in PS6/A1 interferes with iab-2 silencing, resulting in the posterior transformation of PS6/A1 into PS7/A2. In this case, the identity of the affected abdominal segment can easily be recognized in embryos and larvae. Despite the existence of intense transcription of iab-2 in embryos, the dominant gain-of-function phenotype associated with iab-2 misexpression is only detectable in the adult. Thus transcription across the iab regulatory regions appear to interfere with silencing during the late maintenance phase, when the adult structures are forming (Hogga, 2002).

If transcription can interfere with Pc-G silencing, what are the mechanisms responsible for this activity? Factors that affect RNA polymerase II (RNAPII) transcript elongation have been shown to have an effect on chromatin. For example, it has been suggested that histone acetyl transferases (HAT) such as PCAF or ELP3 assist RNAPII in relieving inhibition caused by nucleosome arrays. Although active chromatin requires acetylation of specific lysine residues in the H3 and/or H4 histone tails, the recent purification of Pc complexes suggests that histone deacetylation is required for establishing a stable long-term Pc-G silencing complex. In the case of scsprom (the inserted scs element), perhaps the frequent passage of RNAPII and its associated histone acetylation activities though the PREs interferes with the assembly of the Pc-G silencing. Involvement of acetylated histones in antagonizing PcG-dependent silencing is supported by the findings showing that high levels of acetylated histone H4 are associated with non-repressive PRE sequences. Alternatively, it has been recently found that variant histone H3.3 is deposited on active chromatin during transcription, providing a mechanism for the immediate activation of genes that are silenced by histone modification. It may be possible that transcription across iab-7 (and also iab-8) results in deposition of new nucleosome marked by H3.3, interfering thereby with the maintenance of silencing by the Pc-G complex (Hogga, 2002).

It has been known for a long time that the large cis-regulatory regions of the bithorax complex are transcribed. In blastoderm stage embryos, the iab-2 though iab-8 regions can be divided into three domains, each transcribed in a region that extends from a specific anterior limit to the posterior limit of the segmented part of the embryo. These domains are only broadly defined but their order on the chromosome reflects the anterior limit of expression for each of them. In the light of the current data, it is tempting to speculate that transcription of the iab domains convey a regulatory signal, preventing assembly of the Polycomb-repressing complex on the iab domains that need to remain active. If this is true, transcripts should appear in the anteriormost parasegments/segments where each cis-regulatory domain is activated. However, so far, transcripts in every regulatory region have not been seen; this would account for the sequential activation of each regulatory domain. Moreover, this model predicts that the iab-7 PRE and iab-7 domains should be transcribed from PS12, where iab-7 is first active. So far transcripts across the iab-7 domain have only been detected in PS13 and 14. Thus, it remains unclear whether intergenic transcription plays a role in wild-type animals to create and/or maintain open chromatin, or whether the existence of intergenic transcripts is the consequence of an open structure. However, these experiments strongly suggest that forced transcription through an inactive cis-regulatory domain interferes with the maintenance of silencing, highlighting an incompatibility between transcription and Pc-G mediated silencing. This activity probably reflects a fundamental mechanism to protect an actively transcribed gene from being inactivated by the Pc-G proteins that are present in all cells (Hogga, 2002).

A series of mutations have been recovered in the bithorax complex of Drosophila that transform the first segment of the abdomen into a copy of the second or third abdominal segment. These dominant Ultraabdominal alleles are all associated with P element insertions that are transcribed in the first abdominal segment. The transcripts proceed past the end of the P element for up to 50 kb, extending through the regulatory regions for the second and third abdominal segments. Blocking transcription from the P element promoter reverts the mutant phenotype. Previously identified Ultraabdominal alleles, not associated with P elements, also show abnormal transcription of the same region. The P elements initiate transcripts that proceed through PREs and boundaries, and the phenotypes depend on the production of these transcripts. Other work shows that transcription across the BX-C can relieve silencing, and transcription has been associated with loss of silencing. These observations raise the possibility that non-coding RNAs in wild-type animals may function to activate segmental regulatory regions (Bender, 2002).

Transcription might change the chromosome in several ways. The RNA polymerase II complex involved in elongation includes a histone acetyltransferase that could modify nucleosomes across the transcribed region. The act of transcription might remove bound complexes (such as the Polycomb complex) or prevent their spread along the chromosome. This mechanism was suggested by studies in yeast that have shown that transcription of yeast telomeres relieves the telomere position effect. Transcription might also allow transient access to DNA sequences near the RNA polymerase which might otherwise be covered with nucleosomes or packaged in a 'higher order' structure. A further possibility is that the ectopic RNA product has a function in activation (Bender, 2002).

It is not clear what site or function is affected by the ectopic transcripts. The boundary between the bxd and iab-2 regulatory regions most likely lies just distal to the UabHH1 insertion site; perhaps the ectopic transcription disrupts this boundary. Alternatively, the iab-2 region includes at least one PRE; perhaps transcription across this site relieves the repression imposed by the Polycomb Group. Unfortunately, there is no clear indication from the available BX-C mutations what phenotype to expect from the loss of a PRE (Bender, 2002).

The ectopic RNAs appear not to affect the segmental regulation of the complex early in embryonic development, although the transcripts are abundant in PS6 from stage 10 (elongated germ band) onwards. Misexpression of ABD-A in PS6, which is presumably necessary for the observed segmental transformations, is not seen in embryos except in occasional cells in the central nervous system. Perhaps there is a critical time later in development when ectopic RNA matters, such as the time of abdominal histoblast proliferation in the pupa. Alternatively, continuous transcription might activate abd-A stochastically, so that over time the majority of PS6 cells switch to the active state (Bender, 2002).

The RNA transcripts from UabHH1 are antisense to the normal transcripts of the abd-A gene in PS7-12, and one might expect abd-A expression to be blocked. Indeed, the level of ABD-A protein in UabHH1 embryos is reduced in the PS7 epidermis relative to wild type. ABD-A expression appears normal in the developing central nervous system, and in the epidermis of PS8-PS12, presumably because the UabHH1 transcripts in older embryos are primarily in the epidermis of PS6 and PS7. In UabHH1 larvae, there is also evidence of loss of abd-A function in PS7; the second abdominal setal belt is weakly transformed towards the first. The UabHH1 adults don't show anterior transformation (loss of abd-A function) in PS7, but any such effect would be masked by the strong posterior transformation (gain of abd-A function) (Bender, 2002).

It seems surprising that there are not more gain-of-function alleles in the BX-C or elsewhere due to readthrough from P elements. However, most P element transposons contain selectable marker genes downstream of the P promoter; perhaps these sequences help to terminate transcripts initiated at the P promoter. It is also likely that strong gain-of-function mutations would be dominant lethals. There are a variety of gain-of-function mutations in the BX-C associated with rearrangements, which could mediate their effects by non-coding readthrough transcription from the juxtaposed DNA. Contrabithorax alleles, like Cbx3 and CbxTxt, are good candidates (Bender, 2002).

The dramatic effects of ectopic transcription hint at a function for non-coding transcripts in the wild type BX-C. Non-coding transcripts have been documented in the human ß-globin locus, and such transcription has been correlated with changes in DNaseI sensitivity. Several non-coding transcripts have been described in the BX-C, most notably in the bxd and iab-3 regions. These RNA products appear in blastoderm embryos, at or before the onset of segment-specific expression of the homeotic proteins. Other early RNAs, not associated with BX-C protein products, have been detected by RNA in situs in early embryos (Bender, 2002).

There is, so far, no evidence for a function of these RNAs. A deletion (pbx1) that removes the promoter for the bxd RNA has no effect on the embryonic expression pattern of UBX, although the UBX pattern in imaginal discs is changed. The latter effect of pbx1 may well be due to loss of imaginal disc enhancers, but the bxd RNA could matter for the development of the adult, just as ectopic RNA does. A difference between embryos and larvae has been reported in their requirements for Polycomb Group repression. Perhaps the later mode of Polycomb Group repression is sensitive to and regulated by non-coding transcripts (Bender, 2002).

Epigenetic inheritance to maintain the expression state of the genome is essential during development. In Drosophila, the cis regulatory elements, called the Polycomb Response Elements (PREs) function to mark the epigenetic cellular memory of the corresponding genomic region with the help of PcG and trxG proteins. While the PcG genes code for the repressor proteins, the trxG genes encode activator proteins. The observations that some proteins may function both as PcG and trxG members and that both these groups of proteins act upon common cis elements, indicates at least a partial functional overlap among these proteins. Trl-GAGA was initially identified as a trxG member but later was shown to be essential for PcG function on several PREs. In order to understand how Trl-GAGA functions in PcG context, the interactors of this protein were sought. lola like, aka batman, was identified as a strong interactor of GAGA factor in a yeast two-hybrid screen. lolal also interacts with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7PRE mediated pairing dependent silencing of mini-white transgene. These observations suggest a possible mechanism for how Trl-GAGA plays a role in maintaining the repressed state of target genes involving lolal, which may function as a mediator to recruit PcG complexes (Mishra, 2003).

Genetic interaction studies show that lolal interacts with a variety of PcG and trxG mutations. This underscores the important role of this protein in the regulation of developmental genes. Interestingly, lolal interactions with ph mutation leads to transformation of 2nd (and some times 3rd) leg to 1st leg, an apparent anteriorization type of homeotic transformation in thoracic but in the abdominal region same combination leads to posteriorization type of homeotic transformation, pigmentation of A4 (A4-->A5) reduction in the size of A6 (A6-->A7). However, appearance of sex comb in 2nd and 3rd legs is also known to be due to derepression of Scr in posterior segments thereby explaining this phenotype as due to loss of the repression function of the PcG proteins. Furthermore, trxG and PcG mutations upon interaction with lolal can give a similar phenotype. In lolal context, Asx and trg both show partial A6-->A5 transformation in the abdominal region. Pc is involved in pairing dependent silencing complex recruited by iab-7PRE. ph is also involved in the PS function of iab-7PRE. While it was known that lolal enhances the homeotic phenotype of ph, it is demonstrated that both ph and lolal are involved in establishing the repressive complex at the iab-7PRE. This indicates that lolal and ph function in coordination to set up a repressive complex. Taken together, these results suggest that lolal may be acting along with Trl-GAGA or with other partners in different complexes in a locus or stage specific manner. Depending on the context it could be an activator or repressor function. Since not only 'ON' or 'OFF' but also several 'levels of expression states' for a given hox gene or indeed other regulated loci are maintained, it is likely that a unique combination of trxG and PcG proteins may be needed for each varying level of expression state of a given locus (Mishra, 2003).

The MYST domain acetyltransferase Chameau functions in epigenetic mechanisms of transcriptional repression

Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity. While modification by histone acetyltransferase (HAT) is usually linked to transcriptional activation, evidence is provided for HAT function in several types of epigenetic repression. Chameau (Chm), a new Drosophila member of the MYST HAT family, dominantly suppresses position effect variegation (PEV), is required for the maintenance of Hox gene silencing by Polycomb group (PcG) proteins, and can partially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, in vivo evidence is provided that the acetyltransferase activity of Chm is required in these processes, since a variant protein mutated in the catalytic domain no longer rescues either PEV modification, telomeric silencing of SAS2-deficient yeast cells, or lethality of chm mutant flies. These findings emphasize the role of an acetyltransferase in gene silencing, which supports, according to the histone code hypothesis, the observation that transcription at a particular locus is determined by a precise combination of histone tail modifications rather than by overall acetylation levels (Grienenberger, 2002).

To examine whether Chm and PcG proteins act together to maintain Hox gene repression, the effect of a reduction of chm dosage was tested on homeotic transformations that result from mutations affecting either PcG transregulators or a PRE cis-regulatory element. The first PcG dominant phenotype examined was a T2 into T1 transformation. In the second leg disc of Pc male heterozygotes, derepression of Sex comb reduced leads to the formation on the second leg of a sex comb, a structure normally found on the first leg only. The mutation of one copy of chm significantly enhances this phenotype. chm and PcG gene interactions in the specification of adult abdomen identities were tested. In parasegment 9 (PS9) of males heterozygous for PcXT109 or for the ph410 allele of polyhomeotic (ph), inappropriate expression of Abdominal-B (Abd-B) produces a mild transformation of the fourth abdominal segment into the fifth (A4 into A5), as evidenced by patches of pigmentation in the anterior part of A4. This phenotype, which is never observed in a wild-type context, occurs at low frequency in males heterozygous for chm (less than 1%). Double heterozygotes for chm and for ph or Pc exhibit increased A4 into A5 transformation and/or increased number of transformed individuals, compared to single PcG mutants. Finally, the homeotic transformation induced by a PRE mutation was examined. McpB116 affects Abd-B silencing in PS9, giving rise to incomplete A4 into A5 transformations. This homeotic phenotype is stronger in double heterozygotes for chm14 and McpB116 and becomes further enhanced by the mutation of one copy of Pc. In the various genetic contexts reported here, chm was therefore found to genetically interact with PcG genes and the Mcp element in a positive manner. These synergistic effects strongly suggest that Chm collaborates with PcG proteins for PRE-mediated repression at Hox gene loci (Grienenberger, 2002).

Drosophila Grainyhead specifies late programmes of neural proliferation by regulating the mitotic activity and Hox-dependent apoptosis of neuroblasts: Grh acts upstream of AbdA

The Drosophila central nervous system is generated by stem-cell-like progenitors called neuroblasts. Early in development, neuroblasts switch through a temporal series of transcription factors modulating neuronal fate according to the time of birth. At later stages, it is known that neuroblasts switch on expression of Grainyhead (Grh) and maintain it through many subsequent divisions. The function of this conserved transcription factor is to specify the regionalised patterns of neurogenesis that are characteristic of postembryonic stages. In the thorax, Grh prolongs neural proliferation by maintaining a mitotically active neuroblast. In the abdomen, Grh terminates neural proliferation by regulating the competence of neuroblasts to undergo apoptosis in response to Abdominal-A expression. This study shows how a factor specific to late-stage neural progenitors can regulate the time at which neural proliferation stops, and identifies mechanisms linking it to the Hox axial patterning system (Cenci, 2005).

Thoracic neuroblasts normally continue dividing into pupal stages, stopping at ~120 hours, by which time ~100 adult-specific neurons have been generated. By compromising grh function, it was observed that neurogenesis ceases two days prematurely, at ~72 hours. This limits the average size of neuroblast clones to ~30 cells, indicating that Grh is required to generate 70% of all adult-specific neurons in the thorax (Cenci, 2005).

Four lines of evidence are provided suggesting that the underlying basis for premature cessation of thoracic proliferation in grh mutant clones is reduced mitotic activity of the neuroblast, most probably followed by Hox-independent apoptosis. (1) Although grh mutant neuroblasts are present at 72 hours they are mitotically inactive; (2) by 96 hours, no recognisable grh mutant neuroblasts remain; (3) inhibiting cell-death effector caspases by misexpressing P35 rescues the loss of grh mutant neuroblasts; (4) although misexpression of Hox proteins in thoracic neuroblasts induces apoptosis, Ubx, the resident Hox protein of the posterior thorax, remains excluded from grh mutant neuroblasts at 72 hours. Importantly, the role of Grh in maintaining mitotically-active neuroblasts is not a general 'housekeeping' function but is specific for their age. Thus, wild-type neuroblasts in the early embryo are Grh-negative yet viable and actively dividing. This observation suggests that the late switch to Grh-dependency involves additional factors. These could be intrinsic to the neuroblast or provided by a glial-cell niche. Consistent with the niche idea, neuroblast divisions within the postembryonic brain require DE-cadherin-dependent interactions between glia and neural cells (Cenci, 2005).

In the central abdomen, it has been found that, at 72 hours, many neuroblasts downregulate Grh and become TUNEL positive. When the neuroblast death pathway is blocked in H99 clones, Grh expression continues in mitotically active neuroblasts long after the 72-hour stage. This indicates that abdominal neuroblasts remain in Grh-positive mode during their final division and that Grh is only downregulated after the onset of apoptosis. Moreover, loss of Grh activity leads to the failure of neuroblasts to undergo apoptosis. As these persistent neuroblasts not only survive but also remain actively engaged in the cell cycle, they generate a 3.5-fold excess of cells within each abdominal neuroblast lineage. Together, these findings identify Grh as a terminal neuroblast factor that is an essential component of the abdomen-specific 'stop' programme (Cenci, 2005).

Two different interactions with the Hox gene AbdA underlie the dramatic reversal of Grh function from pro-proliferative in the thorax to anti-proliferative in the abdomen. (1) Grh acts upstream of AbdA to maintain its late phase of expression, and (2) it functions in parallel with AbdA to activate apoptosis. Although the functional significance of grh-dependent AbdA maintenance is not clear, it may be that efficient neuroblast apoptosis requires AbdA levels to remain high for a significant proportion of the interval separating initial AbdA upregulation and the TUNEL-positive stage. More definitively, epistasis tests were used to show that Grh, acting in parallel with AbdA activity, is essential for abdominal neuroblast apoptosis. Thus, when the AbdA-maintenance deficit is rescued using hs-AbdA, neuroblast death remains blocked. Since AbdA is not required to activate neuroblast Grh expression, Grh and AbdA must work in parallel to activate apoptosis. Together with the finding that AbdA is required to activate H99 gene activity, this study demonstrates that inputs from Grh and AbdA are both essential to activate proapoptotic genes and thus trigger neuroblast apoptosis. Whereas the late upregulation of AbdA provides a timing cue to schedule the onset of apoptosis, the much broader phase of Grh expression defines the period of neuroblast competence to respond appropriately to it (Cenci, 2005).

The restricted temporal pattern of Grh expression ensures that competence to undergo AbdA-dependent apoptosis, rather than some other AbdA-dependent output, is only installed at late stages. Consistent with this, neuroblasts in the early embryo that are AbdA positive but Grh negative go on to generate substantial embryonic lineages. Low levels of expression from UAS-grh transgenes make it difficult to test whether Grh is sufficient to confer apoptotic competence to these early embryonic neuroblasts. In the late embryo, however, neuroblasts have already switched on Grh, and, within the central abdomen, all but three undergo abdA-dependent death The observation that reduced neural grh function leads to supernumerary postembryonic neuroblasts positioned outside the vm, vl and dl rows, raises the possibility that Grh is required for all developmentally programmed neuroblast apoptosis (Cenci, 2005).

Polycomb genes interact with the tumor suppressor genes hippo and warts in the maintenance of Drosophila sensory neuron dendrites via regulation of homeobox transcription factors

Dendritic fields are important determinants of neuronal function. However, how neurons establish and then maintain their dendritic fields is not well understood. Polycomb group (PcG) genes are required for maintenance of complete and nonoverlapping dendritic coverage of the larval body wall by Drosophila class IV dendrite arborization (da) neurons. In esc, Su(z)12, or Pc mutants, dendritic fields are established normally, but class IV neurons display a gradual loss of dendritic coverage, while axons remain normal in appearance, demonstrating that PcG genes are specifically required for dendrite maintenance. Both multiprotein Polycomb repressor complexes (PRCs) involved in transcriptional silencing are implicated in regulation of dendrite arborization in class IV da neurons, likely through regulation of homeobox (Hox) transcription factors. Genetic interactions and association between PcG proteins and the tumor suppressor kinase Warts (Wts) is demonstrated, providing evidence for their cooperation in multiple developmental processes including dendrite maintenance (Parrish, 2007).

Dendrite arborization patterns are a hallmark of neuronal type; yet how dendritic arbors are maintained after they initially cover their receptive field is an important question that has received relatively little attention. The Drosophila PNS contains different classes of sensory neurons, each of which has a characteristic dendrite arborization pattern, providing a system for analysis of signals required to achieve specific dendrite arborization patterns. Class IV neurons are notable among sensory neurons because they are the only neurons whose dendrites provide a complete, nonredundant coverage of the body wall. This study found tha the function of Polycomb group genes is required specifically in class IV da neurons to regulate dendrite development. In the absence of PcG gene function, class IV dendrites initially cover the proper receptive field but subsequently fail to maintain their coverage of the field. Time-lapse analysis of dendrite development in esc or Pc mutants suggests that a combination of reduced terminal dendrite growth and increased dendrite retraction likely accounts for the gradual loss of dendritic coverage in these mutants. Maintenance of axonal terminals in class IV da neurons is apparently unaffected by loss of PcG gene function, suggesting that PcG genes function as part of a program that specifically regulates dendrite stability (Parrish, 2007).

Establishment of dendritic territories in class IV neurons is regulated by homotypic repulsion, and this process proceeds normally in the absence of PcG function. In PcG mutants, class IV neurons tile the body wall by 48 h AEL, similar to wild-type controls. However, beginning at 48 h AEL, likely as a result of reduced dendritic growth and increased terminal dendrite retraction, class IV neurons of PcG mutants gradually lose their dendritic coverage. In contrast, the axon projections and terminal axonal arbors of PcG mutants show no obvious defects. Although an early role for PcG genes in regulating axon development cannot be ruled out, MARCM studies showed that PcG genes are required for the maintenance of dendrites but not axons in late larval development. Thus, different genetic programs appear to be responsible for the establishment and maintenance of dendritic fields, and for the maintenance of axons and dendrites (Parrish, 2007).

It is well established that PcG genes participate in regulating several important developmental processes including expression of Hox genes for the specification of segmental identity. In comparison, much less is known about the function of PcG genes in neuronal development. Studies of the expression patterns of PcG genes and the consequences of overexpression of PcG genes suggest that PcG genes may affect the patterning of the vertebrate CNS along the anterior-posterior (AP) axis, analogous to their functions in specifying the body plan. A recent study demonstrates that the PcG gene Polyhomeotic regulates aspects of neuronal diversity in the Drosophila CNS. The current study now links the function of PcG genes to maintenance of dendritic coverage of class IV sensory neurons. Thus it will be interesting to determine whether PcG genes play a conserved role in the regulation of dendrite maintenance (Parrish, 2007).

Since Hox genes function in late aspects of neuronal specification and axon morphogenesis, it seems possible that regulation of Hox genes by PcG genes may be important for aspects of post-mitotic neuronal morphogenesis, including dendrite development. The PcG genes esc and E(z) are required for proper down-regulation of BX-C Hox gene expression in class IV neurons. The timing of this change in BX-C expression corresponds to the time frame during which PcG genes are required for dendritic maintenance. Furthermore, post-mitotic overexpression of BX-C genes in class IV da neurons, but not other classes of da neurons, is sufficient to cause defects in dendrite arborization, thus phenocopying the mutant effects of PcG genes. Finally, it was found that Hox genes are required cell-autonomously for dendrite development in class IV neurons, and loss of Hox gene function causes defects in terminal dendrite dynamics that are opposite to the defects caused by loss of PcG genes. Therefore, it seems likely that PcG genes regulate dendrite maintenance in part by temporally regulating BX-C Hox gene expression (Parrish, 2007).

Several recent studies have focused on the identification of direct targets of PcG-mediated silencing, demonstrating that PcG genes regulate expression of distinct classes of genes in different cellular contexts. During Drosophila development, PRC proteins likely associate with >100 distinct loci, and the chromosome-associate profile of PRC proteins appears dynamic. Therefore, identifying the targets of PcG-mediated silencing in a given developmental process has proven difficult. Thus far, alleles of >20 predicted targets of PcG-mediated silencing have been analyzed for roles in establishment or maintenance of dendritic tiling and a potential role has been found for only Hox genes. Future studies will be required to identify additional targets of PcG-mediated silencing in regulation of dendrite maintenance (Parrish, 2007).

PcG genes are broadly expressed, so it seems likely that interactions with other factors or post-translational mechanisms may be responsible for the cell type-specific activity of PcG genes. Indeed, PcG genes genetically interact with components of the Wts signaling pathway to regulate dendrite development specifically in class IV neurons. Based on the observations that wts mutants also show derepression of Ubx in class IV neurons and that Wts can physically associate with PcG components, it seems likely that Wts may directly or indirectly influence the activity of PcG components. In proliferating cells, Wts phosphorylates the transcriptional coactivator Yorkie to regulate cell cycle progression and apoptosis, demonstrating that Wts can directly influence the activity of transcription factors. In support of a possible role for Wts directly modulating PcG function, several recent reports have documented roles for phosphorylation in regulating PcG function both in Drosophila and in vertebrates. Thus, it is possible that some of the components involved in PcG-mediated silencing are regulated by Wts phosphorylation. Alternatively, association of Wts with PcG proteins may facilitate Wts-mediated phosphorylation of chromatin substrates (Parrish, 2007).

The tumor suppressor kinase Hpo regulates both establishment and maintenance of dendritic tiling in class IV neurons through its interactions with Trc and Wts, respectively, but how Hpo coordinately regulates these downstream signaling pathways is currently unknown. Similar to mutations in wts, mutations in PcG genes interact with mutations in hpo to regulate dendrite maintenance but show no obvious interaction with trc, consistent with the observation that PcG gene function is dispensable for establishment of dendritic tiling. Although it is possible that different upstream signals control Hpo-mediated regulation of establishment and maintenance of dendritic tiling, the nature of such signals remain to be determined. Another possibility is that the activity of the Wts/PcG pathway could be antagonized by additional unknown factors that promote establishment of dendritic tiling (Parrish, 2007).

In addition to their interaction in regulating dendrite maintenance, PcG genes and wts interact to regulate expression of the Hox gene Scr during leg development. This finding suggests that the Hpo/Wts pathway may play a general role in contributing to PcG-mediated regulation of Hox gene expression. The presence of ectopic sex combs provides a very simple and sensitive readout of wts/PcG gene interactions and should form the basis for conducting large-scale genetic screens to identify other genes that interact with wts or PcG genes and participate in this genetic pathway (Parrish, 2007).

Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3: Expression of Ultrabithorax and abdominal-A is affected by dLsd1 depletion

Histone-tail modifications play a fundamental role in the processes that establish chromatin structure and determine gene expression. One such modification, histone methylation, was considered irreversible until the recent discovery of histone demethylases. Lsd1 was the first histone demethylase to be identified (Shi, 2004). Lsd1 is highly conserved, from yeast to humans, but its function has primarily been studied through biochemical approaches. The mammalian ortholog has been shown to demethylate monomethyl- and dimethyl-K4 and -K9 residues of histone H3. This study, along with a second study by Rudolph (2007) describes the effects of Lsd1 (Suppressor of variegation 3-3) mutation in Drosophila. The inactivation of dLsd1 strongly affects the global level of monomethyl- and dimethyl-H3-K4 methylation and results in elevated expression of a subset of genes. dLsd1 is not an essential gene, but animal viability is strongly reduced in mutant animals in a gender-specific manner. Interestingly, dLsd1 mutants are sterile and possess defects in ovary development, indicating that dLsd1 has tissue-specific functions. Mutant alleles of dLsd1 suppress positional-effect variegation, suggesting a disruption of the balance between euchromatin and heterochromatin. Taken together, these results show that dLsd1-mediated H3-K4 demethylation has a significant and specific role in Drosophila development (Di Stefano, 2007).

Su(var)3-3, the Drosophila homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. Su(var)3-3 dictates the distinction between euchromatic and heterochromatic domains during early embryogenesis. Su(var)3-3 mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. Su(var)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of Su(var)3-3/+ females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. These data indicate an early developmental function for the Su(var)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which Su(var)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation (Rudolph, 2007).

The homeobox (Hox) gene locus is subject to extensive H3-K4 methylation by trithorax-group proteins. It was therefore asked whether the expression level of the Hox genes Ultrabithorax (Ubx) and abdominal-A (abdA) is affected by dLsd1 depletion. Ubx- and abdA-mRNA levels increased 2-fold in SL2 cells treated with dLsd1 double-stranded RNA (dsRNA). These changes were specific and were not seen with other control genes (dDP and Hid). To verify the relevance of these observations in vivo, the expression of these genes was compared in wild-type and dLsd1ΔN mutant flies. A significant upregulation of each of these targets was found in dLsd1ΔN mutant flies, confirming the importance of dLsd1-mediated repression in vivo. Intriguingly, it was observe that this upregulation is age dependent: The difference in gene expression is minimal in larval stages, and, consistent with this, the Hox gene-expression pattern in imaginal discs from dLsd1ΔN mutant larvae and in embryos is largely unaltered. However, the level of nAcrβ, Ubx, and Abd-B gradually and significantly increases with age after eclosion, suggesting that dLsd1 function is especially important for the regulation of gene expression in adult tissues (Di Stefano, 2007).

The data support a model in which heterochromatin formation and gene silencing in PEV are defined during early embryonic development of Drosophila. A dynamic balance between HMTases and demethylases controls establishment of the functionally antagonistic histone H3K4 and H3K9 methylation marks at the border region of euchromatin and heterochromatin. In transcriptionally silent cleavage nuclei, chromatin is in a naive state with only little H3K9me2 and with H3K4 methylation completely missing. A dramatic transition of chromatin structure occurs during blastoderm formation and cellularization by establishing H3K4 and H3K9 methylation. In contrast to H3K9 acetylation, which is already found in cleavage chromatin, H3K4 methylation at prospective euchromatin appears first at the end of cleavage in cycle 12. In parallel, di- and trimethylation of H3K9 and HP1 binding establish heterochromatin. Pole cells, which are the primordial germ cells of Drosophila, are in a transcriptionally silent state and show extensive H3K9me2 and H3K9me3. During the definition of the euchromatin-heterochromatin boundaries in blastoderm cells and for the establishment of repressive H3K9 methylation marks in primordial germ cells, the SU(VAR)3-3 demethylase plays an early and inductive regulatory role. SU(VAR)3-3 might also be involved in control of early transcriptional activities within Drosophila pericentromeric sequences preceding heterochromatin formation, as suggested by a model of heterochromatin formation that depends on the RNAi pathway (Rudolph, 2007).

Genetic analysis revealed that SU(VAR)3-3 functions upstream of the H3K9 HMTase SU(VAR)3-9 and the heterochromatin-associated proteins HP1 and SU(VAR)3-7 in control of gene silencing in PEV. Combined with earlier studies of epigenetic interactions, heterochromatic gene silencing is established by a sequential action of SU(VAR)3-3, SU(VAR)3-9, the amount of Y heterochromatin, HP1, and SU(VAR)3-7. RPD3 also acts upstream of SU(VAR)3-9, because Rpd3 mutations dominate the dose-dependent PEV enhancer effect of SU(VAR)3-9. Additional genomic copies of Su(var)3-3 are epistatic to a Rpd3 mutation placing the H3K4 demethylase SU(VAR)3-3 together with RPD3 at the top of a mechanistic hierarchy controlling heterochromatic gene silencing in Drosophila. Such a role is in agreement with the enriched association of SU(VAR)3-3 to prospective heterochromatin in early blastoderm nuclei. In Su(var)3-3 null embryos, there is an extension of H3K4me2 and concomitant reduction of H3K9me3 at prospective heterochromatin, suggesting that SU(VAR)3-3 has a protective function at heterochromatic regions to restrict expansion of H3K4 methylation. Similarly, H3K9 acetylation becomes expanded toward heterochromatin. H3K4 methylation precedes H3K9 methylation in blastoderm nuclei, and both SU(VAR)3-3 and SU(VAR)3-9 are abundant proteins within cleavage chromatin. A developmentally regulated silencing complex between SU(VAR)3-3, RPD3, and SU(VAR)3-9 is therefore likely to dictate the distinction between euchromatic and heterochromatic domains during early embryogenesis. A comparable functional crosstalk between human LSD1 and HDAC1/2, which depends on nucleosomal substrates and the CoREST (see Drosophila CoRest) protein, has been demonstrated in vertebrates (Lee, 2006). The interaction between SU(VAR)3-3 and RPD3 could also explain butyrate sensitivity of Su(var)3-3 mutations. The effect of SU(VAR)3-3 on heterochromatin formation during blastoderm could involve both maternal and zygotic protein. Association of SU(VAR)3-3 with cleavage chromatin is dependent on maternal sources. In contrast, all other effects on gene silencing are zygotically determined, and no maternal effects on PEV were found in any of the Su(var)3-3 mutations. This is also supported by clonal analysis showing early onset and stable maintenance of gene silencing in PEV (Rudolph, 2007).

Histone replacement marks the boundaries of cis-regulatory domains

Cellular memory is maintained at homeotic genes by cis-regulatory elements whose mechanism of action is unknown. Drosophila homeotic gene clusters have been examined by measuring, at high resolution, levels of histone replacement and nucleosome occupancy. Homeotic gene clusters display conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement. Peaks of histone replacement closely correspond to nuclease-hypersensitive sites, binding sites for Polycomb and trithorax group proteins, and sites of nucleosome depletion. These results suggest the existence of a continuous process that disrupts nucleosomes and maintains accessibility of cis-regulatory elements (Mito, 2007).

Chromatin can be differentiated by the replication-independent replacement of one histone variant with another. For example, histone H3.3 is deposited throughout the cell cycle, replacing H3 that is deposited during replication. Unlike replication-coupled assembly of H3, which occurs in gaps between old nucleosomes on daughter helices, the insertion of H3.3 is preceded by disruption of preexisting histones during transcription and other active processes. H3.3 replacement profiles resemble those for RNA polymerase II, which suggests that gradual replacement of H3.3 occurs in the wake of transiting polymerase to repair disrupted chromatin. This study asked whether histone replacement and nucleosome occupancy are also distinctive at cis-regulatory elements (Mito, 2007).

Log-phase Drosophila S2 cells were induced to produce biotin-tagged H3.3 for two or three cell cycles. DNA was extracted from streptavidin pull-down assay and input material, labeled with Cy3 and Cy5 dyes, and cohybridized to microarrays. To provide a standard, biotin-tagged H3-containing chromatin was profiled in parallel. Analysis of H3.3/H3 levels over the entire 3R chromosome arm revealed that the 350-kb bithorax complex (BX-C) region displays the lowest H3.3/H3 ratio of any region of comparable size on 3R, and the Antennapedia homeotic gene complex (ANTP-C) also displays an unusually low H3.3/H3 ratio. Low H3.3/H3 ratios at the homeotic gene clusters are attributable to infrequent histone replacement, and not to low nucleosome occupancy, because H3.3 levels at the BX-C are far below the median for all of 3R, whereas H3 levels are slightly above the median overall. Even the heterochromatic chromosome 4 includes only shorter (100-kb) stretches that are as depleted in H3.3 as the BX-C (Mito, 2007).

A close-up view of the BX-C iab region reveals the presence of several prominent H3.3 peaks. Notably, the seven highest peaks correspond to the functional boundaries of the seven proximal-to-distal cis-regulatory domains that regulate the abd-A (iab2 to iab4) and Abd-B (iab5 to iab8) homeotic genes successively from anterior to posterior in the abdomen. Conspicuous peaks of H3.3 also correspond to the bxd Polycomb response element (PRE) and to promoters within the Abd-B gene, which is known to be active in S2 cells. Therefore, each of the most prominent H3.3 peaks in the region corresponds to a previously defined cis-regulatory element. These findings are likely to be general, because in budding yeast, promoters and boundaries are also sites of intense histone replacement (Mito, 2007).

A characteristic feature of both boundaries and PREs in the BX-C is that they span deoxyribonuclease I (DNaseI)-hypersensitive sites in a variety of cell types, including S2 cells. To better delineate histone replacement patterns in the vicinity of hypersensitive sites, the entire BX-C was tiled at 20-bp resolution. The bxd, Mcp, Fab-7, and Fab-8 PRE-boundaries each encompass conspicuous peaks of H3.3 abundance that closely correspond to all the known nuclease-hypersensitive sites within the region. Nuclease hypersensitivity identifies sites of relatively accessible DNA, so that their correspondences to peaks of histone replacement suggest that continuous disruption of nucleosomes exposes cis-regulatory DNA relative to surrounding regions (Mito, 2007).

PRE-boundary elements in the BX-C and other regions are binding sites for multiple Polycomb group (PcG) proteins, which have been mapped in an S2 cell line at high resolution. If the process that disrupts nucleosomes also facilitates PcG binding, then a correspondence would be expected between peaks of PcG binding and peaks of H3.3. Indeed, when H3.3 profiles were compared with those for Enhancer-of-zeste (EZ) and Posterior-sex-combs (PSC) PcG proteins, all 10 peaks of PcG binding in the abdominal region were found to be local peaks of H3.3. Likewise at the ANTP-C, all 13 peaks of PcG binding in the Scr-Antp region correspond to high levels of H3.3. H3.3 enrichment at PcG-binding sites is not attributable to higher nucleosome occupancy, because essentially identical results were obtained for H3.3/H3 profiles (Mito, 2007).

Not all PREs in the BX-C are found to be sites of PcG binding; for example, neither Fab-7 nor Fab-8 is detectably bound by EZ or PSC. The fact that all PcG sites are peaks of histone replacement, but not vice versa, suggests that histone replacement at PREs and boundaries is constitutive and independent of the expression of the homeotic genes that they regulate. For example, Abd-B is expressed at high levels in S2 cells and displays the typical H3.3 5' peak for an active gene, whereas Ubx and abd-A are nearly inactive, yet the PREs and boundaries regulating all three genes are sites of conspicuous histone replacement over a low background (Mito, 2007).

Histone replacement averaged over the 175 genomewide EZ+PSC peaks outside of the BX-C and ANTP-C was examined and an H3.3 peak was observed centered over the PcG maximum. Therefore, the strong association between PcG protein binding and histone replacement is not limited to homeotic gene clusters. The genomewide H3.3 peak is higher than that for the BX-C and ANTP-C, presumably because other PcG-binding sites are not superimposed over such deep H3.3 valleys (Mito, 2007).

The colocalization of PcG-binding sites and local peaks of H3.3 suggests that the process that disrupts nucleosomes locally maintains the accessibility of cis-regulatory DNA to PcG proteins. If so, then there should be a lower average occupancy of nucleosomes over sites of PcG protein binding than over their surrounding regions. To test this possibility, nucleosomal DNA and fragmented genomic DNA were hybridized on the same microarrays, and nucleosomal/genomic DNA log ratios were measured. Around peak regions of EZ+PSC binding, nucleosomal DNA was clearly depleted on average, similar to the depletion seen for active gene promoters, and essentially the same results were obtained with different methods for genomic DNA fragmentation. It is concluded that the correspondence between histone replacement and nucleosome depletion is a genomewide feature of PcG-binding sites (Mito, 2007).

In Drosophila, many cis-regulatory elements, including PREs and boundaries, are bound by the trxG proteins, Zeste and GAGA factor (GAF). To test the possibility that histone replacement is enhanced and nucleosome occupancy is reduced where Zeste protein preferentially binds, 390 Zeste-binding sites identified by high-resolution chromatin immunoprecipitation (ChIP) combined with tiling microarrays (ChIP-chip profiling) were aligned, and log ratios of H3.3/H3 and nucleosome occupancy were averaged. A prominent maximum of histone replacement and a sharp minimum of nucleosome occupancy was observed centered over the point of alignment. Similar results were obtained for predicted GAF sites, which suggests that nucleosome disruption is a general feature of trxG protein DNA-binding sites. H3.3 enrichment at PcG- and trxG protein-binding sites results from a replication-independent replacement process, because essentially identical profiles were obtained for H3.3core, which lacks the N-terminal tail and does not assemble during replication (Mito, 2007).

Like Fab-7 and Fab-8, heat shock gene promoters are prominent sites of GAF binding, nuclease hypersensitivity, and reduced nucleosome occupancy. Heat shock protein Hsp70 genes are constitutively 'poised' for rapid induction, but do not produce detectable mRNAs in the uninduced state. Hsp70 genes were aligned at their 5' ends and H3.3 and H3 profiles were averaged. For comparison, similarly aligned H3.3 and H3 profiles were averaged for all 2165 genes on 3R with known 5' and 3' ends, divided into quintiles based on expression levels. H3.3 patterns were similar to those of highly active genes, with histone replacement levels peaking on either side of heat shock promoters. As do transcriptionally active gene promoters, heat shock genes display prominent H3.3 and H3 dips in abundance that are attributable to partial nucleosome depletion. Constitutive histone replacement also appears to be a feature of poised promoters in vertebrates, because H3.3 is strongly enriched in the upstream region of the chicken folate receptor gene, regardless of whether the gene is active or inactive (Mito, 2007).

What process maintains the chromatin of cis-regulatory elements in a state of flux? Many DNA-binding and chromatin-binding proteins involved in gene regulation display short residence times on DNA, and some mouse transcription factors show dynamic behavior at their functional binding sites. A model for this process has been proposed, involving alternating cycles of nucleosome disruption by a Brahma-related SWI/SNF chromatin-remodeler and transcription factor binding. The binding of PcG and trxG proteins is also dynamic, and it is proposed that a similar cycle of nucleosome disruption and factor binding takes place at boundaries and PREs. Nucleosome disruption by SWI/SNF remodeling complexes would occasionally evict nucleosomes and transiently expose DNA, which would become available to other diffusible factors, including PcG proteins. The continued local presence of nucleosome remodelers would result in another cycle of remodeling, nucleosome depletion, nuclease hypersensitivity, and histone replacement at the site. This model could account for the diversity of trxG proteins, which include DNA-binding proteins (Zeste and GAF), nucleosome remodelers (Brahma and Kismet), and histone methyltransferases (Trithorax and Ash1) that are specific for H3K4, a modification that is highly enriched on H3.3. The resulting dynamic process would allow for proteins that promote opposite epigenetic outcomes to act at common cis-regulatory sites (Mito, 2007).

H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex

The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a 'stairstep' pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity (Bowman, 2014).

The Polycomb Group repression system is often described as a cellular memory mechanism, which can impose lifelong silencing of a gene in response to a transitory signal. That view seems valid, but the concept of a PcG regulatory domain is much richer. In the PS6 domain of the BX-C, for example, there are many enhancers to drive Ubx expression in specific cells at specific developmental times, all of which are blocked in parasegments one through five, but active in parasegments 6 through 12. Individual enhancers need not include a segmental address that is specified, for example, by gap and pair-rule DNA-binding factors; their function is segmentally restricted by the domain architecture. Indeed, these enhancers will drive expression in a different parasegment when inserted into a different domain (as in the Cbx transposition). Each domain has a distinctive collection of enhancers; the UBX pattern in PS5 is quite different from that in PS6. Thus, there are two developmental programs for Ubx, one in each of these parasegments, without the need for a duplication of the Ubx gene. Other loci with broad regions of H3K27 methylation may likewise be parsed into multiple domains, once histone marks are examined in specific cell types (Bowman, 2014).

The all-or-nothing H3K27me3 coverage of the BX-C parasegmental domains validates and refines the domain model. In particular, K27me3 is uniformly removed across the PS5 and PS7 domains in PS5 and PS7, even though the activated genes in those parasegments (Ubx and abd-A, respectively) are only transcribed in a subset of cells. It is interesting that both PRC1 and PRC2 components have binding patterns that do not fully reflect function (repression and K27 methylation, respectively), indicating the possibility that function of these complexes is regulated separately from binding. The challenges now are to understand how PcG regulated domains are established, differently in different parasegments, and to describe the molecular mechanisms, including changes in chromosome structure, that block gene activity in H3K27 trimethylated domains (Bowman, 2014).

CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing

Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. This study shows that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. It is proposed that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing (Tie, 2009).

The major findings of this work are: (1) that Drosophila CBP acetylates H3K27; (2) that this acetylation requires TRX; and (3) that it prevents H3K27 trimethylation by E(Z) at Polycomb target genes and antagonizes Polycomb silencing. The remarkably complementary developmental profiles of H3K27ac and H3K27me3 (but not H3K27me2) during embryogenesis suggest that the deposition of H3K27me3, which increases steadily after ~4 hours with the onset of Polycomb silencing, occurs at the expense of a substantial fraction of the H3K27ac already present. This suggests that the establishment of Polycomb silencing might require active deacetylation of this pre-existing H3K27ac. The reciprocal effects of knockdown and overexpression of CBP and E(Z) on H3K27 trimethylation and acetylation in bulk chromatin further suggest that the two modifications constitute alternative chromatin states associated with active and inactive genes. Consistent with this, ChIP-chip experiments revealed that H3K27me3 and H3K27ac are mutually exclusive genome wide. Moreover, in S2 cells, the inactive abd-A gene does not have the H3K27ac modification in its promoter region, but acquires it upon RNAi knockdown of E(Z). It will be important to determine whether such a modification switch occurs genome wide after loss of E(Z) (Tie, 2009).

The ability of E(Z) overexpression to suppress the small rough eye phenotype of CBP overexpressers further supports the conclusion that H3K27 trimethylation by E(Z) antagonizes H3K27 acetylation by CBP and suggests that deacetylation of H3K27 by RPD3, and possibly other deacetylases, might be a prerequisite for subsequent methylation by E(Z) and therefore important for reversal of an active state. Conversely, the ability of CBP and TRX overexpression to increase the global H3K27ac level at the expense of H3K27me3 suggests that either active demethylation of H3K27me3 by the H3K27-specific demethylase UTX (Agge, 2007; Lee, 2007; Smith, 2008), or histone replacement (Ahmad, 2002), might be a prerequisite to acetylation by CBP. Indeed, depletion of Drosophila UTX in vivo using a GAL4-inducible UTX RNAi transgene line results in an increase in H3K27me3, as previously reported (Smith, 2008), and in a marked decrease in H3K27ac. These data, together with the evidence of developmentally programmed reversal of Polycomb silencing, now suggest that the widely accepted stability of Polycomb silencing during development might be more dynamically regulated than previously appreciated (Tie, 2009).

This is the first report that CBP/p300 acetylates H3K27. Recombinant Drosophila CBP acetylates H3K27 and K18 in vivo and in vitro. The greatly reduced H3K27ac levels in CBP-depleted S2 cells also strongly suggest that CBP is the major H3K27 acetylase in Drosophila. The conservation of H3K27 acetylation by human p300, together with the reported association of CBP with the TRX homolog MLL in humans (Ernst, 2001), suggest that it is likely to play a similar role in antagonizing Polycomb silencing in mammals (Tie, 2009).

The genome-wide distribution of H3K27ac, as estimated from human ChIP-chip experiments, appears very similar to that of H3K4me3. This suggests that H3K27ac is much more widely distributed than just at Polycomb target genes, which are estimated to number several thousand in mammalian cells and hundreds in Drosophila. Although these numbers could grow with the identification of additional Polycomb-silenced genes in additional cell types, the recently reported strong correlation of H3K27ac with active genes suggests that it plays an additional role(s) in promoting the transcription of active genes, including those that are never targets of Polycomb silencing. (Note that the H3K27ac at non-Polycomb target genes will not be directly affected by global changes in H3K27me3.) Interestingly, like H3K27me3, H3K27ac appears on the transcribed regions of Polycomb target genes, which might reflect a role for H3K27ac in facilitating transcriptional elongation, and, conversely, a role for H3K27me3 in inhibiting elongation. In addition to its anti-silencing role in preventing H3K27 trimethylation, H3K27ac may also serve as a signal for recruitment of other proteins with additional enzyme activities that alter local chromatin structure further to facilitate or promote transcription. Prime candidates are those containing a bromodomain, a conserved acetyl-lysine-binding module present in several dozen chromatin-associated proteins, including a number of TrxG proteins that also antagonize Polycomb silencing (Tie, 2009).

The results presented in this study provide new insight into how TRX and CBP function together to antagonize Polycomb silencing. Robust H3K27 acetylation by CBP is dependent on TRX, suggesting that H3K27ac plays a crucial role in the anti-silencing activity of TRX. Consistent with this, preliminary genetic evidence suggests that the Polycomb phenotypes caused by TRX overexpression are dependent on CBP, as they are suppressed by RNAi knockdown of CBP. The nature of this dependence is currently unknown, but could involve targeting of CBP by TRX or regulation of the H3K27 acetylation activity of CBP by TRX (Tie, 2009).

The physical association of TRX and CBP and the widespread coincidence of H3K27ac and H3K4me3 sites in the human ChIP-chip data further suggest that the two modifications might be coordinately executed by TRX and CBP. However, the results also raise the possibility that H3K4 trimethylation by TRX itself might be less important for antagonizing Polycomb silencing than H3K27 acetylation. This possibility is also suggested by the discovery of Polycomb-silenced genes in ES and human T cells that contain 'bivalent' marks (both H3K4me3 and H3K27me3) in their promoter regions (although the H3K4me3 levels at these inactive genes are typically lower, on average, than they are at active genes, hinting at the possible importance of quantitative effects of the two marks) (Tie, 2009).

A speculative model is proposed for the regulation of Polycomb silencing that incorporates the activities of TRX, CBP, E(Z), RPD3 and UTX. Repressed genes are marked with H3K27me3. H3K27 trimethylation by PRC2 (which can also control DNA methylation in mammals) requires RPD3 (and possibly other histone deacetylases) to deacetylate any pre-existing H3K27ac. H3K27me3 promotes binding of PC-containing PRC1 complexes, which may inhibit H3K27 acetylation and maintain silencing through 'downstream' events, including those promoted by the H2AK119 mono-ubiquitylation mediated by its RING subunit. Conversely, active genes are marked with H3K4me3 and H3K27ac. H3K27 acetylation by CBP is dependent on TRX and possibly other TrxG proteins, as suggested by the observation that H3K27me3 levels are significantly increased on salivary gland polytene chromosomes from trx, ash1 and kis mutants. The current results predict that this increase will be accompanied by a decrease in H3K27ac. Interestingly, ash1 encodes another HMTase that also interacts with CBP and antagonizes Polycomb silencing. Acetylation of H3K27 is likely to also require the K27-specific demethylase UTX when removal of pre-existing H3K27me3 is a prerequisite for acetylation, e.g. for developmentally regulated reversal of Polycomb silencing at the onset of differentiation. H3K27ac prevents H3K27 trimethylation and might also serve as a signal for recruitment of other TrxG proteins with additional chromatin-modifying activities that may protect the H3K27ac modification and also alter local chromatin structure to promote transcription and further inhibit Polycomb silencing (Tie, 2009).

Structure, evolution and function of the bi-directionally transcribed iab-4/iab-8 microRNA locus in arthropods

In Drosophila melanogaster, the iab-4/iab-8 locus encodes bi-directionally transcribed microRNAs that regulate the function of flanking Hox transcription factors. This study showed that bi-directional transcription, temporal and spatial expression patterns and Hox regulatory function of the iab-4/iab-8 locus are conserved between fly and the beetle Tribolium castaneum. Computational predictions suggest iab-4 and iab-8 microRNAs can target common sites, and cell-culture assays confirm that iab-4 and iab-8 function overlaps on Hox target sites in both fly and beetle. However, w key differences were observed in the way Hox genes are targeted. For instance, abd-A transcripts are targeted only by iab-8 in Drosophila, whereas both iab-4 and iab-8 bind to Tribolium abd-A. This evolutionary and functional characterization of a bi-directionally transcribed microRNA establishes the iab-4/iab-8 system as a model for understanding how multiple products from sense and antisense microRNAs target common sites (Hui, 2013).

The iab-4 miRNA locus has some unusual properties: the locus is transcribed in both directions, producing two primary miRNA transcripts and two hairpin precursors. Each precursor is processed to produce two mature miRNAs, one from each arm of each precursor hairpin. Only a handful of other miRNAs have been shown by deep sequencing data to be transcribed in both directions; currently, the miRBase database has only 27 animal examples. This study shows that bi-directional transcription of the iab-4/8 locus and production of miRNAs from both transcripts is conserved in insects. However, the relative abundance of the four mature miRNAs varies significantly between fly and beetle (Hui, 2013).

The four mature miRNAs produced from the iab-4 locus are extremely similar. Indeed, they are all seed-shifted variants of each other. This state is possible only because the mature sequences are partially palindromic. Thus, sense and antisense sequences are highly similar, as are partially complementary mature sequences from opposite arms of the same hairpin. As the mature sequences are closely related, the predicted targets of the four mature products overlap significantly. Previous work suggests that this is an unusual situation: the targets of alternate miRNAs derived from the 5'- and 3'-arms of almost all miRNAs are largely different. It was shown that iab-4-5p and iab-8-5p have more common targets that expected by chance. This functional overlap of antisense products may have facilitated the maintenance of the bi-directionality in the iab-4/iab-8 locus. Indeed, the same pattern was observed in mir-307, the other miRNA locus that produces mature miRNAs from both genomic strands (Hui, 2013).

This analysis of the repression of engineered perfect target sites clearly shows significant cross-targeting for three of the four mature miRNAs. Furthermore, it was shown that the Hox gene Ubx/Utx is a conserved target of both iab-4 and iab-8 miRNAs in both Drosophila and Tribolium. However, between fly and beetle, differences were found in Hox gene targets of iab-4/8 miRNAs and differences in the sites that mediate those targets. For example, abd-A is regulated only by iab-8 miRNAs in Drosophila, whereas both iab-4 and iab-8 miRNAs target abd-A transcripts in Tribolium. There are, therefore, both conserved and variable aspects of the targeting properties of the four mature miRNAs produced from the iab-4/8 locus in insects. The conservation of the Hox genes Ubx and abd-A as targets of the iab-4/8 miRNAs further establishes the ancient connection between the miRNAs of the Hox complex and their role in modulating the function of the Hox genes themselves. No other intergenic miRNA has been linked by genomic position to its function, yet all Hox complex miRNAs (iab-4, mir-196 and mir-10) have been found to modulate Hox gene function (Hui, 2013).

The iab-4/8 locus provides for fundamental insight into the mechanisms of evolution and the function of sense/antisense miRNA pairs. The production of functional products from both strands of the same locus may impose an evolutionary trade-off, driven on one hand by sequence conservation because of structural constraints, and on the other hand by constraints imposed by target specificity. It is proposed that the deep conservation can be explained in part by the common targeting properties of the multiple mature products generated from these two transcripts. Given the functional similarity of the miRNA products of iab-4 and iab-8, the antisense transcription of the locus can be considered analogous to the acquisition of an enhancer by the sense transcript to drive expression and miRNA production in the additional domain. Furthermore, the palindromic nature of the iab-4/iab-8 mature sequences determines that the novel antisense miRNA will share targets with the pre-existing sense miRNA. It is suggestd that this explains the apparent contradiction between extreme conservation of mature miRNA sequences on both arms, yet significant plasticity between organisms as to which arm is the dominant product. However, the evolution of target sites in abd-A demonstrates that functional target sites can be differentially regulated between even closely related species (Hui, 2013).

Return: abdominal-A Transcriptional regulation part 1/2

abdominal-A: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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