Transcriptional Regulation

Giant regulates the establishment of the expression patterns of Antennapedia and Abdominal-B. (Reinitz, 1990).

The Abd-B gene consists of two distinct elements that provide a morphogenetic (m) function in PS 10-13 and a regulatory (r) function in PS 14, where it represses m function (Boulet, 1991).

Drosophila Mi-2 protein binds to a domain in the gap protein Hunchback which is specifically required for the repression of HOX genes. Using LexA-Hb as bait, cDNAs were isolated representing six different genes. dMi-2 contains five conserved sequence motifs that are also present in the two human Mi-2 proteins and in two Caenorhabditis elegans ORFs: two chromodomains, a DNA-stimulated adenosine triphosphatase (ATPase) domain, two PHD finger motifs, a truncated helix-turn-helix motif resembling the DNA-binding domain of c-myb, and a motif with similarity to the first two helices of an HMG domain. dMi-2 homozygotes survive until the first or second larval instar. Mutant embryos and larvae show no obvious mutant phenotypes. Specifically, expression of BXC genes such as Ultrabithorax (Ubx) and Abdominal-B (Abd-B) is completely normal in these mutant embryos. This normal expression may be due to maternally deposited dMi-2 RNAs or proteins that persist through subsequent development. Consistent with this, all early embryos from a dMi-2 deletion stock (including those lacking the gene) show the same high levels of dMi-2 RNA. An attempt was made to generate embryos from mutant dMi-2 germ cells. However, germ cells that are mutant for any of the seven tested dMi-2 alleles fail to develop. This failure can be rescued by a dMi-2 transgene, demonstrating that dMi-2 is essential for the development of germ cells (Kehle, 1998).

dMi-2 protein was tested to see if it participates in PcG repression. As in the case of dMi-2, maternally deposited PcG product often rescues homozygous mutant PcG embryos to a considerable extent. Extensive derepression of HOX genes can be observed if such homozygous embryos are also mutant for another PcG gene. Thus embryos homozygous for the PcG gene Posterior sex combs (Psc) and dMi-2 were examined and it was found that Ubx and Abd-B are derepressed more extensively in this double mutant than in Psc homozygotes alone. A similar result was found if dMi-2 is combined with other PcG mutations; these double mutants consistently lead to much enhanced homeotic transformations compared with the single PcG mutants. Thus, there is a synergy between dMi-2 and PcG genes. dMi-2 behaves like the PcG mutations Enhancer of Polycomb and Suppressor 2 of zeste, neither of which on their own cause a homeotic phenotype but do so in combination with other PcG mutations. This suggests that dMi-2 functions in PcG repression (Kehle, 1998).

It has been proposed that Hb directly or indirectly recruits PcG proteins to DNA to establish PcG silencing of homeotic genes. The present data suggest that dMi-2 might function as a link between Hb and PcG repressors. Although dMi-2 contains two motifs with similarity to DNA-binding domains (the myb and HMG domains), dMi-2 does not seem to bind to DNA on its own. Therefore, Hb may recruit dMi-2 to DNA. Xenopus Mi-2 was recently purified as a subunit of a histone deacetylase complex with nucleosome remodeling activity. In yeast and in vertebrates, several transcription factors repress transcription by recruiting histone deacetylases. It is possible that in Drosophila, nucleosome remodeling and deacetylase activities of a dMi-2 complex, recruited to homeotic genes by Hb, may result in local chromatin changes that allow binding of PcG proteins to the nucleosomal template. Alternatively, the proposed Hb-dMi-2 complex might directly bind a PcG protein and recruit it to DNA. Finally, the involvement of dMi-2 in PcG silencing suggests that this process may involve deacetylation of histones (Kehle, 1998 and references).

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 Polycomb group (PC-G) proteins are responsible for keeping developmental regulators, like homeotic genes, stably and inheritably repressed during Drosophila development. It is thought that the PC-G exerts its function at the higher order chromatin level. The distribution of the PC protein has been mapped in the homeotic bithorax complex (BX-C) of Drosophila tissue culture cells. The PC protein quantitatively covers large regulatory regions of repressed BX-C genes. Conversely, the Abdominal-B gene is active in these cells and the regulatory region is devoid of any bound PC protein (Orlando, 1993).

General transcription factors bind promoters repressed by Polycomb group proteins

To maintain cell identity during development and differentiation, mechanisms of cellular memory have evolved that preserve transcription patterns in an epigenetic manner. The proteins of the Polycomb group (PcG) are part of such a mechanism, maintaining gene silencing. They act as repressive multiprotein complexes that may render target genes inaccessible to the transcriptional machinery, inhibit chromatin remodelling, influence chromosome domain topology and recruit histone deacetylases (HDACs). PcG proteins have also been found to bind to core promoter regions, but the mechanism by which they regulate transcription remains unknown. To address this, formaldehyde-crosslinked chromatin immunoprecipitation (X-ChIP) was used to map TATA-binding protein (TBP), transcription initiation factor IIB (TFIIB) and IIF (TFIIF), and dHDAC1 (RPD3) across several Drosophila promoter regions. Binding of PcG proteins to repressed promoters does not exclude general transcription factors (GTFs) and depletion of PcG proteins by double-stranded RNA interference leads to de-repression of developmentally regulated genes. PcG proteins interact in vitro with GTFs. It is suggested that PcG complexes maintain silencing by inhibiting GTF-mediated activation of transcription (Breiling, 2001).

For X-ChIP analysis of promoter regions, the following PcG target genes were chosen: Abdominal-B (Abd-B, B-promoter), iab-4, abdominal-A (abd-A, AI-promoter) and Ultrabithorax (Ubx), all located in the Bithorax complex (BX-C), engrailed (en) and empty spiracles (ems). Also chosen were RpII140 (the subunit of RNA polymerase II with relative molecular mass 140,000 [Mr 140K]) and brown (bw): these last two do not reside in PC binding sites on polytene chromosomes and thus are most probably not PcG regulated. Expression of these genes in Drosophila SL-2 culture cells was assessed by polymerase chain reaction with reverse transcription (RT-PCR) and it was found that Abd-B and RpII140 are transcribed whereas iab-4, abd-A, Ubx, en, ems and bw are inactive (Breiling, 2001).

Acetylation of histones H3 and H4 is considered to be a mark for ongoing transcription. Thus, the promoters of the genes were screened for the presence of amino-terminally acetylated H4 and H3 by X-ChIP. Two antisera were used, one that recognizes H4 acetylated at lysine 12 and one or more other lysines, and one that recognizes H3 acetylated at lysines 9 and/or 18. H4 was found generally acetylated across the promoter regions analysed, in some cases with reduced levels in upstream and downstream regions. H3 is strongly acetylated in the active Abd-B and RpII140 promoters, whereas the inactive loci (iab-4, abd-A, Ubx, en, ems and bw) showed a decrease (5-10 times less than the H3 signal in the active Abd-B and RpII140 promoters) or absence of acetylation both at the core promoters as well as downstream of the initiator. Thus, H3 is acetylated in the active but underacetylated in the inactive promoters, whereas H4 acetylation shows no such changes. Acetylation of histones H3 and H4 seems to be regulated independently across the BX-C, consistent with results in other systems (Breiling, 2001).

The same promoter regions were analyzed by X-ChIP using antibodies against the PcG proteins Polycomb (PC) and Polyhomeotic (PH), dHDAC1, TBP, TFIIB and TFIIF (RAP 30 subunit, associated with RNA polymerase II). All six proteins were found in the core promoter regions (200 base pairs [bp] around the initiator) of the Abd-B, iab-4, abd-A, Ubx, en and ems transcription units. PC was found in most regions both upstream and downstream of the transcription start site (Breiling, 2001).

The presence of PC and PH at the active Abd-B-B promoter is striking, although precedented. Co-localization of PcG proteins and trithorax-group (trxG) proteins, which have been identified as suppressors of the PcG, has been reported for most PcG-bound regulatory regions of the BX-C, including promoters. PcG and certain trxG proteins might simultaneously be needed for changing and maintaining opposite transcriptional states. Thus, coincidental association of repressors and activators with active genes might act to guarantee regulated levels of transcription (Breiling, 2001).

PcG proteins at repressed promoters may prevent activation of RNA Polymerase II, otherwise committed to transcribe. This hypothesis was tested by dsRNA interference (RNAi), a targeted destruction of messenger RNA, to see whether the inhibition of PcG protein synthesis would lead to de-repression of inactive genes. After prolonged treatment of SL-2 cells with Pc and ph dsRNAs, PC protein is no longer detectable in cellular extracts by Western blotting, and the amount of PH is significantly lower than in non-treated cells. With the same kinetics, PcG-regulated promoters, which are inactive in non-treated cells (iab-4, abd-A, Ubx, en and ems), become de-repressed upon treatment with Pc or ph dsRNAs. In contrast, bw does not show any change of expression state, like Abd-B and RpII140, underlining the specificity of PcG control. Remarkably, an incubation time of 8 days is necessary to observe a significant de-repression of PcG target genes. It appears that PcG proteins are rather stable and cells have to divide several times (eight times assuming a duplication time of roughly 24 h) with inhibited PcG protein synthesis, before an effect on transcription is seen (Breiling, 2001).

The major conclusion from this work is that promoters constitute a key target of PcG function. Evidence is provided that, unexpectedly, GTFs are retained at PcG-repressed promoters and that PcG proteins may function through direct physical interactions with GTFs. This mechanism of transcriptional regulation may provide both transcriptional competence and the flexibility necessary for the rapid re-arrangement of patterns of gene expression in response to developmental signals. Thus, the presence of GTFs and some trxG proteins at PcG-repressed promoters would allow a relatively fast re-activation of these genes, as differentiation processes require. In this context, PcG proteins would need to be continuously present at target gene promoters to constitutively inhibit transcription, a prediction supported by the finding that PcG-repressed genes are re-expressed in cells depleted of PcG proteins by dsRNA interference (Breiling, 2001).

Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants

In both Drosophila and vertebrates, spatially restricted expression of HOX genes is controlled by the Polycomb group (PcG) repressors. Mutants of a novel Drosophila PcG gene, Suppressor of zeste 12 [Su(z)12], exhibit very strong homeotic transformations. Su(z)12 function is required throughout development to maintain the repressed state of HOX genes. Unlike most other PcG mutations, Su(z)12 mutations are strong suppressors of position-effect variegation (PEV), suggesting that Su(z)12 also functions in heterochromatin-mediated repression. Furthermore, Su(z)12 function is required for germ cell development. The Su(z)12 protein is highly conserved in vertebrates and is related to the Arabidopsis proteins EMF2, FIS2 and VRN2. Notably, EMF2 is a repressor of floral homeotic genes. These results suggest that at least some of the regulatory machinery that controls homeotic gene expression is conserved between animals and plants (Birve, 2001).

Su(z)12 hemizygous mutant embryos derived from Su(z)12 mutant germ cells already show very extensive misexpression of Ubx at the extended germ band stage. These animals showed severe homeotic phenotypes with all abdominal, thoracic and several head segments transformed into copies of the eight abdominal segment. This phenotype is consistent with Abd-B being misexpressed in all segments. The strong PcG phenotype of these Su(z)12 mutant embryos is comparable to that of embryos lacking esc or Pc function. Zygotically provided Su(z)12 function is sufficient to prevent the inappropriate activation of HOX genes; Su(z)12 -/+ heterozygotes obtained as the progeny of Su(z)12 mutant germ cells and a wild-type sperm develop into wild-type-looking adults (Birve, 2001).

The requirement for Su(z)12 at later developmental stages was tested by generating Su(z)12 mutant clones in imaginal discs. Assays were performed for HOX gene silencing in such clones by monitoring the expression of the HOX genes Ubx and Abd-B in the imaginal wing disc (where they are normally stably repressed) using antisera against their protein products. In these experiments, the Su(z)12 mutant cells were identified by the absence of a GFP-expressing marker gene. In addition, the Minute technique was used to generate Su(z)12-/Su(z)12-clones that carry two copies of a wild-type Minute allele [i.e., Su(z)12- M+/ Su(z)12- M+], which gives them a growth advantage relative to their Su(z)12- M+/ Su(z)12+ M- neighbors (Birve, 2001).

Cell clones of the different Su(z)12 alleles were examined 96 hours after clone induction. Su(z)12 mutant clones show strong misexpression of both Ubx and Abd-B in most mutant cells. In summary, the PcG phenotypes observed with several Su(z)12 alleles suggest that Su(z)12 is needed throughout development to keep HOX genes repressed. Moreover, these results support the allele classification obtained by the analysis of germ-line clones; namely, that Su(z)122 and Su(z)125 are hypomorphic alleles whereas Su(z)121 and Su(z)124 appear to be stronger alleles (Birve, 2001).

Database searches show that the Su(z)12 protein is highly conserved in vertebrates and, strikingly, that Su(z)12-related proteins also exist in plants. In contrast, the worm and yeast genomes do not seem to encode Su(z)12-related proteins. The function of the highly conserved human homolog of Su(z)12, HsSU(Z)12, is not known but EMF2, FIS2 and VRN2, the three Su(z)12-related proteins in Arabidopsis, have been identified as regulators in plant development. One characteristic feature of all these proteins is a single classical C2H2 zinc finger similar to the fingers found in sequence-specific DNA-binding proteins. Attempts to show any DNA-binding activity of a polypeptide containing the Su(z)12 zinc finger have failed so far. A second stretch of amino acids that is conserved between Su(z)12, HsSU(Z)12, EMF2, VRN2 and FIS2 is located C-terminal to the zinc finger. This part of the protein has been termed the VEFS box [VRN2-EMF2-FIS2-Su(z)12 box]. The predicted protein products encoded by Su(z)123 and Su(z)124 lack both the zinc finger and the VEFS box, whereas the protein encoded by Su(z)121 is predicted to contain the zinc finger but lacks the VEFS box (Birve, 2001).

The Drosophila pho-like gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic in homeotic gene silencing

Polycomb group proteins (PcG) repress homeotic genes in cells where these genes must remain inactive during Drosophila and vertebrate development. This repression depends on cis-acting silencer sequences, called Polycomb group response elements (PREs). Pleiohomeotic (Pho), the only known sequence-specific DNA-binding PcG protein, binds to PREs, but pho mutants show only mild phenotypes compared with other PcG mutants. pho-like, a gene encoding a protein with high similarity to Pho, has been characterized. Pho-like binds to Pho-binding sites in vitro and pho-like; pho double mutants show more severe misexpression of homeotic genes than do the single mutants. These results suggest that Pho and Pho-like act redundantly to repress homeotic genes. The distribution of five PcG proteins on polytene chromosomes was examined in pho-like, pho double mutants. Pc, Psc, Scm, E(z) and Ph remain bound to polytene chromosomes at most sites in the absence of Pho and Pho-like. At a few chromosomal locations, however, some of the PcG proteins are no longer present in the absence of Pho and Pho-like, suggesting that Pho-like and Pho may anchor PcG protein complexes to only a subset of PREs. Alternatively, Pho-like and Pho may not participate in the anchoring of PcG complexes, but may be necessary for transcriptional repression mediated through PREs. In contrast to Pho and Pho-like, removal of Trithorax-like/GAGA factor or Zeste, two other DNA-binding proteins implicated in PRE function, do not cause misexpression of homeotic genes or reporter genes in imaginal discs (Brown, 2003).

pho homozygotes die as pharate adults with weak homeotic transformations, while phol homozygotes survive and are phenotypically normal adults. By contrast, phol; pho double mutants die as third instar larvae and fail to pupate. Examination of phol, pho larvae shows that the brain is smaller than normal, the discs are misshapen and smaller than wild-type discs, and the salivary gland polytene chromosomes are enlarged. The larger salivary gland polytene chromosomes may be due to additional rounds of endoreplication in the double mutants. To test whether phol functions in PcG repression, Ubx and Abd-B expression was examined in wing imaginal discs from single and double mutants of phol and pho. As expected, no Ubx or Abd-B expression was observed in wild-type or phol mutant wing discs. pho mutants showed misexpression of Ubx in a few cells in the wing pouch, but did not misexpress Abd-B. By contrast, phol; pho double mutants strongly misexpress Ubx and Abd-B in the wing disc. This suggests that Phol and Pho redundantly repress homeotic genes in imaginal discs and can partially substitute for each other. Ubx misexpression is confined to the wing pouch in phol; pho double mutants; the lack of Ubx misexpression in more peripheral areas of the disc possibly reflects downregulation by Abd-B, which is strongly misexpressed in these regions of the disc (Brown, 2003).

Whether removal of phol during larval development would also cause derepression of Ubx and Abd-B was tested by generating clones of phol mutant cells in imaginal wing discs of pho mutant larvae. In these experiments, phol mutant cells were identified by the absence of a GFP marker. Strong misexpression of Ubx and Abd-B was observed in double mutant cells in the wing pouch similar to the misexpression observed in wing discs from the phol; pho double mutant larvae. These observations suggest that either Phol or Pho is required throughout development to keep homeotic genes repressed (Brown, 2003).

Drosophila FACT contributes to Hox gene expression through physical and functional interactions with GAGA factor

Chromatin structure plays a critical role in the regulation of transcription. Drosophila GAGA factor directs chromatin remodeling to its binding sites. Drosophila FACT (facilitates chromatin transcription), a heterodimer of dSPT16 and dSSRP1, is associated with GAGA factor through its dSSRP1 subunit, binds to a nucleosome, and facilitates GAGA factor-directed chromatin remodeling. Moreover, genetic interactions between Trithorax-like encoding GAGA factor and spt16 implicate the GAGA factor-FACT complex in expression of Hox genes Ultrabithorax, Sex combs reduced, and Abdominal-B. Chromatin immunoprecipitation experiments indicate the presence of the GAGA factor-FACT complex in the regulatory regions of Ultrabithorax and Abdominal-B. These data illustrate a crucial role of FACT in the modulation of chromatin structure for the regulation of gene expression (Shimojima, 2003).

GAGA factor-dFACT complex was identified by co-immunoprecipitation with epitope tagged GAGA factor. GST pull-down assays show that GAGA factor makes a direct contact with dFACT through its dSSRP1 subunit. Gel electrophoresis mobility shift assays reveal that dFACT binds to the nucleosome. Furthermore, dFACT stimulates GAGA factor-directed chromatin remodeling in the embryonic extract of Drosophila. Based on these data, the following model is proposed for GAGA factor-directed site-specific chromatin remodeling. The GAGA factor-dFACT complex binds to a GAGAG sequence on DNA. dFACT binds to nucleosome and stimulates chromatin remodeling. This allows remodeling in a GAGA factor binding site-dependent manner. Because human FACT binds to histones H2A and H2B (Orphanides, 1999), and the yeast SPN complex enhances DNase I sensitivity of nucleosome in a region where H2A and H2B contact the DNA (Formosa, 2001), it is most likely that FACT binds to DNA at the entry and exit site of the nucleosome through its HMG subunit SSRP1, and then acts to destabilize and remove the H2A/H2B dimers to facilitate chromatin remodeling. However, the H2A/H2B dimers remain associated with the FACT-nucleosome complex through SPT16 such that they can quickly rebind to the H3/H4 tetramer when required. In support of this model, an acidic amino acid stretch found in histone-interacting proteins such as nucleoplasmin and NAP1 is conserved in the C-terminal tail of SPT16. Furthermore, H2B (and probably H2A) has been shown to turn over more rapidly than H3 and H4 during transcription (Shimojima, 2003).

The most interesting finding in this study is the involvement of the GAGA factor-dFACT complex in the regulation of gene expression. The anterior transformation of T3 and A6 in Deltaspt16 Trl double heterozygotes and the binding of the GAGA factor-dFACT complex to the bxd region of Ubx and the iab-6 element of Abd-B in vivo indicate that the complex contributes to the epigenetic maintenance of Hox gene expression. Based on these data, the following scheme is envisioned for the maintenance of the active state. The GAGA factor-dFACT complex induces chromatin remodeling in the regulatory regions of various GAGA factor-dependent genes and potentiates transcription. Whereas the expression of ftz and hsp70 is transient, the active state is maintained in Hox genes such as Ubx, Scr, and Abd-B with the aid of other trx group gene products (Shimojima, 2003).

What is the mechanism underlying the maintenance? Among trx group proteins, BRM constitutes an SWI/SNF-type chromatin remodeling complex. This type of chromatin remodeler possesses a unique ability to act on condensed mitotic chromatin. A sequence-specific regulator, Zeste, has been shown to recruit the BRM complex to its target sites. Functionally distinct chromatin remodeling induced by the GAGA-dFACT and Zeste-BRM complexes may be important to keep the active state through many rounds of cell cycle. In addition to the GAGA factor-dFACT and the BRM complexes, three trx group protein complexes have been identified to date. One is TAC1 consisting of Trx, dCBP, and Sbf1, which acetylates core histones in nucleosomes. Mutations in trx or nejire encoding dCBP have been shown to reduce the expression of Ubx. The others are ASH1 and ASH2 complexes. ASH1 also has been known to interact directly with dCBP. These data suggest that acetylation of core histones or other proteins plays a crucial role in the maintenance of the active state. In support of this hypothesis, hyper-acetylation of H4 has been shown to be a heritable epigenetic mark of the active state. The finding that a counteracting Pc group complex ESC/E(Z) contains histone deacetylase RPD3 is also consistent with this hypothesis. Chromatin remodeling induced by the GAGA factor-d-FACT and the Zeste-BRM complexes might be essential for maintenance of the hyperacetylated state of H4 (Shimojima, 2003).

Acf1 confers unique activities to ACF/CHRAC and promotes the formation rather than disruption of chromatin in vivo thus functioning to silence Abd-B

Chromatin assembly is required for the duplication of chromosomes. ACF (ATP-utilizing chromatin assembly and remodeling factor) catalyzes the ATP-dependent assembly of periodic nucleosome arrays in vitro, and consists of Acf1 and the ISWI ATPase. Acf1 and ISWI are also subunits of CHRAC (chromatin accessibility complex), whose biochemical activities are similar to those of ACF. This study investigated the in vivo function of the Acf1 subunit of ACF/CHRAC in Drosophila. Although most Acf1 null animals die during the larval-pupal transition, Acf1 is not absolutely required for viability. The loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin in embryos. Biochemical experiments with Acf1-deficient embryo extracts further indicate that ACF/CHRAC is a major chromatin assembly factor in Drosophila. The phenotypes of flies lacking Acf1 suggest that ACF/CHRAC promotes the formation of repressive chromatin. The acf1 gene is involved in the establishment and/or maintenance of transcriptional silencing in pericentric heterochromatin and in the chromatin-dependent repression by Polycomb group genes. Moreover, cells in animals lacking Acf1 exhibit an acceleration of progression through S phase, which is consistent with a decrease in chromatin-mediated repression of DNA replication. In addition, acf1 genetically interacts with nap1, which encodes the NAP-1 nucleosome assembly protein. These findings collectively indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus (Fyodorov, 2004).

Eukaryotic DNA is packaged into a periodic nucleoprotein complex termed chromatin. The nucleosome is the basic repeating unit of chromatin, and the nucleosomal core consists of 146 bp of DNA wrapped around an octamer of histones H2A, H2B, H3, and H4. In addition to the core histones, chromatin contains other components such as linker histones and high mobility group proteins. Chromatin is involved in the regulation of transcription and other DNA-directed processes via posttranslational modifications of core histones, the reorganization of nucleosomes by chromatin remodeling factors, and the alteration of higher-order structures (Fyodorov, 2004 and references therein).

The assembly of chromatin is a fundamental biological process that occurs in proliferating cells during DNA replication and in quiescent cells during maintenance and repair of chromosomes. During DNA replication, chromatin structure is transiently disrupted at the replication fork, and the preexisting nucleosomes are segregated randomly between the daughter DNA strands. Then, additional nucleosomes are formed with newly synthesized histones. In this process, it appears that histones H3 and H4 are deposited prior to the incorporation of histones H2A and H2B. Chromatin assembly also occurs in nonreplicating DNA, and several examples of replication-independent assembly of chromatin have been described. These latter processes may occur during histone replacement, DNA repair, and transcription (Fyodorov, 2004 and references therein).

The basic chromatin assembly process is mediated by core histone chaperones and an ATP-utilizing motor protein. The histone chaperones include CAF-1 (chromatin assembly factor-1), NAP-1 (nucleosome assembly protein-1), Asf1 (anti-silencing function-1), nucleoplasmin, N1/N2, and Hir (histone regulatory) proteins. These proteins appear to deliver the histones from the cytoplasm to the sites of chromatin assembly in the nucleus. The ATP-utilizing assembly factor ACF (ATP-utilizing chromatin assembly and remodeling factor) can catalyze the transfer of histones from the chaperones to the DNA to yield periodic nucleosome arrays. The assembly reaction can also be catalyzed by purified RSF (remodeling and spacing factor), which appears to possess both chaperone and motor activities (Fyodorov, 2004).

This work investigates the biological function of ACF. ACF was purified from Drosophila embryos as an activity that mediates the ATP-dependent assembly of regularly spaced nucleosome arrays in vitro. During the assembly process, ACF commits to and translocates along the DNA template. ACF consists of two subunits, Acf1 and ISWI, which cooperatively catalyze nucleosome assembly in conjunction with histone chaperone proteins NAP-1 or CAF-1. Acf1 is the larger subunit of ACF, and it possesses WAC, DDT, WAKZ, PHD finger, and bromo-domain motifs. ISWI belongs to the SNF2-like family of DNA-dependent ATPases, and is a subunit of the ACF, CHRAC (chromatin accessibility complex), NURF, and TRF2 complexes. NURF and TRF2 complexes share only the ISWI subunit with ACF, whereas CHRAC is closely related to ACF. CHRAC was purified on the basis of its ability to increase the access of restriction enzymes to DNA in chromatin, and it consists of Acf1, ISWI, and two small subunits, CHRAC-14 and CHRAC-16, which are detected only during early embryonic development. The biochemical activities of ACF and CHRAC are indistinguishable. These Acf1-containing species will be referred to as 'ACF/CHRAC'. To study the function of ACF/CHRAC in vivo, a genetic analysis of the Drosophila acf1 gene was performed. The results indicate that Acf1 programs ACF/CHRAC to perform functions that are distinct from those of the NURF complex, which shares a common ISWI ATPase subunit with ACF/CHRAC. In addition, the phenotypes of flies lacking Acf1 suggest that ACF/CHRAC does not disrupt chromatin, as might be expected for a nucleosome remodeling factor, but rather promotes the formation of chromatin, as would be expected for a chromatin assembly factor (Fyodorov, 2004).

Polycomb regulation is caused by chromatin-dependent transcriptional silencing. The identity of body segments in Drosophila is specified by homeotic genes of the Antennapedia and bithorax complexes, which are in turn subject to regulation by Polycomb and trithorax group (PcG and trxG) genes. PcG genes encode protein complexes that can maintain chromatin-dependent transcriptional silencing via cis-acting DNA elements termed Polycomb response elements, or PREs (Fyodorov, 2004).

To determine the influence of Acf1 on Polycomb regulation, whether the loss of Acf1 affects transcriptional repression by the Ubx PRE in a PRE-miniwhite reporter gene was examined. In the wild-type control background(acf13/acf13), the expression of the PRE-miniwhite reporter gene was strongly repressed, with pigments limited to a small part of the adult fly eye. In the absence of Acf1 (acf11/acf11), partial activation was observed of the PRE-miniwhite reporter gene with pigments distributed over a larger area of the eye. This observed derepression in the homozygous acf11 background is comparable to derepression in a heterozygous Pc background (Fyodorov, 2004).

Whether acf1 interacts genetically with the segmentation function of Pc was investigatede. The appearance of extra sex combs on distal portions of the second and third legs in F1 males was scored in the progeny from a cross between males with a heterozygous deficiency for Pc (Df(3L)Asc) and females homozygous for acf1 alleles. The mutation of acf1 significantly enhanced this Pc phenotype in a manner similar to that seen with other enhancers of the Pc gene. Whereas only about 18% or 17% of the Df(3L)Asc/+; acf13/+ or Df(3L)Asc/+; acf14/+ males had extra sex combs on second and/or third pairs of legs (from the total number of male progeny scored, 61% or 58% of the Df(3L)Asc/+; acf11/+ or Df(3L)Asc/+; acf12/+ male flies had the extra sex comb phenotype). In addition, >50% of males in the latter two crosses exhibited ectopic pigmentation of their A3 and A4 abdominal tergites, which was never observed in crosses with acf13 or acf14 mothers. These results, combined with the derepression of PRE-mediated miniwhite silencing, demonstrate that acf1 is a Polycomb enhancer and suggest that ACF/CHRAC is involved in the assembly and/or maintenance of repressive chromatin in Polycomb-responsive loci (Fyodorov, 2004).

The identity of Drosophila abdominal segments A5-A8 is determined by homeotic selector genes of the bithorax complex. For instance, in Pc/acf1 males, the posteriorly directed homeotic transformation may be caused by an increase in the expression of the bithorax complex gene Abd-B on loss of Acf1. In contrast, the anterior transformation phenotype of ISWI/+; acf1/acf1 and nap1/nap1; acf1/acf1 animals is reminiscent of mutations in various trithorax group genes, which include the brm and kis genes that encode ATPase subunits of chromatin remodeling complexes. This anterior transformation is likely to result from a decrease in expression of Abd-B on loss of Acf1. These data suggest that Acf1 may be involved in repression or activation of Abd-B in different contexts. Transcriptional repression of Abd-B by Acf1 is consistent with its function in the assembly of repressive chromatin. In fact, genetic evidence in yeast as well as polytene chromosome localization studies in Drosophila primarily implicate ISWI-containing complexes in transcriptional repression in vivo. Transcriptional activation of Abd-B by Acf1 could be due to its chromatin remodeling function, which could potentially facilitate transcription, or to an indirect effect, such as the repression of a transcriptional repressor of Abd-B (Fyodorov, 2004).

Surprisingly, Acf1 is not absolutely required for viability. Chromatin from homozygous acf1 mutant embryos exhibits less nucleosomal periodicity as well as a shorter repeat length than chromatin from wild-type embryos. Extracts from Acf1-deficient embryos assemble nucleosomes in vitro much less efficiently than wild-type extracts, and also that the deficiency in chromatin assembly can be rescued on addition of purified recombinant ACF or Acf1. These findings indicate that ACF/CHRAC is a major chromatin assembly activity in Drosophila, but also that Acf1-deficient flies contain other ATP-utilizing chromatin assembly factor(s) that are able to sustain partial viability (Fyodorov, 2004).

The analysis of the Acf1 null flies revealed that ACF/CHRAC performs different biological functions than NURF, even though ACF/CHRAC and NURF both share a common ISWI ATPase. Hence, the unique subunits of ACF/CHRAC and NURF can program the basic motor function of ISWI to perform specific biological tasks in vivo (Fyodorov, 2004).

ATP-utilizing motor proteins could potentially assemble or disrupt chromatin structure. Through multiple lines of investigation, the function of ACF/CHRAC was studied in vivo. (1) Whether there are genetic interactions between acf1 and nap1 was investigated, because the ACF/CHRAC motor protein and the NAP-1 histone chaperone function together in chromatin assembly in vitro. Double mutant nap1/nap1; acf1/acf1 flies exhibit a homeotic transformation that is not seen in the corresponding single mutant flies. These results are consistent with the biochemical activities of ACF/CHRAC and NAP-1 in the chromatin assembly process (Fyodorov, 2004).

(2) The effect of Acf1 on heterochromatic transcriptional silencing was tested. In these experiments, suppression of pericentric position-effect variegation was detected on loss of Acf1. It was additionally found that Acf1-deficient flies exhibit reduced levels of Polycomb-mediated transcriptional silencing. These findings indicate that ACF/CHRAC is important for the establishment and/or maintenance of repressive chromatin states (Fyodorov, 2004).

(3) Whether Acf1 enhances or disrupts chromatin-mediated repression of DNA replication was investigated. Shortening of S phase was observed in Acf1-deficient embryos and larval neuroblasts, consistent with a role of ACF/CHRAC in the assembly rather than disruption of chromatin in vivo. The effect of chromatin structure on the duration of S phase in larvae was investigated with a deficiency that uncovers the histone gene cluster. These animals contain reduced levels of histones and exhibit an acceleration of late S phase progression in larval neuroblasts relative to that in wild-type flies. Thus, the mutation of acf1 as well as the reduction in the level of histones each correlate with an increase in the rate of S phase progression. These data collectively support a role of Acf1 in the assembly of histones into chromatin (Fyodorov, 2004).

In summary, several independent lines of experimentation implicate Acf1 in the formation of chromatin in vivo. These experiments provide evidence for the function of ACF/CHRAC (and other ATP-utilizing factors) in the assembly of chromatin in conjunction with the NAP-1 histone chaperone. They also include the unexpected finding of a role of ACF/CHRAC in Polycomb-mediated silencing as well as the discovery of mutations (acf1 and Df(2L)DS6) that result in an unusual increase in the rate of S phase. Lastly, the loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin, which support a role of Acf1 in the assembly of repressive chromatin. Hence, the collective biochemical and genetic data indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus (Fyodorov, 2004).

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).

Association of cohesin and Nipped-B with transcriptionally active regions of the Drosophila genome

The cohesin complex is a chromosomal component required for sister chromatid cohesion that is conserved from yeast to man. The similarly conserved Nipped-B protein is needed for cohesin to bind to chromosomes. In higher organisms, Nipped-B and cohesin regulate gene expression and development by unknown mechanisms. Using chromatin immunoprecipitation, it was found that Nipped-B and cohesin bind to the same sites throughout the entire non-repetitive Drosophila genome. They preferentially bind transcribed regions and overlap with RNA polymerase II. This contrasts sharply with yeast, where cohesin binds almost exclusively between genes. Differences in cohesin and Nipped-B binding between Drosophila cell lines often correlate with differences in gene expression. For example, cohesin and Nipped-B bind the Abd-B homeobox gene in cells in which it is transcribed, but not in cells in which it is silenced. They bind to the Abd-B transcription unit and a downstream regulatory region and thus could regulate both transcriptional elongation and activation. It is proposed transcription facilitates cohesin binding, perhaps by unfolding chromatin, and that Nipped-B then regulates gene expression by controlling cohesin dynamics. These mechanisms are likely involved in the etiology of Cornelia de Lange syndrome, in which mutation of one copy of the NIPBL gene encoding the human Nipped-B ortholog causes diverse structural and mental birth defects (Misulovin, 2008).

The studies reported in this paper represent the first large-scale mapping of cohesin binding to a metazoan genome. The cohesin binding regions in Drosophila are much larger on average than in yeast, extending from a few kilobases up to 100 kb or so in length, and cohesin-free regions can extend from several kilobases in size up to a megabase or so. The reasons for the differences in cohesin localization between yeast and Drosophila are unknown, but multiple speculative possibilities can be considered. One is that, in Drosophila, transcription might be needed in many cases to provide a 10-nm chromatin fiber that fits into the 35-nm internal diameter of the cohesin ring, while in yeast, much of the chromosome already has an accessible structure. For instance, the H1 linker histone that helps form higher order chromatin structures is likely present at most nucleosomes in metazoan organisms, while in yeast, the related Hho1 linker histone is present at low levels and does not globally regulate chromatin structure or gene expression. It is also feasible that in yeast, which has a small compact genome, the positions of cohesin binding sites have been evolutionarily optimized to avoid interference with transcription. It is also worth noting that in Drosophila, cohesin peaks occur three- to eightfold less frequently in coding sequences than in intergenic sequences or introns. In yeast, where most genes lack introns, similar preferences would favor binding to intergenic sequences. It is unclear why cohesin prefers noncoding over coding sequences in Drosophila, but it is possible that differences in DNA sequence or binding of other proteins could be critical factor (Misulovin, 2008).

In yeast, cohesin binds more densely around centromeres. In Drosophila, the centromeres are in heterochromatin that consists largely of repetitive sequences. Thus, the studies reported in this paper provide no information regarding the binding of cohesin or Nipped-B binding to centromeres. By immunostaining, cohesin binds to both mitotic and meiotic centromeres in Drosophila. Immunostaining with Nipped-B antibody indicates that Nipped-B colocalizes with cohesin along chromosome arms in both polytene and meiotic chromosomes, but not at centromeres in meiotic chromosomes. Thus, Nipped-B might not be involved in regulating association of cohesin with centromeres during meiosis (Misulovin, 2008).

Based on effects of Nipped-B and cohesin on cut expression in vivo, it was originally proposed that cohesin binding to the cut regulatory region hinders enhancer-promoter interactions and that Nipped-B alleviates this effect by dynamic control of cohesin binding (Dorsett, 2004). The finding that Nipped-B colocalizes with cohesin supports the idea that it dynamically regulates binding. The preferential association of cohesin with transcribed regions suggests additional mechanisms by which cohesin binding might affect transcription, and vice versa. As a general model, it is envisioned that transcription facilitates cohesin binding and that the cohesin that binds affects subsequent transcription. Nipped-B then regulates these effects on transcription by dynamic control of cohesin binding or subunit interaction (Misulovin, 2008).

Features of the cohesin binding to the active Abd-B gene in Sg4 cells raise the possibility that in some cases, cohesin could interfere with both transcriptional elongation and activation. Some cohesin and PolII peaks coincide in both the Abd-B transcription unit and 3' regulatory region, which contains intergenic transcription units likely involved in Abd-B regulation. The cohesin in the regulatory region could hinder Abd-B activation by affecting this intergenic transcription. For instance, in the human β-globin gene, blocking intergenic transcription between the enhancer and promoter by insertion of a transcription terminator or an insulator reduces activation. Genes with distant regulatory elements, such as cut and Ubx, may be more sensitive to Nipped-B dosage because of combined effects on activation and elongation (Misulovin, 2008).

Cohesin might also have positive effects on gene expression in some cases. Although it is unknown if the effect is direct, reduction of Rad21 dosage decreases runx gene expression during early zebrafish development. Similarly, Smc1 homozygous mutant clones in the Drosophila mushroom body show reduced ecdysone receptor (EcR) gene expression, and cohesin binds EcR in all three cell lines examined in this study. These findings do not provide an obvious explanation for how cohesin could directly facilitate gene expression, except the possibility that it might help maintain the chromatin in an unfolded state that is more conducive to transcription. Another possibility is that, in specific cases, cohesin might contribute to chromatin boundary function to block the spread of silencing factors as it does at the HMR silent locus in yeast. There is a cohesin/Nipped-B peak at the Fab-7 boundary element flanking the active Abd-B domain in Sg4 cells, and thus the possibility cannot be ruled out that cohesin plays a role in defining chromatin domains permissive for gene expression (Misulovin, 2008).

The data indicate that cohesin and Nipped-B bind preferentially, but not exclusively, to active genes. It is speculated that transcription facilitates cohesin binding by unfolding chromatin to a 10-nm fiber that can be encircled by cohesin. Based on the anti-correlation with histone H3 lysine 27 trimethylation, it also appears likely that silencing, either by preventing transcription or through an independent effect on chromatin structure, inhibits cohesin binding (Misulovin, 2008).

Transcription is neither necessary nor sufficient for cohesin binding because some poorly expressed genes, such as cut, bind cohesin, and some active genes, such as SA, do not. In the case of cut, PolII binds primarily at the promoter in both Sg4 and BG3 cells. There is little downstream polymerase in the cut transcription unit in either cell type, yet there is substantially more cohesin binding to this region in BG3 cells. Thus, there must be additional factors besides transcription that regulate cohesin binding (Misulovin, 2008).

Association of cohesin and Nipped-B with many genes suggests that the diversity of CdLS phenotypes stems from effects on multiple genes. Many of the genes bound by cohesin in Drosophila cells encode evolutionarily conserved transcription factors and receptors that control limb, organ, peripheral, and central nervous system development. These include the genes encoding the Notch receptor, its Serrate and Delta ligands and Mastermind coactivator, the Thickvein transforming growth factor beta (TGFβ) receptor and the Mad DNA-binding protein that mediates TGFβ signaling, the Patched hedgehog receptor, the Ecdysone receptor, and the Epidermal growth factor receptor. Homeobox genes bound by cohesin include cut, Lim1, Distal-less (Dll), homeobrain (hbn), Abd-B, invected (inv), homothorax (hth), and C15, among others. There are also multiple zinc finger protein genes that bind cohesin, including the pannier (pnr) GATA1 ortholog and its interaction partner u-shaped (ush). In BG3 cells, the entire Enhancer of split gene complex encoding multiple bHLH transcription factors involved in nervous system development is bound by cohesin and Nipped-B (Misulovin, 2008).

The finding that cohesin binding to Abd-B correlates with Abd-B expression and the variation in cohesin binding between the three cell lines indicate that many other genes are also likely to bind cohesin in other cell types. Thus, identification of target genes that cause specific CdLS phenotypes will require mapping cohesin binding and gene expression patterns in affected tissues at critical stages of development. Because many genes are bound by cohesin in each cell type, it is speculated that some of the individual patient phenotypes might stem from simultaneous effects on the expression of multiple genes (Misulovin, 2008).

The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila

Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, the evolution of the genetic switch controlling dimorphic bab expression has been identified and traced. The HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSXF activate bab expression whereas in males DSXM directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits (Williams, 2008).

bab expression in the abdominal epidermis is regulated by two separate CREs, one of which directs gene expression in the anterior abdomen of both sexes, and a second, dimorphic element that regulates female-specific gene expression in segments A5-A7. The dimorphic element, when bound by ABD-B and sex-specific isoforms of the DSX protein, acts as a genetic switch that allows pigmentation in males and represses pigmentation in females. Changes in the activities of both CREs have evolved in the course of the origin of the trait from a monomorphic ancestor. Furthermore, dimorphic CRE function evolved by multiple fine-scale changes within the CRE. These results bear on understanding of how sexually dimorphic traits develop, how new sex- and segment-restricted traits arise, and how CRE functions evolve (Williams, 2008).

Sex-restricted traits are the product of differences in gene expression between sexes, therefore, understanding how such traits develop requires the identification of those genes with sex-limited expression and elucidation of the genetic and molecular mechanisms governing their regulation. This study showed that dimorphic bab expression is regulated by a discrete CRE whose activity is combinatorally regulated by the direct inputs of both region- (ABD-B) and sex-specific (DSX) transcription factors. In females, ABD-B acts in concert with the DSXF isoform through binding sites in the dimorphic element to activate bab expression in the posterior segments. Whereas in males, ABD-B activity is overridden by the repressive activity of the DSXM isoform which binds to the same sites as DSXF and hence, permits the formation of the male-specific posterior pigmentation (Williams, 2008).

The genetic pathways that regulate sex-determination and sexual differentiation differ greatly across the animal kingdom, so this mode of male-specific trait regulation in Drosophila may not apply in detail to other animals. However, the integration of region- and sex-specific regulatory inputs must be a requirement for the production of dimorphic traits. It is suggested that the integration of such combinatorial inputs by cis-regulatory elements, as demonstrated for bab, is a general feature of genetic switches within the pathways regulating the production of dimorphic traits (Williams, 2008).

The origins of sexually dimorphic traits have long been of central interest in evolutionary biology. One of the key questions that Darwin grappled with, as have many others subsequently, was whether dimorphic traits are limited to one sex at their origin, or whether these traits first appear in both sexes and then become restricted to one sex. This question has been particularly important and challenging in terms of genetics and evolutionary theory, as it has not been resolved previously how the effects of mutations could be restricted to one sex (Williams, 2008).

In the simplest genetic scenarios of sexual dimorphism, male-limited traits are the products of the male-limited expression of specific genes. The main evolutionary question then, as it has been phrased in classical genetic terms, is whether male-limited gene expression evolves via: (1) 'alleles' that are expressed only in males; or (2) alleles expressed in both sexes which are then suppressed in females or promoted in males. The elucidation of the regulation and evolution of male-specific pigmentation provides a unique opportunity to reconstruct the genetic path of the evolution of a dimorphic trait (Williams, 2008).

Although posterior male-specific pigmentation is a relatively simple, two-dimensional morphological trait, it is clear that it did not originate via just one of the alternative genetic paths above. Rather, the evolution of this trait has involved three paths: the evolution of male-limited gene expression, of female-limited gene expression, and of non-sex-restricted gene expression. Specifically, this study shows that in the course of the evolution from a monomorphically pigmented ancestor, the activity of the female-specific bab dimorphic CRE expanded into segments A6 and A5 and that the activity of the monomorphic bab anterior CRE retreated from segments A6 and A5 of both sexes. These two combined changes produced the sex-specific repression of bab expression in male segments A5 and A6. In addition, in previous work it was shown that the yellow pigmentation gene gained high-level expression in segments A5 and A6 via the acquisition of ABD-B binding sites in a specific yellow gene CRE, whose activity was male-limited due to repression by Bab (which is apparently indirect) (Williams, 2008).

It is important to underscore that none of the genes in this newly-evolved regulatory circuit are globally restricted in their expression to one sex. Rather, the sex-specific features of their expression are controlled by modular CREs that are physically separate from those controlling gene expression in other developing body regions. The properties of these CREs resolve the question of how the effects of mutations can be restricted to one sex. Namely, mutations in a CRE that is under the direct (the female-specific bab dimorphic element) or indirect (the male-specific yellow CRE) control of an effector of sex determination will have sex-limited effects on gene expression. The findings here are a further demonstration of the general principle of how the modular CREs of pleiotropic genes enable the modification of gene expression in and morphology of one body part independent of other body parts, or in this case, the same body part in the opposite sex (Williams, 2008).

It is also notable that none of the CREs analyzed are new to the dimorphically pigmented melanogaster species group. It is clear, then, that the ancestral dimorphic CRE was active in segment A7 and modified to govern sexually dimorphic pigmentation in segments A6 and A5. Thus, in this example, one path is seen to evolving a new dimorphic trait is via the co-option of genetic components that regulate other pre-existing dimorphic traits (Williams, 2008).

One of the major questions concerning the evolution of gene expression is how new gene expression patterns arise. The two most obvious mechanisms would appear to be the gain of new regulatory elements or the gain of new transcription factor-CRE linkages. While the deep ancestry of the dimorphic element ruled out the former, it was expected that the novel sex- and segment-specific regulation of this CRE by DSX and ABD-B in the D. mel. lineage would require the gain of binding sites for these two transcription factors. However, it was found that the both DSX binding sites and most ABD-B sites were present in D. wil. and other monomorphic species and therefore were present in the last common ancestor of both monomorphic and dimorphic species. Thus, the expansion of the dimorphic CRE activity was not due to the wholesale gain of new DSX and ABD-B binding sites (Williams, 2008).

Rather, it was discovered that the expanded, high level activity of the D. mel. dimorphic CRE in segments A6 and A5, relative to the A7-restricted activity of the D. wil. element, was due to an amalgam of changes involving the number, polarity, and topology of transcription factor binding sites. The evolution of dimorphic CRE activity demonstrates how changes beyond the simple gain or loss of binding sites shape CRE evolution. Similarly, changes in the topology and helical phasing of transcription factor binding sites have shaped the evolution of a genetic switch controlling galactose utilization in yeast (Hittinger, 2007). These studies strongly support the view that the relationship between function and sequence variation in CREs is complex. A vast body of work on eukaryotic and prokaryotic transcriptional regulation has shown that binding site polarity and spacing influences the output of regulatory elements. Therefore, it is suggested that one important, but generally unappreciated, class of functionally relevant mutations in CRE and trait evolution involves sequences outside of transcription factor binding sites. CREs thus present a very large target area for potential functionally relevant mutations that quantitatively modulate gene expression and trait development (Williams, 2008).

Finally, these observations concerning the mechanisms underlying the expansion of dimorphic CRE activity help to shed light on another general aspect of the evolution of animal body plans -- the evolution of segmental traits. A large number of studies have demonstrated that some of the major differences among arthropod and vertebrate body plans have involved evolutionary shifts in the spatial boundaries of gene expression along the main body axis. However, the path by which such gene expression patterns are shifted has not been elucidated in any molecular detail. It is submitted here that the expansion of the activity of the dimorphic element from the A7 segment into A6 and A5 is a model of this process. The remodeling of the dimorphic CRE in the course of evolution illustrates that one way such shifts can be accomplished is through numerous small, quantitative incremental changes in the activity of Hox-regulated CREs (Williams, 2008).

Regulation of Hox gene activity by transcriptional elongation in Drosophila

Hox genes control the anterior-posterior patterning of most metazoan embryos. Their sequential expression is initially established by the segmentation gene cascade in the early Drosophila embryo. The maintenance of these patterns depends on the Polycomb group (PcG) and trithorax group (trxG) complexes during the remainder of the life cycle. This study provides both genetic and molecular evidence that the Hox genes are subject to an additional tier of regulation, i.e., at the level of transcription elongation. Both Ultrabithorax (Ubx) and Abdominal-B (Abd-B) genes contain stalled or paused RNA polymerase II (Pol II) even when silent. The Pol II elongation factors Elongin-A and Cdk9 are essential for optimal Ubx and Abd-B expression. Mitotic recombination assays suggest that these elongation factors are also important for the regulation of Notch-, EGF-, and Dpp-signaling genes. Stalled Pol II persists in tissues where Ubx and Abd-B are silenced by the PcG complex. It is proposed that stalling fosters both the rapid induction and precise silencing of Hox gene expression during development (Chopra, 2009a).

Recent studies suggest that the regulation of polymerase II (Pol II) elongation might be a common feature of developmental gene control in the Drosophila embryo. Chromatin immunoprecipitation (ChIP)-chip assays in cultured cell lines suggest that a significant fraction of all protein-coding genes contain stalled Pol II. As many as 10% of all protein-coding genes in the early Drosophila embryo contain Pol II prior to their expression. Many of these genes are developmental control genes, such as those encoding components of cell-signaling pathways, including Wnt, FGF, and Dpp (TGFβ). Moreover, four of the eight Hox genes in Drosophila appear to contain stalled Pol II (lab, Antp, Ubx, and Abd-B) in the early embryo. This study investigated the role of Pol II elongation factors in Hox gene expression (Chopra, 2009a).

To confirm the preliminary evidence for stalled Pol II at the Ubx and Abd-B loci, conventional ChIP assays were performed with different antibodies against Pol II -- namely, 8WG16, which recognizes the CTD of Pol II, and H14, which recognizes the initiating form (Ser-5 phosphorylation) of Pol II. Both of these antibodies have been used in earlier ChIP as well as in ChIP-chip assays to elucidate and map distinct functions of the Pol II complex. Chromatin crosslinking was performed on 0-2 hr wild-type embryos prior to the onset of Hox gene expression. The chromatin was sonicated and precipitated with anti-Pol II antibodies, and then the extracted DNA was used as a template for PCR amplification. Hsp70 was used as a control because it represents the prototypic example of paused Pol II. As expected, the hsp70 promoter region contains strong Pol II signals with both the 8WG16 and H14 antibodies, indicating that an initiated Pol II is bound to the hsp70 promoter prior to heat shock induction. The Ubx and Abd-B promoter regions also exhibit strong signals, whereas PCR amplification performed with exonic probes failed to detect Pol II binding within the main body of the transcription unit. The presence of the H14 signal at these promoters suggests that Ser5 of the Pol II CTD is phosphorylated (initiated Pol II) prior to the activation of Ubx and Abd-B expression. As predicted from the previous ChIP-chip assays, the abd-A promoter region lacks Pol II (Chopra, 2009a).

The preceding studies suggest that Ubx and Abd-B contain a stalled form of Pol II in early embryos. Additional assays were done to investigate Pol II binding in wing and haltere imaginal discs. The hsp70 promoter region contains strong Pol II signals in both wing and haltere discs, consistent with previous studies suggesting that the gene is stably paused in most or all tissues prior to induction by heat shoc. The ChIP assays also identify strong Pol II signals in the Ubx promoter region of wing discs, where the gene is silenced by the PcG complex. In contrast, a probe directed against exon 1 failed to detect significant levels of Pol II within the main body of the transcription unit (Chopra, 2009a).

Very different results were obtained with haltere discs, in which Ubx is strongly expressed and the resulting Ubx repressor inhibits wing development. In this case, strong Pol II signals are detected in both the promoter region and exon, as would be expected for an actively expressed gene. These findings were strengthened by the use of qPCR assays. For these experiments, ChIP assays were done with a cocktail of Pol II antibodies (both 8WG16 and H14). Pol II signals are detected in both the promoter region and exon of the Ubx locus in haltere discs, where the gene is active. In contrast, there are substantially higher levels of Pol II in the promoter region than exon in wing discs where Ubx is silent. Permanganate protection assays are consistent with the occurrence of paused Pol II located between +18 and +35 bp downstream of the Ubx transcription start site (Chopra, 2009a).

Abd-B also exhibits higher levels of Pol II binding in the promoter region as compared with exon 1. However, unlike Ubx, Abd-B is silent in both the wing and haltere discs, so it is not surprising that Pol II is not significantly detected in exon 1 in either tissue. As seen in early embryos, the promoter region of abd-A lacks significant binding of Pol II in wing discs (Chopra, 2009a).

Pol II stalling raises the possibility that Ubx might be regulated at the level of transcriptional elongation. A number of elongation factors have been identified in cell culture assays, including negative elongation factors (NELF) A-E, ELONGIN-A (Elo-A), suppressor of termination (SPT) 4 and 5, and cyclin-dependent kinase 9 (CDK9). Reduced levels of Ubx+ activity cause a slight transformation of halteres into wings because Ubx functions as a repressor of wing development in the halteres. It was reasoned that, if Ubx is regulated at the level of Pol II elongation, then reduced levels of critical elongation factors should enhance the patterning defects observed in weak Ubx mutants (Chopra, 2009a).

mutations in four different elongation factors were specifically examined: Elo-A, Cdk9, Spt4, and Spt5. Cdk9 has been shown to be a critical activator of paused Pol II at the hsp70 promoter. Heterozygotes for each mutation were examined in a Ubx1/+ background, which displays a weak expansion of the halteres. Elo-A/+; Ubx1/+ double heterozygotes display an enhanced transformation of halteres into wings. In particular, several wing-like bristles appear at the leading margin of the halteres. A similar phenotype was observed for Cdk9/+; Ubx1/+ double heterozygotes. Spt4 mutations cause a slight suppression of the Ubx1/+ phenotype, consistent with their dual activities in both attenuating and augmenting Pol II elongation (Chopra, 2009a).

Cdk9 and Elo-A are thought to regulate distinct aspects of Pol II elongation. The Cdk9 kinase phosphorylates Ser-2 of the Pol II CTD, which is critical for the release of Pol II from the pause site in the hsp70 promoter. Inhibition of Cdk9 activity causes a global reduction in Ser-2 phosphorylation. In contrast, Elo-A appears to act at a later point of Pol II elongation after release from the pause site. Mutations in Cdk9 and Elo-A cause an additive enhancement in the Ubx1/+ phenotype. Triple heterozygotes display an expansion in the overall size of the haltere, and the anterior margin contains a series of bristles like those seen in wings. This phenotype suggests that diminished levels of Cdk9 and Elo-A cause significant reductions in Ubx+ activity (Chopra, 2009a).

ChIP-chip and conventional ChIP assays suggest that the Abd-B promoter region might also contain a stalled form of Pol II. As seen for Ubx, reduced levels of Cdk9 and Elo-A cause significant enhancements in the Abd-BM1/+ mutant phenotype. In particular, Abd-BM1/+ heterozygotes display a weak transformation of posterior abdominal segments into anterior segments, particularly the seventh abdominal segment (A7) into A6 (ectopic partial pigmentation) and A6 to A5 (ectopic bristles in A6 sternite). These phenotypes are augmented by reductions in either Cdk9 or Elo-A activity. Double heterozygotes display a more complete A7-to-A6 transformation, as well as an increase in the number of bristles in A6, suggesting a more severe A6-to-A5 transformation. These segmental transformations are weakly enhanced (not suppressed) by lower levels of Spt4 and Spt5. In contrast, mutations in the negative elongation factor Nelf-E strongly suppress the Abd-BM1 phenotype, which is consistent with enhanced transcription of Abd-B. Triple heterozygotes, Abd-BM1/+; Cdk9/+; Elo-A/+, display an even more dramatic transformation of A7 to A6 and A6 to A5. Thus, as seen for Ubx, reduced levels of Cdk9 and Elo-A cause a significant diminishment in Abd-B+ gene activity (Chopra, 2009a).

Stalled Pol II appears to be disproportionately associated with developmental control genes as compared with 'housekeeping' genes that control cell metabolism and proliferation. A substantial fraction of stalled genes exhibit localized patterns of expression during embryogenesis, such as Hox genes and genes encoding components of signaling pathways (e.g., Dpp, FGF, Notch, etc.). Therefore, the possibility was explored that elimination of Cdk9 and Elo-A activity via the production of mitotic clones might produce specific developmental defects in adult appendages. In these experiments, there is no perturbation of Ubx or Abd-B activity. Cdk9 and Elo-A activities are disrupted in an otherwise wild-type background (Chopra, 2009a).

The localized loss of Cdk9 or Elo-A activity in the haltere discs leads to weak wing transformation phenotypes, similar to those seen for reductions in Ubx. In particular, there is an expansion in the size of the halteres, and wing-like bristles appear at the margins. At least some of these phenotypes appear to arise from the specific loss of Ubx expression. Haltere discs containing clonal patches of Cdk9/Cdk9 tissue (identified by the loss of GFP expression) display localized reductions in Ubx activity, as judged by the use of an anti-Ubx antibody. This observation suggests that Ubx transcription is particularly sensitive to diminished activities of Pol II elongation factors, which is consistent with the evidence that the Ubx promoter region contains stalled Pol II (Chopra, 2009a).

Cdk9 and Elo-A mitotic clones produce a variety of patterning defects in the wing and notum. Most notably, there is notching of the wing margins, ectopic wing veins, short crossveins, and both losses and duplications of macrochaete in the notum. These phenotypes might arise from perturbations in Notch, EGF, and Dpp (TGFβ) signaling. Genes encoding components of each of these pathways appear to contain stalled Pol II in early embryos (Chopra, 2009a).

This study has presented evidence that the elongation factors Cdk9 and Elo-A are essential for optimal expression of at least a subset of Drosophila Hox genes, particularly Ubx+ activity in the developing halteres. Small patches of Elo-A/Elo-A or Cdk9/Cdk9 mutant tissue also cause specific patterning defects in the wings and notum. Both Pol II elongation factors are probably required for normal expression of a great number of genes in the Drosophila genome. Indeed, both elongation genes are essential, and every attempt to create large mitotic clones resulted in larval lethality. Such lethality presumably reflects the general role of Elo-A and Cdk9 in gene expression. Previous studies have documented the general importance of the elongation factors ELL and Elo-A in Drosophila larval development and metamorphosis. Nonetheless, it would appear that a small number of patterning genes, including Ubx, are particularly sensitive to the loss of Elo-A and Cdk9 activity (Chopra, 2009a).

It has been extensively argued that Polycomb might mediate repression by propagating an inactive form of chromatin, for example, by methylation of H3K27 followed by recruitment of HP1 or other proteins that package chromatin in an inactive state. However, the demonstration that TBP and Pol II are present in the Ubx proximal promoter in wing imaginal discs suggests that PcG silencing does not render the chromatin inaccessible for the binding of even large protein complexes. Instead, it is proposed that paused Pol II could contribute to PcG silencing by excluding the binding of additional Pol II complexes. Such occlusion by steric hindrance might help reduce transcriptional noise and thereby maintain Ubx repression. Mutations in the elongation factor, ELL [Su(Tpl)], suppress Scr phenotypes caused by the Pc4 Polycomb mutant, raising the possibility that Pol II elongation factors somehow communicate with the PcG-silencing complex. It is proposed that stalling might serve the dual role of fostering both silencing and rapid induction and thereby provide a sharp on/off switch in Hox regulation (Chopra, 2009a).

Interactions among Polycomb domains are guided by chromosome architecture

Polycomb group (PcG) proteins bind and regulate hundreds of genes. Previous evidence has suggested that long-range chromatin interactions may contribute to the regulation of PcG target genes. This study adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue. The results demonstrate that PcG target genes interact extensively with each other in nuclear space. These interactions are highly specific for PcG target genes, because non-target genes with either low or high expression show distinct interactions. Notably, interactions are mostly limited to genes on the same chromosome arm, and it was demonstrated that a topological rather than a sequence-based mechanism is responsible for this constraint. These results demonstrate that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture (Tolhuis, 2011).

This study successfully adapted the 4C method to systematically map long-range chromatin contacts with limited material from a single fly tissue. With this method, interactions were detect between the ANT-C and BX-C in central brain. This observation is in good agreement with earlier microscopic reports, underscoring the strength of the method. Importantly, it was further demonstrated that not only the two Homeotic gene clusters, but also many other PcG target genes interact, suggesting that long-range chromatin contacts between PcDs are common in central brain tissue. The control fragments (wntD, CG5107, Crc, and RpII140), which do not reside in PcDs, have interactions that are distinct from PcDs, emphasizing the specificity of the findings (Tolhuis, 2011).

PcG targets show a strong preference for interaction with other PcG targets, suggesting that PcG proteins help to establish these interactions. This is in line with earlier in vivo and in vitro results that indicated that PcG proteins can keep certain DNA sequences together. However, interactions among PcG target genes are constrained by overall chromosome architecture, because the data demonstrate that loci need to be on the same chromosome arm for efficient interaction (Tolhuis, 2011).

Discrete interaction domains (DIDs) range in size from 6 to 600 kb, with an average of ~170 kb. Thus, highly local strong enrichments are found as well as moderate enrichments over larger regions, which may reflect different types of long-range interactions. It is emphasized that interactions as detected by 4C technically represent events of molecular proximity of DNA sequences, and not necessarily physical binding. Based on the current data it is therefore not possible to identify within the DIDs sequence elements that may mediate direct contact with other DIDs. It is conceivable that contacts between PcDs may occur at any position within the PcDs; if PcG protein complexes have an intrinsic propensity to aggregate, as has been observed in vitro, then large PcDs may have a higher chance of interacting with each other due to their larger ‘sticky’ surface area (Tolhuis, 2011).

The 4C method is a cell population based assay that only detects the most frequent interactions in the population. Previous 4C studies in mammalian cells suggested an extensive network of long-range interactions. The current data also suggests an extensive network among interacting PcDs. However, microscopic studies in mammalian cells revealed that specific long-range interactions occur only in a small proportion of the cells. Likewise, only a proportion of D. melanogaster cells show contacts between the two Homeotic clusters. Therefore, 4C data have to be carefully interpreted, because the identified interactions are in part stochastic and do not all occur simultaneously. As a consequence, it is not known how many PcDs interact in a single cell, but the most common interactions are known in the population of larval brain cells (Tolhuis, 2011).

Interphase chromosomes in most eukaryotes occupy distinct 'territories' inside the nucleus, with only a limited degree of intermingling. Although some studies have reported interactions between some loci that are on two different chromosomes (interchromosomal), unbiased 4C mapping in mouse tissues has indicated that interactions within the same chromosome (intrachromosomal) occur much more frequently than between different chromosomes. In addition, a recent genome-wide map of chromatin interactions in human cells showed that intrachromosomal interactions occur with higher frequency than interchromosomal contacts. The current data are in agreement with these observations, and show that most interactions are even limited to single chromosome arms, at least in D. melanogaster larval brain. Since the experiments indicate that a topological mechanism prevents interactions between the two arms of a chromosome, it is proposed that each arm (rather than the chromosome as a whole) forms a distinct territory. This is consistent with early microscopy studies of chromosome architecture in D. melanogaster, which suggested that chromosome arms are units of spatial organization (Tolhuis, 2011).

What topological mechanism may limit contacts between the two chromosome arms? About ~16 Mbp of pericentric heterochromatin are located in between the two euchromatic arms of chromosome 3. This heterochromatin region could act as a long spacer and prevent efficient interactions between DNA fragments that are located on either chromosome arm. However, the data show that interactions within one arm can span even longer distances, such as between Ptx1 and grn (~22.7 Mbp). Another explanation may be that the pericentric regions of all chromosomes assemble into a nuclear compartment, called chromocenter. This large structure could physically obstruct interactions between chromosome arms (Tolhuis, 2011).

Previous reports have demonstrated that certain PcG-bound PREs can pair in trans (i.e. when they are located on different chromosomes). First, a Fab7 PRE-element integrated on the X chromosome (Fab-X) was often found in close spatial proximity to the endogenous Fab-7 in the BX-C (chromosome 3R), although this phenomenon appears to be tissue-specific and dependent on the transgene integration site. Second, a microscopy assay based on Lac repressor/operator recognition showed that the Mcp PRE-element is able to pair with copies of that same element inserted at remote sites in the genome either in cis (i.e. when they are located on the same chromosome) or in trans. The 4C experiments also identified cases of trans interactions between endogenous loci, although they occur with low frequency (approximately 5% occurs in trans) (Tolhuis, 2011).

Although rare, such interactions between loci on different chromosome arms are of interest, because they indicate that the topological constraints imposed by chromosome architecture can in principle be overcome. In mammalian cells, there is evidence that the relative position of a gene locus within its chromosome territory (CT) influences its ability to form either cis- or trans-interactions. Peripheral regions of mammalian CTs intermingle their chromatin, which may allow for interactions between chromosomes. Indeed, more trans contacts are identified by 4C using a bait that often resides in the CT periphery compared to a bait located in the interior of a CT. Likewise, activation of the HoxB gene cluster during differentiation coincides with relocation away from its CT interior, and the active HoxB1 gene more frequently contacts sequences on other chromosomes compared to the inactive gene. In line with this, a varying degree of trans interactions are observed among eight bait sequences, suggesting distinct capacities to contact chromatin on other chromosomes. The trh gene has the strongest capacity to contact other chromosome arms. Interestingly, trh is located within 500 Kbp of the telomere of chromosome 3L, and 5 out of 6 contacts in trans occur within 500 Kbp of other telomeres. Thus, interactions between chromosome arms may be possible if loci are favorably positioned on the edge of chromosome (arm) territories, which could be the case for telomeric sequences in larval brain cells (Tolhuis, 2011).

The experiments with strain In(3LR)sep showed dramatic changes in interactions, such as loss of contacts between the Homeotic gene clusters and gain of contacts with other PcDs. Despite these changed interactions, no convincing evidence was found for global gene expression alterations on the In(3LR)sep chromosome. This raises the question: how relevant are long-range chromatin contacts between PcG-target genes for regulation of expression?

The lack of detectable expression changes may indicate that long-range interactions have only quantitatively subtle effect on the regulation of gene expression. Nevertheless, such subtle effects on gene expression could be very important for long-term viability and species survival. In(3LR)sep animals do suffer from an overall reduced viability during several stages of development, which may indicate a generally reduced fitness, possibly due to a dimly altered regulation of gene expression (Tolhuis, 2011).

Alternatively, PcG gene regulation may not be affected in strain In(3LR)sep, because Abd-B and Antp, although they no longer interact with each other, still prefer to interact with other PcDs, suggesting that it is not relevant which PcDs interact. In such a model, the complement of all interactions contributes to PcG-mediated gene silencing in a population of cells (Tolhuis, 2011).

Finally, it is interesting to note that over ~100 million years of evolution of the Drosophila genus, exchange of genes between chromosome arms has been rare despite extensive rearrangements within each arm. Chromosome arm territories ensure that genes within a single arm are relatively close compared to genes on other arms, which may have resulted in an increased chance of rearrangements within one arm. Alternatively, the importance of long-range interactions among sets of genes, which are topologically limited to the same arm, may have contributed to the selective pressure that has led to this remarkable conservation of the gene complement of each chromosome arm (Tolhuis, 2011).

The histone H3-K27 demethylase Utx regulates HOX gene expression in Drosophila in a temporally restricted manner

Trimethylation of histone H3 at lysine 27 (H3-K27me3) by Polycomb repressive complex 2 (PRC2) is a key step for transcriptional repression by the Polycomb system. Demethylation of H3-K27me3 by Utx and/or its paralogs has consequently been proposed to be important for counteracting Polycomb repression. To study the phenotype of Drosophila mutants that lack H3-K27me3 demethylase activity, UtxΔ), a deletion allele of the single Drosophila Utx gene, was created. UtxΔ homozygotes that contain maternally deposited wild-type Utx protein develop into adults with normal epidermal morphology but die shortly after hatching. By contrast, UtxΔ homozygotes that are derived from Utx mutant germ cells and therefore lack both maternal and zygotic Utx protein, die as larvae and show partial loss of expression of HOX genes (Ubx and Abd-B) in tissues in which these genes are normally active. This phenotype classifies Utx as a trithorax group regulator. It is proposed that Utx is needed in the early embryo to prevent inappropriate installment of long-term Polycomb repression at HOX genes in cells in which these genes must be kept active. In contrast to PRC2, which is essential for, and continuously required during, germ cell, embryonic and larval development, Utx therefore appears to have a more limited and specific function during development. This argues against a continuous interplay between H3-K27me3 methylation and demethylation in the control of gene transcription in Drosophila. Furthermore, this analyses do not support the recent proposal that Utx would regulate cell proliferation in Drosophila as Utx mutant cells generated in wild-type animals proliferate like wild-type cells (Copur, 2013).

Architectural protein Pita cooperates with dCTCF in organization of functional boundaries in Bithorax Complex

Boundaries in the Bithorax Complex (BX-C) of Drosophila delimit autonomous regulatory domains that drive parasegment-specific expression of homeotic genes. BX-C boundaries have two critical functions: they must block crosstalk between adjacent regulatory domains, and at the same time facilitate boundary bypass. The C2H2 zinc finger Pita protein binds to several BX-C boundaries including Fab-7 and Mcp. To study Pita functions, a boundary replacement strategy was used by substituting modified DNAs for the Fab-7 boundary, which is located between the iab-6 and iab-7 regulatory domains. Multimerized Pita sites block iab-6<-->ab-7 crosstalk but fail to support iab-6 regulation of Abd-B (bypass). In the case of Fab-7 a novel sensitized background was used to show that the two Pita sites contribute its boundary function. Although Mcp is from BX-C, it does not function appropriately when substituted for Fab-7; it blocks crosstalk but does not support bypass. Mutation of the Mcp Pita site disrupts blocking activity and also eliminates dCTCF binding. In contrast, mutation of the Mcp dCTCF site does not affect Pita binding, and this mutant boundary retains partial function (Kyrchanova, 2017).

Previous studies on the Pita (also known as Spotted Dick) protein suggested that it is a transcriptional activator and showed that the replication defects in pita mutants and in RNAi knockdowns were due to a reduction in the expression of the replication origin protein Orc4. The experiments presented in this study, together with previous studies (Maksimenko, 2015), indicate that pita has an additional, if not an entirely different, function, which is chromosome architecture. This paper details the evidence in favor of this conclusion, and also discuss the implications of findings for boundary function in the context of BX-C (Kyrchanova, 2017).

Boundary replacement experiments provide compelling evidence that the zinc-finger protein Pita functions just like other insulator/architectural proteins. When placed in the context of Fab-7, multimerized Pita-binding sites block crosstalk between iab-6 and iab-7, but are not permissive for the regulatory interactions between iab-6 and the Abd-B gene. In this respect, the functioning of the multimerized Pita-binding sites is similar to that observed when multimerized sites for 'canonical' boundary factors, dCTCF and Su(Hw), are substituted for the Fab-7 boundary. In the context of Fab-7, they also block crosstalk between iab-6 and iab-7, but do not support bypass (Kyrchanova, 2017).

The boundary functions of the Pita protein are also supported by experiments testing its activity in a native context. For Fab-7, there are two Pita-binding sites in the HS2 hypersensitive region. Since previous studies have shown that HS1 is sufficient for full boundary activity, when the iab-7 PRE (HS3) is present, it is clear that Pita function is redundant. This was confirmed by introducing mutations in the two Pita-binding sites in a Fab-7 boundary, HS1+2+3, that lacks the '*' nuclease-hypersensitive site, but contains the iab-7 PRE. However, a different result was obtained in the context of a sensitized replacement, HS1+2, in which the iab-7 PRE (HS3) is deleted. In this sensitized background, the two Pita-binding sites in HS2 are essential for boundary activity (Kyrchanova, 2017).

Interestingly, the sensitized HS1+2 Fab-7 replacement has unprecedented properties. Unlike previously described Fab-7 mutations, which are dominant, the boundary defects of HS1+HS2 can be fully complemented by a wild-type boundary in trans. Additionally, as a homozygote, it has differential effects on the specification of dorsal and ventral tissues. The A6 (PS11) sternite is missing in HS1+2 males. This gain-of-function transformation indicates that boundary activity is disrupted in the cells that give rise to this ventral cuticular structure. By contrast, the A6 tergite is not only nearly normal in size, but is also properly specified. This finding means that boundary activity is largely retained in the PS11 cells that give rise to dorsal cuticle structures (Kyrchanova, 2017).

It is also worth noting that HS1+2 is very different from mutations that delete the iab-7 PRE (HS3) but retain the entire Fab-7 boundary. First, the vast majority of homozygous iab-7 PRE (HS3) deletion males are indistinguishable from wild type, arguing that the HS3 deletion retains full boundary function. Second, in a few of the males (~2.5%), small sections of the dorsal A6 tergite are missing. This phenotype is most readily explained by a loss of PRE silencing, and consequent gain-of-function transformation, in a subset of the cells that give rise to the dorsal cuticle. As the HS1+2 replacement differs from all of the iab-7 PRE (HS3) deletions isolated previously in that it lacks 'HS*', it would appear that this part of the boundary contains binding motifs for factors that are important for boundary function specifically in ventral tissues (Kyrchanova, 2017).

This would not be the only Fab-7 boundary factor that has 'developmentally' restricted activity. The two large complexes known to be important for Fab-7 HS1 boundary function, Elba and LBC, are active at different stages of development; the former in early embryos and the latter from mid-embryogenesis onwards. The fact that there is likely to be yet another boundary factor whose activity is developmentally restricted, fits with the idea that boundary function in flies can be subject to stage- and/or tissue-specific regulation (Magbanua, 2015; Kyrchanova, 2017 and references therein).

One of the paradoxes posed by the BX-C boundaries is that six of the nine regulatory domains in the complex are separated from their homeotic target genes by one or more boundaries. Consequently, these boundaries must, on the one hand, block regulatory interactions between adjacent domains and, on the other, facilitate boundary bypass. One of models to explain these two contradictory activities is that BX-C boundaries have unique properties, i.e. they are designed to block interactions between enhancers/silencers, but not between enhancers/silencers and promoters. This model gained currency from replacement experiments, which showed that the BX-C boundary Fab-8 can substitute for Fab-7, while two heterologous boundaries cannot. A prediction of the model is that other BX-C boundaries could also substitute for Fab-7. However, contrary to this prediction, the current experiments indicate that Mcp340 boundary behaves like the heterologous fly boundaries -- it blocks both crosstalk and bypass (Kyrchanova, 2017).

Analysis of the effects of mutations in the Pita and dCTCF sites of the Mcp340 boundary suggest that there is a complicated relationship between blocking crosstalk and blocking or enabling bypass. Although mutations in the Pita and dCTCF disrupt the functioning of Mcp340 replacement, the actual consequences of each mutation are quite distinct. In the case of the Pita mutation, loss of Pita binding was found to lead to a substantial reduction in the binding of dCTCF. This means that for this particular boundary, Pita association is required to recruit dCTCF. The requirement is not, however, reciprocal: deleting the Mcp dCTCF site has no effect on Pita association (Kyrchanova, 2017).

Correlated with the differential effects on protein binding, the M340ΔPita and M340ΔCTCF mutants have quite different phenotypes. The phenotype (mixed gain and loss of function) of the former resembles a classic Fab-7 boundary deletion in which the iab-7 PRE (HS3) is retained. In contrast, the phenotype of the latter is a mixture of gain and loss of function, together with cuticle that has morphological features identical to that in A6 (PS11) of wild-type flies. The presence of cuticle that has the proper PS11 identity argues that M340ΔCTCF retains residual boundary function that, in a subset of cells, is sufficient to not only block crosstalk between iab-6 and iab-7, but is also able to facilitate iab-6 bypass (Kyrchanova, 2017).

A simple interpretation of this finding is that the Pita protein differs from dCTCF in that it blocks crosstalk but can facilitate bypass. However, this simple model is not supported by other findings. First, as noted above, just like multimerized dCTCF sites, multimerized Pita sites block both crosstalk and bypass when substituted for Fab-7. Second, the two Pita sites in Fab-7 are not in themselves sufficient for blocking crosstalk. Third, the Fab-8 boundary, which has two dCTCF sites, but no sites for Pita, has both blocking and bypass activity when substituted for Fab-7. Moreover, these dCTCF sites appear to contribute to the bypass activity of the Fab-8 replacement. Thus, a more likely hypothesis is that there are other, as yet unidentified, factors that are bound to Mcp340 and contribute to the blocking and (newly acquired) bypass activities of the M340ΔCTCF mutant, in addition to the Pita protein. Taken together with the finding that reversing Fab-8 eliminates bypass activity (Kyrchanova, 2016), the current experiments with Mcp suggest that there may not be a common mechanism for generating both blocking and bypass activity. Rather, each BX-C boundary would appear to deploy distinct mechanisms that are adapted for their specific context within the complex (Kyrchanova, 2017).

Based on RNAi knockdown experiments in S2 cells, it has been suggested that Pita is a transcriptional activator and that it could play a crucial role in coordinating S phase progression. This idea was supported by experiments showing that the replication defects induced by Pita depletion are caused by a reduction in Orc4 expression. However, only 32 genes are downregulated (and 10 upregulated) after Pita RNAi, and most appear to have nothing to do with replication. Furthermore, as there are several thousand Pita sites in the genome, the number of affected genes is surprisingly low. In this light, an obvious question is whether blocking, instead of transcriptional activation, might account for the effects of Pita depletion on the Orc4 transcription? Although this study did not investigate how Pita functions in the S2 cells, there are reasons to think that this is a distinct possibility. ChIP experiments have shown that Pita binds to a region upstream of the Orc4 gene in S2 cells. ModEncode ChIP experiments indicate that there is a large PcG silenced domain just beyond this Pita site. Thus, an alternative possibility is that Orc4 expression is reduced when Pita is depleted, because the gene is silenced by the PcG spreading. Several of the other Pita transcriptional targets are also close to the PcG domains, and could be silenced in a similar manner (Kyrchanova, 2017).

Distinct elements confer the blocking and bypass functions of the Bithorax Fab-8 boundary

Boundaries in the Drosophila bithorax complex (BX-C) enable the regulatory domains that drive parasegment specific expression of the three Hox genes to function autonomously. The four regulatory domains (iab-5, iab-6, iab-7 and iab-8) that control the expression of the Abdominal-B (Abd-B) gene are located downstream of the transcription unit and are delimited by the Mcp, Fab-6, Fab-7 and Fab-8 boundaries. These boundaries function to block crosstalk between neighboring regulatory domains. In addition, three of the boundaries (Fab-6, Fab-7 and Fab-8) must also have bypass activity so that regulatory domains distal to the boundaries can contact the Abd-B promoter. In these studies a functional dissection was undertaken of the Fab-8 boundary using a boundary replacement strategy. The studies indicate that the Fab-8 boundary has two separable sub-elements. The distal sub-element blocks crosstalk, but can not support bypass. The proximal sub-element has only minimal blocking activity but is able to mediate bypass. A large multiprotein complex, the LBC, binds to sequences in the proximal sub-element and contributes to its bypass activity. The same LBC complex has been implicated in the bypass activity of the Fab-7 boundary (Kyrchanova, 2019).

Abdominal-B: Biological Overview | Evolutionary Homologs | Promoter Structure | Targets of activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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