Abdominal-B
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).
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).
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).
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).
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).
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).
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).
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).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Abdominal-B:
Biological Overview
| Evolutionary Homologs
| Promoter Structure
| Targets of activity
| Protein Interactions
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.