Ultrabithorax
Polycomb mediates repression of Ubx (McCall, 1994), while Trithorax acts positively to maintain Ubx expression (Breen, 1993).
Silencing also occurs in imaginal disc cells. Fragments which mediate silencing in anterior regions of imaginal discs contain embryonic silencers and HB target sites. bxd is not under hb control itself, but the silencing activity of BXD depends on combination with
fragments containing HB protein binding sites. Since silencing by BXD also requires Polycomb function, this suggests that BXD contains target sites for PC or for PC-like proteins (Christen, 1994).
Trithorax complex genes, absent, small, or homeotic discs 1 and 2 interact with homeotic genes. The consequences of ash1 and ash2 mutations on the expression of homeotic selector genes in imaginal discs demonstrate that both ash1 and ash2 are trans-regulatory elements of homeotic selector gene regulation. Hypomorphic ash1 mutations cause variegated expression of Antennapedia, Sex combs reduced, Ultrabithorax, and engrailed. (LaJeunesse, 1995).
The ash2 transcript detected in null mutant
larvae is interpreted as a maternally derived product. This suggests
that the zygotic requirement for ash2 begins during the
third larval instar and is consistent with phenotypes of
imaginal discs from mutant larvae. For example, in haltere discs from mid third instar mutant larvae there is no expression of an Antennapedia
reporter gene and nearly uniform accumulation of Ultrabithorax protein
as in normal haltere discs, whereas
in haltere discs from late third instar mutant larvae
there is ectopic expression of an Antennapedia reporter
gene and patchy accumulation of Ultrabithorax protein. Finding the first detectable 53kDa Ash2 protein in late third instar larvae
suggests that whatever activity is responsible for producing this smaller Ash2 product is not present before the
end of the third larval instar. The fact that the final
Ash2 protein is significantly different in larvae and
pupae suggests that it has different functions in larvae
and pupae. This may represent a novel mechanism for
a single gene product to have multiple functions (Adamson, 1996).
A regulatory element in the Ubx gene responds to Pc-G and trx-G genes. Transposons, genetically engineered pieces of DNA carrying the regulatory element to new ectopic sites, create new binding sites for Pc-G products in the new sites in which they integrate. The transposons carry Pc-G response elements (PRE), DNA regions responsive to the repressive affect of Pc-G genes. PREs and Pc-G proteins establish a repressive complex that keeps itself and other distal enhancers repressed in cells where they were first active and then repressed, and maintains this repressed state over many cell divisions. PRE functions to silence these remote enhancers or to maintain expression regulated by trx-group products. Hunchback mediates repression at the PRE. The trx-G products stimulate the expression of separate and distinct enhancers, active in imaginal discs (Chan, C-S. 1994).
Newer evidence maintains that silencing of late Ubx enhancers by PRE is Hunchback-independent. If a PRE is combined with late enhancers, including a Wingless responsive CNS element or imaginal disc enhancers, repression of a reporter gene can be established everywhere in the embryo, irrespective of the presence or absence of Hunchback protein. If, however, these late enhancers are combined with a Ubx early enhancer, one that is silenced early, as well as with a PRE, then repression is established only where the reporter gene was inactive at early stages. These results imply that the Polycomb complex is not dependent on Hunchback, and suggest that the pattern of silencing reflects rather the level of activity of the gene at the time the Polycomb repressive protein complex is formed (Poux, 1996).
moira (mor) is a member of the trithorax group of homeotic gene regulators in Drosophila. moira is required for the function of multiple homeotic
genes of the Antennapedia and bithorax complexes (HOM genes) in most imaginal
tissues. Heterozygous mor mutations suppress the following Polycomb-induced phenotypes: The requirement for moira function is at the level of transcription. The ability of moira mutations to supppress Antp homeotic phenotypes is dependent on the promoter.
moira is also required for transcription of the engrailed segmentation gene in the
imaginal wing disc. Because homozygous mor clones have phenotypes similar to those seen in clones of cells that have lost en function, en transcription was examined in clones of cells in the posterior wing. In the absence of transcriptional activation by mor, the pattern of en is altered. Greatly reduced en expression is found in wing clones. The abnormalities caused by the loss of moira function in germ
cells suggest that at least one other target gene requires moira for normal oogenesis (Brizuela, 1997).
Arresting cell division using the string mutation or blocking DNA
replication with aphidicolin failed to prevent ectopic expression of the homeotic gene Ultrabithorax
in Polycomb mutants. Thus, even in the absence of DNA replication, Pc is required to maintain spatially
restricted patterns of homeotic gene expression (Gould, 1990).
The wave of differentiation that traverses the Drosophila eye disc requires rapid transitions in gene expression that are controlled by a number of signaling molecules also required in other developmental processes. A mosaic genetic screen has been used to systematically identify autosomal genes required for the normal pattern of photoreceptor differentiation, independent of their requirements for viability. In addition to genes known to be important for eye development and to known and novel components of the Hedgehog, Decapentaplegic, Wingless, Epidermal growth factor receptor, and Notch signaling pathways, several members of the Polycomb and trithorax classes of genes, encoding general transcriptional regulators, were identified. Mutations in these genes disrupt the transitions between zones along the anterior-posterior axis of the eye disc that express different combinations of transcription factors. Different trithorax group genes have very different mutant phenotypes, indicating that target genes differ in their requirements for chromatin remodeling, histone modification, and coactivation factors (Janody, 2004).
Very similar phenotypes were observed in clones mutant for Pc or E(z), which encode components of two distinct complexes implicated in transcriptional repression. Although likely null alleles for both genes were used, the phenotype of E(z) clones appeared slightly stronger, with a greater likelihood of derepressing hth in posterior regions of the eye disc. The E(z) protein has been shown to act as a histone methyltransferase for H3 K27 within a complex that also includes Extra sex combs (Esc), Suppressor of zeste 12 [Su(z)12], and the histone-binding protein NURF-55. esc appears to act only early in embryonic development, while E(z) and Su(z)12 are continuously required to repress inappropriate homeotic gene expression in wing imaginal discs. The core PRC1 complex contains Pc, as well as Ph, Psc, and dRing1, and can prevent SWI/SNF complexes from binding to a chromatin template. Pc, Psc, and ph are all required to prevent homeotic gene misexpression in wing discs; however, Psc and ph act redundantly with closely related adjacent genes. The two complexes are thought to be linked through binding of the Pc chromodomain to K27-methylated H3. The stronger phenotype of E(z) mutations in the eye disc might suggest that methylation of H3 K27 can recruit other proteins in addition to Pc (Janody, 2004).
In the eye disc, loss of E(z) or Pc leads to misexpression of the homeotic gene Ubx, but this does not seem to account for the entire phenotype. Although Ubx is sufficient to turn on tsh ectopically, misexpression of hth and tsh can occur in E(z) or Pc clones in which Ubx is not misexpressed. This suggests that hth and tsh are either direct targets of Pc/E(z)-mediated repression or targets of a downstream gene other than Ubx, possibly one of the homeotic genes not examined. Tsh misexpression would be sufficient to explain the suppression of photoreceptor differentiation in clones close to the morphogenetic furrow, since it is able to maintain expression of Hth and Ey and, in combination with them, to repress eya. Misexpression of Tsh can also account for overgrowth of Pc or E(z) mutant cells at the posterior margin of the eye disc (Janody, 2004).
Loss of maternal brahma function blocks
oogenesis; individuals homozygous for extreme brm alleles die as late embryos with no obvious pattern
defects (Brizuella, 1994). Since it has not been possible to generate embryos lacking both
maternal and zygotic brm function, the exact role of brm in embryonic development is not clear.
Information concerning the role of brm after embryogenesis has been derived primarily from the
analysis of hypomorphic brm alleles. Individuals trans-heterozygous for certain combinations of brm
alleles survive to adulthood and exhibit developmental abnormalities similar to those arising from
reduced expression of Antp-C and Bx-C genes, including the transformation of first legs to second legs
and the fifth abdominal segment to a more anterior identity (Brizuella, 1994). Because the
effect of complete loss of brm function had not been examined, it was unclear whether brm is also involved in other processes. To clarify the role of brm in Drosophila development, mosaic analysis has been used to determine the null phenotype of brm mutations. As an
alternative approach, site-directed mutagenesis was used to generate dominant-negative brm mutations
and investigate the functions of evolutionarily conserved domains within the Brm protein (Elfring, 1998).
A dominant-negative brm mutation (DNbrm) was generated by replacing a conserved lysine in the ATP-binding site of the Brm protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the high molecular weight (2 million Daltons) Brm complex. Expression of the dominant-negative Brm protein causes
peripheral nervous system defects, homeotic transformations, and decreased viability. Individuals bearing
one or two copies of the dominant-negative Brm are viable, but frequently exhibit partial transformations
of haltere to wing, as evidenced by an increase in haltere size and the appearance of ectopic bristles on
the capitellum. Approximately one third of dominant-negative Brm adults exhibit this
transformation, which is presumably caused by the decreased expression of the Ultrabithorax
gene. Increasing the ratio of dominant-negative Brm to wild-type Brm to 2:1 is lethal. Thus,
the dominant-negative brm mutation behaves as an antimorphic allele of brm. Expression of the dominant-negative Brm protein in patterns identical to the segmentation genes hairy or engrailed has no effect on embryonic viability or segmentation.
The lack of an embryonic phenotype resulting from embryonic expression of the dominant-negative Brm protein may be caused by the high maternal expression of wild-type Brm protein, which is sufficient to allow
embryogenesis to proceed to near completion in the absence of zygotic brm function. Expression of the dominant-negative protein in imaginal tissues after embryogenesis leads to greatly reduced viability.
Individuals reared at 20ƒ display partial transformation of first leg
to second leg, as evidenced by a reduction in the number of sex comb teeth on the first leg. This phenotype is also seen in adults trans-heterozygous for hypomorphic brm alleles and is presumably caused by decreased expression of the Sex combs
reduced (Scr) gene. Adults reared at 20ƒ also display twinning of mechanosensory bristles, a phenotype similar to that observed in clones of brm2 tissue.
Expression of the dominant negative protein also has dramatic effects on the
size and morphology of the wing; mutant wings are reduced in size, and the L5 and the posterior
cross-vein (PCV) are usually absent. Defects in the campaniform sensilla, a
class of sensory organs important for flight, are also observed with high frequency. These defects fall into four classes:
missing sensilla, duplication or triplication of sensilla, transformation of sensilla into bristles, and the
appearance of ectopic sensilla. Ectopic sensilla and bristles are observed most
frequently on the L3 vein. Three sensilla (L3-1, L3-2, and L3-3) and no bristles are normally found on
this vein. By contrast, approximately one-half of mutant wings display one or two additional sensilla
on L3. Ectopic bristles are observed on this vein in approximately one-fifth of mutant wings (Elfring, 1998).
Transvection is the phenomenon by which the expression of a gene can be controlled by its homologous counterpart in trans,
presumably due to pairing of alleles in diploid interphase cells. Transvection or trans-sensing phenomena have been reported
for several loci in Drosophila, the most thoroughly studied of which is the Bithorax-Complex (BX-C). It is not known how
early trans-sensing occurs nor the extent or duration of the underlying physical interactions. The physical
proximity of homologous genes of the BX-C during Drosophila embryogenesis has been investigated by applying fluorescent in situ
hybridization techniques together with high-resolution confocal light microscopy and digital image processing. The association
of homologous alleles of the BX-C starts in nuclear division cycle 13, reaches a plateau of 70% in postgastrulating embryos,
and is not perturbed by the transcriptional state of the genes throughout embryogenesis.
After gastrulation, the pairing frequency of the Ubx and Abd-B
loci increased to 60-70%. Pairing frequencies never reach 100%,
indicating that the homologous associations are in equilibrium with a dissociated state. The effects of
translocations and a zeste protein null mutation, both of which strongly diminish transvection phenotypes, have been investigated as to the extent of
diploid homolog pairing. Although translocating one allele of the BX-C from the right arm of chromosome 3 to the left arm
of chromosome 3 or to the X chromosome abolishes trans-regulation of the Ultrabithorax gene, pairing of homologous alleles
surprisingly is reduced to only 20-30%. A zeste protein null mutation neither delays the onset of pairing nor leads to
unpairing of the homologous alleles (Gemkow, 1998).
The data support a model for trans-sensing based on physical
proximity and association of the homologous alleles. The
translocation mutants that were investigated demonstrate that
pairing can occur between segments of non-homologous
chromosomes, albeit with a reduced frequency compared to
that for the sites on homologous chromosomes. It is
concluded that the efficacy of the trans-sensing effect is
dependent on the sensitivity of the phenotype to the
transcriptional level of the gene in question and to the stability
or affinity of the specific paired locus.
What model might be consistent with these data and those of
others? In particular, what mechanism is responsible for the
stable pairing of chromosomes? The information that loci on
extreme positions of the 3R chromosome (near the centromere,
central to the chromosome arm and close to the telomere) show
the same frequency and onset of pairing argues against a zipper
mechanism by which a centromeric association leads to a linear
pairing along the chromosome to the telomere. The equal
pairing frequencies observed for the translocation to the X
chromosome and the opposite arm of chromosome 3 also
argue against the centromere having a dominant role in the
pairing mechanism and furthermore require flexibility in the
chromosomal arms with respect to each other. In addition, the
pairing properties of chromosome 4 argue against an earlier
association of centromeric heterochromatin than euchromatic
loci or a dominant role of heterochromatin-binding proteins in
the recognition process. The heterochromatin-binding protein,
HP1, does not appear in embryos until nuclear cycle 10,
increasing dramatically in cycle 14.
Although HP1 has been implicated as a protein causing
heterochromatin aggregation, the data do not support a model
by which heterochromatin regions would preferentially interact
and drive homologue recognition and pairing. The HP1-
binding loci remain independent in diploid embryonic nuclei
even as late as stage 12. Distinct centromeres can be discerned
in many stage 14 diploid nuclei in which chromosome 4 lies
outside the regions occupied by the other centromeres (Gemkow, 1998).
No
preferential disposition of the unpaired BX-C is seen in the lateral
dimension of the nucleus in cycle 14 embryos. In cycle 14
embryos, the largest interallelic distance measured for probes
from the BX-C is close to the diameter of the nucleus but is not
as large as the axial dimension of the nucleus. This is most easily
explained by the fact that, following the rapid early division
cycles, the chromosomes are all still oriented with their
centromeres at the apical surface, although no
chromocenter exists. Decondensation of the chromosomes in
this orientation predetermines a preferred axial disposition of the
locus. That this interpretation is probably correct is supported by results for chromosome 4, which always lies on the apical
surface in blastoderm embryos. These observations are compatible with a multipoint
recognition of sequences dispersed along the chromosome,
resulting in globally stable interactions despite relatively
unstable (short lifetime) individual associations. The
combination of numerous such associations would lead to a
sudden increase in the overall stability of the paired
chromosome, i.e. to a highly cooperative pairing along the
whole chromosome once a threshold number of interactions
was established. If the pairing is driven by associations of the
chromosomes through protein-protein interactions then each
individual paired site will have a finite binding constant and be
in equilibrium with its unpaired state. Thus, if any
individual locus (in this case, the genes of the BX-C) is probed there will
be a probability (or frequency) of dissociation (in this case
about 30%-35%). The fact that the paired state
predominates and that the distance between unpaired loci
becomes greatly reduced at later times during embryogenesis
is consistent with this model. The
paired alleles on the chromosome may be likened to the buttons on a shirt or blouse. Any one button may become unfastened yet the
whole garment will not open. In addition, the probability of
the unpaired region to pair again will be much higher than in
the original recognition step since the local concentration, i.e.
the total volume available to the homologous loci is reduced
by some orders of magnitude due to the associations of sites
flanking the test locus (Gemkow, 1998).
Several conclusions can be derived from these data: (1) the
homology search occurs with approximately the same kinetics
for the BX-C as for chromosome 4, as well as other sites on
chromosome 3 as soon as the cell cycle is lengthened beyond
successive S and M phases, (2) the frequency of pairing in
postmitotic embryos reflects the affinity of the recognition
elements scaled by the size of the chromosomal target locus,
and (3) pairing or association of the chromosomes may be
mediated through protein-protein interactions.
To address this latter point, a protein was investigated that
influences transvection of some genes (in particular white,
yellow, Ubx and decapentaplegic), shows self-association, and binds at several hundred loci on
polytene chromosomes: the protein Zeste. It has been argued that the self-association tendency
of this protein promotes associations of Zeste-binding sites
in both cis and trans. In addition, there are a number of clustered
Zeste-binding sites in the upstream control region for the
Ubx gene, the
locus that was investigated in these experiments. The effect of a zeste null
mutation on pairing at the Ubx locus was of interest, since this
mutant changes the Ubx phenotypes in transvection-sensitive
experiments. A higher variation of the pairing
frequencies was observed but a similar mean was found for both the onset and extent of
pairing, when compared to wild type; that is, mutant embryos were
detected with pairing frequencies as low as 50%. Local decondensation of the fluorescent signals was detected in some of
the chromosomes at the BX-C in zeste mutants. It is
possible that the Polycomb group protein complexes
themselves, which assemble at the PREs in the BX-C, are a stronger determinant of the local pairing
affinity at this locus. Such a possibility is supported by the
observation of the trans interactions of PREs.
The present hypothesis is that many specific protein-protein
interactions are responsible for recognition and pairing along
the chromosomes and that they comprise proteins that have
other functions such as enhancers or repressors. There is clear indication from data on mitotic recombination, that the length of time
between mitoses sets the lower limit on the frequency of
pairing. According to the model presented here,
sites with strong protein-protein interactions would act as
nucleating, sites but stable homolog associations along the
length of the chromatids would only occur by multipoint
recognition and interaction (Gemkow, 1998).
Drosophila Iswi, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF,
CHRAC, and ACF. To clarify the biological functions of Iswi, null and dominant-negative Iswi mutations were generated and characterized. Iswi
mutations affect both cell viability and gene expression during Drosophila development. Iswi mutations also cause striking alterations in the structure of the male X
chromosome. The Iswi protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for Iswi in transcriptional
repression. These findings reveal novel functions for the Iswi ATPase and underscore its importance in chromatin remodeling in vivo (Deuring, 2000).
To determine when Iswi is required during development, the lethal phase and phenotype of Iswi null mutants were examined. Individuals heterozygous for ISWI1 or ISWI2 are viable and phenotypically normal. ISWI1/Df(2R)vg-C individuals die during late larval or early pupal development and display no obvious homeotic transformations or other pattern defects. Similar results were obtained for both ISWI2/Df(2R)vg-C and ISWI1/ISWI2 individuals (Deuring, 2000).
In vitro studies have suggested that Iswi plays an important role in transcription by facilitating the interaction of transcription factors with chromatin. One of the best candidates for a transcription factor that requires Iswi for its activity is the GAGA factor. GAGA factor binds to GA-rich sequences near the promoters of a wide variety of Drosophila genes and is thought to activate transcription by altering local chromatin structure. As the ATPase subunit of NURF, Iswi assists the GAGA factor to remodel chromatin in vitro, suggesting that the two proteins may act in concert to modulate chromatin structure in vivo as well. To examine possible interactions between Iswi and GAGA factor in vivo, the phenotypes of mutations in the two genes have been compared. GAGA factor is encoded by Trithorax-like (Trl), a member of the trithorax group of homeotic gene activators. Trl mutations enhance mutations in trithorax and cause homeotic transformations resulting from the decreased transcription of homeotic genes. Trl mutations also enhance position effect variegation, suggesting that GAGA factor antagonizes the assembly or function of heterochromatin. Unlike Trl mutations, Iswi mutations fail to enhance or suppress position effect variegation. No dominant interactions could be detected between mutations in Iswi and other genes, including Trl, other trithorax group genes (trithorax and brm), and Polycomb, a repressor of homeotic genes that is thought to act at the level of chromatin structure. These data suggest that Iswi and GAGA factor play distinct roles in chromatin remodeling in vivo (Deuring, 2000).
To investigate the role of Iswi in transcriptional activation in vivo, the effect of Iswi mutations on the expression of two targets of the GAGA factor were examined: the segmentation gene engrailed (en) and the homeotic gene Ultrabithorax (Ubx). The expression of En protein is reduced dramatically in imaginal discs of ISWI1/ISWI2 mutant larvae. Similar results are observed for Ubx. These data suggest that Iswi is essential for the expression of both en and Ubx in imaginal discs, although the possibility that this interaction is indirect cannot be ruled out (Deuring, 2000).
To directly observe interactions between Iswi and chromatin in vivo, the distribution of Iswi protein on salivary gland polytene chromosomes in third instar larvae was examined by immunofluorescence microscopy. Consistent with a fairly general role in transcription or other processes, Iswi protein is present at a large number of euchromatic sites in the polytene chromosomes. The same pattern was observed using whole sera and affinity-purified antibodies. The chromosomal distribution of Iswi protein is not appreciably altered following heat shock (Deuring, 2000).
Iswi protein is also associated with a subset of heterochromatin, as evidenced by punctate staining at the chromocenter. It is difficult to analyze the distribution of heterochromatic proteins on salivary gland chromosomes, since heterochromatic sequences are underreplicated in polytene tissues. To more accurately map the regions of heterochromatin with which Iswi interacts, the distribution of Iswi protein on mitotic chromosomes from larval neuroblasts was examined. On mitotic chromosomes, Iswi protein is abundantly present on the euchromatic arms of all chromosomes and is concentrated in regions of heterochromatin enriched with middle-repetitive sequences. For example, on the heterochromatic Y chromosome, Iswi is concentrated in the h11–13 region, which is composed almost entirely of middle repetitive DNA families. By contrast, little Iswi protein is detected in regions containing predominantly satellite DNA. The distributions of Iswi and GAGA factor on polytene and mitotic chromosomes were determined by double-label immunofluorescence microscopy. Both GAGA factor and Iswi are associated with hundreds of sites in the euchromatin of polytene chromosomes, but the distributions of the two proteins do not overlap extensively. Even greater differences in the distributions of the two proteins were observed in mitotic chromosomes where the GAGA factor, but not Iswi, is associated with GAGA-satellite sequences. The lack of extensive colocalization does not rule out an interaction between Iswi and GAGA at specific loci, but it does suggest that Iswi and GAGA are not obligatory partners (Deuring, 2000).
The decrease in en and Ubx expression in Iswi mutant larvae is consistent with reports that Iswi is involved in transcriptional activation in vitro. Consequently, it was not anticipated that the distributions of Iswi and RNA Pol II on salivary gland polytene chromosomes would be mutually exclusive. The preferential association of Iswi with transcriptionally inactive regions suggests that Iswi may create changes in chromatin structure that are not conducive to RNA Pol II transcription in vivo. Although there is no direct evidence that Iswi represses transcription, such a function would be consistent with the proposal that Iswi acts antagonistically toward histone acetyltransferases to compact chromatin structure. Based on these observations, further investigation of the role of Iswi in transcriptional repression is clearly warranted (Deuring, 2000).
How can the distributions of Iswi and RNA Pol II on polytene chromosomes be reconciled with the effect of Iswi mutations on gene expression in imaginal discs and the ability of Iswi complexes to activate transcription in vitro? One possibility is that Iswi has roles in both transcriptional repression and activation. NURF, ACF, and CHRAC were purified from Drosophila embryo extracts, and nothing is known about the nature or relative abundance of Iswi complexes in larvae. Perhaps only one Iswi complex is associated with transcriptionally inactive chromatin in the larval salivary gland, while others are either less abundant or transiently interact with chromatin to activate transcription. It is also possible that the interaction of Iswi with en and Ubx is indirect. For instance, the decreased expression of the two genes may be a secondary consequence of reduced cell viability in Iswi mutant larvae (Deuring, 2000).
The nucleosome remodeling factor (NURF) is one of several ISWI-containing protein complexes that catalyze ATP-dependent nucleosome sliding and facilitate transcription of chromatin in vitro. To establish the physiological requirements of NURF, and to distinguish NURF genetically from other ISWI-containing complexes, mutations were isolated in the gene encoding the large NURF subunit, nurf301. NURF is shown to be required for transcription activation in vivo. In animals lacking NURF301, heat-shock transcription factor binding to and transcription of the hsp70 and hsp26 genes are impaired. Additionally, NURF is shown to be required for homeotic gene expression. Consistent with this, nurf301 mutants recapitulate the phenotypes of Enhancer of bithorax, a positive regulator of the Bithorax-Complex previously localized to the same genetic interval. Finally, mutants in NURF subunits exhibit neoplastic transformation of larval blood cells that causes melanotic tumors to form (Badenhorst, 2002).
ISWI, the catalytic subunit of NURF, is required for expression of the homeotic gene engrailed (en). However, ISWI is also a component of two other chromatin remodeling complexes, ACF and
CHRAC. To resolve which ISWI-containing complex is required for homeotic gene expression, expression of Ultrabithorax (Ubx) and engrailed (en) were examined in nurf301 mutant animals. When both copies of nurf301 are mutated, in homozygous mutant
nurf3011 larvae, expression of the Ubx protein becomes undetectable. The normal expression of Ubx in the haltere and third leg discs of wild-type third instar larvae is absent in nurf301 mutant animals. Expression of the homeotic gene en requires nurf301. The normal expression of En in the posterior compartment of imaginal discs is abolished in nurf3012 mutants. Semiquantitative RT-PCR analysis confirms that Ubx and en transcript levels are reduced in nurf301 mutant animals. These results confirm that the defects in homeotic transcription seen in iswi mutants are caused by abrogated NURF function (Badenhorst, 2002).
A positive regulator of the Bithorax-Complex, E(bx), has
been localized genetically to 61A, the same cytological interval as nurf301. However, unlike numerous regulators of the
BX-C, E(bx) had not been cloned. Since NURF is required for
expression of Ubx, whether nurf301
corresponds to E(bx) was tested. Both alleles of E(bx) were no
longer extant, so whether the mutations that were isolated in
nurf301 recapitulated the published morphological properties
of E(bx) mutants was tested (Badenhorst, 2002).
nurf301 mutants, like E(bx), increase
the severity of bithorax (bx) mutant phenotypes.
bx is a DNA regulatory element required for correct expression
of Ubx in regions that give rise to the third (T3), but not
second thoracic segment (T2) of the adult fly. This expression distinguishes T3 from T2 identity. Loss or reduction of Ubx levels in bx mutant animals (Ubx6.28/bx34e and
Ubx6.28/bx8 mutant combinations causes a homeotic transformation of the third thoracic segment to the anterior second thoracic segment. Thus, the third thoracic segment, which is normally vestigial and naked, is transformed into the second thoracic segment, increasing its size and causing sensory bristles to develop. Moreover, the
haltere (T3) is transformed toward wing fate (T2), manifested by
increases in size and the development of bristles. The strength of
these transformations is increased when one copy of E(bx) also is removed. Mutation of one copy of nurf301 similarly enhances bx phenotypes. With one
copy of either the nurf3011, nurf3012 or a
deficiency that removes nurf301 -- Df(3L)3643 -- the
strength of the transformation is enhanced. nurf301 enhances both bx34e and bx8 mutations (Badenhorst, 2002).
Although NURF is required for expression of the homeotic genes in
imaginal discs, neither E(bx) nor nurf301 homozygous
mutant larvae display obvious homeotic transformations of the larval cuticle. The absence of mutant larval cuticle phenotypes is likely due
to the large maternal dowry of nurf301 transcript contributed to embryos. Larval cuticular patterning is established before these transcripts have dissipated. Attempts were made to generate embryos lacking the maternal nurf301 contribution through use of the dominant female sterile technique. Although germ-line clones were produced using the parental chromosome, it was not possible to recover germ-line clones using nurf3011. Like ISWI, NURF301 is required for ovary development (Badenhorst, 2002).
An important question is how NURF is recruited to target sites in vivo.
Four genes were shown in this study to be dependent on nurf301 for expression: Ubx, en, hsp26, and
hsp70. All contain multiple binding sites for the GAGA factor,
which is genetically required for their correct expression. On the Drosophila hsp70 and hsp26 promoters, (GA.CT)n cognate elements (to which the GAGA factor binds) are required for HSF-binding.
When these sequences are deleted, HSF-binding to transgenes in polytene
chromosomes is impaired, consistent with the defects seen in nurf301 mutant animals. It is therefore compelling that recent biochemical studies show that NURF and the GAGA factor bind to each other in crude extracts, and that purified NURF301 and GAGA factor interact directly in vitro. The principal interacting domains map to an N-terminal
region of NURF301 and a stretch flanking the Zn finger DNA-binding
motif of GAGA factor. These data suggest that NURF
is recruited by the GAGA factor through specific, direct interactions
with the NURF301 subunit, to catalyze local sliding of nucleosomes at
bx, en, hsp26, and hsp70 promoters,
increasing accessibility to sequence-specific transcription factors and
RNA polymerase II. Curiously, though, reduction of nurf301
levels fails to enhance phenotypes of mutations in Trithorax-like, the gene that encodes the GAGA factor (Badenhorst, 2002).
Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region
from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE
activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments.
PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites
but can still bind PcG complexes in vitro. The GAGA factor is a component of at least some types of PcG complexes and
may participate in the assembly of PcG complexes at PREs (Horard, 2000).
Dissection of the PRE reveals that it is
a compound region containing several sequences that are able (to
different extents) to induce variegated expression of the
miniwhite gene, respond to PcG mutations, and create new
binding sites for PcG proteins on polytene chromosomes. The separate
fragments are definitely weaker in activity than the whole. A single
copy of a fragment containing different restriction enzyme fragments (BP, AB, and part of HA) silences very
effectively, indicating that the different sequences
normally cooperate to achieve more complete silencing to a degree that
is not attained by multiple tandem copies of one fragment. The
different subfragments most likely contribute complementary functions,
but it has not been possible to demonstrate that different PcG proteins
interact with different subfragments. As with the entire PRE, the
response to different PcG mutations depends strongly on the site of
insertion of the transposon construct. The genomic context makes
therefore a strong contribution not only to the strength of the
silencing but also to the relative importance of the different PcG
components of the silencing complex. The activity of PRE-containing
transposons inserted at different sites suggests that this contribution
is due not only to sequences flanking the insertion site but also to
the interaction in trans with other genomic PRE sites (Horard, 2000).
Only one of the three subfragments tested in embryos, BP, was able to
maintain repression of the Ubx-lacZ reporter gene. This could be due simply to the relative PRE strengths of the different fragments. That is, increasing the number of copies of the other fragments might achieve the same silencing strength. Another
possibility is that the complex formed at the BP fragment is
qualitatively different from that recruited by the other fragments; for
example, it might be able to recruit PcG proteins sufficiently early in embryonic development to have an effect on the Ubx-lacZ
gene, while other PRE fragments might be able to institute silencing only at later stages. Different affinities for PcG complexes could also
account for the different abilities to create binding sites for PcG
proteins on polytene chromosomes. However, the fact that the PF
fragment, though able to induce variegation at a high rate and to bind
PcG proteins on polytenes, failed to show any detectable PcG complex
formation in the immunoprecipitation assays suggests that the nature
and composition of the complexes and/or the mode and timing of their
recruitment are likely to differ for the different fragments (Horard, 2000).
The in vitro experiments show that GAGAG-containing sequences (GAGAG is the consensus binding site for GAGA protein) are binding sites for PcG complexes and that the GAGA factor is associated with PcG complexes present in the nuclear extracts. Ion exchange chromatography of nuclear extracts
confirms that, while PcG proteins elute over a broad range of salt
concentrations, the in vitro binding activity constitutes a small
minority and copurifies with the GAGA factor. The multiplicity and heterogeneity
of PcG complexes present in nuclear extracts would not be detected in
affinity-based purification schemes. In contrast, the GAGA factor, along with Polyhomeotic, is found in a multiprotein complex that binds in vitro to PRE regions corresponding to ours. The possibility that some other PcG protein also
recognizes the GAGA consensus sequence cannot be excluded, but the association of the GAGA factor with PcG complexes shows that it is most likely involved in at
least one mode of PcG binding to PRE DNA. Does this reflect a role for
the GAGA factor in PcG silencing in vivo? The GAGA factor was
originally identified as a transcription-stimulating factor both in
vivo and in vitro and was classified as a trxG protein because it
stimulated the activity of homeotic genes while its mutants had
phenotypes indicative of homeotic insufficiency.
However, some evidence suggests that it can also be associated with
repressive functions. The GAGA factor, together with another activator,
NTF-1, also binds to an 11-bp element required for the repression of
tailless by the torso-dependent pathway. Evidence that it might be involved in PRE function consists of the fact that GAGA
mutations decrease the silencing effected by the Fab-7 PRE.
In the Ubx gene, the bxd PRE region contains the
largest concentration of GAGA binding sites. If each continuous
G(AG)n stretch is taken as one binding site, the 1-kb interval containing the
core of the PRE contains 13 sites while the next highest concentration
(8 sites) is found in a 1-kb region containing the bx PRE
(not to be confused with the BX enhancer). Chromatin cross-linking and
immunoprecipitation experiments confirm that these regions bind the
GAGA factor in vivo. The results suggest that at these
sites the GAGA factor is not an antagonist of silencing and is not
simply an accessory or a facilitator of PcG complex formation but may,
in concert with other factors, contribute to targeting PcG complexes (Horard, 2000).
It was surprising, in view of in vitro results, that the effects of
Trl mutations on either miniwhite variegation or
the silencing of the Ubx-lacZ reporter are sporadic and
strongly dependent on the insertion site. One possible explanation is
that, in vivo, the GAGA factor is only one of a set of DNA-binding
recruiting proteins and that, while it contributes to, it is not
essential for, the assembly of PcG complexes. Chromatographic
fractionation of nuclear extracts indicates in fact that only a
fraction of the PcG complexes present in embryonic extracts are
associated with the GAGA factor. Furthermore, embryos contain an important maternal supply of
the GAGA factor, which would mask the effect of a reduced zygotic contribution. Later, other recruiting factors might be involved. Finally, the results cannot exclude the possibility that, although the GAGA factor is (1) a component of PcG complexes, (2) can target their binding in vitro, and (3) is apparently important for the function of the Fab-7 PRE, it is not primarily involved in
recruitment at the bxd PRE. Instead, its role might be
primarily architectural. The GAGA factor binds to DNA as a multimer that recognizes clustered GAGA consensus sequences, and it has been argued that such binding would be
expected to bend DNA in a way incompatible with nucleosome assembly.
GAGA binding would then clear the PRE core of nucleosomes and bend it
to facilitate interactions among other DNA-binding components (Horard, 2000).
The presence of
GAGA binding sites alone appears to be sufficient in vitro to bind a
PcG complex since not only the PRE fragments but also the
Ubx promoter and the hsp70 promoter bind, though they have no known PcG silencing activity in vivo.
In addition, a GAGA-containing oligonucleotide also binds efficiently
to PcG complexes. Nevertheless, GAGA protein binding to a DNA sequence is not sufficient to recruit PcG complexes in vivo. Clearly the in
vitro binding reaction does not reflect the in vivo activity. The most
probable explanation of this discrepancy is that the binding detected
in vitro is due to complexes that are preassembled in vivo and are then
dissociated from the chromatin during the preparation of nuclear
extracts. If the nature and composition of PcG complexes are templated
by the PREs at which they are assembled, GAGA-containing PcG complexes
would be efficiently targeted to GAGA binding sites in vitro while, in
vivo, complex formation would require the de novo recruitment and
assembly of PcG complexes, involving other DNA binding components or
cofactors. This interpretation is favored because it would also explain
the variable compositions of PcG complexes detected at different
chromosomal sites. In vivo, the large majority of GAGA binding sites
visible on polytene chromosomes are not associated with PcG binding,
suggesting that only a small fraction of the GAGA protein is involved
in PcG complexes. This interpretation also accounts for the fact that
the LexA-GAGA protein cannot recruit PcG complexes to LexA binding
sites. It is also noted that the target of PcG
complexes in vivo is chromatin, not naked DNA. The presence of
nucleosomes might normally increase the selectivity, allowing PcG
complexes to assemble only at sites where other recruiting or
architectural proteins are also bound (Horard, 2000).
In view of these results, the existence of GAGA sites at the
Ubx promoter raises other possibilities. In the presence of
a PRE, a GAGA factor bound at the Ubx promoter might
participate in the silencing activity by interacting with
GAGA-containing PcG complexes recruited at the PRE, mediating or
contributing to promoter silencing. Both the hsp70 and
hsp26 promoters are efficiently repressed by the presence of
a PRE in the same transposon construct. The GAGA factor might contribute to silencing in these cases also. The miniwhite gene, which is also silenced by the PRE,
does not contain typical clustered GAGA sites in its promoter region
but only a few scattered sites in the transcribed region. The
expression of the miniwhite gene is strongly dependent on
the site of insertion and on distant enhancers within or outside of the
transposon construct. The silencing of these enhancers might be in part
responsible for the effect of the PRE on miniwhite
expression. Alternatively, other proteins binding to the
miniwhite promoter region might interact with PcG complexes (Horard, 2000).
The immunoprecipitation experiments
also detected binding that is not competed by GAGA oligonucleotides
with PRE fragments that do not contain consensus GAGA binding sites.
This implies that other recognition sequences and other DNA-binding
proteins are involved in these cases. The recent discovery that Pleiohomeotic (a Drosophila PcG protein homolog of the mammalian YY-1
factor) binds to DNA suggests that it might be one such recruiter of
PcG complexes. There are in fact a number of putative
Pho binding sites with the minimal consensus GCCAT in the PRE region:
one in AB, two in BP (a third site is destroyed by the BglI
cleavage), and three in the PF fragment. These bind Pho protein in
vitro and are important for PRE activity in vivo. However, none are found in the HH or HA fragments; hence these presumably depend on other recruiting proteins. However, the PF fragment, though it contains three putative Pho sites, is conspicuous for its inability to bind PcG complexes in extracts, suggesting that Pho is either not present in the complex containing Pc and Psc or
does not interact directly with it. The fact that the mammalian Pho
homolog YY-1 causes sharp bends in the DNA raises the
possibility that Pho too might serve a primarily architectural role
without necessarily interacting directly with PcG complexes (Horard, 2000).
Although PF does not contain GAGA sites, it is almost as effective in
inducing PcG-dependent variegation of the miniwhite gene as
the BP fragment and it can generate new PcG binding sites at the site
of insertion on polytene chromosomes. Yet PF cannot maintain repression
of the Ubx-lacZ reporter gene in embryos. One possible
explanation for these results is that PF is the target for yet another
PcG recruiting mechanism that either functions poorly under these in
vitro binding conditions or depends on proteins that are not present in
the embryonic extracts. The fact that the PF fragment can recruit
silencing complexes in larval cells but cannot maintain repression in
the embryo would be consistent with a requirement for proteins present
only at later developmental stages. Another possible explanation is
that PF does interact with certain PcG complexes which do not include
PC or PSC and hence escaped detection (Horard, 2000).
The picture of the PRE that emerges from these experiments is that of a
mosaic of multiple interaction sites which may require different
DNA-binding proteins to recruit PcG components. A similar conclusion
has been reached, based on deletions that
abolish the activity of the bxd PRE and by an in vitro binding approach similar to that reported in this study.
GAGA sites are associated with some PREs but not others (e.g., the
Mcp PRE). If the GAGA factor acts as a recruiting protein, it is most likely only one of many possible recruiters. Different recruiters might interact specifically with different PcG proteins, accounting for the fact that the binding sites for different PcG proteins on polytene chromosomes do not completely coincide.
Nevertheless, the ability of PcG proteins to interact with one another
or to enter into a chain of recruitment means that, in
most cases, strong binding sites for one PcG protein will be able to
recruit at least to some degree the other PcG proteins. The difference between direct and indirect recruitment may be responsible for the fact
that a strong chromosomal binding site for one PcG protein is sometimes
a weak binding site for another PcG protein (Horard, 2000 and references therein).
Trithorax (Trx) is a member of the trithorax group (trxG) of epigenetic regulators; these proteins are required to maintain active states of Hox gene expression during development. A trithorax acetylation complex (TAC1) has been purified that contains Trx, dCBP, and Sbf1. Like CBP, TAC1 acetylates core histones in nucleosomes, suggesting that this activity may be important for epigenetic maintenance of gene activity. dCBP and Sbf1 associate with specific sites on salivary gland polytene chromosomes, colocalizing with many Trx binding sites. One of these is the site of the Hox gene Ultrabithorax (Ubx). Mutations in either trx or the gene encoding dCBP reduce expression of the endogenous Ubx gene as well as of transgenes driven by the bxd regulatory region of Ubx. Thus Trx, dCBP, and Sbf1 are closely linked, physically and functionally, in the maintenance of Hox gene expression (Petruk, 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).
The Drosophila Polycomb Group (PcG) proteins are
required for stable long term transcriptional silencing of
the homeotic genes. Among PcG genes, esc is unique
in being critically required for establishment of PcG-mediated
silencing during early embryogenesis, but not for
its subsequent maintenance throughout development. Esc has been shown to be physically associated
with the PcG protein E(Z). Esc,
together with E(z), is present in a 600 kDa complex that is
distinct from complexes containing other PcG proteins. This Esc complex has been purified and it also
contains the histone deacetylase Rpd3 and the histone-binding
protein p55 (Chromatin assembly factor 1 subunit), which is also a component of the
chromatin remodeling complex NURF and the chromatin
assembly complex CAF-1. The association of Esc and
E(z) with p55 and Rpd3 is conserved in mammals. Rpd3 is required for silencing mediated by a
Polycomb response element (PRE) in vivo and E(z)
and Rpd3 are bound to the Ubx PRE in vivo, suggesting
that they both act directly at the PRE. It is proposed that histone
deacetylation by this complex is a prerequisite for
establishment of stable long-term silencing by other
continuously required PcG complexes (Tie, 2001).
The presence of Rpd3 in the Esc complex suggests that
histone deacetylation is an intrinsic activity of the Esc
complex and that Rpd3 is required for PRE-mediated
silencing. The related mammalian EED complex has been
shown to contain the Rpd3 homologs HDAC1 and HDAC2,
and immunoprecipitates containing this complex can
deacetylate a histone H4 tail-peptide in vitro. In yeast, Rpd3-dependent repression in vivo has
been shown to be associated with deacetylation of histones H4
and H3.
Which nucleosomes would be deacetylated by the Esc
complex? Histone deacetylation by yeast Rpd3 appears to be
highly localized, extending only one or two nucleosomes from
a site to which it is recruited. Since components of the Esc complex
are physically associated with the Ubx PRE in vivo, Esc-mediated
deacetylation may be restricted to nucleosomes
comprising and immediately adjacent to PREs. Nucleosomes
outside the PRE might also be targeted if the PRE has long-range
interactions with the promoter or if the Esc complex
itself also binds to the promoter or other regions outside the Ubx
PRE, a possibility that the data presented here do not rule out.
Although an effect is observed of several Rpd3 mutations on
silencing of a PRE-mini-white reporter, which is an extremely
sensitive assay, PcG phenotypes have not been reported for
Rpd3 mutants. A hypomorphic Rpd3 allele associated with the
insertion of a P-element transposon in the noncoding 5'
untranslated region has been analyzed in the most detail.
Homozygous mutant embryos derived from germline clones of
this allele do not exhibit PcG phenotypes, but have a pair-rule
phenotype similar to that of ftz mutants. Abundant ubiquitously
distributed Rpd3 RNA and protein of maternal origin are
detectable in early (0-2 hour) wild-type embryos, but are
reduced no more than fivefold in these Rpd3 mutant embryos
derived from germline clones. By stage 9-10, the level of
maternally derived Rpd3 RNA and protein is greatly
diminished. Localized zygotic expression of Rpd3 becomes
detectable in the brain and ventral nervous system of wild-type
embryos, but is not detectable in these mutant embryos, suggesting that this
Rpd3 allele may have a stronger effect on zygotic expression
than maternal expression. If Rpd3 protein derived from
maternally synthesized RNA is sufficient to promote
development of a normal cuticular phenotype, then it remains
possible these mutant embryos may contain sufficient
maternally derived protein to do so and that germline clones of
a true null Rpd3 allele would display PcG phenotypes.
Alternatively, it is possible that the function of Rpd3 in the
Esc complex is not absolutely essential for Esc-dependent
silencing or is redundant, i.e. when eliminated, it can be
compensated by another histone deacetylase, either one
normally associated with the Esc complex or a related one that
can associate with the complex in the absence of Rpd3. A
number of other histone deacetylases have been identified in
Drosophila and at least two are reported to be ubiquitously
distributed in the early embryo (Tie, 2001).
The 600 kDa Esc complex is distinct from complexes
containing PC and other PcG proteins. This suggests that the
Esc complex and other PcG complexes are likely to have
separate functions. Furthermore, in embryos lacking any
functional Esc protein, some weak residual Pc-dependent
silencing activity is still detected, also
supporting separate, if interdependent, functions. Similar
conclusions have been drawn for the homologous mammalian
PcG complexes, which have been reported to be expressed in temporally distinct stages of B cell
differentiation, further suggesting they have distinct functions. In Drosophila, derepression of
homeotic genes is detected slightly earlier in Esc mutants than
in other PcG mutants, raising the possibility
that Esc complex function might be required earlier than other
PcG complexes. However, unlike the apparent temporal
separation of the homologous complexes during mammalian B
cell development, both Esc- and PC-containing complexes are
present together throughout most of embryogenesis, before
Esc disappears, and E(z), like other PcG proteins, is required
continuously throughout development. The phenotypic
similarities between Esc, E(z) and other PcG mutants, the
genetic interactions among them and their common association
with PREs, suggests that their functions, however distinct at
the biochemical level, are interdependent (Tie, 2001).
What role might Esc-mediated histone deacetylation play
in PcG silencing? Given the critical early requirement for Esc,
Esc-mediated deacetylation of PRE-associated nucleosomes
might be an essential prerequisite for the initial binding of one
or more components of PRC1 or other PcG complexes to
PREs. A schematic model is presented for such a function
of the Esc complex in which Esc complex-mediated
deacetylation of PRE associated histones is a critical step in
establishing stable long-term PcG silencing. Alternatively, the
Esc complex may be required for events subsequent to the
initial binding of other PcG proteins to a PRE, perhaps for their
assembly into active silencing complexes or for interaction of
PRE-bound PcG complexes with the promoter. Indeed,
repression of a reporter gene by a tethered GAL4-Pc fusion
protein remains dependent on endogenous Esc and E(z) as
well as other PcG proteins. This indicates that,
at least for PC, constitutive binding to DNA does not bypass
the requirement for Esc and E(z). This also suggests that
while the biochemical evidence reveals no stable direct association of the
Esc complex with other PcG complexes, it remains possible
that there is a transient or less stable association in vivo that is
essential for establishing PcG silencing (Tie, 2001).
Polycomb Group complexes assemble at polycomb
response elements (PREs) in vivo and silence genes in the
surrounding chromatin. To study the recruitment of
silencing complexes, various Polycomb
Group (PcG) proteins have been targeted by fusing them to the LexA DNA
binding domain. When LexA-Pc, -Psc, -Ph or -Su(z)2
are targeted to a reporter gene, they recruit functional
PcG-silencing complexes that recapitulate the silencing
behavior of a PRE: silencing is sensitive to the state of
activity of the target chromatin. When the target is
transcriptionally active, silencing is not established but
when the target is not active at syncytial blastoderm, it
becomes silenced. The repressed state persists through
embryonic development but cannot be maintained in larval
imaginal discs even when the LexA-PcG fusion is
constitutively expressed, suggesting a discontinuity in the
mechanism of repression. These proteins also interact with
other PC-containing complexes in embryonic nuclear
extracts. In contrast LexA-Pho is neither able to silence
nor to interact with Pc-containing complexes. Analysis of
pho mutant embryos and of PRE constructs whose Pho-binding
sites are mutated suggests that, while Pho is
important for silencing in imaginal discs, it is not necessary
for embryonic PcG silencing (Poux, 2001).
These results show that several PcG proteins, targeted by fusion
to a DNA-binding domain, can recruit a repressive PcG
complex. Several new
conclusions can be drawn from these experiments. (1) The
recruitment of the silencing complex cannot occur before
blastoderm. The alpha1-tubulin-LexA-Pc construct reveals that
even when the protein is deposited in the egg during oogenesis,
as well as being zygotically expressed, it does not block the
initiation of transcription from the Ubx promoter. These results
suggest that the LexA-PcG protein cannot establish repression
at this early stage, just as the endogenous PcG proteins known
to be present in the normal pre-blastoderm embryo do not
prevent the initiation of Ubx transcription. Thus, PcG silencing
directed by a PRE or by LexA-Pc appears only to set in after
blastoderm. One possible explanation is that the assembly of a
functional PcG complex at the PRE or at the LexA-binding
sites is a multistep process that requires time and is not
accomplished until after blastoderm. Another interesting
possibility is that the state of the chromatin in nuclei whose
very rapid nuclear divisions are just beginning to slow down,
cannot yet support the establishment of PcG silencing, for
example, because the nucleosomes still bear the deposition-associated histone acetylation pattern. A similar argument
might explain why centric heterochromatin is not detectable
until blastoderm (Poux, 2001).
(2) The repression established by the LexA-PcG protein
is not unconditional but it is sensitive to the state of activity of
the target. Like a genuine PRE complex, the LexA-PcG protein
establishes silencing only in cells in which the reporter gene is
inactive, thus discriminating between active and inactive
chromatin targets. The later-acting Ubx H1 enhancer can still
function but only in the progeny of cells that were active at
early times. The fact that, like the endogenous PRE, the action of the
LexA-PcG protein distinguishes between active and silent
chromatin, indicates that the discrimination occurs after the
binding of the first PcG protein. The sensitive step could be the
assembly of a sufficient nucleus of PcG proteins or still later,
the involvement of other factors that effect the silencing. It has been shown that Pc-containing complex purified from
embryonic nuclear extracts can prevent chromatin remodelling
in vitro if it is bound to chromatin before the addition of
purified SWI/SNF complex but not if it is added
simultaneously or afterwards. If the
activation of the Ubx-lacZ reporter gene by the enhancers
involves the recruitment of the Drosophila SWI/SNF complex,
this observation could help to understand how the LexA
binding sites function as a genuine synthetic PRE possessing
at least one aspect of the cellular memory displayed by
endogenous PREs (Poux, 2001).
(3) Nevertheless, the LexA-binding sites do not constitute a fully
functional PRE. Silencing by LexA-PcG proteins is much less
effective during larval development. It is possible that the
activity of the Ubx H1 enhancer in imaginal discs is more difficult
to repress, e.g. because of very high activator concentration.
More likely, once the H1 enhancer is active, it is much more
difficult to repress by LexA-Pc induced at later times. It cannot be
explained, however, why neither daily heat shocks, nor
constitutive expression of LexA-Pc can maintain a continuity
between embryonic silencing and larval silencing. Repeated
heat shocks do eventually reduce the level of expression of the
reporter in imaginal discs but the memory of the early domains
of repression is lost. The apparent discontinuity in PcG
silencing between the embryo and the larva, suggests that there
might be a real mechanistic difference between the two states.
There are also differences in the
maintenance properties of embryos and larvae. One possible explanation is that additional proteins,
recruited at a true PRE but not by the LexA fusion proteins,
might be necessary for continuous repression at postembryonic
developmental stages (Poux, 2001).
PcG proteins Psc, Su(z)2 and Ph are as effective as Pc in
recruiting a silencing complex, implying that any one of the
'core' PcG proteins can reconstitute a silencing complex. In
contrast, LexA-Gaga and -Pho do not silence the reporter
gene. That LexA-Gaga does not silence is hardly surprising.
Many promoters, including the hsp70 promoter, the alpha1-tubulin
promoter or the Ubx promoter itself, contain Gaga-binding
sites but are not thereby targets for PcG silencing in vivo. The possibility that the LexA-Gaga fusion
protein is defective in some respect cannot be excluded, although it is able to bind
to the normal endogenous sites on polytene chromosomes and
participate in PcG complexes. Most likely, however, Gaga/Trithorax-like
factor by itself cannot recruit PcG complexes. It is supposed that,
like many other nuclear factors, Gaga can have either a
stimulating or a repressing activity, depending on the binding
of other proteins. In the chromatin context of the PRE,
however, Gaga interacts with PcG proteins to form a stable
complex (Poux, 2001).
The Pho protein binds to DNA sequences contained in the
bxd PRE and has been proposed as a major recruiter of PcG
complexes. Mutating the Pho binding sites in the bxd PRE causes loss of
silencing in imaginal discs. In the experiments carried out in this study, however, LexA-Pho is unable to silence the
reporter gene. Since LexA-Pho efficiently rescues pho
mutants in vivo, it is concluded that LexA-Pho is fully functional and that
Pho cannot, by itself, recruit PcG complexes able to silence
the reporter gene either in the embryo or in imaginal discs (Poux, 2001).
The ability of LexA fusion proteins to assemble PcG
complexes that can be targeted to LexA-binding sites allow for the exploration in vitro of the composition of the complexes. It is
important to note that what is seen in the immunoprecipitation
experiments is the bulk of PcG complexes that contain the
LexA-Pc protein. It is not possible to distinguish between complexes
recruited by the LexA-Pc protein at the LexA-binding site and
complexes formed at other PREs, to which the LexA-Pc
protein has been recruited. These experiments therefore do not
necessarily reflect the nature of the complex formed at the
reporter gene, which is limited to what components can be
directly or indirectly recruited by the LexA fusion protein.
Nevertheless, the in vitro binding experiments reveal the ability
of the LexA fusion protein to participate in complexes
containing other PcG proteins (Poux, 2001).
The binding experiments show that the four 'core' proteins
Pc, Psc, Su(z)2 and Ph are recruited to PcG complexes and
are themselves able to recruit repressive complexes. It cannot be
shown that all four proteins are simultaneously associated in one
complex but it is very likely that at least Pc, Psc and Ph can
participate in the same complex. Similar
experiments have also shown that Gaga factor is associated
with at least some PcG complexes, although
it is not itself able to recruit them to the reporter gene. In contrast,
Pho neither recruits any of the core PcG proteins nor
participates in the majority of PcG complexes present in
embryonic extracts. Conceivably, when the LexA-binding
domain is bound to DNA, it might hinder the access of other
PcG proteins to the Pho moiety. It is thought that this is unlikely
because even wild-type Pho does not seem to be associated with
PcG proteins. A PRE fragment containing three Pho binding
sites but no GAGA sites does not bind PC-containing complexes
in vitro, although it interacts readily with
endogenous Pho or LexA-Pho in the same embryonic extracts,
indicating that also endogenous Pho is not stably associated
with PcG complexes. Similarly, Pho is not detected in a
purified PRC1 complex (Poux, 2001).
There remains the possibility that Pho is not the protein that
binds in vivo to the Pho consensus binding sites in the PRE.
These sites
in the bxd PRE are important for silencing in imaginal discs. Formally, this does not exclude the
possibility that these are binding sites for some other protein
required for silencing. More likely, Pho does intervene at the
PRE but not as a direct recruiter of PcG complexes. Its role
might be to alter the chromatin conformation at the PRE and
facilitate the interactions between different components
necessary for effective silencing. Mammalian YY1 has been
shown to act as a repressor as well as an activator, depending
on the context. It
can interact directly with histone deacetylases and,
presumably, contribute directly to repression. In addition, it may be a component of some mammalian
PcG complexes. In Drosophila, Pho
might make a similar contribution to silencing, at least when
it is bound in the context of the PRE. The domain of YY1 that
interacts with HDAC is not conserved in Pho but a different
sequence motif might preserve the ability to interact with
Drosophila histone deacetylase complexes (Poux, 2001).
These experiments, which show no obvious Ubx derepression in
pho1/pho1 embryos and no derepression when all pho consensus sites in the PRE are mutated, support the idea that
neither Pho nor its binding sites play an essential role in
embryonic PcG silencing. A very
limited derepression of abdA and AbdB has been reported in pho mutant embryos. This effect, involving ectopic expression
in a few cells of PS5, might also be interpreted as an indirect
consequence of segmentation defects frequently observed in
pho mutants rather than a real
derepression. In any case, the available evidence excludes a
major role for Pho as a recruiter of PcG complexes. However, Pho-binding sites are necessary for silencing in
larval imaginal discs and pho function is
required for normal imaginal segmental identity. These results
suggest that this function is required to stabilize PcG silencing
in late larval and pupal development, and that its essential role
in embryonic development is unrelated to homeotic regulation. The establishment and maintenance of silencing complexes at
a PRE is clearly a complicated multistage process and the
nature of the initial recruiters is still unknown (Poux, 2001).
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 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).
Deacetylation of the N-terminal tails of core histones plays a crucial role in gene silencing. Rpd3 and Hda1 represent two major types of genes encoding trichostatin A-sensitive histone deacetylases. Drosophila Rpd3, referred to here by its alternative name HDAC1, interacts cooperatively with Polycomb group repressors in silencing the homeotic genes that are essential for axial patterning of body segments. The biochemical copurification and cytological colocalization of HDAC1 and Polycomb group repressors strongly suggest that HDAC1 is a component of the silencing complex for chromatin modification on specific regulatory regions of homeotic genes (Chang, 2001).
To demonstrate that the effect of Hdac1 mutations is exerted at the level of expression of homeotic genes, the expressions were examined of Sex combs reduced (Scr) and Ultrabithorax (Ubx) proteins in wild-type and Pc mutant imaginal discs. Scr proteins normally are expressed at high levels in the first leg discs, but are not expressed in the second and third leg discs. In Pc4 mutant heterozygotes, however, Scr proteins also can be detected at low levels in second and third leg discs. Consistent with the increase in ectopic sex comb teeth, dramatic increases in the levels of Scr proteins are observed in the second and third leg discs from Pc4 mutant heterozygotes that were also heterozygous for any of the Hdac1 alleles except Hdac1326. In addition, Ubx proteins are marginally detectable only in the peripodial membranes of imaginal wing discs of wild-type or Pc4 mutant heterozygous larvae. In larvae heterozygous for both Pc4 and an Hdac1 mutation, high levels of Ubx proteins are observed in the medial sections of the wing discs proper. In contrast to the lack of ectopic Scr expression in Pc4 heterozygotes carrying the Hdac1326 allele, a much stronger effect on ectopic Ubx expression is observed; Ubx protein levels in both first and second leg discs are increased substantially. It is highly likely that the expanded Ubx expression reduces Scr expression, resulting in suppressed Pc phenotype (i.e., reduced numbers of ectopic sex comb teeth) in Pc4/Hdac1326 trans-heterozygotes. These results strongly suggest that Hdac1 acts cooperatively with Pc to repress homeotic genes during larval and pupal development (Chang, 2001).
Experiments also were performed to explore the role of Hdac1 in regulating the embryonic expressions of two homeotic genes, Abd-B and Ubx. Abd-B proteins normally are expressed in a graded fashion in the posterior part of ventral nerve cord, starting from parasegment 10 (PS10). Although this pattern is not altered in homozygous Hdac1303 mutants, significant levels of Abd-B proteins are observed in more anterior parasegments of homozygous Psce24 mutants. Much higher levels of ectopic Abd-B proteins are found in Psce24 Hdac1303 double mutants, indicating a synergistic effect of Hdac1 and Psc on Abd-B repression. Consistent (but less striking) effects also are observed on Ubx protein levels. The anterior boundary of the Ubx expression domain is PS5, with the exception of a small cluster of cells in the middle of PS4 that also express Ubx proteins. Although homozygous Psce24 mutants only show sporadic low levels of Ubx expression in more anterior parasegments, Psce24 Hdac1303 double mutants show significantly higher levels of ectopic Ubx expression in more cells. In PS5, more cells with higher levels of Ubx proteins are observed in the double mutants than in either of the single mutants. In contrast, Ubx expression is reduced substantially in the abdominal parasegments of the double mutants compared with that in the single mutants, presumably reflecting Ubx repression by more extensive ectopic expression of Abd-B and possibly ABD-A. These data indicate that Hdac1 is essential for homeotic gene silencing in embryos (Chang, 2001).
Given the genetic and biochemical interactions between Hdac1 and Pc, it might be anticipated that a fraction of HDAC1 would colocalize with Pc-G complexes on polytene chromosomes. Approximately 100 common binding sites have been identified for several Pc-G proteins. At least 70% of these sites (identified by staining with PSC mAbs) also stain with the HDAC1 antibody, including the Antennapedia complex at 84AB and the bithorax complex at 89E. These results suggest that HDAC1 proteins act together with a substantial fraction of the Pc-G complex. However, the relative intensities of the signals for PSC and HDAC1 at these sites do not always correlate, suggesting a regulatory, rather than a constitutive function. Furthermore, HDAC1 is much more widely distributed along the chromosomes than is PSC, consistent with its role in global gene regulation and/or chromatin structure (Chang, 2001).
The colocalizations of HDAC1 and PSC were examined further on polytene chromosomes from a transgenic line that carries a Ubx upstream cis-regulatory region (i.e., bxd-14) inserted at 62A. This insert contains a functional PRE and creates a new Pc-G-binding site. Staining with both PSC and HDAC1 antibodies reveals that a new PSC site coincides with a new HDAC1-binding site. This new binding site is beside an HDAC1 site present in the wild-type chromosome, creating a broader signal of HDAC1 at this site. These results strongly suggest that HDAC1 and Pc-G proteins are recruited to this ectopic PRE (Chang, 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).
The kinetics of HOX gene derepression was examined in Su(z)121 and Su(z)124 mutant clones by analyzing Ubx expression 24, 48 and 72 hours after clone induction. The Minute technique was used in these experiments. 24 hours after clone induction, Ubx is still tightly repressed. 48 hours after clone induction, Su(z)121 mutant clones show misexpression of Ubx protein in the wing pouch but Ubx is still stably silenced in other parts of the wing disc. In Su(z)124 mutant clones, Ubx is still stably silenced 48 hours after clone induction except in a few clones in the center of the pouch where weak Ubx signal was detected. Finally, 72 hours after clone induction, repression of Ubx is lost in most Su(z)121 and Su(z)124 mutant clones in the pouch, in the latter case Ubx is still silenced in some parts of the disc. This slow and gradual loss of silencing is comparable to the kinetics of HOX gene derepression in Pc, Pcl, Scm or Sce mutant clones (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).
During late embryogenesis, the expression domains of homeotic genes are maintained by two groups of ubiquitously
expressed regulators: the Polycomb repressors and the Trithorax activators. It is not known how the activities of the
two maintenance systems are initially targeted to the correct genes. Zeste and GAGA are sequence-specific DNA-binding proteins that are Trithorax group activators of the homeotic gene Ultrabithorax. Zeste and GAGA DNA-binding sites at the proximal promoter are also required to maintain, but
not to initiate, repression of Ubx. Furthermore, the repression mediated by Zeste DNA-binding site is abolished in zeste null embryos. These data
imply that Zeste and probably GAGA mediate Polycomb repression. A model is presented in which the dual transcriptional activities of Zeste and
GAGA are an essential component of the mechanism that chooses which maintenance system is to be targeted to a given promoter (Hur, 2002).
Zeste, GAGA and a third transcription factor, NTF-1 (Grainy head), activate promoter constructs of the Ubx gene in embryos via an intermingled cluster of sites between nucleotides -200 to -31. However, the constructs that were used in these experiments contain only a small subset of the Ubx cis regulatory region, and while they reproduce many features of Ubx expression, they do not respond to Polycomb repression when inserted at many chromosomal locations. Consequently, they have not permitted a rigorous analysis of the role of the proximal promoter factors in maintaining repression. To address this question, larger constructs have been used that contain the 22 kb of DNA upstream of the Ubx mRNA start site. These constructs do not suffer from significant position effect variation; they more closely approximate the expression pattern of the endogenous Ubx gene than the shorter constructs; they maintain efficient repression in late embryos as shown by the lack of ß-galactosidase reporter gene expression in more anterior and posterior regions, and they are genetically under the control of PcG genes (Hur, 2002).
Deletion of nucleotides -200 to -31 essentially abolishes transcription from the large Ubx promoter constructs, indicating a crucial role for factors binding to the proximal promoter. To determine the role of each factor separately, three constructs were prepared, each containing binding sites for either Zeste, GAGA or NTF-1 inserted between the deletion end points of the above construct. Importantly, biochemical, in vivo u.v. crosslinking, and genetic experiments strongly suggest that the DNA-binding sites used in these constructs are recognized only by their cognate factor, and not by any other sequence-specific DNA-binding activities. Binding sites for each factor separately activate transcription of the large constructs during late embryogenesis. Strikingly, constructs containing only GAGA- or Zeste-binding sites at the proximal promoter are not expressed in the anterior or posterior of the embryo, whereas constructs bearing only NTF-1 sites are strongly transcribed in these terminal regions (Hur, 2002).
Ectopic expression of Ubx in anterior and posterior regions is generally caused by a failure of the initiating repressors or the Polycomb maintenance system. One interpretation of this result is that Zeste and GAGA are required for at least one form of repression, while NTF-1 is not. It is also possible, however, that Zeste and GAGA are not repressors. Instead, it may be that they are unable to activate expression in anterior or posterior regions, even though they are expressed at similar levels throughout the embryo. To distinguish between these two possibilities, constructs were examined that contained either Zeste and NTF-1 sites or GAGA and NTF-1 sites. These constructs are expressed in the central region of the embryo; but, importantly, they are not significantly expressed in anterior or posterior regions. Since NTF-1 can activate Ubx transcription in these terminal regions, the absence of terminal expression is consistent with GAGA and Zeste directly repressing transcription in addition to their activation function (Hur, 2002).
To establish decisively if Zeste and GAGA are repressors, it was desirable to use a genetic test. Unfortunately, GAGA is a lethal gene and a broadly acting regulator required for expression of transcription factors that regulate Ubx in early embryos. Thus, it has not been possible to determine genetically whether GAGA is a direct repressor of Ubx. By contrast, zeste is a largely redundant gene. zeste null embryos and flies are essentially wild type, and the endogenous Ubx gene is expressed normally in these animals; but because the 22UZ transgenes lack the cis regulatory elements through which factors that redundantly share the function of zeste act, these transgenes should be regulated by zeste (Hur, 2002).
Consistent with this idea, transgenes containing only Zeste sites at the proximal promoter fail to express in zeste mutant embryos, whereas constructs containing only GAGA or NTF-1 binding sites are expressed in this same genetic background. Thus, this genetic experiment confirms that Zeste bound at the proximal promoter is required to activate transcription of the 22UZ constructs in the normal domain of Ubx expression. To test the role of Zeste in repression, constructs containing binding sites for both Zeste and NTF-1 at the proximal promoter were compared in wild type and zeste mutant embryos. In the normal domain of Ubx expression, these constructs are expressed at similar levels in mutant and wild-type embryos. Importantly, these constructs are derepressed in anterior and posterior regions of embryos lacking zeste. Thus, Zeste actively represses transcription in terminal regions of the embryo via binding sites at the proximal promoter (Hur, 2002).
To distinguish if Zeste is required for the initiation or the maintenance of repression, expression of the 22UZ ZESTE/NTF-1 construct was examined at an earlier stage. In embryos that lack zeste, the 22UZ ZESTE/NTF-1 transgene is almost fully repressed in anterior and posterior regions at this earlier stage. Only weak derepression is observed in a few isolated cells. Thus, the transiently expressed factors that initiate repression in the early embryo must be active, and the extensive derepression observed later must be due to a failure in the maintenance system (Hur, 2002).
The PcG genes are an essential part of system that maintains repression of the endogenous Ubx gene. To confirm that these genes also act on these transgenes, the 22UZ Zeste and 22UZ GAGA constructs were crossed into PcG mutant embryos. Both transgenes are derepressed in late stage embryos lacking the Polycomb gene. Similar results were obtained in embryos lacking another PcG gene, extra sex combs. Thus, Zeste -- and probably also GAGA -- act together with the Polycomb system to maintain repression of Ubx (Hur, 2002).
It is suspected that GAGA and Zeste have redundant, overlapping functions in maintaining repression because the 22UZ Native construct, which contains Zeste, GAGA and NTF-1 sites, is not derepressed in zeste mutant embryos, which contrasts with the behavior of the 22UZ ZESTE/NTF-1 construct. Such redundancy in repression would parallel the known redundancy between these two transcription factors in activating Ubx in the central portions of the animal, and helps explain the previous lack of evidence that Zeste and GAGA are repressors (Hur, 2002).
The data presented in this paper are consistent with the earlier genetic data that suggested that some trxG and PcG proteins may have dual activities. Further support for this idea comes from recent biochemical experiments that have shown that GAGA is complexed with two PcG proteins in Drosophila nuclear extracts and Zeste is part of a multisubunit complex that contains Polycomb. In addition, PcG proteins are frequently associated in vivo with promoter regions that include Zeste or GAGA DNA recognition sites, including the Ubx proximal promoter examined in this paper. Most PcG proteins do not recognize specific DNA sequences; thus, the interaction with Zeste and GAGA may serve to recruit PcG proteins to promoters (Hur, 2002).
But is it essential that some proteins, such as Zeste and GAGA, participate in both repression and activation, or is it mere coincidence? This joint participation may be essential. At the transition between the initiating repressors and the Polycomb system, one possibility is it that Polycomb proteins are recruited to or activated on only those genes that are bound by initiating repressors; the initiating repressors may physically bind to PcG proteins to recruit them. However, Polycomb repression can be established on Ubx promoter constructs that lack initiating repressors elements, provided that initiating enhancer elements are also absent. In other words, at the transition between the establishment and maintenance of the Ubx expression pattern, the Polycomb systems reads the absence of activation, rather than the presence of repression or repressors (Hur, 2002).
Endogenous Zeste protein binds to Ubx promoter constructs in vivo whether they are transcribed or not. It is suggested that in the early embryo in the cells in which Ubx is activated, Zeste is complexed, directly or indirectly, with initiating activators on the Ubx promoter. These complexes mask surfaces on Zeste that would otherwise be bound by components of the Polycomb system. By contrast, in those cells where Ubx is not activated, Zeste is still bound to the promoter but is not a part of an activating complex. Surfaces on Zeste protein would then be exposed and could serve as the signal that the Polycomb system reads to initiate the maintenance phase of repression. The dual activities of Zeste and GAGA could be a key to understanding this fascinating regulatory mechanism (Hur, 2002).
Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity. While modification by histone acetyltransferase (HAT) is usually linked to transcriptional activation, evidence is provided for HAT function in several types of epigenetic repression. Chameau (Chm), a new Drosophila member of the MYST HAT family, dominantly suppresses position effect variegation (PEV), is required for the maintenance of Hox gene silencing by Polycomb group (PcG) proteins, and can partially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, in vivo evidence is provided that the acetyltransferase activity of Chm is required in these processes, since a variant protein mutated in the catalytic domain no longer rescues either PEV modification, telomeric silencing of SAS2-deficient yeast cells, or lethality of chm mutant flies. These findings emphasize the role of an acetyltransferase in gene silencing, which supports, according to the histone code hypothesis, the observation that transcription at a particular locus is determined by a precise combination of histone tail modifications rather than by overall acetylation levels (Grienenberger, 2002).
Direct evidence for a role of Chm in Hox gene silencing was obtained from the examination of Ubx expression in imaginal discs. Whereas Ubx is not detected in the columnar epithelium of a wild-type wing disc, derepression is observed in few cells from discs heterozygous for Pc, and a more extended activation occurs in discs heterozygous for both chm and Pc. These results confirm that Chm and PcG proteins act together to repress Hox genes. No misexpression of Ubx could be detected, however, in discs from chm homozygous larvae. Thus, chm can be classified as an enhancer of PcG mutations instead of a novel PcG gene (Grienenberger, 2002).
The establishment and maintenance of mitotic and meiotic stable (epigenetic) transcription patterns is fundamental for cell determination and function. Epigenetic regulation of transcription is mediated by epigenetic activators and repressors, and may require the establishment, 'spreading' and maintenance of epigenetic signals. Although these signals remain unclear, it has been proposed that chromatin structure and consequently post-translational modification of histones may have an important role in epigenetic gene expression. The epigenetic activator Ash1 is a multi-catalytic histone
methyl-transferase (HMTase) that methylates lysine residues 4 and 9 in H3 and 20 in H4. Transcriptional activation by Ash1 coincides with methylation of these three lysine residues at the promoter of Ash1 target genes. The methylation pattern placed by Ash1 may serve as a binding surface for a chromatin remodelling complex containing the epigenetic activator Brahma (Brm), an ATPase, and inhibits the interaction of epigenetic repressors with chromatin. Chromatin immunoprecipitation indicates that epigenetic activation of Ultrabithorax transcription in Drosophila coincides with trivalent methylation by Ash1 and recruitment of Brm. Thus, histone methylation by Ash1 may provide a specific signal for the establishment of epigenetic, active transcription patterns (Beisel, 2002).
Polycomb group (PcG) proteins play important roles in
maintaining the silent state of HOX genes. Recent studies have
implicated histone methylation in long-term gene silencing. However, a
connection between PcG-mediated gene silencing and histone methylation
has not been established. This study reports the purification and
characterization of an EED-EZH2 complex, the human counterpart of the
Drosophila ESC-E(Z) complex. The
complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, it is shown that
H3-K27 methylation colocalizes with, and is dependent on, E(z)
binding at an Ultrabithorax (Ubx) Polycomb
response element (PRE), and that this methylation correlates with
Ubx repression. Methylation on H3-K27 facilitates binding of
Polycomb (Pc), a component of the PRC1 complex, to histone H3
amino-terminal tail. Thus, these studies establish a link between
histone methylation and PcG-mediated gene silencing (Cao, 2002).
To understand the functional relation between E(z)-mediated H3-K27
methylation and HOX gene silencing, a study was carried out of E(z) binding, H3-K27 methylation, and recruitment of PC, a core component of the PRC1
complex, to the major PRE of the Ubx gene in
S2 tissue culture cells by chromatin immunoprecipitation (ChIP). Consistent with the involvement of E(z) in H3-K27 methylation, ChIP
analysis of a 4.4-kb region that includes this PRE showed
precise colocalization of E(z) binding and H3-K27 methylation. In contrast, similar colocalization was not observed for
mK9, indicating that H3-K9 methylation, or at least K9-dimethylation,
is independent of E(z) binding. To further verify the importance of E(z)
binding for H3-K27 methylation, attempts were made to disrupt Esc-E(z)
complex activity using RNA interference (RNAi). It was reasoned that
depletion of the Esc protein, a direct binding partner of E(z) and a
component of the Esc-E(z) complex, would result in disruption of PRE
binding by E(z). Depletion of Esc with RNAi results in greatly reduced PRE binding by E(z), loss of H3-K27 methylation, and concomitant loss
of PC binding. Depletion of PC in S2 cells has
been shown to result in derepression of Ubx.
Therefore, these data collectively suggest that the Esc-E(z) complex is
critical not only for H3-K27 methylation, but also for PC binding to
the PRE region, and that H3-K27 methylation is associated with
Ubx repression (Cao, 2002).
To examine the relation between E(z) binding, H3-K27 methylation, and
Ubx gene repression in vivo, wing imaginal discs were dissected from homozygous E(z)61 larvae that had
been either reared continuously at 18°C or shifted from 18° to
29°C ~48 hours before dissection, and analyzed E(z) binding and
H3-K27 methylation in the same Ubx PRE region by ChIP. Consistent with previous studies demonstrating disruption of polytene chromosome binding by both E(z)61 and PC proteins at 29°C, ChIP analysis showed loss of E(z)61 and PC binding to this PRE at restrictive temperature.
In addition, H3-K27 methylation colocalizes with E(z) binding at
permissive temperature, but is lost along with E(z) binding at 29°C.
In contrast, similar changes in H3-K9 methylation were not observed
under the same conditions. Under normal conditions,
Ubx is not expressed in wing discs due to PcG-mediated
silencing. Similar inactivation of an
E(z) temperature-sensitive allele during larval
development has been shown to result in derepression of Ubx
in wing discs. Thus, Ubx PRE-associated
nucleosomes appear to be targeted by E(z)-mediated H3-K27 methylation,
which correlates with PC binding and repression of Ubx.
Collectively, these data suggest that H3-K27 methylation plays an
important role in the maintenance of Ubx gene silencing (Cao, 2002).
Polycomb group proteins act through Polycomb group response elements (PREs) to maintain silencing at homeotic loci. The minimal 1.5-kb bithoraxoid (bxd) PRE contains a region required for pairing-sensitive repression and flanking regions required for maintenance of embryonic silencing. Little is known about the identity of specific sequences necessary for function of the flanking regions. Using gel mobility shift analysis, DNA binding activities have been identified that interact specifically with a multipartite 70-bp fragment (MHS-70) downstream of the pairing-sensitive sequence. Deletion of MHS-70 in the context of a 5.1-kb bxd Polycomb group response element derepresses maintenance of silencing in embryos. A partially purified binding activity requires multiple, nonoverlapping d(GA)(3) repeats for MHS-70 binding in vitro. Mutation of d(GA)(3) repeats within MHS-70 in the context of the 5.1-kb bxd PRE destabilizes maintenance of silencing in a subset of cells in vivo but gives weaker derepression than deletion of MHS-70. These results suggest that d(GA)(3) repeats are important for silencing but that other sequences within MHS-70 also contribute to silencing. Antibody supershift assays and Western analyses show that distinct isoforms of Polyhomeotic and two proteins that recognize d(GA)(3) repeats, the Trl/GAGA factor and Pipsqueak (Psq), are present in the MHS-70 binding activity. Mutations in Trl and psq enhance homeotic phenotypes of ph, indicating that Trl/GAGA factor and Psq are enhancers of Polycomb that have sequence-specific DNA binding activity. These studies demonstrate that site-specific recognition of the bxd PRE by d(GA)(n) repeat binding activities mediates PcG-dependent silencing (Hodgson, 2001).
pipsqueak is a sequence-specific DNA binding protein that targets a Polycomb group protein complex to Polycomb response elements (PREs). The Polycomb (Pc) group (Pc-G) of repressors is essential for
transcriptional silencing of homeotic genes that determine the axial development of metazoan animals. It is generally believed that the multimeric complexes formed by these proteins nucleate certain chromatin structures to silence promoter activity upon binding to PREs. Little is known, however, about the molecular mechanism involved in sequence-specific binding of these complexes. An immunoaffinity-purified Pc protein complex has been shown to contains a DNA binding activity specific to the (GA)n motif in a PRE from the bithoraxoid region of Ultrabithorax. This activity can be attributed primarily to the large protein isoform encoded by pipsqueak (psq) instead of to the well-characterized GAGA factor Trithorax-like. The functional relevance of psq to the silencing mechanism is strongly supported by its synergistic interactions with a subset of Pc-G that cause misexpression of homeotic genes (Huang, 2002).
An ~440-bp DNA fragment from the bithoraxoid region of Ubx can recapitulate both positive and negative effects of trx and Pc, respectively. Immunoaffinity chromatography has been used to purify tagged Pc-G complexes and then their DNA binding activity was assayed. The (GA)n motif in this fragment has been found to be the primary binding site for the Pc-G complexes. Several lines of evidence are presented to show that the DNA binding protein for the Ubx PRE is encoded by pipsqueak (Huang, 2002).
Several lines of evidence are provided to show that a novel DNA binding factor encoded by psq is a constituent of CHRASCH (chromatin-associated silencing complex for homeotics), a previously characterized major Pc-G protein complex. Since CHRASCH also contains a histone modification factor, HDAC1, it is suggested that this complex may represent a fully functional entity that can nucleate certain chromatin structures at and around specific sequences (i.e., PRE) of homeotic genes (Huang, 2002).
The bxd region has been extensively examined for polycomb response elements. Although different fragments ranging from ~400 bp to ~1 kb have been studied, they share a common region represented almost entirely by the B-151 fragment analyzed in this study. Among the three binding motifs of this fragment, it was found that the (GA)n motif represents the most prominent binding site for CHRASCH. In recent studies, the role of this motif in silencing has been demonstrated in transgenic flies. Thus, it is believed that this motif plays a critical role in anchoring one of the major Pc-G complexes (i.e., CHRASCH). These results, however, are not mutually exclusive to the possibility that other motifs may be required for different functional aspects of PRE (Huang, 2002).
Repression and activation of the expression of homeotic genes are maintained by proteins encoded by the Polycomb group (PcG) and trithorax group (trxG) genes. Complexes formed by these proteins are targeted by PcG or trxG response elements (PREs/TREs), which share binding sites for several of the same factors. The repressive class II PcG complex PRC1 has more than 30 protein subunits, including 5 that have been genetically defined as PcG proteins: Polycomb (Pc), Posterior sex combs (PSC), Polyhomeotic (Ph), dRING1, and, at substoichiometric levels, sex combs on midlegs (SCM).
GAGA factor and Zeste bind specifically to PREs/TREs and have been shown to act as both activators and repressors. Purified proteins and complexes have been reconstituted from recombinant subunits to characterize the effects of GAGA and Zeste proteins on PcG function using a defined in vitro system. Zeste directly associates with the PRC1 core complex (PCC) and enhances the inhibitory activity of this complex on all templates, with a preference for templates with Zeste binding sites. GAGA does not stably associate with PCC, but nucleosomal templates bound by GAGA are more efficiently bound and more efficiently inhibited by PCC. Thus Zeste and GAGA factor use distinct means to increase repression mediated by PRC1 (Mulholland, 2003).
To demonstrate directly that GAGA enhances template recognition by PCC, a recruitment assay was developed. Ubx5S DNA, consisting of a portion of the Ubx promoter with known high-affinity Zeste binding sites, was biotinylated, assembled into chromatin, and immobilized on streptavidin-coated magnetic beads. Nucleosomal arrays were incubated with either buffer only or GAGA for 15 min at 30°C prior to addition of PCC. Following a 20-min binding period, array-bound beads and all material bound to them were separated magnetically from unbound protein. Western blot analysis demonstrates that PCC and GAGA components bound to the nucleosomal array-bead complex and that PCC association is increased on arrays bound by GAGA. GAGA and PCC bind only minimally to unconjugated beads. Increasing amounts of competitor nucleosomal array result in a loss of PCC association with the array-bead complex, but do not affect PCC association with the GAGA array-bead complex. These results demonstrate that PCC has a higher affinity for nucleosomal templates bound by the GAGA factor. Prebinding the GAGA{Delta}POZ protein does not lead to increased recruitment of PCC (Mulholland, 2003).
These experiments show that a template prebound by GAGA factor is more efficiently bound and repressed by PCC than an unbound template. GAGA factor might recruit or stabilize PCC binding by directly interacting with its subunits, or GAGA might alter the template in a manner that favors PCC binding. GAGA factor oligomers have been shown to be able to bind multiple templates simultaneously, bringing them together. Binding by GAGA factor might create a network of templates that is more efficiently bound and recognized by PCC than an individual template might be (Mulholland, 2003).
In the defined in vitro system used in this study, both Zeste and GAGA factor can enhance the activity of a PRC1 core complex to repress remodeling of a nucleosomal template. Zeste binds directly to these PcG proteins to generally increase their repressive function, and prebinding GAGA factor to the template recruits the PRC1 core to that template. Previous genetic and mechanistic studies have suggested that regulation of PRC1 repression is a complicated process involving targeting by sequence-specific DNA binding proteins, covalent modification of histone tails, and perhaps targeting by siRNAs. The differences in function of GAGA and Zeste suggest that their role in PcG repression is more complex than previously suspected. One hypothesis for how sequence-specific factors establish PcG repression is that they create a binding surface with greater affinity for PRC1. Although these experiments with GAGA factor are consistent with this hypothesis, the experiments with Zeste suggest that additional mechanisms contribute to targeting by sequence-specific factors. Zeste binds tightly to the core components of PRC1 and enhances their activity even when templates do not contain targeting sequences. This might be important in facilitating the ability of PRC1 repression to spread away from PRE elements, and thus may facilitate the repression of large domains by PRC1 (Mulholland, 2003).
Although originally identified as an activator, Zeste can also function in vivo as a repressor. For example, in zeste mutant flies, a transgene containing the Ubx promoter modified to contain only Zeste binding sites is derepressed in the anterior and posterior segments of the embryo. Two distinct substitution mutants of zeste express proteins that repress rather than activate the white gene but retain activator function required for transvection, suggesting that Zeste has both inherent activation and repression activities that can be separated. It is possible that, when incorporated into PRC1, Zeste is configured so as to only display surfaces responsible for repression (Mulholland, 2003).
It is widely believed that PcG activity is targeted and maintained throughout the course of development by multiple systems. For instance, ESC/E(z) can methylate H3 K27, and PC can bind to this modification, suggesting that a methylation mark might also play a key role in targeting PRC1 and/or in regulating the spread of PRC1 activity. The combined effects of factors such as GAGA that target PRC1 activity, factors such as Zeste that augment PRC1 activity, and other systems such as those for covalent modification of histones might be necessary for faithful maintenance of PRC1 association with a template. It is likely that further mechanisms, such as RNAi, also contribute (Mulholland, 2003).
These multiple mechanisms might be additive or synergistic. Additionally, redundancy between them would provide a fail-safe scheme for maintenance of repression. For instance, if methylation at H3 K27 and increased function by Zeste each were sufficient to establish repression by PRC1, then repression could be established even if one or the other were to fail. Consistent with this hypothesis of redundant function, experiments were performed in which both Zeste and GAGA were present; no significant additive or synergistic effects on PCC function was seen. The establishment of defined in vitro systems, such as used here, will aid in unraveling the connections between the different mechanisms that contribute to regulation of PcG function (Mulholland, 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
Ubx regulation: Table of contents
moira mutations suppress the derepression phenotypes caused by mutations in another Pc group gene, Polycomblike. moira mutant clones in the haltere differentiate large bristles, characteristic of the anterior wing margin, and often lead to absence or duplication of halteres. Homozygous mor mutations in the posterior wing result in a distorted wing shape; the venation is disrupted and large socketed bristles appear along the posterior wing margin. Leg clones result in the femur and tibia being short and twisted and enlargement of the tarsal segment. Clones of the head cause the shape of the head to be abnormal in the dorsal region and sometimes cause the ocellus to be abnormal or absent. Embryos homozygous for moira mutations have defects in head structures, including truncated lateralgraten and defects in the mouth hooks and dorsal bridge. The first and second midgut constrictions are shifted posterior to their wild-type positions (Brizuela, 1997).