cut
cut could be activated directly by AS-C genes and Krüppel. However, expression of achaete-scute complex genes and Krüppel in external sense organs is only transient, while expression of cut is sustained. cut activity is not required to sustain its own expression (Jack, 1995). The activity of daughterless and AS-C is necessary to specify external sense organs, even when cut is overexpressed (Blocklinger, 1991).
The gene atonal (ato) and the genes of the achaete-scute complex (AS-C), all proneural genes, are required for the
selection of sense organ precursors. The proneural genes also endow these precursors with sense organ subtype
information. In most of the ectoderm, atonal is required for precursors of chordotonal sense organs,
whereas AS-C genes are required for those of most external sense organs, such as bristles. Previous misexpression experiments have shown that many ectopic external sense organs are formed upon atonal misexpression, as well as chordotonal organs. There are even some ectodermal areas in which atonal seems capable of only yielding external sense organs. The suggestion has been that ato is less capable that AS-C of giving subtype information. But there is also a suggestion that ato's lack of specificity may be an artifact of the nature of misexpression given by heatshock-inducible constructs. To address the
question of how proneural genes influence subtype identity, the Gal4/UAS
system of misexpression was employed. Unlike previous misexpression experiments, it was found that under specific
conditions of misexpression, atonal shows high subtype specificity for ectopic sense organ formation.
Moreover, atonal can even transform wild-type external sense organs into chordotonal organs, although
scute cannot perform the reciprocal transformation. This evidence demonstrates that atonal's subtype
determining role is not to directly activate chordotonal fate, but to repress the activation of cut, a gene
that is necessary for external sense organ fate, thereby freeing its precursors to follow the alternative
chordotonal organ fate. The realization that ato's subtype determining role is not to activate chordotonal genes but to inhibit cut expression simplifies the apparent complication that ato is required for a variety of sense organs: not only for chordotonal organs but also for R8 photoreceptors and even for a subset of external sense organs (some antennal olfactory receptors). Whereas all AS-C-dependent elements express cut, the unifying theme to these ato-dependent sensory elements is that none of them express cut (and therefore the olfactory organs are distinct from the majority of external sense organs). Therefore, the differential role of proneural genes in subtype determination is to specify not external sense organs vs. chordotonal organs, but Cut-positive vs. Cut-negative sense organs. The exact subtype fate of ato-dependent SOPs is not controlled by ato but presumably depends on the local context. In AS-C dependent SOPs, AS-C genes activate neural precursor genes that determine SOP fate. These genes would normally confer chordotonal fate, except that AS-C genes also activate cut, which inhibits chordotonal fate and activates external sense organ fate. In ato-dependent SOPs, there is still the potential for cut expression, perhaps because of the co-expresssion of AS-C, but this is countered by ato. This allows the SOPs to take on the alternative chordotonal fate (Jarman, 1998).
Where it is expressed, the gene cut differentitates the external sense organs, from the chordotonal organs (where it is not expressed). Among the external sense organs, pox-n is expressed in only poly-innervated organs where it induces cut and differentiates these from the mono-innervated organs. pox-n expression does not depend on Cut, but Pox-n induces cut in poly-innervated external sense organs. The local within the large cut regulatory region that is controlled by Pox-n has been established (Verwoort, 1995).
The Drosophila antenna is a highly derived appendage required for a variety of sensory functions including olfaction and audition. To investigate how this complex structure is patterned, the specific functions of genes required for antenna development were examined. The nuclear factors, Homothorax, Distal-less and Spineless, are each required for particular aspects of antennal fate. Coexpression of Homothorax, necessary for nuclear localization of its ubiquitously expressed partner Extradenticle with Distal-less is required to establish antenna fate. This study tests which antenna patterning genes are targets of Homothorax, Distal-less and/or Spineless. Antennal expression of dachshund, atonal, spalt, and cut requires Homothorax and/or Distal-less, but not Spineless. It is concluded that Distal-less and Homothorax specify antenna fates via regulation of multiple genes. Phenotypic consequences of losing either dachshund or spalt and spalt-related from the antenna are reported. dachshund and spalt/spalt-related are essential for proper joint formation between particular antennal segments. Furthermore, the spalt/spalt-related null antennae are defective in hearing. Hearing defects are also associated with the human diseases Split Hand/Split Foot Malformation and Townes-Brocks Syndrome, which are linked to human homologs of Distal-less and spalt, respectively. It is therefore proposed that there are significant genetic similarities between the auditory organs of humans and flies (Dong, 2002).
In contrast, there are other genes expressed in both antenna and leg precursors that have distinct patterns in the two appendages. Among these are dac, ato, ct and ss. The domain of dac expression in the antenna (a3) is much smaller than in the leg where it is expressed in multiple segments. The function of dac in antennal development has not been described previously (Dong, 2002).
cut, which is required for differentiation of external sensory (ES) class neurons, is expressed throughout the presumptive proximal antenna (a1 and a2) and head capsule but is expressed in small clusters of cells throughout the leg disc (Dong, 2002).
Expression of the homeodomain transcription factor encoded by ct almost completely fills the hth expression domain of the third instar antennal disc. In contrast, the ct and hth expression patterns in the leg disc are distinct from one another. This makes ct a strong antenna-specific candidate target for Hth. The antennal expression of ct is lost in hth null clones indicating that ct is indeed downstream of hth. To test whether the a2 expression of ct also requires Dll, ct expression was examined in Dll mutants. ct expression is not reduced in Dll null clones or in Dll hypomorphs. Therefore, although Dll and hth are both required for antennal fate, cut is an antenna-specific target of Hth activation that is independent of Dll. As with other antenna-specific targets of Dll and Hth, ct expression is also not lost in ss null antenna (Dong, 2002).
Krüppel, caudal and cut are expressed in the Malpighian tubules before and during differentiation. Two of the genes, Krüppel and cut, are known to be required for development of the tubules. The absence of maternal and zygotic caudal function reduces their normal growth and elongation. Normal Krüppel function, which is known to be required for caudal expression, is also required for cut expression, while cut and caudal are expressed independently of each other. Loss of Krüppel activity confers hindgut characteristics on those cells that normally form the Malpighian tubules. Loss of cut function alters the expression of some markers but not others. The pathway of tissue specific gene regulation, apparently, branches beyond Krüppel to form, at a minimum, a cut and a caudal branch (Liu, 1992).
Genetic evidence is presented showing that lines, a Drosophila segment polarity gene that has yet to be cloned, is required for the function of the Abdominal-B protein. In lines mutant embryos Abdominal-B protein expression is normal but is incapable of promoting its normal function: formation of the posterior spiracles and specification of an eighth abdominal denticle belt. The tail and A8 segment of lin embryos are highly abnormal. The A8 denticle belt is replaced by naked cuticle that occasionally forms a few denticles less pigmented than the normal ventral denticles. This abnormal A8 cuticle does not resemble the cuticle of any region of the wild-type or of the lin mutant embryo. The absence of anal pads and the abnormal hindgut suggests abnormal development of abdominal segment 11, however, other aspects of the tail development are normal, such as the formation of an anal tuft. In lin embryos the sensory organs are formed at roughly correct positions but have an abnormal shape (Castelli-Gair, 1998).
The Abd-B gene directs the formation of the posterior spiracles by controlling downstream target genes. The defects associated with lines mutation arise because in lines mutant embryos the Abdominal-B protein cannot activate its direct target empty spiracles (ems) or other downstream genes, such as cut(ct) and spalt(sal), while it can still function as a repressor of Ultrabithorax and abdominal-A. ems is one gene required for the formation of posterior spiracles. ems expression in the posterior spiracles is regulated by Abd-B. In lin embryos the transcription of ems is not activated in the posterior spiracles, showing that lin is required for Abd-B to activate its direct downstream target. The other putative Abd-B downstream targets (cut and spalt are also required for the normal development of the posterior spiracles. The activation of ct and sal in the anlage of the posterior spiracles requires Abd-B function but their activation remains independent of one another and of ems, suggesting that all three genes are independently controlled by Abd-B. In lin mutants neither ct nor sal are activated in the anlage of the posterior spiracles. These results show that in lin mutant embryos, Abd-B is incapable of activating some of its targets. The requirement of lines for Abd-B function is not a specific property of the A8 segment. In wild-type embryos, ectopic Abd-B expression using the GAL4 targeting system results in the formation of ectopic posterior spiracles in segments anterior to A8. In contrast, ectopic Abd-B expression in lin mutants does not form ectopic posterior spiracles showing that no matter where the Abd-B protein is expressed in the embryo it requires lines to be fully functional (Castelli-Gair, 1998).
The effect of lin on Abd-B can be explained at the molecular level if lin is required for protein posttranscriptional modification or as a transcriptional cofactor of Abd-B. There is some evidence that the Abd-B protein is posttranslationally modified. If Lin were mediating this process, it would imply that such posttranscriptional modification is functional in vivo. Alternatively if Lines is a transcriptional cofactor of Abd-B, Lines would be interacting with Abd-B in a similar way to that proposed for Extradenticle with Ubx and Abd-A, or Ftz-F1 with Ftz. It is interesting that Exd does not have any effect on Abd-B protein binding or function, and that lin is specific for Abd-B but not for the other Hox genes tested. This suggests that different HOX proteins use different cofactors that contribute to the DNA binding specificity of the HOX proteins (Castelli-Gair, 1998).
The development of the posterior spiracles of Drosophila may serve as a model to link patterning genes and morphogenesis. A genetic cascade of transcription factors downstream
of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic
mechanisms. The inner cells that give rise to the spiracular chamber invaginate by elongating into 'bottle-shaped' cells. The surrounding cells give rise to a protruding
stigmatophore by changing their relative positions in a process similar to convergent extension. In the larvae the spiracular chamber forms a very refractile filter, the filzkorper. The opening of the spiracular chamber, the stigma, is surrounded by four sensory organs; the spiracular hairs. Clones labeling the spiracular hairs show that each one is formed by four cells related by lineage, two neural and two support cells, the typical structure of a type I external sensory organ. When the larva is buried in the semi-liquid medium on which it feeds, the stigmatophore periscopes out of the medium allowing the larva to continue breathing. The genetic cascades regulating spiracular chamber, stigmatophore,
and trachea morphogenesis are different but coordinated to form a functional tracheal system. In the posterior spiracle, this coordination involves the control of the
initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly. As a result, the opening of the tracheal system shifts back
from the spiracular branch of the trachea into the posterior spiracle cells (Hu, 1999).
Downstream of Abd-B the cascade can be subdivided into various levels. The activation of six genes -- cut, empty spiracles (ems), nubbin (nub), klumpfuss (klu), and spalt (sal) -- does not require expression any of the other genes studied, suggesting that these six genes are at the top of the cascade under Abd-B regulation. The cut, ems, nub, and klu genes are expressed in the spiracular chamber in overlapping patterns. The sal gene is not expressed in the spiracular chamber but in the cells that surround it and will form the stigmatophore. The exclusion of sal from the spiracular chamber is partly due to repression by cut, because in cut mutants sal is expressed at low levels in the internal part of the spiracle. Downstream of these putative Abd-B targets other genes are activated. These include the transcription factors grainyhead (grh), trachealess (trh) and engrailed (en) (Hu, 1999).
The spiracle phenotypes in mutants for the early Abd-B downstream genes have been analyzed. In sal mutants the stigmatophore does not form, resulting in embryos with a normal spiraclular chamber that does not protrude. Conversely, mutations in ems and cut affect the spiracular chamber but not the stigmatophore. Mutations for ems result in a spiracular chamber that lacks a filzkorper and is not connected to the trachea. In cut mutants the filzkorper is almost completely missing, but the trachea is still connected to the spiracular chamber and the spiracular hairs are also missing. In trh mutants, where the tracheal pits do not form and there is no tracheal network, the spiracular chamber cells still invaginate, forming a filzkorper. However, this filzkorper is shorter than that of the wild type probably due to a secondary requirement of trh, which is also expressed in the spiracular chamber cells. These results show that the spiracular chamber, the stigmatophore, and the trachea develop independently of one another. No phenotypes for either klu or nub could be detected, indicting that although these genes are expressed in the spiracle, they are either redundant or their function is not required for spiracle morphogenesis (Hu, 1999).
The cut gene is the best marker for cells that will make the spiracular chamber because cut is expressed in these cells when they are still on the surface and continues being expressed after spiracle invagination. At stage 11 cut is expressed in a group of about 70 cells arranged as a two-dimensional sheet. Most of these cells are posterior to the A8 tracheal pit although a few coexpress both tracheal and spiracular markers. These cells are located in the dorsal half of the anterior compartment of A8; at this stage these cells have a shape similar to that of cells at homologous positions in more anterior segments (Hu, 1999).
To follow the movements of the spiracular chamber cells as they invaginate, constructs were examined that were made with particular enhancers of the cut, ems, and grh genes, each of which drive expression of beta-gal in a subset of cells that express the cut gene at stage 11. These enhancers do not drive the whole spiracular expression of their genes, but are good tools for studying cell specification and the morphogenetic movements of the posterior spiracle cells. The expression of cut in the posterior spiracle is controlled by at least three different enhancers, two of which have been used in this study. From stage 13, the ct-A4.2 enhancer marks the precursors of the four spiracular hairs. The grh-D4 enhancer of the grh gene is expressed in a single group of cells in this area. The expression of ems in the spiracle is driven by at least by one enhancer: ems-1.2. From stage 11 this enhancer marks a group of cells abutting the tracheal pit. Double stainings of the cut-D2.3, ems-1.2, and grh-D4 lacZ constructs show that they are expressed in non-overlapping subsets of cells. The correlation of the expression of these three constructs allows the fate mapping of the spiracular chamber primordium when it is a two dimensional sheet of cells. The different spatial expression of these enhancers at stage 11 shows that the two-dimensional sheet of cells is already patterned and that the cells invaginate to precise positions during development (Hu, 1999 and references therein).
The connection of the posterior spiracle to the trachea is a regulated event. In mutants for the Drosophila FGF and FGF-receptor homologs branchless and breathless the tracheal pits do invaginate, but since they do not migrate toward one another, they do not form a continuous network. In contrast, in btl mutants, the posterior spiracle connects normally to the A8 spiracular branch of the trachea. In mutants for Abd-B the stigma of A8 does not slide posteriorly, but stays in the same position as in anterior abdominal segments, where the spiracular branch attaches to the outside epidermis. The contribution of the ems gene to coordination of morphogenetic movements has been examined. The spiracle-trachea connection occurs in cut and sal mutants but not in ems mutants. In ems mutants,
invagination of the spiracle cells adjacent to the trachea does not occur, but more posterior cells of the spiracle invaginate normally. The elongation does not occur simultaneously in all cells, but starts in the more anterior ones and, in general, the invaginating cells keep contact with the external surface of the embryo. This results in the cells that have invaginated earlier being deeper in the spiracular chamber and more elongated. The defective invagination in ems mutants results in a spiracle without a
lumen and with the tracheal opening located outside it. The results show that cell elongation and formation of a lumen are two independently controlled processes. The spiracles provide a good model for the study of cellular and molecular mechanisms controlling cell shape and cell rearrangements, two mechanisms which are used during the morphogenesis of a variety of organisms (Hu, 1999).
The Drosophila melanogaster Nipped-B protein facilitates
transcriptional activation of the cut and Ultrabithorax
genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of
Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces
pombe are required for sister chromatid cohesion during mitosis.
The evolutionarily conserved Cohesin protein complex mediates sister
chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to
associate with chromosomes. This study shows that Nipped-B is also
required for sister chromatid cohesion but that, opposite the
effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits
long-range activation of cut. To explain these findings, a model is proposed based on the chromatin domain boundary activities of
Cohesin in which Nipped-B facilitates cut activation by
alleviating Cohesin-mediated blocking of enhancer-promoter communication (Rollins, 2004).
These experiments addressed two questions: (1) does Nipped-B, in addition to
facilitating remote activation of cut and Ultrabithorax,
participate in mitotic sister chromatid cohesion, and (2) does Cohesin
participate in long-range activation of cut? The first was motivated by
the sequence similarity of Nipped-B to yeast adherins required for sister
chromatid cohesion, and the second was motivated by the published observations
that the yeast adherins are required for the Cohesin complex to associate with
chromosomes. The results indicate that the cooperation between adherins and
Cohesin that occurs in yeast is conserved in Drosophila. The findings
also indicate, however, that the SA/Scc3 subunit of Cohesin opposes Nipped-B in
long-range activation of the cut gene (Rollins, 2004).
The high rate of precocious sister chromatid separation (PSCS)
in homozygous and heteroallelic Nipped-B mutants
and the increased lethality of Rad21/Scc1 RNAi in flies heterozygous for
a Nipped-B mutation observed in this study are consistent with the findings that
the yeast Scc2 and Mis4 homologues of Nipped-B are required for Cohesin to
associate with chromosomes.
Although PSCS was detected in third-instar neuroblasts in RNAi experiments,
it is unlikely that the synthetic lethality of a heterozygous Nipped-B
mutation and Rad21/Scc1 RNAi is caused by changes in long range gene
activation, because as discussed below, Nipped-B and Cohesin appear to have
opposing roles in gene activation. In the RNAi experiments, it is possible that
the pupal lethality is caused by PSCS in a subpopulation of critical cells, but
the possibility cannot be ruled out that Nipped-B and Cohesin cooperate with each
other in other essential functions (Rollins, 2004).
No physical association between the yeast adherins and Cohesin has been
detected, nor do they colocalize on chromosomes. No
chromosomal population of Nipped-B could be detected,
but if Nipped-B acts primarily as a
chaperone for loading Cohesin onto chromosomes, only a small fraction of
Nipped-B may be transiently interacting with chromosomes at any time. The
mechanisms by which yeast adherins facilitate Cohesin chromosome binding are
unclear, but the synthetic lethality between a heterozygous Nipped-B
mutation and Rad21/Scc1 RNAi observed here indicates that this functional
connection is conserved in metazoans. In budding yeast, Cohesin begins to
associate with chromosomes in late G1, while in fission yeast,
Caenorhabditis elegans, Drosophila, and mammalian cells it begins
to associate in telophase. Nipped-B is detected in the nucleus
at all stages that have a nuclear membrane, indicating that it could be involved
in Cohesin chromosomal association beginning in telophase and thus could
influence all potential interphase functions of Cohesin, in addition to sister
chromatid cohesion (Rollins, 2004).
The finding that SA/Scc3 RNAi reduces the severity of the
ctK wing-nicking phenotype indicates that the SA/Scc3
component of Cohesin inhibits cut expression. This is the opposite to the
role of Nipped-B at cut. Multiple Nipped-B mutations were
recovered in a screen for mutations that increase the severity of a wing-nicking
phenotype displayed by a cut allele with a weak gypsy insulator insertion.
Reduced mRNA levels indicated that some of these
Nipped-B mutations are loss-of-function alleles, and viability of
homozygous Nipped-B mutants was rescued by a transgene expressing a
Nipped-B cDNA from a Chip gene promoter. Thus, Nipped-B protein facilitates activation of cut
by the wing margin enhancer (Rollins, 2004).
The effect of Nipped-B on cut expression is likely direct.
Nipped-B does not regulate cut by altering the
activities of known cut regulators because it is most limiting for
cut expression when there is a gypsy insertion at cut while the
other known regulators are more limiting with other types of cut
mutations. Moreover, heterozygous Nipped-B loss-of-function alleles
reduce cut expression, and partial reduction of Nipped-B is unlikely to
cause an equal or greater change in the expression of another cut
regulator. Although the effects of Nipped-B on gene expression were most
apparent with gypsy insertion alleles of cut, a measurable effect was
observed in heterozygous females with a wild-type cut allele and an
allele in which the wing margin enhancer is deleted. Thus, Nipped-B also
facilitates the activation of wild-type cut (Rollins, 2004).
All three SA/Scc3 RNAi insertions and one of three Rad21/Scc1
insertions reduced the number of nicks displayed by the ctK
gypsy insertion. It is thought likely that the Cohesin complex, and not just one
or two of its subunits, is responsible for reducing cut expression. Scc1
and Scc3 operate together as a unit in both Drosophila and C.
elegans. Thus, it is unlikely
that they work independently of each other in regulating gene expression.
Indeed, Rad21/Scc1 RNAi in cultured Drosophila cells reduces both
Rad21/Scc1 and SA/Scc3 proteins, and data presented here
indicate that Rad21/Scc1 and SA/Scc3 may regulate each other's transcript
levels. However, the possibility cannot be ruled out that Rad21/Scc1 and SA/Scc3
work independently of the Smc1 and Cap/Smc3 Cohesin subunits, which form another
stable subcomplex. Initial
attempts to reduce expression of the SMC subunits by RNAi were unsuccessful (Rollins, 2004).
It is unlikely that the effects of SA/Scc3 on cut expression occur by
reducing the expression of a cut activator. The small reductions in
SA/Scc3 expression in these experiments are unlikely to cause equal or
larger changes in the activities of other cut regulators. Also effects are not seen
of Cohesin RNAi on ct53d, which has a small
deletion in the enhancer and is affected by all known cut regulators
except Nipped-B. It is most
likely, therefore, that SA/Scc3 acts directly at cut or by reducing the
ability of Nipped-B to facilitate activation (Rollins, 2004).
The possibility that the negative effect of SA/Scc3 on cut expression may
be specific to gypsy insertion alleles cannot be ruled out, it is thought
improbable. The negative effect is likely to be related to the positive effect
of Nipped-B, and Nipped-B facilitates the expression of wild-type cut.
If SA/Scc3 does specifically affect gypsy insertion
alleles, however, it may interact with the gypsy insulator and contribute to
enhancer blocking. This is consistent with evidence that Cohesin functions at
chromosomal boundaries in yeast. Certain Smc1 and Smc3 mutations
reduce the ability of a boundary that flanks the HMR silent mating-type locus to
block the spread of gene-silencing Sir protein complexes, and Scc1 associates with this boundary.
It has also been proposed that Cohesin binding sites are
boundaries that control the extent of chromosome loop formation by Condensin. This proposal is based in part on the observation that
Cohesin is needed to reestablish chromosome condensation upon returning
temperature-sensitive Condensin mutants to the permissive temperature. In
Drosophila, the gypsy insulator partially blocks the negative effects of
heterochromatin on the expression of a euchromatic gene, suggesting that it has
boundary activity, and in yeast, the Su(Hw) protein that
binds the gypsy insulator also blocks the spread of gene-silencing complexes. If SA/Scc3 or Cohesin increases insulation by gypsy,
Nipped-B could facilitate activation by reducing their association with the
insulator (Rollins, 2004).
A more general version of the 'Cohesin insulator' model is preferred, in which
native Cohesin binding sites in the 85-kb region separating the wing margin
enhancer from the cut promoter act as insulators and impede the formation
of structures needed to bring the wing margin enhancer close to the promoter.
In yeast, Cohesin binds every 10 kb or so along
the chromosomes. The spacing of Cohesin in Drosophila has not been
investigated, but multiple complexes could bind in the 85-kb interval between
the wing margin enhancer and the cut promoter. Assuming that
Nipped-B, perhaps by opening the Cohesin ring, facilitates both the loading and
the removal of Cohesin from chromosomes, could explain how Nipped-B
facilitates the activation of wild-type cut. By opening the Cohesin ring,
Nipped-B would help achieve equilibrium between the bound and unbound states by
providing opportunities to load or remove Cohesin from chromosomes. This
mechanism would be distinct from proteolytic removal of Cohesin by separase at
the metaphase-to-anaphase transition but could be involved in the removal of
Cohesin from chromosome arms in prophase. In heterozygous Nipped-B
mutants, which retain substantial Nipped-B activity, the equilibrium endpoint
would not be altered, but it might take longer to achieve equilibrium. Thus, reduced Cohesin binding to chromosomes would not be expected,
but the lower
Nipped-B levels would reduce the windows of opportunity for removal of Cohesin
needed to allow long-range activation. This model also predicts that Nipped-B
does not have to stably associate with chromosomes, which could explain why no
chromosomally bound Nipped-B was detected by immunostaining (Rollins, 2004).
Finally, in a simple indirect model it could be supposed that, similar to its
role in loading Cohesin, Nipped-B could also facilitate chromosomal binding of
another protein complex that assists long-range enhancer-promoter interactions.
In this case, there would be competition between Cohesin and the long-range
activation complex for Nipped-B, and reduction of Cohesin would make Nipped-B
more available to facilitate long-range activation. This and the insulator model
described above are not mutually exclusive, but both explain how Nipped-B
cooperates with Cohesin in sister chromatid cohesion but opposes the effect of
Cohesin proteins on cut expression (Rollins, 2004).
The cohesin protein complex is a conserved structural component of
chromosomes. Cohesin binds numerous sites along interphase chromosomes and is
essential for sister chromatid cohesion and DNA repair. This study tests the idea
that cohesin also regulates gene expression. This idea arose from the finding
that the Drosophila Nipped-B protein, a functional homolog of the
yeast Scc2 factor that loads cohesin onto chromosomes, facilitates the
transcriptional activation of certain genes by enhancers located many
kilobases away from their promoters. Cohesin binds between a
remote wing margin enhancer and the promoter at the cut locus in
cultured cells, and reducing the dosage of the Smc1 cohesin subunit
increases cut expression in the developing wing margin. cut
expression is increased by a unique pds5 gene
mutation (see CG17509) that reduces the binding of cohesin to chromosomes. On the basis of
these results, it is posited that cohesin inhibits long-range activation of the
Drosophila cut gene, and that Nipped-B facilitates activation by
regulating cohesin-chromosome binding. Such effects of cohesin on gene
expression could be responsible for many of the developmental deficits that
occur in Cornelia de Lange syndrome, which is caused by mutations in the human
homolog of Nipped-B (Dorsett, 2005).
To identify general facilitators of enhancer-promoter communication,
genetic screens were conducted to isolate factors that support activation of
the cut gene by a wing margin-specific enhancer located 85 kbp
upstream of the promoter. The region between this enhancer and the promoter contains
many enhancers that activate cut in specific tissues during
embryogenesis and larval development. In addition to tissue-specific activators that
bind to the wing margin enhancer, these screens identified two proteins, Chip
and Nipped-B, that are expressed in virtually all cells, and facilitate the
expression of diverse genes. Chip interacts with many DNA-binding proteins,
and likely supports the cooperative binding of proteins to enhancers and to
sites between enhancers and promoters (Dorsett, 2005).
Nipped-B functions by a different mechanism. Unlike other cut
regulators, Nipped-B is more limiting for cut expression when
enhancer-promoter communication is partially compromised by a weak
gypsy insulator than it is when the enhancer is partially inactivated
by a small deletion, leading to the idea that Nipped-B specifically
facilitates enhancer-promoter communication (Rollins, 1999).
Nipped-B homologs in Saccharomyces cerevisiae, S. pombe and
Xenopus (Scc2, Mis4 and Xscc2), known collectively as adherins, load
the cohesin protein complex onto chromosomes
(Ciosk, 2000; Tomonaga, 2000; Gillespie, 2004; Takahash, 2004; reviewed by Dorsett, 2004),
Nipped-B is required for sister chromatid cohesion, and thus is a functional
adherin (Rollins, 2004). The fact that Nipped-B is an adherin raises the critical
question, addressed here, of whether or not cohesin plays a role in
enhancer-promoter communication. In all metazoans examined, cohesin loading
starts in late anaphase, and it is not removed from the chromosome arms until
prophase. Cohesin, therefore, is a structural component of chromosomes during
interphase, when gene expression occurs (Dorsett, 2005).
Cohesin consists of two Smc proteins, Smc1 and Smc3, and two accessory
subunits, Rad21 (Mcd1/Scc1) and Stromalin (Scc3/SA). Cohesin
forms a ring-like structure. One idea is that adherins, such as Nipped-B, temporarily
open the ring and allow it to encircle the chromosome
(Arumugam, 2003). It is
proposed that cohesin encircles both sister chromatids after DNA replication
to establish cohesion. Cohesin binds every 10 kbp or so along the chromosome
arms in yeast. If it binds at a similar density in metazoans, it could
potentially affect the expression of many genes (Dorsett, 2005).
Determining if the effects of Nipped-B on gene expression are mediated
through cohesin is pertinent to Cornelia de Lange syndrome (CdLS, OMIM
#122470), which is caused by heterozygous loss-of-function mutations in the
human homolog of Nipped-B, Nipped-B-Like (NIPBL, GenBank
Accession Number NM_133433 (Krantz, 2004; Tonkin,
2004). CdLS results in numerous birth defects, including slow
physical and mental growth, upper limb deformities, gastroesophageal and
cardiac abnormalities. These developmental deficits likely reflect changes in
gene expression similar to those caused by heterozygous Nipped-B
mutations (Dorsett, 2005).
This study examines binding of cohesin to the cut gene, and the
effects that the Pds5 sister chromatid cohesion factor has on cut
expression and cohesin binding to chromosomes. The results are consistent with
the idea that cohesin inhibits the activation of cut by the wing
margin enhancer (Dorsett, 2005).
RNAi experiments have shown that slightly reducing the Stromalin
(Scc3) and Rad21 (Mcd1/Scc1) subunits of cohesin increased expression of the
cut gene in the developing wing margin
(Rollins, 2004). To
determine if the Smc1 cohesin subunit plays a similar role, a null
allele of the smc1 gene was generated by excision of a viable P
transposon insertion near the transcription start site. The
smc1exc46 allele is recessive lethal and chromosome
squashes show precocious sister chromatid separation (Dorsett, 2005).
The effects of smc1exc46 on the mutant phenotype
displayed by the ctK gypsy transposon insertion
allele of cut were used to determine changes in cut
expression. The ctK gypsy insulator partially blocks activation
of cut by the wing margin enhancer, causing a scalloped wing
phenotype sensitive to the dosage of factors that regulate cut
(Gause, 2001; Rollins, 2004). A
decrease in cut expression increases nicks in the wing margin, and an
increase in expression leads to fewer nicks. The wing-nicking assay is a
highly specific and sensitive measure of the activation of cut by the
wing margin enhancer, in the developing margin cells of the wing discs during
the 24-hour period centered around pupariation (Dorsett, 2005).
In repeated experiments, the heterozygous smc1exc46
mutation reduced the number of ctK wing margin nicks
relative to the number observed with the heterozygous parental chromosome.
The difference was
significant. It is concluded that, similar to the effects
of reducing the Stromalin (Scc3) and Rad21 (Mcd1/Scc1) cohesin subunits,
reducing the levels of the Smc1 subunit increases cut expression.
Because all three cohesin subunits have a similar effect, it is concluded that the
cohesin complex inhibits cut expression (Dorsett, 2005).
Mutations in genes that modulate cohesin activity for
effects on cut expression were tested. The separation anxiety
(san) and deco (eco, as listed in FlyBase) genes encode
putative acetyltransferase proteins that are required for sister chromatid
cohesion and the association of cohesin with centromeric regions
(Williams, 2003). Separase (Sse) encodes a protease that cleaves cohesin to
permit sister chromatid separation.
Mutations in these genes did not have significant effects on the
ctK mutant phenotype. It is possible that
heterozygosity for these mutations do not sufficiently alter cohesin activity
to change cut expression. Alternatively, these proteins may affect
cohesin only at the centromere (Dorsett, 2005).
If cohesin directly regulates cut, it would be expected to bind to
the cut locus. Salivary gland polytene chromosomes
were immunostained with anti-Smc1 and anti-Stromalin (Scc3) to determine whether
cohesin binds to cut. In wild type, Stromalin and Smc1 co-localize on polytene
chromosomes as expected. Distinct regions of cohesin staining were seen in both bands
and interbands. It is concluded that
cohesin binds many sites along all chromosome arms. Staining was observed
in some chromosomal puffs, suggesting that cohesin
associates with transcribed loci. To test this, it was determined whether cohesin
binds to heat-shock puffs. After 20 minutes of heat shock at 37°C, cohesin
staining was observed in the 93D puff, but not in the others.
Thus, cohesin localization does not correlate with transcription (Dorsett, 2005).
The examination of several nuclei showed that the chromosome band
containing the cut locus (7B3-4) consistently displayed cohesin
staining. This band contains 150 kbp of DNA, and four genes other than cut,
in the regulatory region between the wing margin enhancer and the cut
promoter. At least three of these genes are testis specific.
Salivary glands do not express cut, but because cohesin is a
constitutive chromosomal component, and probably binds to cut in most
cells, these data are consistent with the view that the effects of cohesin on
cut expression in the wing margin are direct (Dorsett, 2005).
Further support is provided by chromatin immunoprecipitation experiments, which show that
cohesin binds to the regulatory region of cut in Drosophila
cultured Kc cells of embryonic origin. An 85-kbp region was examined
encompassing the wing margin enhancer and the promoter; four
cohesin-binding sites were detected. Two binding sites
were centered 0.5- and 4-kbp upstream of the promoter, one was centered about
30.5-kbp upstream of the promoter, and another small broad peak was 68-kbp
upstream of the promoter. The same sites were seen with both antisera, and
neither pre-immune serum showed enrichment of any sequences. Thus, in addition
to the non-dividing polytene salivary cells, cohesin also binds cut
in predominantly diploid dividing cells of embryonic origin. Based on the
assumption that cohesin is a constitutive chromosomal component, and the
finding that it binds to the cut locus in two very different cell
types, it is posited that cohesin also binds cut in the developing wing margin cells and that the effects of cohesin on cut expression in the
developing wing are direct (Dorsett, 2005).
The possibility was considered other factors recruited by cohesin could inhibit cut activation. In fungi, the Pds5 (Spo76) protein is required for sister chromatid cohesion. Pds5 associates with cohesin sites on chromosomes, and requires cohesin for association (Dorsett, 2005 and references therein).
By sequence analysis, the CG17509 gene was identified as the likely pds5 homolog. The P{EPgy2}CG17509EY06473 transposon insertion in the first exon is homozygous viable. It was mobilized to generate two recessive lethal mutations, pds5e3 and pds5e6, that fail to complement each other, and a deletion of the region [Df(2R)BSC39]. Both homozygous mutants and the heteroallelic combination are lethal in late third instar to early pupal stages of development. Late third instar larvae of both mutants display small or missing imaginal discs, and the larval brains are approximately half the volume of wild type, consistent with a mitotic defect (Dorsett, 2005).
Neuroblast metaphase nuclei from mutant third instar larvae were examined for
cohesion defects. Despite examining more than 30 metaphases from each, no normal
metaphases were found in the pds5 mutants. Nearly all displayed
aneuploidy, and most displayed precocious sister chromatid separation. By
contrast, 15.4% of the pds5e3/+ heterozygote metaphases
showed aneuploidy and 12.8% showed sister chromatid separation, similar to the
frequencies observed with wild-type neuroblasts. It is
concluded that the pds5e3 and pds5e6
mutations affect chromosome segregation and sister chromatid cohesion, and
that CG17509 encodes a functional Pds5 homolog (Dorsett, 2005).
The effect of the pds5 mutations on the ctK phenotype was tested relative to the viable P element insertion
used to generate them. Unexpectedly, the two mutations had different effects. The pds5e3 mutation slightly increased the number of wing
margin nicks, indicating that it decreased cut expression, whereas
pds5e6 increased cut expression. Although
a small increase in wing margin nicks was consistently observed with
pds5e3, the wing nicking was not significantly different
from that seen with the parental chromosome. The decreased
nicking associated with the pds5e6 allele, however, was
significantly different from that of the parental chromosome. It is
concluded that the pds5e6 mutation dominantly increases
cut expression, and that the pds5e3 mutation may
cause a small decrease (Dorsett, 2005).
pds5 expression was examined in the two pds5 mutants to
determine why they have different effects on cut expression. Northern
blots revealed a pds5 transcript of the expected size (4.6 kb) in embryos prior to zygotic gene expression, indicating that it is maternal. The transcript was present at 10- to 25-fold lower levels in larvae. To avoid detecting maternal pds5 mRNA, the transcripts produced in
pds5e3 and pds5e6 second instar
larvae were examined. No pds5 transcripts were detected in homozygous
pds5e3 mutants, but a shorter
transcript (3.65 kb) was seen at levels similar to those of wild type in both
heterozygous and homozygous pds5e6 mutants. A wild-type sized
transcript was present in heterozygous pds5e6 mutants, but
was undetectable in homozygotes. The viable parental P insertion line used to
generate both lethal pds5 alleles produced wild-type levels of a
wild-type sized transcript (Dorsett, 2005).
The Northern blots indicate that pds5e3 is a null
allele, and PCR analysis of mutant genomic DNA revealed that sequences
starting at the P insertion site and extending upstream of the transcription
start site are missing. Thus, a reduction in Pds5 dosage
slightly decreases cut expression. It is concluded, therefore, that
wild-type Pds5 does not contribute to the inhibition of cut
expression by cohesin, but may slightly decrease the inhibitory effect (Dorsett, 2005).
The presence of a new transcript in the pds5e6 mutant
suggested that it could produce a mutant protein lacking an activity crucial
for sister chromatid cohesion that somehow interferes with the inhibition of
cut by cohesin. PCR analysis of pds5e6 genomic
DNA revealed that the region from the P insertion site through exon 5 is
missing. 5' RACE analysis of the pds5e6 transcript
shows that it starts 67 nucleotides upstream of the wild-type start site
predicted by EST analysis. The pds5e6 transcript extends from the start site to the P insertion site. The next 17 nucleotides are from the end
of the P insertion, followed by 12 nucleotides of internal P sequence fused to
the pds5 sequence 11 nucleotides downstream of the exon 6 5'
splice site. The exon 6 sequences present in the mutant transcript contain six in-frame AUG codons, two of which match a consensus (RNVATGR) for Drosophila translation initiation sites. Thus the pds5e6 mutant transcript
encodes a protein lacking the N terminus (Dorsett, 2005).
The effects of the two pds5 mutations on cut expression
correlate with a difference in cohesin binding to chromosomes. Although the salivary glands of the homozygous pds5 mutants are substantially reduced, polytene chromosomes were obtained from both. Morphology was altered enough to make it difficult to identify specific loci. Nevertheless, individual chromosome
tips could be identified, and developmental puffs, including the puff at 2B,
were present in both mutants, indicating that the chromosomes are transcribed.
In size-matched third instars, the pds5e3 mutant
chromosomes were thicker than wild type, and the pds5e6
mutant chromosomes were thinner. The pds5e3 null allele did not reduce staining for Smc1 or Stromalin, although the pattern appeared less discrete. By contrast, pds5e6 mutant chromosomes showed strongly reduced staining for Smc1 and Stromalin. Loss of cohesin staining was observed in multiple nuclei from
multiple pds5e6 salivary glands. These results indicate
that the pds5e6 mutant either blocks loading of cohesin
onto chromosomes, or facilitates removal. The reduction of cohesin binding
caused by pds5e6, which dominantly increases cut
expression, is consistent with the hypothesis that cohesin inhibits
cut expression (Dorsett, 2005).
The demonstration that cohesin binds to the cut locus in polytene
chromosomes, and to multiple sites between the remote wing margin enhancer and
the promoter in cultured cells, supports the hypothesis that the effects of
cohesin on cut expression in the wing margin are direct. The wing
margin enhancer does not activate cut in salivary glands or Kc cells,
but it is not technically feasible to examine the association of cohesin with
cut in the developing margin cells in which the wing margin enhancer
functions. Based on the association of cohesin with cut in two
diverse cell types, it is posited that cohesin also binds cut in the
developing wing margin cells, and inhibits activation by the wing margin
enhancer. Such a direct effect of cohesin could explain why small reductions
(<20%) in cohesin subunits induced by RNAi have detectable effects on
cut expression (Dorsett, 2005).
Both pds5 mutations tested cause similar sister chromatid cohesion
defects, but only pds5e6 reduces the binding of cohesin to
chromosomes and increases cut expression. This provides additional
evidence that binding of cohesin to chromosomes is required for it to inhibit
cut activation, and also shows that Pds5 itself is not required to
inhibit gene expression (Dorsett, 2005).
The negative effects of cohesin on cut expression raise the
possibility that cohesin contributes to the silencing of euchromatic genes
placed in heterochromatin. Cohesin binds more densely in centromeric
heterochromatin in yeasts and metazoans, and, in S. pombe,
heterochromatin proteins recruit cohesin.
Moreover, RNAi-mediated silencing of a non-centromeric gene in S.
pombe causes the recruitment of heterochromatin proteins and cohesin to
the silenced gene (Dorsett, 2005).
The idea is favored that cohesin inhibits enhancer-promoter communication in
cut. This idea originates from the allele-specific effects of
Nipped-B mutations on cut expression. The known cut
regulators required for activation by the wing margin enhancer, including
scalloped, vestigial, mastermind, Chip, Nipped-A and
l(2)41Af, all display different cut allele specificities
than Nipped-B. In contrast to these factors, Nipped-B is most limiting
when enhancer-promoter communication is partially compromised by a weak
gypsy insulator, suggesting that Nipped-B facilitates long-range
communication (Dorsett, 2005).
Binding of cohesin to multiple sites between the wing margin enhancer and
the cut promoter is consistent with the hypothesis that Nipped-B
facilitates enhancer-promoter communication by regulating the binding of
cohesin to chromosomes. To explain how Nipped-B aids activation, it is theorized
that Nipped-B can remove cohesin from chromosomes. In a simple model, Nipped-B
facilitates the cohesin-binding equilibrium. When Nipped-B is partially
reduced but not abolished, it takes longer to achieve equilibrium, but the
extent of cohesin binding is not altered and there is little effect on sister
chromatid cohesion. The reduced cohesin on-off rates, however, would diminish
the opportunities for gene activation that require cohesin removal or
repositioning (Dorsett, 2005).
It is not known how cohesin inhibits long-range activation, but mechanisms can be
envisioned for various long-range activation models. For example,
cohesin could inhibit the folding or looping of the chromosome that is
required to bring the enhancer into contact with the promoter. Alternatively,
cohesin could block a Chip-mediated spread of protein binding between the
enhancer and promoter in linking models for long-range activation, or
block the transfer or tracking of RNA polymerase from the enhancer to the
promoter, as appears to occur in the chicken beta-globin gene locus (Dorsett, 2005).
This study found that Drosophila Pds5 is required for sister chromatid
cohesion, consistent with studies on fungal Pds5. To
explain the effects of the pds5e6 mutation on cohesin
chromosome binding and cut expression, it is proposed that it produces a
mutant protein that blocks cohesin binding, or causes cohesin to be released
from chromosomes. This agrees with observations on vertebrate and S.
pombe Pds5 suggesting that Pds5 has both positive and negative effects on
cohesion, possibly by regulating the association of cohesin with chromosomes (Dorsett, 2005).
Vertebrates contain two Pds5 isoforms that associate with chromosomal
cohesin. Reduction of Pds5 partially decreases sister chromatid cohesion.
Consistent with the finding that the pds5e3 null mutation
does not reduce the binding of cohesin to polytene chromosomes, and with
previous work on S. cerevisiae and S. pombe Pds5,
Xenopus Pds5 is not required for the binding of cohesin to
chromosomes. One report suggested that S. cerevisiae Pds5 is
required for the association of cohesin with chromosomes, but it
is possible that this discrepancy might be caused by differences in the mutant
alleles, similar to the differences found between pds5e3
and pds5e6 (Dorsett, 2005 and references therein).
Depletion of Pds5 from Xenopus extracts increases the amount of
cohesin associated with chromatin. A similar increase in cohesin binding could explain the slight decrease in cut expression caused by the
pds5e3 null allele. Consistent with the idea that
wild-type Pds5 partially reduces cohesin binding, deletion of S. pombe
pds5 partially suppresses a temperature-sensitive mutation in
mis4, which encodes the homolog of the Nipped-B and Scc2
cohesin-loading factors (Dorsett, 2005 and references therein).
Because wild-type Pds5 appears to partially reduce the binding of cohesin
to chromosomes, it is speculated that the pds5e6 mutation
increases this activity, which may be related to the cohesin-loading function
of Nipped-B/Scc2. Scc2 interacts with cohesin, and is thought to open the
cohesin ring. In synchronized yeast cells, cohesin loads at Scc2-binding
sites and translocates away. Like Nipped-B/Scc2, Pds5 contains several HEAT repeats, and thus might also open the cohesin ring during DNA replication to allow it to encompass both sister chromatids. It could play a similar role in the snap model, in which cohesin complexes bound to the two sisters
interlock to hold the sisters together. If the pds5e6
mutant protein interacts with cohesin non-productively, it could block access
to Nipped-B and prevent loading. Alternatively, when the mutant Pds5 attempts
to establish cohesion, it might fail, releasing cohesin from the chromosome.
Wild-type Pds5 might partially reduce cohesin binding by competing with
Nipped-B for cohesin, or by occasionally failing to establish cohesion (Dorsett, 2005).
The effects of cohesin on cut expression are likely pertinent to
the etiology of Cornelia de Lange syndrome (CdLS). CdLS is
caused by mutations in the Nipped-B-Like (NIPBL) human
homolog of Nipped-B. Most missense mutations that cause CdLS affect residues conserved in Nipped-B. CdLS is characterized by several physical and mental deficits, including slow growth, mental retardation, and upper limb,
gastroesophageal and cardiac deformities.
Heterozygous loss-of-function NIPBL mutations cause CdLS, and thus
the developmental changes likely reflect gene expression effects similar to
those caused by heterozygous Nipped-B mutations. At
least some birth defects in CdLS, such as limb truncations and cardiac
abnormalities, could be caused by changes in expression of the known homeotic
genes. The observations presented here indicate that cohesin likely plays a
role in CdLS by inhibiting the long-range gene control of homeotic genes (Dorsett, 2005).
The possibility that some developmental changes in CdLS reflect reduced
sister chromatid cohesion cannot be ruled out. A recent study found evidence
for cohesion deficits in 41% of CdLS patients compared with 9% of controls.
Also, the autosomal recessive Roberts syndrome has some similarities to CdLS, and is caused by mutations in a human homolog of the Eco1/Eso1/Deco cohesion factor. Cells from Roberts patients display defects in sister chromatid cohesion. Homozygous Drosophila deco1 mutants appear to affect cohesin binding only at centromeric regions, and, as described above, dominant
effects of deco mutations on cut gene expression are not seen, leading to the the idea that most CdLS developmental deficits reflect changes in gene expression instead of in sister chromatid cohesion (Dorsett, 2005).
Drosophila putzig was identified as a member of the TRF2-DREF complex that is involved in core promoter selection. Additionally, putzig regulates Notch signaling, however independently of DREF. This study shows that Putzig associates with the NURF complex. Loss of any NURF component including the NURF-specific subunit Nurf 301 impedes binding of Putzig to Notch target genes, including cut, Enhancer of split and vestigial, suggesting that NURF recruits Putzig to these sites. Accordingly, Putzig can be copurified with any NURF member. Moreover, Nurf 301 mutants show reduced Notch target gene activity and enhance Notch mutant phenotypes. These data suggest a novel Putzig-NURF chromatin complex required for epigenetic activation of Notch targets (Kugler, 2010).
Putzig is a component of a large multiprotein complex that includes the TATA-box-binding-protein-related factor 2 (TRF2) and the DNA-replication related element (DRE) binding factor DREF. The TRF2-DREF complex has been associated with the transcriptional regulation of replication-related genes that contain DREF binding sites. Accordingly, Pzg acts as a positive regulator of cell cycle and replication-related genes. In addition to this, Pzg is also required for Notch target gene activation in a DREF-independent manner. Presumably, Pzg functions at the level of chromatin activation, because the open chromatin structure typical of active Notch target genes is no longer detectable in a pzg mutant background (Kugler, 2010).
The TRF2-DREF complex consists of more than a dozen of proteins and the biochemical function of most of them remains still elusive. Interestingly, it also contains three members of the nucleosome remodeling factor (NURF), imitation switch (ISWI), Nurf 55 and Nurf 38. NURF is a multisubunit complex that has been associated with chromatin activation and repression. NURF triggers nucleosome sliding thereby provoking changes in the dynamic properties of the chromatin. The subunit ISWI is a member of the SWI2/SNF ATPase family and is thought to provide energy for nucleosome remodeling. Nurf 38 encodes an inorganic pyrophosphatase, which catalyzes the incorporation of nucleotides into a growing nucleic acid chain during transcription, replication, and DNA repair mechanisms. Nurf 55 harbors WD-40 repeats, which allow interaction with other proteins and protein complexes. The fourth and largest subunit Nurf 301 is specific to the NURF complex, whereas all other members are shared with other chromatin modifying complexes. Accordingly, Nurf 301 is not a component of the TRF2-DREF complex. Nurf 301 exhibits a number of protein motifs that typify transcription factors and other chromatin modifying proteins. In addition, the N-terminal region of Nurf 301 shows homology to the DNA-binding protein HMGA (high mobility group A) implying that Nurf 301 mediates the contact with the DNA or provides a platform to recruit other transcription factors. In this context it has already been shown that Nurf 301 is required for the transcriptional activation for example of homeotic genes and notably of Ecdyson-receptor (EcR) and Wingless target genes (Kugler, 2010).
The DREF independence of Pzg during the activation of Notch target genes raised the possibility that it may instead involve the NURF complex for chromatin activation. This study provides evidence for a functional interplay between Pzg and the NURF complex with regard to Notch target gene activation. Coimmunoprecipitations revealed that Pzg is present in protein complexes containing the known NURF subunits. Moreover, Pzg binding on Notch target genes is neither detectable in mutants of the NURF-specific subunit Nurf301, nor in mutants affecting other subunits of NURF. In addition, Nurf301 is required for Notch target gene expression, which is impaired in Nurf301 mutant cell clones. Consistent with this, Nurf301 mutants enhance the Notch mutant wing phenotype, strongly arguing for an involvement of the NURF complex in Pzg-mediated epigenetic Notch target gene activation (Kugler, 2010).
This work shows that Pzg is associated with at least two different types of protein complexes that are involved in transcriptional activation: the TRF2-DREF complex and the NURF complex. Interestingly, these two complexes share several members apart from Pzg despite their different roles in core promoter selection versus nucleosome sliding and chromatin activation. However, the specific role for Pzg in the promotion of Notch target gene transcription involves NURF and not the TRF2-DREF complex. Notably, NURF also promotes efficient expression of a subset of Wingless target genes. In this case, a direct interaction between ISWI and Armadillo, the major transcriptional coactivator of Wingless targets, was shown. There is no indication however, that pzg is involved in the regulation of wg, suggesting that the NURF complex recruits Pzg only onto specific promotors. Furthermore, the NURF subunit Nurf 301 contacts the Ecdysone receptor (EcR), thereby modulating the activity of ecdysone signaling during the larval and pupal stages of Drosophila development. How is NURF recruited to Notch target sites? Notch target gene activation involves a ternary complex containing the DNA-binding protein Suppressor of Hairless [Su(H)], intracellular Notch, and Mastermind, plus other more general coactivators. There is no indication of a direct contact of Pzg to either Notch or Su(H), tested by coimmunoprecipitations as well as yeast two-hybrid assays. However, contacts between the other components, notably Mastermind or ISWI cannot be excluded. Mastermind has been shown to interact with several chromatin modifying proteins, for example, with the histone acetyltransferase p300 or with cyclin-dependent kinase 8 (Kugler, 2010).
Several studies in Drosophila and vertebrates have shown that many Notch-responsive target genes are regulated by combinatorial signal inputs, which need the Notch ternary complex and additional cooperators bound to sites nearby. In contrast to cofactors within the transactivation complex, these other factors do not physically interact with the Notch ternary complex but instead synergize during transcriptional activation at Notch target gene promoters. It is conceivable, that a Pzg-NURF complex is likewise needed in conjunction with the Notch transactivator complex for full Notch target gene expression (Kugler, 2010).
It is well established, that chromatin modification complexes share several components. For example, ISWI is not only contained in NURF and TRF2-DREF complexes but also in chromatin-remodeling and assembly factor (CHRAC) and ATP-utilizing chromatin-remodeling and assembly factor (ACF) in Drosophila, where it serves to increase the accessibility of nucleosomal DNA. Nurf 55, also known as CAF-1, forms a stable complex with Drosophila Myb and E2F2/RBf and regulates the transcription of several developmentally important genes. Like ISWI and Nurf 55, also Nurf 38 is present in the TRF2-DREF complex. Pzg is contained within the TRF2-DREF and within the NURF complex serving the activation of proliferation related genes and N target genes, respectively. Not all NURF complexes, however, require pzg, for example, as during the activation of Wg target genes. Sharing components raises the question, how specificity of the different complexes is achieved. Obviously, specificity is mediated either by unique subunits or by certain combinations of shared subunits. These subunits may specifically modulate the activity of the ATPase subunit or, more likely, may help to target the remodeling complexes to particular promoters. Two members of the NURF complex, ISWI and Nurf 301, have been shown to directly target transcription factors. It is tempting to speculate, that Pzg might be a specific cofactor needed to realize some of the operation spectrum of NURF, notably during the epigenetic regulation of Notch target genes (Kugler, 2010).
Continued: Cut Transcriptional regulation part 3/3 | back to
part 1/3
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