Targets of Activity

Irregular facets (If) is a dominant mutation of Drosophila that results in small eyes with fused ommatidia. Previous results showed that the gene Krüppel (Kr), which is best known for its early segmentation function, is expressed ectopically in If mutant eye discs. However, it was not known whether ectopic Kr activity is either the cause or the result of the If mutation. This study shows that If is a gain-of-function allele of Kr. The If mutation was used in a genetic screen to identify dominant enhancers and suppressors of Kr activity on the third chromosome. Of 30 identified Kr-interacting loci, two were cloned, and whether they also represent components of a natural Kr-dependent developmental pathway of the embryo was tested. The two genes, eyelid (eld) and extramacrochaetae (emc), which encode a Bright family-type DNA binding protein and a helix-loop-helix factor, respectively, are necessary to achieve the singling-out of a unique Kr-expressing cell during the development of the Malpighian tubules, the excretory organs of the fly. The results indicate that the Kr gain-of-function mutation If provides a tool to identify genes that are active during eye development and that a number of them function also in the control of Kr-dependent developmental processes (Carrera, 1998).

Kr expression defines the Malpighian tubule anlage at late blastoderm stage and becomes restricted to a ring of cells at the midgut/hindgut boundary from where Kr-expressing Malpighian tubule precursors evert. Previous studies have shown that the specification of Malpighian tubule fate and the segregation of the cells depend on Kr expression in the Malpighian tubule anlage. In Kr-deficient embryos, the respective cells become part of the hindgut epithelium (Carrera, 1998).

Once the tubules evert, Kr expression becomes restricted to a single cell, termed the "tip mother cell". The singling-out process of this cell from an equivalence group of Malpighian tubule precursors involves the activated Notch pathway, which restricts the proneural bHLH proteins encoded by the achaete-scute-complex (ASC) genes to the tip mother cell. In this cell, the ASC proteins act in concert with bHLH protein encoded by daughterless (da) to maintain Kr expression. The tip mother cell divides once, and the daughters give rise to the tip cell, which controls proliferation during tubule elongation and differentiates neuronal characteristics, and an excretory cell, termed "satellite cell". The satellite cell loses Kr expression in a Notch-dependent manner, whereas Kr expression is maintained in the tip cell until the end of embryogenesis (Carrera, 1998).

emc expression accompanies Malpighian tubule development in a manner similar to Kr expression. However, once the tip cell is formed, the patterns of expression become complementary, meaning that emc expression continues in all cells of the elongating Malpighian tubules except in the tip cell. To test whether the complementary patterns of Kr and emc expression reflect a regulatory effect of emc on Kr, as indicated during eye development in the If mutant, Kr expression was examined in the Malpighian tubules of emc mutant embryos. Multiple Kr-expressing cells are seen in emc mutant Malpighian tubules. This finding is consistent with the previous finding that emc mutant embryos develop multiple tip cells and that each of them continues to express achaete. Virtually the same observations have been made with Notch mutants, and Notch acts toward restricting the activity of the proneural bHLH proteins, which are required to maintain Kr expression first in the tip mother cell and subsequently in the tip cell. However, although the activated Notch pathway acts through transcriptional repression of the ASC genes, emc protein antagonizes proneural bHLH activities by sequestering the proteins as heterodimers that are incapable of binding to DNA. The results are therefore consistent with the proposal that emc functions in the control of Kr expression by antagonizing proneural bHLH activities that are required to maintain Kr expression in the tip mother cell (Carrera, 1998).

The Eld protein shows a nuclear localization, consistent with its suspected function as a transcription factor. It appears to act in multiple signaling pathways because it antagonizes wingless activity, suppresses Ras1 activity in the eye, and blocks Notch-dependent neuronal differentiation. During Malpighian tubule development, eld is expressed in a restriced area of the everting precursors that corresponds to the equivalence group of cells expressing the proneural genes (Carrera, 1998).

eld mutant embryos exert a distinct phenotype during Malpighian tubule development that is linked to Kr activity. Whereas the anlage and the four tubules evert normally, each tubule develops two instead of the normal one tip cell. Tip cell development is under the control of Kr activity, so it was next asked whether and when Kr expression is altered in eld mutant embryos. In correspondence with the mutant phenotype, the initial expression of Kr, including its restriction to the tip mother cell, appears to be normal. However, once the tip mother cell has undergone division, two instead of only one of the daughter cells maintain Kr expression. This indicates that eld activity is necessary to prevent Kr expression in the sibling of the tip cell and allows for its differentiation into a satellite cell. Thus, although emc is necessary for the restriction of Kr to the tip mother cell, eld functions specifically at the subsequent step during Malpighian tubule development where an alternative and Kr-dependent cell fate decision is taken between the daughters of the tip mother cell (Carrera, 1998).

Notch signaling is required first for the selection of the tip mother cell and subsequently for the distinction between its daughters to either develop a tip cell or a satellite cell. Consistently, in Notch mutant embryos, all cells of the proneural equivalence group develop first into tip mother cells; these cells divide and subsequently develop into the multiple tip cells that continue Kr expression. In contrast, only two tip cells were found in eld mutants. This finding implies that, if eld acts in a Notch-dependent manner and/or mediates Notch signaling, its activity is required only for the second of the two Notch-dependent differentiation steps during Malpighian tubule development. Thus, eld participates as an optional component in the Notch-signaling pathway and is needed to prevent, directly or indirectly, the maintenance of Kr expression in the satellite cell that would otherwise develop into a second tip cell (Carrera, 1998).

The results of this study demonstrate that gene activities that were identified via an artificial experimental situation, namely the ectopic expression of Kr in the developing eye disc, can lead to the identification of integral components of a Kr-dependent developmental pathway during embryogenesis. In the eye imaginal disc, emc suppresses Kr activity whereas eld has an opposite effect, but both act during embryonic Malpighian tubule development as negative regulators of Kr. No explanation is available for this phenomenon. It could mean, in negative terms, that the Kr misexpression screen turned up dosage-sensitive genes affecting cell fate that were several steps downstream from Kr activity and thus have no direct interaction with Kr. Thus, each gene identified in the modifier screen represents a candidate gene that needs to be evaluated critically through additional criteria as outlined here for eld and emc. The additional screening is essential to distinguish between direct Kr interactors and genes that mediate different read-outs of the Kr pathway in cells that have a different organ or tissue competence. However, in view of the fragmentary information concerning the spatial and temporal control of postblastodermal Kr expression and in view of the fact that the few Kr target genes of Kr were identified by molecular approaches, this experimental strategy to assess components of a Kr-dependent regulatory circuitry seems a valid one (Carrera, 1998).

The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).

Loss of osa can induce phenotypes similar to those caused by ectopic wg expression. Conversely, overexpression of full-length, wild-type Osa (UAS-Osa) results in dominant, gain-of-function phenotypes that often resemble those caused by loss of wg function. However, osa appears to be epistatic to wg, and loss of osa function does not induce ectopic expression of wg. Therefore, the wg gain-of-function phenotypes caused by osa loss of function are likely to result from de-repression of downstream target genes of Wg. To investigate this, the regulation of nubbin (nub) was examined. nub encodes a POU domain protein that is required for the growth and patterning of the wing and is expressed throughout the wing primordium (or wing pouch) in third-instar wing discs. wg signaling is both necessary and sufficient for the expression of nub, since ectopic expression of wg or ectopic activation of the wg pathway can induce ectopic expression of nub, whereas blocking transmission of the wg signal in the wing pouch represses the endogenous expression of nub (Collins, 2000).

nub is ectopically expressed in wing discs that are transheterozygous for null and hypomorphic alleles of osa. An activated form of Armadillo causes similar ectopic nub expression by ectopic activation of the wg pathway. Conversely, the endogenous expression of nub is reduced along the anterior/posterior (A/P) boundary when UAS-Osa is expressed there with a decapentaplegic (dpp)-Gal4 driver. A similar loss of nub expression is caused by the expression of a dominant negative form of Pangolin (Pan) that can no longer bind Arm to activate gene expression (DN-Pan). When Osa and DN-Pan are coexpressed with dpp-Gal4, they acted synergistically to cause a severe reduction in nub expression (Collins, 2000).

In addition to its role in transmitting the wg signal, Arm binds directly to cadherins and is required for the formation of adherens junctions. Over-expression of Drosophila E-Cadherin (DE-Cadherin) can sequester Arm at the plasma membrane and prevent it from participating in Wg signaling; this results in the induction of wg-like phenotypes. When DE-Cadherin (UAS-Cad) is overexpressed in the dorsal compartment of the wing disc with an apterous (ap)-Gal4 driver, dorsal expression of nub is lost and the growth of the wing pouch is reduced. Reduction of osa function in discs expressing UAS-Cad restores more normal nub expression and growth. Furthermore, the ectopic nub expression normally seen in osaeld308/osa4H discs is suppressed by the expression of UAS-Cad in the dorsal compartment. Thus, the level of nub expression is determined by the relative levels of Arm and Osa when either of these levels is reduced. To increase the levels, UAS-Osa is expressed with ap-Gal4, causing a strong reduction of nub expression in the dorsal wing pouch. Expression of DeltaArm with the same Gal4 driver causes nub to be expressed in almost the entire wing disc. The normal domain of nub expression is restored when UAS-Osa and DeltaArm are coexpressed. Taken together, these data demonstrate that Osa is required for the repression of a wg-dependent gene in vivo. Alterations in the dosage of osa can modulate the expression of wg-dependent genes even in the presence of an activated form of Arm or a dominant negative form of Pan, suggesting that Osa does not act upstream of Arm. Alterations in the level of active Pan/Arm complexes can also modulate nub expression in osa mutants; thus, lack of osa does not make Wg target genes entirely independent of Arm (Collins, 2000).

The ARID DNA-binding domain of Osa fused to the repressor domain of Engrailed (UAS-OsaRD) or the activation domain of VP-16 (UAS-OsaAD) can target these domains to genes normally regulated by osa in vivo. The ectopic expression of nub in osaeld308/osa4H wing discs can be prevented by expression of either UAS-Osa or UAS-OsaRD with ap-GAL4. This suggests that Osa functions as a repressor of transcription in the regulation of Wg target genes (Collins, 2000).

To test whether Osa is acting directly on Wg target genes or regulating the expression of some other gene that interacts with the wg pathway, attempts have been made to determine at what level in the wg pathway Osa acts. In third-instar wing discs, wg is expressed in a narrow stripe of cells that straddles the dorsal/ventral (DV) boundary of the wing pouch and directs growth and patterning of the wing blade with respect to the DV axis. Cells adjacent to the DV boundary respond to the wg signal by posttranscriptionally up-regulating cytosolic Arm. Arm then translocates to the nucleus and binds to Pan to activate the expression of downstream target genes such as Distal-less (Dll). When an activated form of the protein kinase Sgg, which constitutively targets Arm for degradation (UAS-Sgg*) is expressed in the dorsal compartment using the ap-Gal4 driver, Arm is not up-regulated. Expression of UAS-Osa in the dorsal compartment similarly prevents the expression of Dll on the dorsal side of the DV boundary. However, these cells still respond to the Wg signal by up-regulating cytosolic Arm. Therefore, Osa represses Wg target genes without affecting the up-regulation of Arm. This places the activity of Osa in the nucleus and argues that Osa may directly repress the expression of Wg target genes (Collins, 2000).

To test the requirements for Osa to repress the expression of Wg target genes, the expression of a lacZ reporter gene driven by a well-characterized wg-responsive enhancer was examined. The midgut enhancer (UbxB) of the Ultrabithorax (Ubx) promoter drives lacZ expression in the embryonic midgut in a pattern that is dependent on both wg and decapentaplegic (dpp). In wild-type embryos UbxB-lacZ is expressed primarily in parasegment (ps) 6, 7, and 8, with weaker expression in ps 3. This expression is de-repressed in embryos lacking the maternal contribution of osa, such that the expression of lacZ expands anteriorly as far as ps 3. Similarly expanded expression is induced by ectopic expression of wg in the mesoderm using 24B-Gal4. Conversely, expression of UAS-Osa or UAS-DN-Pan in the mesoderm represses the expression of UbxB-lacZ. However, neither wg nor dpp is ectopically expressed in the midgut in embryos lacking maternal osa (Collins, 2000).

When the dpp response element in UbxB is mutated (UbxBC), the expression of the lacZ reporter is severely reduced; only weak levels of lacZ expression are detectable in ps 8. Expression of UbxBC-lacZ is unchanged in the absence of maternal osa, suggesting that the dpp response element is still required for the expression of the reporter construct in the absence of Osa. When one of the two wg response elements in UbxB is mutated (UbxB4), the expression of lacZ is reduced in wild-type embryos. However, removal of maternal osa allows an expansion of UbxB4-lacZ expression. This suggests that lack of osa can compensate for a reduction in the responsiveness of the promoter to Wg but not to Dpp. Furthermore, the expression of wild-type UbxB-lacZ is also de-repressed in embryos lacking maternal osa even in the presence of DN-Pan. These data argue that Osa functions specifically to repress the activation of the UbxB enhancer by the Wg pathway (Collins, 2000).

Osa functions as a component of Brm chromatin-remodeling complexes and might be acting through the Brm complex to repress Wg target genes. Other components of the Brm complex were therefore tested for genetic interactions with the wg pathway. Blocking Wg signaling at the wing margin by expressing UAS-Sgg* with vg-Gal4 causes a reduction in wing growth and a loss of the wing margin. These phenotypes are strongly enhanced in flies heterozygous for wg or in those that coexpress UAS-Osa; they are suppressed in flies heterozygous for axin (a negative regulator of Wg signaling or osa. The effects of UAS-Sgg* expression are also suppressed by the loss of one copy of brm or moira (mor), which encodes an essential component of the Brm complex, or by coexpression of a dominant negative form of Brm (DN-Brm). In contrast, two other trithorax group genes [trithorax (trx) and absent, small, or homeotic discs 2 (ash2)] that encode components of other nuclear complexes thought to regulate chromatin structure, failed to modify the UAS-Sgg* phenotype (Collins, 2000).

This demonstrates that there is a specific genetic interaction between the wg pathway and components of Brm complexes and suggests that these complexes are required for the repression of Wg target genes. Indeed, the wg-dependent gene nub is ectopically expressed in wing discs that contain large clones of cells mutant for brm or mor or that expressed DN-Brm in the dorsal compartment. Furthermore, the loss of nub expression caused by expression of UAS-Osa with ap-GAL4 is rescued by coexpression of DN-Brm, indicating that Brm activity is required for the repression of Wg target genes by Osa. The Wg-dependent UbxB-lacZ reporter is also de-repressed in embryos that express DN-Brm, and coexpression of DN-Brm can rescue the loss of UbxB-lacZ expression caused by DN-Pan. These results suggest that Osa acts through the Brm chromatin-remodeling complex to prevent the expression of Wg target genes (Collins, 2000).

In addition to transducing the wg signal in a complex with Arm, Pan is also required for the active repression of Wg target genes in the absence of the Wg signal. This repression requires the association of Pan with the corepressor Groucho (Gro). Gro functionally interacts with the histone deacetylase Rpd3, and this interaction is important for at least some of the repressive activity of Gro. Thus, both Osa-containing Brm complexes and Pan/Gro/Rpd3 complexes repress the expression of Wg target genes and probably mediate this repression by altering the local chromatin architecture at the promoters of these genes. Consistent with this, reduction of gro or rpd3 dosage reduces the ability of Osa to repress nub. The loss of nub expression caused by expression of UAS-Osa with ap-Gal4 is significantly rescued in wing discs homozygous for a hypomorphic allele of rpd3. Also, larvae transheterozygous for osaeld308 and groE48 often ectopically express nub in the wing disc, and 40% of transheterozygous adults have notum-to-wing transformations. These phenotypes are not seen when osa or gro single mutants are crossed to wild-type flies (Collins, 2000).

Whereas many of the genes regulated by the wg pathway require wg for their expression, several genes appear to be repressed by high levels of wg signaling. To determine the effect of Osa on the expression of genes that are normally repressed by wg, the expression of dpp in leg discs was examined with altered dosage of osa. In third-instar leg discs, wg and dpp are expressed along the A/P boundary in the ventral and dorsal compartment, respectively, and mutually antagonize each other's expression. dpp expression is repressed when UAS-Osa is expressed in a broad central domain of the leg disc with a Dll-Gal4 driver and dpp is ectopically expressed in the ventral compartment in osaeld308/osa4H leg discs. Clones of cells mutant for osa can also induce leg duplications in the ventral compartment of the leg, consistent with the ectopic expression of dpp. Thus, in addition to repressing the expression of genes that are normally activated by the wg signal, Osa is also required for the repression of at least one of the genes that are repressed by wg (Collins, 2000).

The most likely explanation of these data is that Osa functions to directly repress Wg target gene expression, with such target genes being defined by their inclusion of a Pan-binding site. Osa function is not exclusive to the Wg signaling pathway; Osa also functions as a promoter specific activator of Antennapedia expression and as a coactivator for Zeste and likely represses E2F-mediated gene expression. Furthermore, the expression of even-skipped is perturbed in embryos lacking maternal osa, a phenotype that precedes the expression of wg in the embryo. However, the strong correlation of the expression of Wg target genes with the level of Osa suggests that counteracting Osa activity is an important function of the Wg pathway (Collins, 2000).

Osa functions as a component of Brm chromatin-remodeling complexes. These complexes and closely related complexes in other species such as the yeast SWI/SNF complex catalyze an ATP-dependent alteration in the structure of nucleosomal DNA that can run in either direction to render the DNA either more or less accessible to binding by transcription factors. Whereas chromatin-remodeling complexes are generally thought to promote gene expression, recent reports have demonstrated that they are also required for the repression of some genes. Genome-wide analysis shows that more genes have elevated than reduced expression in a swi2 mutant yeast strain, and some of these genes are directly repressed by SWI/SNF. The hBRM complex in humans has been shown to cooperate with the retinoblastoma protein (Rb) to repress E2F-1-mediated activation. Furthermore, brm, mor, and osa have been identified as enhancers of an E2F gain-of-function phenotype, suggesting that Brm complexes also repress E2F activation in Drosophila (Collins, 2000 and references therein).

Because Osa can antagonize Brm complex function in some tissues, it has been thought possible that Brm complex activity could be required for the expression of Wg target genes and that Osa might be a negative regulator of Brm complex function. However, the findings that the effects of blocking the wg pathway at the wing margin can be suppressed by reducing the dosage of brm or mor and that nub and UbxB-lacZ are ectopically expressed when brm or mor function is lost suggest that Brm complexes are required for the repression, rather than the activation, of Wg target genes. Furthermore, expression of a form of Brm that has a mutation in its ATP-binding site also induces ectopic expression of nub and UbxB-lacZ and can rescue the loss of nub expression caused by overexpression of Osa. Because the ATPase activity of Brm is required for the chromatin-remodeling activity of the Brm complex, this suggests that chromatin remodeling by the Brm complex is necessary for Osa to repress the expression of Wg target genes (Collins, 2000 and references therein).

In addition to activating gene expression by recruiting Arm to the promoters of wg-responsive genes, Pan also represses these same genes by recruiting the transcriptional corepressor Gro. Interestingly, Gro has been shown to interact with the N-terminal tail of histone H3 and with the histone deacetylase Rpd3, and it has therefore been proposed that Gro mediates repression by altering chromatin structure. Consistent with this, a strong genetic interaction exists between osa and gro that suggests that their activities in repressing Wg target genes are closely related. Although it has not previously been reported that Rpd3 functions in the repression of wg target genes, reducing the function of rpd3 can partly rescue the loss of nub expression caused by the overexpression of Osa. Rpd3 is therefore important for the repression of Wg target genes; testing whether it is essential awaits the isolation of null alleles (Collins, 2000).

The loss of either osa or gro leads to ectopic expression of Wg target genes; thus, the activity of one is not sufficient to repress the expression of these genes without the activity of the other. Osa and Gro may, therefore, be mediating the same repressive event rather than acting in parallel. Interestingly, human SWI/SNF forms a repressor complex with Rb and the histone deacetylase HDAC. This complex interacts with the cyclin E promoter through the binding of Rb to E2F-1 and represses E2F-1 activation of cyclin E expression. This suggests the intriguing possibility that Osa and the Brm complex function in a larger repressor complex containing Gro and Rpd3 and that this complex is recruited to Wg target genes though the binding of Gro to Pan. However, Gro acts as a corepressor for a large number of transcription factors, and Osa cannot be required for all repression mediated by Gro because loss of osa does not result in neurogenic phenotypes like those caused by the loss of gro. Further research is needed to determine if Gro and/or Rpd3 can directly interact with components of the Brm complex and, if so, what determines the specificity of this interaction (Collins, 2000 and references therein).

The mechanism by which Wg signaling leads to the active repression of genes such as dpp is not fully understood, although it is counteracted by Sgg. However, the observation that dpp expression is repressed by Osa suggests that other factors may allow Wg signaling to reinforce repressive chromatin modeling by the Brahma complex on such promoters (Collins, 2000).

A model for the regulation of gene expression by components of the Wg pathway is presented. The chromatin remodeling activity of the OsaBrm complex is required to maintain the chromatin at the promoters of wg-responsive genes in a repressive conformation. This would prohibit the association of other transcription factors with their binding sites and prevent the recruitment of components of the basal transcription machinery. Osa/Brm complexes may be recruited to Wg-responsive genes through an association with Pan/Gro/Rpd3 complexes. In response to the Wg signal, Arm is stabilized and accumulates in the cytosol. This accumulation of cytosolic Arm permits Arm to translocate to the nucleus and displace Gro from Pan and, in so doing, relieve the repression mediated by Gro, Rpd3, and Osa/Brm complexes. Arm may also promote a more open chromatin conformation by recruiting the HAT activity of dCBP, thus permitting the association of other transcription factors with their binding sites. Also, the stimulation of the DNA-bending activity of Pan by Arm may bring distantly spaced transcription factors into juxtaposition to promote the activation of gene expression. In the absence of osa, the chromatin is maintained in a more open and less repressive conformation. This would permit other transcription factors to interact with their binding sites at lower concentrations than would otherwise be possible. Under these conditions, the low levels of Arm that are always present in the cell may be sufficient to promote the activation of gene expression without the Wg signal (Collins, 2000).

Transcriptional regulation of the Drosophila moira and osa genes by the DREF pathway

The DNA replication-related element binding factor (DREF) plays an important role in regulation of cell proliferation in Drosophila, binding to DRE and activating transcription of genes carrying this element in their promoter regions. Overexpression of DREF in eye imaginal discs induces a rough eye phenotype in adults, which can be suppressed by half dose reduction of the osa or moira (mor) genes encoding subunits of the BRM complex. This ATP-dependent chromatin remodeling complex is known to control gene expression and the cell cycle. In the 5' flanking regions of the osa and mor genes, DRE and DRE-like sequences exist which contribute to their promoter activities. Expression levels and promoter activities of osa and mor are decreased in DREF knockdown cells and the results in vitro and in cultured cells indicate that transcription of osa and mor is regulated by the DRE/DREF regulatory pathway. In addition, mRNA levels of other BRM complex subunits and a target gene, string/cdc25, were found to be decreased by knockdown of DREF. These results indicate that DREF is involved in regulation of the BRM complex and thereby the cell cycle (Nakamura, 2008).

This study demonstrated that both osa and mor are DREF target genes. Thus osa and mor promoters exhibited decreased activities when carrying mutations in their DREs and after knockdown of DREF in cultured cells. In addition, levels of osa and mor mRNAs were reduced in DREF knockdown cells. Third, DREF can bind to DREs of osa and mor in vitro, and binding of DREF to the genomic regions containing DREs of both genes was observed in cultured cells. These results showed that DRE and DREF are important for osa and mor promoter activation. Promoters having mutations in all DREs of both osa and mor genes, however, still retained some activity. It is therefore possible that another element(s) and/or unknown factor(s) regulated by DREF are involved in osa and mor transcriptional activation. The observed rescue of the DREF-induced rough eye phenotype by a reduction in the osa and moire gene dosage is consistent with the idea that the osa and moire gene transcription is activated by DREF. However,the possibility cannot be excluded that the rescue could also be affected by a mechanism involving protein-protein interactions between DREF and BAP/PBAP at the promoters of cell cycle-regulated genes. Further analyses are necessary to address this point (Nakamura, 2008).

Both osa and mor encode components of the BRM complex, which is a SWI/SNF type ATP-dependent chromatin remodeling complex conserved from yeast to human, with two forms, BAP and PBAP. Osa is a signature subunit of BAP, while PBAP contains Polybromo and BAP170 in its place. Localization patterns of Osa and Polybromo on polytene chromosomes differ, though several sites overlap. Whole-genome expression analysis also demonstrated that BAP and PBAP differentially regulate gene expression. For example, Osa negatively regulates expression of the Wingless-target genes and the achaete/scute gene. Osa, Polybromo and BAP170 are all required for function of BRM complex. It is thought that Osa functions in recruitment of BAP to its target genes. Mor, a subunit common to both BAP and PBAP, is presumed to be essential for complex integrity, since its absence results in degradation of both forms. SRG3, which is a homolog of Mor in mammals, also acts for complex stabilization by protecting against proteasomal degradation. Therefore, Osa and Mor are essential subunits for function and stabilization of BRM complexes and DREF may control integrity of the BRM complex through activating osa and mor gene expression (Nakamura, 2008).

BAP and PBAP share seven subunits, Brm, Mor, Snr1, BAP111, BAP60, BAP55 and Actin. Brm is a catalytic subunit harboring the ATPase domain and it was previously reported that reduction of the brm gene dose suppressed the DREF-induced rough eye phenotype. It was also found the the mRNA level of brm is decreased in DREF knockdown cells. However, DRE-like sequences in the second intron, do not appear to function as regulatory elements, since DREF does not bind to the genomic region containing these sites in vivo. DREF may therefore indirectly control brm gene expression (Nakamura, 2008).

The genes coding for BAP55 and BAP60, common subunits for BAP and PBAP, also contain DRE or DRE-like sequences in their 5' flanking regions and are affected by DREF knockdown. DREF binds to the genomic regions containing their DREs in vivo and it is, therefore, possible that BAP55 and BAP60 are directly regulated by DREF. Furthermore, the PBAP-specific subunit BAP170 carries a DRE in its 5' flanking region. Reduction of mRNA levels of osa, polybromo and BAP170 in DREF knockdown cells also is evidence that DREF contributes to the transcriptional regulation of both BAP and PBAP complexes. Therefore, DREF may regulate expression of genes coding for most subunits for both BAP and PBAP complexes and influence expression of many genes through chromatin remodeling (Nakamura, 2008).

It has been reported that OSA-containing BAP complexes are necessary for G2/M progression through stg promoter activation while PBAP complexes are not. stg encodes a CDC25 phosphatase, which is required for G2/M progression. It is well known that DREF predominantly regulates the transcription of DNA replication-related genes. Reduced stg mRNA has been reported in DREF-eliminated cells and this study also observed reduction of stg mRNA levels in DREF knockdown cells, as with brm, osa and mor. In addition to regulation of S phase entry, DREF thus appears to play an important role in G2/M transition by activating the BAP complex to promote cell cycling. Two DRE-like sequences were found in the stg gene upstream region, -219 to -212 (5'-aATCGATg) and -591 to -584 (5'-TATCGATt). Therefore, DREF could regulate stg gene expression directly via binding to DRE-like and/or indirectly via activation of genes coding for BAP complexes. Further analysis is necessary to distinguish these possibilities (Nakamura, 2008).

BRM complexes are thought to inhibit S phase entry and mutations of brm, osa and mor suppress the rough eye phenotype induced by E2F/DP/p35 overexpression. The rough eye phenotype of a cyclin E hypomorphic mutant was also suppressed by BRM complex mutation through increase in the S phase. Therefore, BRM complexes appear to negatively regulate S phase entry, while DREF activates E2F gene transcription and promotes G1/S progression. Although osa is ubiquitously expressed in eye imaginal discs, it is most intensely expressed anterior to the morphogenic furrow where cells enter the G1 phase. Similarly, DREF is strongly expressed in this region. It is conceivable that DREF simultaneously activates both positive and negative regulators of G1/S progression. This kind of regulation may be necessary for fine tuning of cell cycle progression to inhibit excess S phase induction (Nakamura, 2008).

Osa, a subunit of the BAP chromatin-remodelling complex, participates in the regulation of gene expression in response to EGFR signalling in the Drosophila wing

Gene expression is regulated in part by protein complexes containing ATP-dependent chromatin-remodelling factors of the SWI/SNF family. In Drosophila there is only one SWI/SNF protein, named Brahma, which forms the catalytic subunit of two complexes composed of different proteins. The protein Osa defines the Bramha associated protein (BAP) complex, and the proteins Polybromo and Bap170 are only present in the complex named PBAP. This work analysed the functional requirements of Osa during Drosophila wing development, and found that osa is needed for cell growth and survival in the wing imaginal disc, and for the correct patterning of sensory organs, veins and the wing margin. Other members of the BAP complex, such as Snr1, Bap55, Moira (Mor) and Brahma, also share these functions of Osa. Focus was placed on the requirement of Osa during the formation of the wing veins. Genetic interactions between osa alleles and mutations affecting the activity of the EGFR pathway suggest that one aspect of Osa is intimately related to the response to EGFR activity. Thus, loss of osa and EGFR signalling results in similar wing vein phenotypes, and osa alleles enhance the loss of veins caused by reduced EGFR activity. In addition, Osa is required for the expression of several targets of EGFR signalling, such as Delta, rhomboid and argos. It is suggested that one role of Osa and Brm in the wing is to establish a chromatin environment in the regulatory regions of EGFR target genes, making them available for both activators and repressors and facilitating transcription in response to EGFR signalling (Terriente-Félix, 2009).

Chromatin structure is critical to modulate gene expression during development, and is affected by a variety of alterations such as histone modification, DNA methylation and changes in conformation. Proteins related to Drosophila Brm, such as yeast SNF2 modify chromatin in an ATP-dependent manner, causing repositioning of nucleosomes along the DNA and re-distribution of histone proteins between nucleosomes. The SWI/SNF complexes are conserved in all eukaryotes, and display specific interactions with distinct transcription factors to regulate different subsets of genes. There are several examples where sequence-specific transcription factors interact specifically with SWI/SNF complexes. For example, the ATPase BRG1 binds Zn-finger proteins and hBRM interacts specifically with CBF-1/Su(H), which recruits hBRM to Notch target promoters such as those of HES1 and HES5 (Terriente-Félix, 2009).

A key aspect in the analysis of Brm function is the identification of targets accounting for the functions of the complex. A necessary step in this analysis is the description of its functional requirements using genetic approaches; which helps to identify the specific processes affected by loss of BAP function. The current data indicate that Osa is required during wing disc development for cell viability, cell proliferation, and for the formation of wing veins and the wing margin. Interestingly, increased expression of Osa in the wing also causes phenotypes related to wing growth and patterning, such as reduced wing size, ectopic sensory organs and hairs and the formation of extra vein tissue in most interveins. This analysis focused mostly on Osa, and this raises the question of whether its requirement reflects the function of the BAP complex. This is the most likely scenario, because the preliminary analysis of other BAP members, such as Snr1, Bap55, Mor and Brm uncovers similar phenotypes in the wing. Thus, lowering Snr1, Bap55 or Mor levels reduces wing size, disrupts the wing epithelium and causes the differentiation of ectopic sensory organs and hairs. These wings also display loss of veins, and in general the overall phenotypes are similar to those of loss of Osa. The phenotype of iRNA expression directed against brm is much milder, perhaps due to a lower efficiency of this construct, but still these wings show a loss of veins phenotype. The reduction of Bap170, a member of the PBAP complex, causes the formation of ectopic veins, which is the opposite phenotype to loss of function in osa and in other members that are present in both the BAP and PBAP complexes. Thus, although Brm is the catalytic subunit in both BAP and PBAP, these complexes could act in opposite manners on the same target genes at least during wing vein formation (Terriente-Félix, 2009).

Some Osa requirements can be explained by modifications in the transcriptional response to the activity of the Wg signalling pathway and by effects on wg expression. The function of Wg is required for the formation of the wing margin, including the development of sensory organs and veins along the anterior wing margin. In the absence of Wg signalling the wing margin does not form, and when Wg signalling is inappropriately activated ectopic sensory organs and hairs differentiate throughout the wing blade. In addition to affecting the response to Wg signalling, Osa is also required for the expression of wg along the dorso-ventral boundary. This requirement might be related to Notch signalling in these cells, and explains why the remnants of wing tissue formed in osa mutant wings do not form the wing margin or ectopic sensory organs (Terriente-Félix, 2009).

This study focused on the characterisation of Osa during the formation of the longitudinal wing veins. This process is independent of Wg signalling, and requires the activities of the Notch, Dpp and EGFR signalling pathways. Osa is needed for the expression of bs in the interveins, because bs is not expressed in cells mutant for osa. The regulation of bs expression involves the activity of Ash2 and the function of the Hh and Dpp pathways. It is suggested that Osa participates in the activation of bs facilitating the availability of its regulatory regions to these activators. This aspect of Osa function does not explain the phenotype of loss of veins characteristic of osa mutant cells, because the loss of Bs expression is normally associated with the differentiation of ectopic veins. The only context where bs mutant cells differentiate as interveins is when the activity of the EGFR pathway is reduced. Therefore, it is suggested that loss of bs expression is accompanied in osa mutant cells by a failure in the response to EGFR activity, leading to the differentiation of intervein tissue. Interestingly, the expression of bs is also severely reduced when Osa is present at higher than normal levels, and in this case loss of Bs is accompanied, as expected, by the formation of ectopic veins. The effects of increased Osa on bs expression can also be explained if Osa facilitates EGFR activity, because this pathway mediates the repression of bs in the proveins. In both cases, the common aspect mediated by Osa might be to regulate bs expression in collaboration with its transcriptional activators and repressors (Terriente-Félix, 2009).

Because the failure of osa mutant cells to differentiate the veins is not due to changes in bs expression, nor to changes in the expression of provein genes such as kni and caup, the search for Osa candidate targets was narrowed to the EGFR pathway. Several results suggest a close relationship between Osa and EGFR signalling in the wing. First, the phenotypes of changing osa expression in the veins are very similar to those resulting from the same manipulation in EGFR activity. Thus, a reduction in any core component of the EGFR pathway eliminates the veins, whereas the increase in EGFR signalling activity causes the formation of extra veins in intervein territories. Second, genetic interactions were observed between osa and several components of the EGFR pathway compatible with a function of Osa promoting EGFR activity in the veins. Finally, the extra veins caused by excess of Osa are suppressed when the activity of EGFR is reduced, indicating that Osa cannot substitute for EGFR activity. The changes in vein and intervein expression patterns are already detected in the wing disc, before other signalling pathways, such as Dpp, act to promote vein formation. Taken together, these observations suggest that Osa facilitates the response to EGFR activity in the wing disc, but cannot promote the transcription of EGFR targets in the absence of EGFR signalling (Terriente-Félix, 2009).

The changes in the expression of EGFR target genes observed in osa mutant cells or in osa gain-of-function experiments are compatible with a direct function of Osa/BAP is the transcriptional regulation of EGFR targets such as Dl, rho and aos. How Osa and the BAP complex are targeted to specific genomic regions is not entirely clear, although it is likely that sequence-specific transcription factors are involved in this process. Transcription in response to EGFR signalling is mediated by proteins belonging to the ETS family, such as Pointed-P2, Pointed-P1 and Yan in Drosophila. However, these genes are not required during wing vein formation, suggesting that other ETS proteins or uncharacterised transcription factors bring about interactions between the regulatory regions of EGFR target genes and the BAP complex (Terriente-Félix, 2009).

It is unlikely that Osa participates in any step of the EGFR pathway previous to the transcription of its target genes. It was noticed, however, that the expression of dP-ERK, a direct read-out of the pathway activity, is also affected in osa mutant cells. Thus, these cells frequently fail to express normal levels of dP-ERK, a result indicating that EGFR activity is reduced. The most likely explanation for this observation is that, in the wing, the EGFR pathway is engaged in a positive feedback loop mediated by the activation of rho expression, which maintains EGFR activity in cells where it has already been activated. Thus, loss of osa leads to a failure to express rho and subsequently to a reduction in the activity of the pathway detected as a loss of dP-ERK expression. There is one experimental situation in which Osa function appears to be dispensable for the expression of EGFR target genes. Thus, when a constitutive active form of Ras, RasV12, is driven in the wing, the augmented expression of Dl and aos, and the accumulation of dP-ERK are not affected by a reduction in Osa levels. It is possible that in this situation of strong and constitutive activity of the pathway, the possible modifications to chromatin structure brought about by Osa/BAP on EGFR target genes are not necessary, perhaps because at this level of EGFR activation the transcriptional repressors antagonising EGFR target gene transcription, such as Cic and Gro, are inactivated by the pathway, and this might make dispensable the function of Osa (Terriente-Félix, 2009).

It is not entirely clear to what extent the link observed between BAP function and EGFR signalling during wing disc development is conserved in other developmental systems and in other organisms. Some phenotypes of osa and brm alleles described in the eye disc, such as the loss of photoreceptor cells, are also observed upon a reduction in EGFR activity. Similarly, the loss of distal growth in the legs is also characteristic of reduced EGFR activity. These data are indicative of a general requirement for Osa in the expression of EGFR target genes at least in imaginal discs. The genetic approach that was used identifies transcription downstream of EGFR signalling as a relevant in vivo function of BAP complexes. Subsequent biochemical analysis should determine whether the functional interactions observed are mediated by direct binding of BAP to the regulatory regions of bs and other EGFR target genes (Terriente-Félix, 2009).

Protein Interactions

The Drosophila osa gene, like yeast SWI1, encodes an AT-rich interaction (ARID) domain protein. Genetic and biochemical evidence is presented that Osa is a component of the Brahma complex, the Drosophila homolog of SWI/SNF. To determine whether Osa is associated with the high molecular weight Brm complex, Schneider cell nuclear extracts were fractionated through a glycerol gradient and immunoblotted with antibodies against the various proteins. Osa, Brm and Snr1 co-sediment in the bottom third of the gradient, suggesting that they are part of a large protein complex. Although Osa and Brm are present in similar fractions, Snr1 sediments in the bottom half of the gradient and could also be part of another complex that does not contain Osa or Brm. Alternatively, the anti-Snr1 antibody might be much more sensitive, detecting very low levels of the Snr1 protein. When glycerol gradient fractions are immunoprecipitated with anti-Osa antibody, Osa, Brm and Snr1 co-precipitate in the same region of the gradient in which they co-sediment. ISWI and Ash2 both show broad sedimentation patterns, appearing in the bottom half of the gradient, but neither protein is immunoprecipitated from the gradient fractions with anti-Osa antibody. Thus, in vivo, Osa is found in a large complex with Brm and Snr1, but does not bind to proteins in other chromatin remodeling complexes. The ARID domain of Osa binds DNA without sequence specificity in vitro, but the domain is sufficient to direct transcriptional regulatory domains to specific target genes in vivo. Endogenous Osa appears to promote the activation of some of these genes. Some Brahma-containing complexes do not contain Osa and Osa is not required to localize Brahma to chromatin. These data suggest that Osa modulates the function of the Brahma complex (Collins, 1999).

osa genetically interacts with trithorax group genes. Ectopic expression of a dominant-negative form of Brm with a mutation in the ATP binding site (UAS-brmK804R) disrupts many developmental processes. An optomotor-blind (omb)-GAL4 driver was used to direct expression of UAS-brmK804R in the central region of the wing disc; this results in loss of the distal wing margin, formation of ectopic campaniform sensillae and wing margin bristles, and disruptions in wing vein morphology. These phenotypes are strongly enhanced in animals heterozygous for osa. Expression of UAS-brmK804R at the wing margin using vestigial (vg)-GAL4 results in the loss of the proximal, posterior wing margin, a phenotype that is again enhanced in osa heterozygotes. The effect of increasing osa dosage was tested by co-expressing a full-length osa transcript under the control of the same vg-GAL4 driver, and this completely rescues the dominant-negative Brm phenotype. Interestingly, ectopic expression of osa alone with vg-Gal4 induces a dominant loss of proximal wing hinge structures, and this phenotype is also rescued in animals co-expressing osa and dominant-negative brm. This suggests that the functions of Osa and Brm are closely related, because a reduction in the activity of one can compensate for an excess of the other (Collins, 1999).

Ectopic expression of Osa in eye imaginal discs using eyeless (ey)-GAL4 results in a variable reduction in eye size. Rather than the expected suppression, an enhancement of this phenotype has been observed in flies that either co-express dominant-negative Brm or are heterozygous for brm. The eye phenotype is also enhanced by mor and SNF5-related 1 (Snr1), both of which encode components of the Brm complex. However, reducing the dosage of the trithorax group genes trx, ash1 or ash2 does not enhance the Osa overexpression phenotype. As expected, a reduction in osa dosage suppresses the small eye phenotype. Clones of mor mutant cells in the eye disc exhibit a severe reduction in growth, which is partially rescued if the cells are also mutant for osa. Taken together, these data demonstrate that osa shows strong and specific genetic interactions with components of the Brm complex. However, in the wing, osa appears to act in concert with brm, whereas in the eye, osa opposes the functions of brm, snr1 and mor (Collins, 1999).

Brm-related complexes are thought to promote transcription by altering the architecture of nucleosomal DNA, thus generating a conformation that is more favorable to binding by transcription factors and the basal transcriptional machinery. Some genes, such as even-skipped, show reduced levels of expression in osa mutant embryos, supporting the role of Osa as an activator of gene expression. However, other genes, such as engrailed, show expanded domains of expression in osa mutants. These genes could be directly activated or repressed by Osa, or their changes in expression level could be secondarily due to the regulation of other transcription factors by Osa. The lack of specificity of DNA binding by Osa in vitro prevents the demonstration of direct action of Osa by altering Osa binding sites in the promoters of potential target genes. As an alternative approach, attempts were made to preserve Osa's target specificity in vivo and to determine the effect of making it an obligate activator or repressor of transcription. Either the exogenous activator domain of VP16 or the repressor domain of Engrailed was fused to the DNA-binding domain of Osa. The effects of misexpressing these activator (UAS-osaAD) and repressor (UAS-osaRD) forms of Osa under the control of the GAL4-responsive UAS sequences were compared with those caused by misexpressing the full-length wild-type Osa protein. The Osa DNA-binding domain appears to be sufficient for chromosomal localization of these fusion proteins, because an antibody to VP16 detects the OsaAD protein along the length of polytene chromosomes (Collins, 1999).

The notum of the adult fly contains a regular pattern of small (microchaetae) and large (macrochaetae) bristles. Expression of the osa transgenes in the developing notum using a GAL4 insertion in the pannier (pnr) gene results in a dominant alteration of bristle formation. Ectopic expression of osa causes the loss of both micro- and macro-chaetae, and defects in the midline of the notum, scutellum and abdomen. Expression of UAS-osaAD with the same GAL4 driver leads to a very similar phenotype, and co-expression of UAS-osa and UAS-osaAD induces a stronger, apparently additive phenotype. Expression of UAS-osaRD with pnr-GAL4 has the opposite effect, inducing the formation of ectopic macrochaetae on the notum. Co-expression of UAS-osa with UAS-osaRD rescues the bristle loss phenotype caused by the expression of UAS-osa alone. Thus, targeting an activation domain to Osa-regulated genes has an effect similar to overexpression of the full-length protein, while a repressor domain has the opposite effect (Collins, 1999).

In the wing, expression of UAS-osaRD with omb-GAL4 produces ectopic campaniform sensillae and wing margin bristles. This phenotype is enhanced in flies heterozygous for osa, suggesting that it results from interference with wild-type osa function. It is also very similar to the effect of expression of dominant-negative brm. Expression of UAS-osaAD causes the opposite phenotype, loss of campaniform sensillae. Expression of full-length osa with this driver results in dominant pupal lethality; although a small number of flies expressing osa eclose, their wings are deformed, making a phenotypic comparison difficult. The observation that UAS-osaAD and UAS-osaRD cause specific phenotypes in the developing wing disc, related to those caused by full-length Osa, implies that the DNA-binding domain of Osa has functional specificity in spite of its lack of DNA sequence specificity in vitro. Binding to other proteins could contribute to its ability to act on specific promoters. Expressing the DNA-binding domain alone has no effect, suggesting that its promoter interactions are not strong enough to compete significantly with endogenous Osa. The similar effects of UAS-osa and UAS-osaAD and opposite effects of UAS-osaRD also indicate that, in the wing imaginal disc, Osa functions as an activator of gene expression (Collins, 1999).

The SWI/SNF family of ATP-dependent chromatin-remodeling factors plays a central role in eukaryotic transcriptional regulation. In yeast and human cells, two subclasses have been recognized: one comprises yeast SWI/SNF and human BAF, and the other includes yeast RSC and human PBAF. Therefore, it was puzzling that Drosophila appeared to contain only a single SWI/SNF-type remodeler, the Brahma (BRM) complex. This study reports the identification of two novel BRM complex-associated proteins: Drosophila Polybromo and BAP170, a conserved protein not described previously. Biochemical analysis established that Drosophila contains two distinct BRM complexes: (1) the BAP complex, defined by the presence of Osa and the absence of Polybromo and BAP170, and (2) the PBAP complex, containing Polybromo and BAP170 but lacking Osa. Determination of the genome-wide distributions of Osa and Polybromo on larval salivary gland polytene chromosomes revealed that BAP and PBAP display overlapping but distinct distribution patterns. Both complexes associate predominantly with regions of open, hyperacetylated chromatin but are largely excluded from Polycomb-bound repressive chromatin. It is concluded that, like yeast and human cells, Drosophila cells express two distinct subclasses of the SWI/SNF family. These results support a close reciprocity of chromatin regulation by ATP-dependent remodelers and histone-modifying enzymes (Mohrmann, 2004).

The chromatin-remodeling Protein Osa interacts with CyclinE in Drosophila eye imaginal discs

Coordinating cell proliferation and differentiation is essential during organogenesis. In Drosophila, the photoreceptor, pigment, and support cells of the eye are specified in an orchestrated wave as the morphogenetic furrow passes across the eye imaginal disc. Cells anterior of the furrow are not yet differentiated and remain mitotically active, while most cells in the furrow arrest at G1 and adopt specific ommatidial fates. Microarray expression analysis was used to monitor changes in transcription at the furrow, and genes were identified whose expression correlates with either proliferation or fate specification. Some of these are members of the Polycomb and Trithorax families that encode epigenetic regulators. Osa is one; it associates with components of the Drosophila SWI/SNF chromatin-remodeling complex. Studies of this Trithorax factor in eye development implicate Osa as a regulator of the cell cycle: Osa overexpression caused a small-eye phenotype, a reduced number of M- and S-phase cells in eye imaginal discs, and a delay in morphogenetic furrow progression. In addition, evidence is provided that Osa interacts genetically and biochemically with CyclinE. The results suggest a dual mechanism of Osa function in transcriptional regulation and cell cycle control (Baig, 2010).

This work applied DNA microarray hybridization to investigate the differences between mitotically active anterior and differentiating posterior eye-disc cells. Advantage was taken of the program of ommatidial differentiation to identify genes with essential roles at the stage of eye development when logarithmic growth transitions to mitotic arrest and adoption of specific cell types. Several recent studies have cataloged transcripts in whole eye discs with SAGE or DNA microarray hybridization, but this is the first genomic analysis that combines an analysis of purified posterior eye-disc fragments with mutant conditions that alter the program of photoreceptor differentiation. 866 transcripts were identified with differential anterior or posterior expression. Supporting the validity of this approach, functions that correlate with the mitotic activity of committed, but still undifferentiated, anterior cells segregate to the 'anterior' group, while neuronal functions are overrepresented in the 'posterior' group. This analysis and a recent SAGE-based investigation of regional differences in expression levels in eye imaginal discs identified several chromatin factors including PcG and TrxG members and proteins involved in heterochromatization, suggesting that chromatin-based transcriptional regulation plays a role in regional specific cell functions in eye development (Baig, 2010).

This study investigated the role of the BAP chromatin-remodeling complex subunit Osa at the MF. Several observations link the TrxG factor Osa to cell cycle control. First, the BAP components osa and moira have been implicated in a regulatory network of cell proliferation and cell cycle progression by evidence that they are transcriptional targets of the DNA replication-related element-binding factor (Nakamura, 2008). Second, phenotypes of osa mutant cells suggest that Osa is required for both differentiation and proliferation. Finally, by analyzing the osa overexpression phenotype, evidence was found for genetic and biochemical interaction of Osa with DmCycE. Interestingly, whereas expression of cell cycle regulators such as string/cdc25 is dependent on Osa's chromatin-remodeling function, the reduction in cell cycle progression that results from overexpression of Osa appears to be independent of string/cdc25 and CycE transcription rates. These results support a dual mechanism to link chromatin remodeling with cell cycle control (Baig, 2010).

CycE function appears to be modulated by BAP, the Osa-containing form of the SWI/SNF complex. Genetic interactions of CycE with several core components of both the BAP and PBAP forms of the SWI/SNF complex have been described previously. Consistent with the current observations, these studies also detected a genetic interaction between osa and CycE. Furthermore, a direct or indirect physical association between CycE and SWI/SNF components was detected by co-immunoprecipitation with Brm or Snr1. These results now show that CycE also immunoprecipitates with the BAP signature protein Osa. Although the PBAP signature proteins Polybromo or BAP170 were not tested in this study, the Osa overexpression small-eye phenotype and lack of cell cycle defects in single and double mutants for Polybromo and BAP170 suggest that the cell cycle function is specific to the BAP version of the Drosophila SWI/SNF complexes (Baig, 2010).

CycE-SWI/SNF complex interactions appear to be evolutionarily conserved since BRG1 (Brahma Related Gene 1, one of two mammalian orthologs of Drosophila Brm) and BAF155 (orthologous to Moira) copurify with CycE from human cells. In addition, expression of the SWI/SNF complex components BRG1 or INI/hSNF5 (orthologous to Snf1) causes G1 cell cycle arrest in human tissue culture cells. Interestingly, the cell cycle arrest can be rescued by co-expression of hCycE or hCycD1, respectively. These data are therefore consistent with a function of the Drosophila BAP and human SWI/SNF-like complexes as cell cycle regulators. Furthermore, the genetic and biochemical interaction data suggest that this function requires Cyclin activity (Baig, 2010).

Chromatin-remodeling activity and the function of SWI/SNF in cell cycle regulation must be tightly controlled to assure proper development and to prevent the transition of normal cells into cancer cells. The current findings are consistent with a function of Osa in negatively controlling cell cycle progression. A fine-tuned balance of repressive and activating signals seems to coordinate cell cycle progression by controlling Osa protein levels and downstream events such as CycE interaction or string/cdc25 expression. The elevated Osa protein levels anterior to the MF that are observed in normal development might reflect the contribution of Osa in the transition of these cells into a G1-arrested state. The G1 arrest of these cells requires the function of several signaling pathways: Hh, Dpp, Wg, Egfr, and Notch. By downregulating CycE activity, the increased Osa protein levels in these cells might contribute to counteracting the mitogenic activity of these signaling pathways that is observed in other developmental contexts (Baig, 2010).

Genetic interactions between osa and components of the Wg signal transduction pathway were also detected. These interactions could be a consequence of the small size of the eye field in Osa-overexpressing discs, since the signaling molecule Wg is normally expressed in lateral positions and has a locally restricted negative effect on Dpp-mediated MF progression. If relative Wg signaling increases in the abnormally small eye, repression of Dpp function in medial cells should increase. This model is supported by the weak dpp expression in the small discs and by the half-moon shape of the MF in Osa discs. The MF bends posteriorly in the Osa-overexpressing discs (indicating that the retarding effect is strongest in lateral positions), whereas the MF points anteriorly in wg mutant discs (presumably due to the missing repressive Wg effects in lateral positions). Partial rescue of Osa overexpression by impaired Wg signaling is consistent with this model. On the basis of these findings it is speculated that the posterior position of the MF that is caused by Osa overexpression is a manifestation of a developmental delay in eye development due to inhibition of cell proliferation and the resulting relative increase of the repressive Wg signal on dpp expression (Baig, 2010).

However, there are alternative regulatory possibilities in which the interplay of Osa and Wg signaling involves mutual transcriptional regulation and/or coregulation of common target genes at the transcriptional level. In Drosophila, expression of an activated form of the Wg signaling component Armadillo causes a small-eye phenotype that is suppressed by lowering the dosage of functional brm. Furthermore, Osa has been characterized as an antagonist of Wg signaling in wing development by inhibiting the expression of Wg target genes. A suppression of the Osa small-eye phenotype by Wg pathway mutants was detected, suggesting that Wg signaling acts synergistically with Osa in this system. These findings point at context-dependent features that appear to differ between wing and eye development. Such context-dependent functions have been reported earlier even between different cell populations of wing imaginal discs. For example, Wg signaling represses Drosophila Myc (DMyc) expression in the presumptive wing margin. In this area of the disc, repression of DMyc promotes G1 arrest via the regulation of the Drosophila retinoblastoma family (Rbf) protein, while forced expression of DMyc promotes cell cycle progression by inducing CycE expression. In contrast, Wg signaling in the hinge region of the wing imaginal disc has the opposite effect on cell proliferation. As these examples illustrate, it is difficult to generalize the relation between Osa, Wg signaling, and Myc function. However, a possible contribution of DMyc regulation to the Osa overexpression small-eye phenotype provides an interesting possibility. Observations in other systems support a role of SWI/SNF function in transcriptional regulation of cell cycle genes. In vertebrates, direct transcriptional regulation of Cyclins by SWI/SNF complex components has been implicated, and mammalian BRG1 and β-catenin (the vertebrate ortholog of Armadillo) interact with each other to activate Wnt target genes. In Drosophila, only a single osa gene exists, and it is involved in both activation and repression of target genes. In mammals, the two Osa orthologs BAF250a/b seem to have antagonistic functions in activating or repressing cell-cycle-specific genes such as cdc2, cyclin E, and c-Myc, and this regulation involves binding to the promoter sequences (Baig, 2010).

No significant changes were detected in DmCycE transcript or protein levels in osa and other BAP component mutants; instead, biochemical interaction was detected between Osa and DmCycE. To date, the functional consequence surrounding the association of Cyclin/Cdk complexes with chromatin-remodeling complexes remains unclear. Although different Cyclins possess distinct functions and tissue specificities, several reports describe roles for different CDK/cyclin complexes in transcription and RNA splicing. In many cases, CDK/cyclin complexes regulate the activity of components of the transcription machinery or other factors in a cell-cycle-dependent manner. Along these lines, CycE/CDK2 phosphorylates NPAT (nuclear protein mapped to the AT locus), which in turn activates replication-dependent transcription of histones. This function is stimulated by CycE binding to the histone genes in human tissue culture cells. It is conceivable that the kinase activity of CycE/Cdk2 modulates the activity of the BAP chromatin-remodeling complexes in a cell-cycle-dependent manner as it has been demonstrated for human Brm, BRG1, or BAF155 (Baig, 2010).

Mammalian SWI/SNF--a subunit BAF250/ARID1 is an E3 ubiquitin ligase that targets histone H2B

The mammalian SWI/SNF chromatin-remodeling complex facilitates DNA access by transcription factors and the transcription machinery. The characteristic member of human SWI/SNF-A is BAF250/ARID1 (homolog of Drosophila Osa), of which there are two isoforms, BAF250a/ARID1a and BAF250b/ARID1b. This study reports that BAF250b complexes purified from mammalian cells contain elongin C (Elo C), a BC box binding component of an E3 ubiquitin ligase. BAF250b was found to have a BC box motif, associate with Elo C in a BC box-dependent manner, and, together with cullin 2 and Roc1, assemble into an E3 ubiquitin ligase. The BAF250b BC box mutant protein was unstable in vivo and was autoubiquitinated in a manner similar to that for the VHL BC box mutants. The discovery that BAF250 is part of an E3 ubiquitin ligase adds an enzymatic function to the chromatin-remodeling complex SWI/SNF-A. The immunopurified BAF250b E3 ubiquitin ligase was found to target histone H2B (see Drosophila H2B) at lysine 120 for monoubiquitination in vitro. To date, all H2B monoubiquitination was attributed to the human homolog of yeast Bre1 (RNF20/40). Mutation of Drosophila osa, the homolog of BAF250, or depletion of BAF250 by RNA interference (RNAi) in cultured human cells resulted in global decreases in monoubiquitinated H2B, implicating BAF250 in the cross talk of histone modifications (Li, 2010).

This study has shown that BAF250b interacts with Elo C and Cul2. The interaction with Elo C and Cul2 is dependent on a BC box in the CTD of BAF250b, whereas the Cul2 interaction requires both ARID and the BC box. In vivo, the single amino acid substitution in the BC box (BC*) resulted in the degradation of the BC box mutant BAF250b through autoubiquitination. The Cul2-dependent regulation of BC* supports the idea that BAF250b serves as an E3 ubiquitin ligase substrate recognition module in vivo. Histone H2B has been determined to be a substrate for the BAF250 complex in a nucleosomal context. It is proposed that this complex is assembled in a manner similar to that for the well-characterized VHL complex, which targets HIF1α. The ARID and the CTD of BAF250 have been shown to be important for transcriptional activation. BAF250CTD was also shown to interact with the glucocorticoid receptor to activate transcription. It is possible that the coactivator function of BAF250 is in part mediated through its association with Elo C and Cul2 (Li, 2010).

Until now, all ubiquitinated H2B (H2B-Ub) was thought to arise from the action of the heterodimeric RNF20/40 E3 ubiquitin ligase, although it has been noted that depletion of RNF20 by RNAi affected transcription of only a subset of genes. H2B-Ub has been shown to be required for transcriptional activation in vitro and associates with transcriptionally active genes in vivo. RNF20 is sufficient for ubiquitin ligase activity in vitro and shares approximately 30% homology with S. cerevisiae Bre1, which performs the analogous function in yeast. Interestingly, the yeast BAF250 ortholog Swi1 lacks an identifiable BC box necessary for interaction with Elo B/C. The Elo B/C interaction domain and the Swi1 ARID are also not highly conserved. Swi1 has not been identified in genetic screens for factors affecting H2B monoubiquitination, and multiple groups have reported on the elimination of H2B-Ub upon deletion of bre1 in yeast. Thus, the probability of Swi1 possessing E3 ubiquitin ligase activity seems low. Because yeast is a unicellular organism, the layers of epigenetic regulation necessary in mammals would not be required. The BAF250 E3 ubiquitin ligase activity therefore may reflect an evolutionary adaptation unique to the demands of developmental regulation in multicellular organisms (Li, 2010).

The BAF250-Elo B/C-Cul2-Roc1 complex would also be regulated by assembly and neddylation of the cullin subunit, whereas RNF20/RNF40 is presumably constitutively active. Dynamic regulation of the BAF250 E3 ubiquitin ligase assembly and activity by neddylation, the COP9 signalosome, and TIP120A/CAND1 complexes would allow the type of temporal regulation necessary in early development. Recent studies of developing cells have demonstrated that temporal regulation of different transcription factor complexes is important to cell fate decisions (Li, 2010).

Both BAF250a and BAF250b are expressed in mammalian embryonic stem cells. Chromatin immunoprecipitation studies suggest that Nanog, Oct4, and Sox2 occupy the BAF250b promoter, while E2F4 occupies the BAF250a promoter. The dependence of mammalian embryonic stem cells on BAF250a and -b argues that these are genuine trxG members. Although trxG and PcG proteins have antagonistic roles in controlling HOX gene expression, many of the trxG and PcG proteins studied have chromatin-remodeling and chromatin-modifying capabilities. Posttranslational modifications of histones, such as H3 lysine-4 trimethylation (H3K4me3) and H2B monoubiquitination (H2B-Ub), show a positive correlation with transcription activation. H2B-Ub is required for transcription from a chromatinized template in vitro and regulates H3K4me3 levels in vivo. In contrast, H2A monoubiquitination is considered a transcriptionally repressive mark set by Ring1a/b, part of Polycomb repressor complex 1. In accordance with Osa's antagonistic role in regard to Ring1a/b in HOX gene regulation, this study shows that BAF250a is a positive regulator of HoxA9 and that the BAF250b complex is specific for histone H2B in a nucleosomal context. Indeed, in vitro ubiquitination assays and in vivo knockdown experiments indicate that H2B is a target of the BAF250 E3 ubiquitin ligase. BAF250 is not essential to SWI/SNF targeting in vivo or to chromatin remodeling in vitro. Thus, it is not thought that BAF250 siRNA knockdown has disrupted SWI/SNF remodeling activity in general. Furthermore, Osa and Cul2 synergistically interact in genetic analysis carried out with the Drosophila wing discs, confirming the importance of association with Cul2 to the function of Osa in vivo. The discovery that BAF250 associates with Elo B/C, Cul2, and Roc1 to form an E3 ubiquitin ligase that specifically monoubiquitinates histone H2B in a nucleosomal context provides a mechanistic explanation for Osa's classification as a trxG member (Li, 2010).

The identification of a novel ubiquitination pathway mediated by the chromatin-remodeling complex SWI/SNF-A raises several important questions. Is the H2B-Ub mark set before or after remodeling? Does H2B-Ub affect chromatin remodeling? Is the H2B ubiquitination mediated by BAF250 gene specific? If so, what distinguishes such genes from those targeted by the E3 ligase RNF20/40? Genome-wide analysis of the occupancy of BAF250, RNF20/40, and H2B-Ub will help to identify genes targeted by the ubiquitin ligase activity of BAF250, which would facilitate future biochemical studies (Li, 2010).

Akirin specifies NF-kappaB selectivity of Drosophila innate immune response via chromatin remodeling

The network of NF-kappaB-dependent transcription that activates both pro- and anti-inflammatory genes in mammals is still unclear. As NF-kappaB factors are evolutionarily conserved, Drosophila was used to understand this network. The NF-kappaB transcription factor Relish activates effector gene expression following Gram-negative bacterial immune challenge. This study shows, using a genome-wide approach, that the conserved nuclear protein Akirin is a NF-kappaB co-factor required for the activation of a subset of Relish-dependent genes correlating with the presence of H3K4ac epigenetic marks. A large-scale unbiased proteomic analysis revealed that Akirin orchestrates NF-kappaB transcriptional selectivity through the recruitment of the Osa-containing-SWI/SNF-like Brahma complex (BAP). Immune challenge in Drosophila shows that Akirin is required for the transcription of a subset of effector genes, but dispensable for the transcription of genes that are negative regulators of the innate immune response. Therefore, Akirins act as molecular selectors specifying the choice between subsets of NF-kappaB target genes. The discovery of this mechanism, conserved in mammals, paves the way for the establishment of more specific and less toxic anti-inflammatory drugs targeting pro-inflammatory genes (Bonnay, 2014).

An ancient Pygo-dependent Wnt enhanceosome integrated by Chip/LDB-SSDP

TCF/LEF factors (see Drosophila Pangolin) are ancient context-dependent enhancer-binding proteins that are activated by β-catenin (see Drosophila Armadillo) following Wnt signaling. They control embryonic development and adult stem cell compartments, and their dysregulation often causes cancer. β-catenin-dependent transcription relies on the NPF motif of Pygo proteins. This study used a proteomics approach to discover the Chip/LDB-SSDP (ChiLS) complex as the ligand specifically binding to NPF. ChiLS also recognizes NPF motifs in other nuclear factors including Runt/RUNX2 and Drosophila ARID1, and binds to Groucho/TLE. Studies of Wnt-responsive dTCF enhancers in the Drosophila embryonic midgut indicate how these factors interact to form the Wnt enhanceosome, primed for Wnt responses by Pygo. Together with previous evidence, this study indicates that ChiLS confers context-dependence on TCF/LEF by integrating multiple inputs from lineage and signal-responsive factors, including enhanceosome switch-off by Notch. Its pivotal function in embryos and stem cells explain why its integrity is crucial in the avoidance of cancer (Fiedler, 2015).

TCF/LEF factors (TCFs) were discovered as context-dependent architectural factors without intrinsic transactivation potential that bind to the T cell receptor α (TCRα) enhancer via their high mobility group (HMG) domain. They facilitate complex assemblies with other nearby enhancer-binding proteins, including the signal-responsive CRE-binding factor (CREB) and the lineage-specific RUNX1 (also called Acute Myeloid Leukemia 1, AML1). Their activity further depends on β-catenin, a transcriptional co-factor activated by Wnt signaling, an ancient signaling pathway that controls animal development and stem cell compartments, and whose dysregulation often causes cancer. The context-dependence of TCFs is also apparent in other systems, for example in the embryonic midgut of Drosophila where dTCF integrates multiple signaling inputs with lineage-specific cues during endoderm induction. The molecular basis for this context-dependence remains unexplained (Fiedler, 2015).

In the absence of signaling, T cell factors (TCFs) are bound by the Groucho/Transducin-like Enhancer-of-split (Groucho/TLE) proteins, a family of co-repressors that silence TCF enhancers by recruiting histone deacetylases (HDACs) and by 'blanketing' them with inactive chromatin. TLEs are displaced from TCFs by β-catenin following Wnt signaling, however this is not achieved by competitive binding but depends on other factors. One of these is Pygopus (Pygo), a conserved nuclear Wnt signaling factor that recruits Armadillo (Drosophila β-catenin) via the Legless/BCL9 adaptor to promote TCF-dependent transcription. Intriguingly, Pygo is largely dispensable in the absence of Groucho, which implicates this protein in alleviating Groucho-dependent repression of Wg targets (Fiedler, 2015).

Pygo has a PHD and an N-terminal asparagine proline phenylalanine (NPF) motif, each essential for development and tissue patterning. Much is known about the PHD finger, which binds to Legless/BCL9 and to histone H3 tail methylated at lysine 4 via opposite surfaces that are connected by allosteric communication. By contrast, the NPF ligand is unknown, but two contrasting models have been proposed for its function (1">Figure 1) (Fiedler, 2015).

This study used a proteomics approach to discover that the NPF ligand is an ancient protein complex composed of Chip/LDB (LIM-domain-binding protein) and single-stranded DNA-binding protein (SSDP), also called SSBP. This complex controls remote Wnt- and Notch-responsive enhancers of homeobox genes in flies (Bronstein, 2011), and remote enhancers of globin and other erythroid genes in mammals, integrating lineage-specific inputs from LIM-homeobox (LHX) proteins and other enhancer-binding proteins. Using nuclear magnetic resonance (NMR) spectroscopy, this study demonstrated that Chip/LDB-SSDP (ChiLS) binds directly and specifically to Pygo NPFs, and also to NPF motifs in Runt-related transcription factors (RUNX) proteins and Osa (Drosophila ARID1), whose relevance is shown by functional analysis of Drosophila midgut enhancers. Furthermore, Groucho was identified as another new ligand of ChiLS by mass spectroscopy. This study thus define the core components of a Wnt enhanceosome assembled at TCF enhancers via Groucho/TLE and RUNX, primed for timely Wnt responses by ChiLS-associated Pygo. The pivotal role of ChiLS in integrating the Wnt enhanceosome provides a molecular explanation for the context-dependence of TCFs (Fiedler, 2015).

The discovery of ChiLS as the NPF ligand of Pygo proteins led to the definition of the core components of a multi-protein complex tethered to TCF enhancers via Groucho/TLE and RUNX, and slated for subsequent Wnt responses by Pygo (see Model of the Wnt enhanceosome). ChiLS also contacts additional enhancer-binding proteins via its LID, including lineage-specific and other signal-responsive factors, thereby integrating multiple position-specific inputs into TCF enhancers, which provides a molecular explanation for the context-dependence of TCF/LEF. This complex will be referred to as the Wnt enhanceosome since it shares fundamental features with the paradigmatic interferon β-responsive enhanceosome (Panne, 2007). Its components are conserved in placozoa, arguably the most primitive animals without axis and tissues with only a handful of signaling pathways including Wnt, Notch and TGF-β/SMAD, suggesting that the Wnt enhanceosome emerged as the ur-module integrating signal-responses (Fiedler, 2015).

Other proteins have been reported to interact with the Pygo N-terminus, but none of these recognize NPF. It is noted that this N-terminus is composed of low-complexity (intrinsically disordered) sequences that are prone to non-specific binding (Fiedler, 2015).

NPF is a versatile endocytosis motif that binds to structurally distinct domains, including eps15 homology (EH) domains in epsin15 homology domain (EHD) protein. Indeed, EHDs were consistently identified in lysate-based pull-downs with triple-NPF baits. EHDs are predominantly cytoplasmic, and do not interact with nuclear Pygo upon co-expression, nor are any of the Drosophila EHDs required for Wg signaling in S2 cells. ChiLS is the first nuclear NPF-binding factor (Fiedler, 2015).

NPF binding to ChiLS appears to depend on the same residues as NPF binding to EHD domains, that is, on the aromatic residue at +2, the invariant P at +1, N (or G) at 0 and NPF-adjacent residues, including negative charges at +3 and +4 (whereby a positive charge at +3 abolishes binding to EHD). Indeed, an intramolecular interaction between the +3 side-chain and that of N predisposes NPF to adopt a type 1 β-turn conformation, which increases its affinity to the EHD pocket, while the -1 residue undergoes an intermolecular interaction with this pocket. ChiLS also shows a preference for small residues at -1 and -2, similarly to N-terminal EHDs although RUNX seems to differ at -1 and -2 from Pygo and MACC1 (F/L A/E/D vs S A, respectively) (Fiedler, 2015).

Groucho/TLE is recruited to TCF via its Q domain, which tetramerizes. Intriguingly, the short segment that links two Q domain dimers into a tetramer is deleted in a dTCF-specific groucho allele that abolishes dTCF binding and Wg responses, suggesting that TCF may normally bind to a Groucho/TLE tetramer (Fiedler, 2015).

Groucho/TLE uses its second domain, the WD40 propeller, to bind to other enhancer-binding proteins on Wnt-responsive enhancers, most notably to the C-terminal WRPY motif of RUNX proteins (common partners of TCFs in Wnt-responsive enhancers). This interaction can occur simultaneously with the WD40-dependent binding to ChiLS, given the tetramer structure of Groucho/TLE. In turn, RUNX uses its DNA-binding Runt domain to interact with HMG domains of TCFs, and to recruit ChiLS. RUNX thus appears to be the keystone of the Wnt enhanceosome since it binds to the enhancer directly while undergoing simultaneous interactions with Groucho/TLE (through its C-terminal WRPY motif), TCF and ChiLS (though its Runt domain) (Fiedler, 2015).

In line with this, Runt has pioneering functions in the early Drosophila embryo, shortly after the onset of zygotic transcription, and in the naïve endoderm as soon as this germlayer is formed, in each case prior to the first Wg signaling events. RUNX paralogs also have pioneer-like functions in specifying cell lineages, that is, definitive hematopoiesis in flies and mammals (Fiedler, 2015).

The model predicts that ChiLS, once tethered to the enhanceosome core complex, recruits Pygo via NPF to prime the enhancer for Wnt responses (see Model of the Wnt enhanceosome). Given the dimer-tetramer architecture of ChiLS, its binding to Pygo can occur simultaneously to its NPF-dependent binding to RUNX. In turn, tethering Pygo to the Wnt enhanceosome may require Pygo's binding to methylated histone H3 tail, similarly to Groucho/TLE whose tethering to enhancers depends on binding to hypoacetylated histone H3 and H4 tails. Interestingly, Pygo's histone binding requires at least one methyl group at K4-the hallmark of poised enhancers. Indeed, Drosophila Pygo is highly unorthodox due to an architectural change in its histone-binding surface that allows it to recognize asymmetrically di-methylated arginine 2-a hallmark of silent chromatin. Thus, the rare unorthodox Pygo proteins may recognize silent enhancers even earlier, long before their activation, consistent with the early embryonic function of Pygo, prior to Wg signaling (Fiedler, 2015).

Overcoming the OFF state imposed on the enhancer by Groucho/TLE involves Pygo-dependent capturing of β-catenin/Armadillo, which recruits various transcriptional co-activators to its C-terminus. Although these include CREB-binding protein (CBP), a histone acetyl transferase, its tethering to TCF enhancers is likely to co-depend on CRE-binding factors (CREB, c-Fos) and SMAD which synergize with Armadillo to activate these enhancers-similarly to the interferon-β enhanceosome where CBP recruitment also co-depends on multiple enhancer-binding proteins (Panne, 2007). The ensuing acetylation of the Wnt enhancer chromatin could promote the eviction of Groucho/TLE whose chromatin anchoring is blocked by acetylation of histone H3 and H4 tails, thus initiating the ON state (Fiedler, 2015).

Osa antagonizes Wg responses throughout development, and represses UbxB through its CRE, which also mediates repression in response to high Wg signaling. Osa could therefore terminate enhancer activity, by displacing HAT-recruiting enhancer-binding proteins such as CREB and c-Fos from CREs and by cooperating with repressive enhancer-binding proteins such as Brinker (a Groucho-recruiting repressor that displaces SMAD from UbxB) to re-recruit Groucho/TLE to the enhancer, thereby re-establishing its OFF state. Notably, Osa binds Chip, to repress various Wg and ChiLS targets including achaete-scute and dLMO (Fiedler, 2015).

Therefore, ChiLS is not only a coincidence detector of multiple enhancer-binding proteins and NPF proteins, but also a switch module that exchanges positively- and negatively-acting enhancer-binding proteins (through LID) and NPF factors, to confer signal-induced activation, or re-repression. Its stoichiometry and modularity renders it ideally suited to both tasks. It is noted that the interferon-β enhanceosome does not contain a similar integrating module, perhaps because it is dedicated to a single signaling input (Fiedler, 2015).

ChiLS is essential for activation of master-regulatory genes in the early embryo, similarly to DNA-binding pioneer factors such as Zelda (in the Drosophila embryo) or FoxA (in the mammalian endoderm) which render enhancers accessible to enhancer-binding proteins. Moreover, ChiLS maintains HOX gene expression throughout development, enabling Wg to sustain HOX autoregulation, a mechanism commonly observed to ensure coordinate expression of HOX genes in groups of cells (Fiedler, 2015).

Another hallmark of pioneer factors is that they initiate communication with the basal transcription machinery associated with the promoter. Chip is thought to facilitate enhancer-promoter communication, possibly by bridging enhancers and promoters through self-association. Indeed, Ldb1 occupies both remote enhancers and transcription start sites (e.g., of globin genes and c-Myb), likely looping enhancers to the basal transcription machinery at promoters which requires self-association, but possibly also other factors (such as cohesin, or mediator) (Fiedler, 2015).

It is noted that the chromatin association of Ldb1 has typically been studied in erythroid progenitors or differentiated erythroid cells, following activation of erythoid-specific genes. It would be interesting (if technically challenging) to examine primitive cells, to determine whether ChiLS is associated exclusively with poised enhancers prior to cell specification or signal responses (Fiedler, 2015).

Previous genetic analysis in Drosophila has linked chip predominantly to Notch-regulated processes. Likewise, groucho was initially thought to be dedicated to repression downstream of Notch, before its role in antagonizing TCF and Wnt responses emerged. Moreover, Lozenge facilitates Notch responses in the developing eye, and in hematocytes. Indeed, the first links between Groucho/TLE, RUNX and nuclear Wnt components came from physical interactions, as in the case of ChiLS. The current work indicates that these nuclear Notch signaling components constitute the Wnt enhanceosome. Although the most compelling evidence for this notion is based on physical interactions, the genetic evidence from Drosophila is consistent with a role of ChiLS in Wg responses (Bronstein, 2010). Indeed, mouse Ldb1 has been implicated in Wnt-related processes, based on phenotypic analysis of Ldb1 knock-out embryos and tissues. Notably, Ldb1 has wide-spread roles in various murine stem cell compartments that are controlled by Wnt signaling (Fiedler, 2015).

An interesting corollary is that the Wnt enhanceosome may be switchable to Notch-responsive, by NPF factor exchange and/or LMO-mediated enhancer-binding protein exchange at ChiLS. Hairy/Enhancer-of-split (HES) repressors could be pivotal for this switch: these bHLH factors are universally induced by Notch signaling, and they bind to ChiLS enhancers to re-recruit Groucho/TLE via their WRPW motifs. HES repressors may thus be capable of re-establishing the OFF state on Wnt enhancers in response to Notch (Fiedler, 2015).

Notably, restoring a high histone-binding affinity in Drosophila Pygo by reversing the architectural change in its histone-binding surface towards human renders it hyperactive towards both Wg and Notch targets even though pygo is not normally required for Notch responses in flies. Humanized Pygo may thus resist the Notch-mediated shut-down of the Wnt enhanceosome, owing to its elevated histone affinity that boosts its enhancer tethering, which could delay its eviction from the enhanceosome by repressive NPF factors. The apparent Notch-responsiveness of the Wnt enhanceosome supports the notion that orthodox Pygo proteins (as found in most animals and humans) confer both Wnt and Notch responses (Fiedler, 2015).

Previous genetic studies have shown that the components of the Wnt enhanceosome (e.g., TCF, RUNX, ChiLS and LHX) have pivotal roles in stem cell compartments, as already mentioned, suggesting a universal function of this enhanceosome in stem cells. It is therefore hardly surprising that its dysregulation, that is, by hyperactive β-catenin, is a root cause of cancer, most notably colorectal cancer but also other epithelial cancers. Indeed, genetic evidence implicates almost every one of its components (as inferred from the fly counterparts) in cancer: AML1 and RUNX3 are tumour suppressors whose inactivation is prevalent in myeloid and lymphocytic leukemias, and in a wide range of solid tumors including colorectal cancer, respectively. Likewise, ARID1A is a wide-spread tumor suppressor frequently inactivated in various epithelial cancers. Furthermore, many T-cell acute leukemias can be attributed to inappropriate expression of LMO2. Intriguingly, AML1 and ARID1A behave as haplo-insufficient tumor suppressors, consistent with the notion that these factors compete with activating NPF factors such as Pygo2, RUNX2 and possibly MACC1 (predictive of metastatic colorectal cancer) for binding to ChiLS, which will be interesting to test in future. The case is compelling that the functional integrity of the Wnt enhanceosome is crucial for the avoidance of cancer (Fiedler, 2015).

osa/eyelid: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.