Imitation SWI

DEVELOPMENTAL BIOLOGY

Embryonic

Iswi transcripts are uniform during the blastoderm and germ band extention but become restricted to the ventral nerve cord and brain. Iswi transcripts are also detected in the embryonic gonads following germ band retraction (Elfring, 1994).

Effects of Mutation

Drosophila Iswi, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF, CHRAC, and ACF. To clarify the biological functions of Iswi, null and dominant-negative Iswi mutations were generated and characterized. Iswi mutations affect both cell viability and gene expression during Drosophila development. Iswi mutations also cause striking alterations in the structure of the male X chromosome. The Iswi protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for Iswi in transcriptional repression. These findings reveal novel functions for the Iswi ATPase and underscore its importance in chromatin remodeling in vivo (Deuring, 2000).

To determine when Iswi is required during development, the lethal phase and phenotype of Iswi null mutants were examined. Individuals heterozygous for ISWI¹ or ISWI² are viable and phenotypically normal. ISWI¹/Df(2R)vg-C individuals die during late larval or early pupal development and display no obvious homeotic transformations or other pattern defects. Similar results were obtained for both ISWI²/Df(2R)vg-C and ISWI¹/ISWI² individuals (Deuring, 2000).

Since Iswi homozygotes die as late larvae or early pupae, somatic clonal analysis was used to investigate the role of Iswi during later stages of development. Clones of homozygous ISWI² mutant tissue were generated in heterozygous larvae using the FLP-FRT technique. As an internal control for effects on cell viability or division, the size, frequency, and phenotype of Iswi mutant clones were compared in the presence or absence of an insertion of a rescuing Iswi⁺ transgene on the third chromosome (P[w⁺ Iswi⁺-6HIS-HA]19–2; see below). Surprisingly, clones of Iswi mutant tissue were observed in all body segments, although both the size and frequency of clones were reduced in the genitalia, head, and thoracic segments, relative to controls. No homeotic transformations or other defects were observed in clones of Iswi mutant tissue in any body segment (Deuring, 2000).

Since Iswi is expressed at high levels in the female germline, it was suspected that maternally contributed Iswi gene products might partially compensate for the loss of zygotic Iswi expression. To investigate this possibility, germline clones lacking a functional Iswi gene were produced in females using the FLP-FRT dominant female-sterile technique. The dominant female-sterile mutation ovo^D1 blocks oogenesis. Expression of FLP recombinase in FRT P[ovo^D1]/FRT ISWI² larvae produces germline clones that lack the ovo^D1 mutation and are homozygous for ISWI². In control flies of the genotype FRT P[ovo^D1]/FRT, germline mosaic females were recovered at 100% efficiency (86 fertile females out of 86 examined). In contrast, the induction of clones in FRT P[ovo^D1]/FRT ISWI² females did not restore fertility, indicating that loss of maternal Iswi function blocks oogenesis. To determine which step in oogenesis is blocked by the loss of Iswi, the ovaries of ISWI² germline mosaic females were examined. ovo^D1 blocks oogenesis prior to vitellogenesis (the beginning of stage 8 of egg development). The egg chambers of ovaries from ISWI² germline mosaics were indistinguishable from those of ovo^D1 heterozygotes, even though clone induction in control females is highly efficient. These data indicate that Iswi is essential for an early stage of oogenesis (Deuring, 2000).

Since it is not possible to generate individuals lacking both maternal and zygotic Iswi function, an alternative approach to analyze the role of Iswi during Drosophila development was sought. Engineered, dominant-negative mutations have proven to be quite useful for studying the function of SWI2/SNF2 family members. Mutations in the ATP-binding site of several of these proteins eliminate their function but do not prevent interactions with other proteins. As a result, they have strong, dominant-negative effects when expressed in vivo. Site-directed mutagenesis has been used to create an Iswi protein in which the conserved lysine in the ATP-binding site is replaced with an arginine. This K159R substitution eliminates the ATPase and chromatin-remodeling activities of Iswi in vitro. As anticipated, a transgene expressing the ISWI^K159R protein under the control of the Iswi promoter was unable to rescue the recessive lethality of either ISWI¹ or ISWI². However, this mutation did not alter either the stability of the Iswi protein or its incorporation into high molecular weight complexes. The ISWI^K159R mutation should therefore behave as a strong, dominant-negative allele (Deuring, 2000).

The effect of expressing high levels of the ISWI^K159R protein in vivo was examined using the GAL4 system. The ISWI^K159R gene was placed under the control of the GAL4-regulated promoter. Widespread expression of the ISWI^K159R protein is lethal. By contrast, individuals expressing ISWI^K159R in more restricted patterns often survive to adulthood, allowing for an examination of adult phenotypes resulting from the loss of Iswi function. Individuals expressing ISWI^K159R in a pattern identical to that of the eyeless gene develop into adults with eyes that are dramatically reduced in size or absent. Individuals that expressed ISWI^K159R in the sensory organ precursor cells that give rise to the peripheral nervous system (under the control of a scabrous-GAL4 transgene) developed into adults lacking multiple mechanosensory bristles. These phenotypes are the result of decreased Iswi activity, since they are strongly enhanced by ISWI². The severe loss of adult structures resulting from expression of the ISWI^K159R protein indicates that Iswi is essential for either cell viability or division (Deuring, 2000).

In vitro studies have suggested that Iswi plays an important role in transcription by facilitating the interaction of transcription factors with chromatin. One of the best candidates for a transcription factor that requires Iswi for its activity is the GAGA factor. GAGA factor binds to GA-rich sequences near the promoters of a wide variety of Drosophila genes and is thought to activate transcription by altering local chromatin structure. As the ATPase subunit of NURF, Iswi assists the GAGA factor to remodel chromatin in vitro, suggesting that the two proteins may act in concert to modulate chromatin structure in vivo as well. To examine possible interactions between Iswi and GAGA factor in vivo, the phenotypes of mutations in the two genes have been compared. GAGA factor is encoded by Trithorax-like (Trl), a member of the trithorax group of homeotic gene activators. Trl mutations enhance mutations in trithorax and cause homeotic transformations resulting from the decreased transcription of homeotic genes. Trl mutations also enhance position effect variegation, suggesting that GAGA factor antagonizes the assembly or function of heterochromatin. Unlike Trl mutations, Iswi mutations fail to enhance or suppress position effect variegation. No dominant interactions could be detected between mutations in Iswi and other genes, including Trl, other trithorax group genes (trithorax and brm), and Polycomb, a repressor of homeotic genes that is thought to act at the level of chromatin structure. These data suggest that Iswi and GAGA factor play distinct roles in chromatin remodeling in vivo (Deuring, 2000).

To investigate the role of Iswi in transcriptional activation in vivo, the effect of Iswi mutations on the expression of two targets of the GAGA factor were examined: the segmentation gene engrailed (en) and the homeotic gene Ultrabithorax (Ubx). The expression of En protein is reduced dramatically in imaginal discs of ISWI¹/ISWI² mutant larvae. Similar results are observed for Ubx. These data suggest that Iswi is essential for the expression of both en and Ubx in imaginal discs, although the possibility that this interaction is indirect cannot be ruled out (Deuring, 2000).

To directly observe interactions between Iswi and chromatin in vivo, the distribution of Iswi protein on salivary gland polytene chromosomes in third instar larvae was examined by immunofluorescence microscopy. Consistent with a fairly general role in transcription or other processes, Iswi protein is present at a large number of euchromatic sites in the polytene chromosomes. The same pattern was observed using whole sera and affinity-purified antibodies. The chromosomal distribution of Iswi protein is not appreciably altered following heat shock (Deuring, 2000).

Iswi protein is also associated with a subset of heterochromatin, as evidenced by punctate staining at the chromocenter. It is difficult to analyze the distribution of heterochromatic proteins on salivary gland chromosomes, since heterochromatic sequences are underreplicated in polytene tissues. To more accurately map the regions of heterochromatin with which Iswi interacts, the distribution of Iswi protein on mitotic chromosomes from larval neuroblasts was examined. On mitotic chromosomes, Iswi protein is abundantly present on the euchromatic arms of all chromosomes and is concentrated in regions of heterochromatin enriched with middle-repetitive sequences. For example, on the heterochromatic Y chromosome, Iswi is concentrated in the h11–13 region, which is composed almost entirely of middle repetitive DNA families. By contrast, little Iswi protein is detected in regions containing predominantly satellite DNA. The distributions of Iswi and GAGA factor on polytene and mitotic chromosomes were determined by double-label immunofluorescence microscopy. Both GAGA factor and Iswi are associated with hundreds of sites in the euchromatin of polytene chromosomes, but the distributions of the two proteins do not overlap extensively. Even greater differences in the distributions of the two proteins were observed in mitotic chromosomes where the GAGA factor, but not Iswi, is associated with GAGA-satellite sequences. The lack of extensive colocalization does not rule out an interaction between Iswi and GAGA at specific loci, but it does suggest that Iswi and GAGA are not obligatory partners (Deuring, 2000).

To determine whether Iswi is associated with actively transcribed genes, the distribution of Iswi and the second largest subunit of RNA polymerase II (subunit IIc) on salivary gland polytene chromosomes were compared by double-label immunofluoresence microscopy. Subunit IIc is an essential component of RNA Pol II and should therefore be associated with both paused and elongating forms of the enzyme. Surprisingly, the distributions of the two proteins are predominantly nonoverlapping. Thus, Iswi is preferentially associated with regions that are not actively transcribed by RNA Pol II in the larval salivary gland (Deuring, 2000).

The survival of Iswi mutants until early pupal development allowed an examination of the effect of Iswi mutations on chromosome structure in vivo. Striking defects are observed in the organization of the salivary gland polytene chromosomes of Iswi mutant larvae. The structure of the single X chromosome of male mutant larvae is much shorter and broader than normal. This alteration in the structure of the X chromosome is highly penetrant and never observed in the polytene chromosomes of female mutant larvae. Loss of Iswi function had more subtle effects on the structure of the autosomes in both male and female larvae. The autosomes are often thinner than normal, which could be due to alterations in DNA replication or chromatin assembly resulting from loss of Iswi activity. By contrast, the structure of mitotic chromosomes prepared from neuroblasts of third instar larvae hemizygous for either ISWI¹ or ISWI² appear relatively normal (Deuring, 2000).

The results reported here provide the first evidence that the Iswi ATPase plays an essential role in vivo. Loss of Iswi function leads to reduced cell viability, decreased expression of segmentation and homeotic genes in imaginal discs, and global alterations in chromosome structure. It is noteworthy that these findings differ from recent studies of Iswi in Saccharomyces cerevisiae. Yeast contain two genes highly related to Drosophila Iswi: ISW1, and ISW2. Like Drosophila Iswi, yeast ISW1 and ISW2 are subunits of protein complexes that carry out ATP-dependent chromatin-remodeling reactions in vitro. However, complete loss of either ISW1 or ISW2 function in yeast does not affect viability, although isw1 isw2 double mutants exhibit subtle growth defects under conditions of stress. This may be due to redundancy with CHD1, another SWI2/SNF2 family member, since isw1, isw2, and chd1 triple mutants exhibit temperature-sensitive synthetic lethality (Deuring, 2000).

Based on findings of redundancy between ISW1, ISW2, and CHD1 in yeast, it is possible that Iswi and other SWI2/SNF2 family members have some overlapping functions in Drosophila. More than eight Drosophila members of the SWI2/SNF2 family have been identified. These include BRM, the ATPase subunit of a SWI/SNF-like complex. Although these genetic studies have not provided evidence of any redundancy between Iswi and other members of the SWI2/SNF2 ATPase family, the possibility merits further investigation (Deuring, 2000).

The altered appearance of the male X chromosome in Iswi mutant larvae provides dramatic evidence of a role for Iswi in the modulation of higher order chromatin structure. Although the molecular basis of this phenotype is unclear, it is likely to reflect a unique structural feature of the male X chromosome that renders it more sensitive to the loss of Iswi function. One candidate for such a feature is the hyperacetylation of lysine 16 of histone H4, which requires the activity of the dosage compensation machinery and the MOF histone acetyltransferase. Mutations in genes required for dosage compensation cause the male X chromosome to appear more condensed than normal. By contrast, mutations in Iswi cause the male X chromosome to appear much less condensed than normal. These observations suggest that gene products involved in dosage compensation and Iswi have opposite effects on higher order chromatin structure (Deuring, 2000).

What is the relationship, if any, between Iswi and histone acetylation? The effect of Iswi mutations on the structure of the male X chromosome could be explained if the acetylation of lysine 16 of histone H4 renders chromatin less susceptible to chromatin compaction mediated by the Iswi ATPase. Intact histone tails are required for Iswi activity in vitro because both the ATPase activity and the mononucleosome-remodeling activity of the NURF complex are severely reduced when nucleosomes lacking histone tails are used as substrates (Deuring, 2000). The tail of histone H4 appears to be particularly important for the interaction between Iswi and nucleosomes, since the ATPase activity of recombinant Iswi protein is stimulated by nucleosomes lacking the tails of histones H2A, H2B, and H3, but not H4 (P. Becker, personal communication to Deuring, 2000).

Although the acetylation state of histone tails has not been shown to alter the ATPase activity of the NURF complex in vitro, it remains possible that the effect of Iswi on higher order chromatin structure is sensitive to the acetylation of specific lysine residues. This possibility is consistent with a proposal that the acetylation of the N-terminal tail of histone H4 disrupts interactions between nucleosomes. Another potential link between Iswi and histone acetylation was provided by the characterization of Acf1. Acf1 (the largest subunit of ACF) contains a bromodomain, a conserved domain that was recently found to specifically bind peptides corresponding to acetylated histone tails (Deuring, 2000).

The decrease in en and Ubx expression in Iswi mutant larvae is consistent with reports that Iswi is involved in transcriptional activation in vitro. Consequently, it was not anticipated that the distributions of Iswi and RNA Pol II on salivary gland polytene chromosomes would be mutually exclusive. The preferential association of Iswi with transcriptionally inactive regions suggests that Iswi may create changes in chromatin structure that are not conducive to RNA Pol II transcription in vivo. Although there is no direct evidence that Iswi represses transcription, such a function would be consistent with the proposal that Iswi acts antagonistically toward histone acetyltransferases to compact chromatin structure. Based on these observations, further investigation of the role of Iswi in transcriptional repression is clearly warranted (Deuring, 2000).

How can the distributions of Iswi and RNA Pol II on polytene chromosomes be reconciled with the effect of Iswi mutations on gene expression in imaginal discs and the ability of Iswi complexes to activate transcription in vitro? One possibility is that Iswi has roles in both transcriptional repression and activation. NURF, ACF, and CHRAC were purified from Drosophila embryo extracts, and nothing is known about the nature or relative abundance of Iswi complexes in larvae. Perhaps only one Iswi complex is associated with transcriptionally inactive chromatin in the larval salivary gland, while others are either less abundant or transiently interact with chromatin to activate transcription. It is also possible that the interaction of Iswi with en and Ubx is indirect. For instance, the decreased expression of the two genes may be a secondary consequence of reduced cell viability in Iswi mutant larvae (Deuring, 2000).

These studies do not address the specific roles of NURF, ACF, and CHRAC, since Iswi mutations should eliminate the activity of each of these complexes. The isolation and analysis of additional Iswi mutations, as well as the further analysis of genes encoding Iswi-associated proteins, will be necessary to clarify the individual functions of the Iswi-containing complexes in chromatin remodeling and transcription in vivo (Deuring, 2000).

Mutations in Drosophila Iswi, a member of the SWI2/SNF2 family of chromatin remodeling ATPases, alter the global architecture of the male X chromosome. The transcription of genes on this chromosome is increased 2-fold relative to females due to dosage compensation, a process involving the acetylation of histone H4 at lysine 16 (H4K16). Blocking H4K16 acetylation suppresses the X chromosome defects resulting from loss of Iswi function in males. In contrast, the forced acetylation of H4K16 in Iswi mutant females causes X chromosome defects indistinguishable from those seen in Iswi mutant males. Increased expression of MOF, the histone acetyltransferase that acetylates H4K16, strongly enhances phenotypes resulting from the partial loss of Iswi function. Peptide competition assays have revealed that H4K16 acetylation reduces the ability of Iswi to interact productively with its substrate. These findings suggest that H4K16 acetylation directly counteracts chromatin compaction mediated by the Iswi ATPase (Corona, 2002).

Toutatis, a TIP5-related protein, positively regulates Pannier function during Drosophila neural development

The GATA factor Pannier (Pnr) activates proneural expression through binding to a remote enhancer of the achaete-scute (ac-sc) complex. Chip associates both with Pnr and with the (Ac-Sc)-Daughterless heterodimer bound to the ac-sc promoters to give a proneural complex that facilitates enhancer-promoter communication during development. Using a yeast two-hybrid screening, Toutatis (Tou; see Teutates the supposed deified spirit of male tribal unity in ancient Celtic polytheism, best known under the name Toutatis, through the Gaulish catchphrase "By Toutatis!", invented for the Asterix comics by Goscinni and Uderzo), which physically interacts with both Pnr and Chip, was identified. Loss-of-function and gain-of-function experiments indicate that Tou cooperates with Pnr and Chip during neural development. Tou shares functional domains with chromatin remodelling proteins, including TIP5 (termination factor TTFI-interacting protein 5) of NoRC (nucleolar remodelling complex), which mediates repression of RNA polymerase 1 transcription. In contrast, Tou acts positively to activate proneural gene expression. Moreover, Iswi associates with Tou, Pnr and Chip, and is also required during Pnr-driven neural development. The results suggest that Tou and Iswi may belong to a complex that directly regulates the activity of Pnr and Chip during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).

Transcriptional activation of many developmentally regulated genes is mediated by proteins binding to enhancers scattered over the genome, raising the question on how long-range activation is restricted to the relevant target promoter. Numerous studies have highlighted the essential role of boundaries, which maintain domains independent of their surrounding (Vanolst, 2005).

The patterning of the large sensory bristles (macrochaetae) on the thorax of Drosophila melanogaster is a powerful model to study how enhancers communicate with promoters during regulation of gene expression. Each macrochaeta derives from a precursor cell selected from a group of equivalent ac-sc-expressing cells, the proneural cluster. ac and sc encode basic helix-loop-helix proteins (bHLH) that heterodimerize with Daughterless (Da) to activate expression of downstream genes required for neural fate. Transcription of ac and sc in the different sites of the imaginal disc is initiated by enhancers of the ac-sc complex and the expression is maintained throughout development by autoregulation mediated by the (Ac-Sc)-Da heterodimers binding to E boxes within the ac-sc promoters. Each enhancer interacts with specific transcription factors that are expressed in broader domains than the proneural clusters and define the bristle prepattern. Thus, the GATA factor Pannier (Pnr) binds to the dorsocentral (DC) enhancer and activates proneural expression to promote development of DC sensory organs. The Drosophila LIM-domain-binding protein 1 (Ldb1), Chip physically interacts both with Pnr and the (Ac-Sc)-Da heterodimer to give a multiprotein proneural complex which facilitates the enhancer-promoter communication (Vanolst, 2005 and references therein).

Chromatin plays a crucial role in control of eukaryotic gene expression and is a highly dynamic structure at promoters. In Drosophila, the polycomb (Pc) group and the trithorax (Trx) group proteins are chromatin components that maintain stable states of gene expression and are involved in various complexes. The Pc group proteins are required to maintain repression of homeotic genes such as Ultrabithorax, presumably by inducing a repressive chromatin structure. Members of the Trx group were identified by their ability to suppress dominant Polycomb phenotypes. Evidence has been provided that enhancer-promoter communication during Pnr-driven proneural development is negatively regulated by the Brahma (Brm) chromatin remodelling complex, homologous to the yeast SWI/SNF complex (Vanolst, 2005).

Evidence is presented that Toutatis (Tou), a protein that associates both with Pnr and Chip and that positively regulates activity of the proneural complex encompassing Pnr and Chip during enhancer-promoter communication. Tou has been previously identified in a genetic screen for dominant modifiers of the extra-sex-combs phenotype displayed by mutant of polyhomeotic (ph), a member of the Pc group in Drosophila. Tou shares functional domains with Acf1, a subunit of both the human and Drosophila ACF (ATP-utilizing chromatin assembly and remodelling factor) and CHRAC (chromatin accessibility complex), and with TIP5 of NoRC (nucleolar remodelling complex). Hence, Tou regulates activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling. Moreover, Iswi, a highly conserved member of the SWI2/SNF2 family of ATPases, is also necessary for activation of ac-sc and neural development. Since Iswi is shown to physically interact with Tou, Pnr and Chip, it is suggested that a complex encompassing Tou and Iswi directly regulates activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).

In Drosophila, Chip has been postulated to be a facilitator required both for activity of the DC enhancer of the ac-sc complex. Enhancer-promoter communication at the ac-sc complex is negatively regulated by the Brm complex whose activity is targeted to the ac-sc promoter sequences through dimerization of the Osa subunit with both Pnr and Chip. The Brm complex is thought to remodel chromatin in a way that represses transcription (Vanolst, 2005).

Tou and Iswi appear to act together as subunits of a multiprotein complex to positively regulate activity of Pnr and Chip during enhancer-promoter communication. Tou and Iswi therefore display opposite activity to that of the Brm complex, raising questions about their molecular function during neural development. Tou shares essential functional domains with members of the WAL family of chromatin remodelling proteins, including Acf1 of ACF and CHRAC. Importantly, Acf1 and TIP5 associate in vivo with Iswi, showing that Iswi can mediate both activation and repression of gene expression. Tou positively regulates Pnr/Chip function during the period of ac-sc expression in neural development, and it associates with Iswi. Since Iswi also positively regulates Pnr/Chip function, it is hypothesized that a complex encompassing Tou and Iswi acts during long-range activation of proneural expression, possibly through chromatin remodelling. Further studies will help to resolve this issue (Vanolst, 2005).

Interestingly, Chip and Pnr seem to play similar roles both during recruitment of the Brm complex and recruitment of Tou and Iswi, since they dimerize with Osa, Tou and Iswi. In addition, Pnr and Chip apparently cooperate to strengthen the physical association with Osa and Tou. However, Osa, on the one hand, and Tou and Iswi, on the other, display antagonistic activities during neural development. Since they are ubiquitously expressed, accurate regulation of ac-sc expression would require a strict control of the stoichiometry between Osa, Tou and Iswi. It remains to be investigated whether the functional antagonism between Osa and Tou/Iswi relies on a molecular competition for association with Pnr and Chip. Determination of this would require a complete molecular definition of the putative complex encompassing Tou and Iswi, together with a full understanding of how this complex and the Brm complex molecularly interact with the proneural complex to regulate enhancer-promoter communication during development (Vanolst, 2005).

Biochemical analysis of Iswi and Iswi-containing complexes, together with genetic studies of Iswi and associated proteins in flies and in budding yeast, has revealed roles for Iswi in a wide variety of nuclear processes, including transcriptional regulation, chromosome organization and DNA replication. Accordingly, Iswi was found to be a subunit of various complexes, including NURF (nucleosome remodelling factor), ACF and CHRAC. Iswi-containing complexes were primarily recognized as factors that facilitate in vitro transcription from chromatin templates. However, genetic analysis in Drosophila and in Saccharomyces cerevisiae have provided evidence that Iswi-containing complexes are involved in both transcriptional activation and repression in vivo. For example, immunostaining of Drosophila polytene chromosomes of salivary glands showed that Iswi is associated with hundreds of euchromatic sites in a pattern that is non-overlapping with RNA polymerase II. It suggests that Iswi may play a general role in transcriptional repression. In contrast, it was also demonstrated that expression of engrailed and Ultrabithorax are severely compromised in Iswi-mutant Drosophila larvae. Recent studies have also shown that a mouse Iswi-containing complex, NoRC, plays an essential role during repression of transcription of the rDNA locus by RNA polymerase I. Tou, a protein that is structurally related to the TIP5 subunit of NoRC. Tou positively regulates enhancer-promoter communication during Pnr-driven proneural development and its activity is targeted to the ac-sc promoter sequences through dimerization with Pnr and Chip. Evidence is provided that Iswi is required during neural development. Overexpression of Iswi^K159R in the precursor cells of the sensory organs using the sca^Gal4 driver leads to flies lacking multiple bristles, suggesting that Iswi functions late during neural development, essential for either cell viability or division of the precursor cell. Using the Iswi¹/Iswi² transheterozygous combination and individuals overexpressing Iswi^K159R in earlier stages of development and in less restricted patterns, it has been shown that Iswi also regulates ac-sc expression. Interestingly, the regulation is probably direct since Iswi associates with the transcription factors Pnr and Chip, known to promote ac-sc expression at the DC site. Since Iswi interacts with Tou, it is proposed that Tou and Iswi may positively regulate activity of Pnr and Chip during enhancer-promoter communication, possibly as subunits of a multiprotein complex involved in chromatin remodelling (Vanolst, 2005).

The nucleosome remodeling factor ISWI functionally interacts with an evolutionarily conserved network of cellular factors

ISWI is an evolutionarily conserved ATP-dependent chromatin remodeling factor playing central roles in DNA replication, RNA transcription, and chromosome organization. The variety of biological functions dependent on ISWI suggests that its activity could be highly regulated. To identify factors that antagonize ISWI activity a novel in vivo eye-based assay was developed to screen for genetic suppressors of ISWI. This screen revealed that ISWI interacts with an evolutionarily conserved network of cellular and nuclear factors that escaped previous genetic and biochemical analyses (Arancio, 2010).

To identify novel factors working in antagonism with ISWI, a new in vivo assay was developed that allowed screening for genetic suppressors of eye phenotypes caused by true loss-of-function ISWI alleles. Advantage was taken of the Ey-Gal4, UAS-Flip (EGUF) approach to produce flies with eyes composed exclusively of mitotic clones that have lost ISWI function. Loss of ISWI in the eye caused reduced rough eyes, eye color variegation, and loss of cell identity. The ISWI-EGUF eye phenotypes were employed to set up a dominant modifier screen to isolate factors antagonizing ISWI activity in vivo. Employing classic gene network bioinformatics analysis, the results of this screen were combined with those obtained in two others screens conducted in Drosophila and in Caenorhabditis elegans, where an ISWI allele and its worm ortholog were isolated. The combination of genetic and bioinformatics approaches employed resulted in the identification of an evolutionarily conserved network of modifiers of ISWI eye phenotypes, which included several potential antagonists of ISWI function. This analysis revealed new roles for ISWI in cell cycle progression as well as unanticipated mechanisms by which its activity could be regulated, shedding new light into the evolutionarily conserved physiological function of ISWI family members in cell cycle regulation (Arancio, 2010).

The ISWI chromatin remodeler organizes the hsrω ncRNA-containing omega speckle nuclear compartments

The complexity in composition and function of the eukaryotic nucleus is achieved through its organization in specialized nuclear compartments. The Drosophila chromatin remodeling ATPase ISWI plays evolutionarily conserved roles in chromatin organization. Interestingly, ISWI genetically interacts with the hsrω gene, encoding multiple non-coding RNAs (ncRNA) essential, among other functions, for the assembly and organization of the omega speckles. The nucleoplasmic omega speckles play important functions in RNA metabolism, in normal and stressed cells, by regulating availability of hnRNPs and some other RNA processing proteins. Chromatin remodelers, as well as nuclear speckles and their associated ncRNAs, are emerging as important components of gene regulatory networks, although their functional connections have remained poorly defined. This study provides multiple lines of evidence showing that the hsrω ncRNA interacts in vivo and in vitro with ISWI, regulating its ATPase activity. Remarkably, it was found that the organization of nucleoplasmic omega speckles depends on ISWI function. These findings highlight a novel role for chromatin remodelers in organization of nucleoplasmic compartments, providing the first example of interaction between an ATP-dependent chromatin remodeler and a large ncRNA (Onorati, 2011).

Factors that coordinate nuclear activities occurring on chromatin and the nucleoplasmic compartments remain unidentified and uncharacterized. Therefore, an important open question in nuclear organization field is how nuclear speckles localize and organize themselves near transcriptionally active genes to cross talk with chromatin factors for processing of the nascent RNAs. These data indicate that ISWI may provide a functional 'bridge' between chromatin and nuclear speckle compartments. Indeed, ISWI can directly or indirectly contact the omega speckles in intact nuclei, through hsrω-n ncRNA or some of the associated hnRNPs. Confocal analysis suggested a functional 'bridge' between a chromatin factor (ISWI) and nucleoplasmic omega speckle components (hsrω ncRNA and hnRNPs). However, not all omega speckles show partial overlap with ISWI. Indeed, these molecular “bridges” between chromatin and nucleoplasm are probably transient, since time-lapse movies on live cells with fluorescently tagged chromatin and omega-speckle components clearly show very high mobility of these speckles, which probably may explain the absence of classic co-localization between ISWI and omega speckle components. (Onorati, 2011).

The observed direct physical interaction between ISWI and hsrω-n ncRNA together with the stimulation of ISWI-ATPase activity in light of the partial overlap revealed by confocal microscopy suggests that ISWI may interact with hsrω-forming speckles only transiently, probably to help the hsrω ncRNA to properly associate with or release the various omega speckle-associated hnRNPs. Loss of ISWI may impair the correct maturation, organization or localization of omega speckles resulting in an observed omega “trail” phenotype (Onorati, 2011).

The data also provide a possible explanation for the suppression of ISWI defects by hsrω-RNAi. In ISWI mutants carrying normal levels of hsrω transcripts, the limited maternally derived ISWI is shared between chromatin remodelling and omega speckle organization reactions so that its sub-threshold levels in either compartments severely compromises both functions. However, when hsrω transcript levels are reduced by RNAi in ISWI null background, most of the maternal ISWI may become available for chromatin remodelling reactions, so that a minimal threshold level of chromosome organization can be achieved. This would permit initiation of close to normal developmental gene activity programs resulting in suppression of the ISWI eye and chromosome defects or in the postponement of the larval lethality to pupal stage. Additionally, it is known that when hsrω ncRNA is down-regulated through RNAi, levels of free hnRNPs and other chromatin factors (i.e., CBP) are also elevated. Therefore, the possibility that these changes may also counteract ISWI defects by as yet unknown mechanisms cannot be excluded (Onorati, 2011).

This work provides the first example of modulation of an ATP-dependent chromatin remodeler by a ncRNA, and is the first in vivo and in vitro demonstration of a role of chromatin remodeler in organization of a nuclear compartment. However, the mechanism underlying stimulation of the ATPase activity of ISWI by the hsrω-n ncRNA, which may facilitate the organization of omega speckles, remains to be understood. Given the evolutionary derivation of the ISWI ATPase-domain from RNA-helicase-domains, a provocative hypothesis is that ISWI could 'remodel' speckles by structurally helping the assembly or release of specific hnRNPs with the hsrω-n ncRNA to generate mature omega speckles. Chromatin remodelers, nuclear speckles and their associated long ncRNAs are emerging as essential components of gene regulatory networks, and their deregulation may underlie complex diseases. The functional homology of the human noncoding sat III transcripts with the Drosophila hsrω ncRNA (Jolly, 2006), highlights the relevance and translational significance of studies unraveling the functional connections between ncRNA-containing nuclear compartments and chromatin remodelers. (Onorati, 2011).

The ATPase domain of ISWI is an autonomous nucleosome remodeling machine

ISWI slides nucleosomes along DNA, enabling the structural changes of chromatin required for the regulated use of eukaryotic genomes. Prominent mechanistic models imply cooperation of the ISWI ATPase domain with a C-terminal DNA-binding function residing in the HAND-SANT-SLIDE (HSS) domain. Contrary to these models, this study shows by quantitative biochemical means that all fundamental aspects of nucleosome remodeling are contained within the compact ATPase module of Drosophila ISWI. This domain can independently associate with DNA and nucleosomes, which in turn activate ATP turnover by inducing a conformational change in the enzyme, and it can autonomously reposition nucleosomes. The role of the HSS domain is to increase the affinity and specificity for nucleosomes. Nucleosome-remodeling enzymes may thus have evolved directly from ancestral helicase-type motors, and peripheral domains have furnished regulatory capabilities that bias the remodeling reaction toward different structural outcomes (Mueller-Planitz, 2013).

Several current models ascribe critical functions to the HSS domain during remodeling. The HSS domain was suggested to bind and release DNA and drag it into the nucleosome upon cues from the ATPase domain, to form channels for nucleosomal DNA or to stabilize high-energy structures such as DNA bulges off the histone surface. Notably, this study found that ISWI lacking its HSS domain still remodeled nucleosomes, although the reaction proceeded an order of magnitude more slowly. This defect, however, was accounted for by a proportionally decreased ATP turnover. It is therefore concluded that the HSS domain is not an integral component of the motor core of ISWI (Mueller-Planitz, 2013).

Whereas passive secondary roles of the HSS during remodeling are fully consistent with the results, the ATPase data do not favor models that postulate active coordination, that is, transduction of energy, between the ATPase and the HSS domains. Steady-state ATP hydrolysis parameters (k_cat/K_m,obs) of ligand-free, DNA-bound and nucleosome-bound ISWI largely remained unaffected when the HSS was deleted. Notably, the characteristic biphasic ATP concentration dependence of hydrolysis was preserved when the HSS domain was missing. It remains possible, though, that energy is transduced only after the rate-limiting step of ATP hydrolysis, because steady-state measurements are blind to that regime (Mueller-Planitz, 2013).

The autonomy of the ATPase domain does not appear to be a specialty of ISWI, because Chd1 derivatives that lack their C-terminal DNA-binding domain can still slide nucleosomes. This commonality adds to the growing list of shared functional properties of ISWI and Chd1 remodelers. In fact, substantial parts of both enzymes are also structurally related. Chd1 harbors a SANT-SLIDE domain in place of the HSS domain of ISWI, and both enzymes contain the bridge motif adjacent to the conserved ATPase domain. Although the N-terminal parts of both enzymes lack any apparent homology, they nevertheless may perform similar functions (Mueller-Planitz, 2013).

How does ISWI remodel nucleosomes without the involvement of the HSS domain? Previous studies placed the ATPase region of several remodelers close to superhelix location 2 (SHL2) of the nucleosome, whereas the HSS domain of ISWI was found to bind the linker DNA. As ISWI_26-648 discriminates between nucleosomes and DNA and is sensitive to the H4 tail, at least a fraction of ISWI_26-648 can productively bind at SHL2 (Mueller-Planitz, 2013).

Strong histone-DNA contacts are present around SHL2. Weakening the strongest contacts is expected to be rate limiting for remodeling. This could occur when the binding energy of the remodeler toward the nucleosome is exploited or when the ATPase domain tries to translocate on DNA while interacting with histones, for example, at the H4 tail. Translocation puts a strain on the nucleosome, caused either by the presence of excess DNA or by a change in the twist of the DNA, which locally destabilizes histone-DNA interactions. The ATPase domain may even be strong enough to pump more DNA toward the dyad than the nucleosomal surface can accommodate, causing it to detach and bulge out. The latter model is difficult to envision for remodeling by the truncated ISWI enzyme, owing to a lack of domains that help form and stabilize the bulge (Mueller-Planitz, 2013).

Once key contacts between histones and DNA are weakened, alternative sets of histone-DNA contacts might become energetically more preferable, leading to a repositioning of the histones relative to DNA. DNA-histone contacts may adjust concertedly or, perhaps more probably, only locally, such that the strain propagates in multiple steps around the nucleosome (Mueller-Planitz, 2013).

Accessory domains may have evolved to optimize catalysis and modulate the outcome of the reaction, which explains their diversity among remodeling machines. This study has shown that, consistent with previous findings, the HSS domain increased the affinity of ISWI toward DNA, a feature that is expected to enhance processivity. In agreement with cross-linking results, evidence was obtained for direct contacts between the HSS domain and the NCP. This interaction was a major source for specificity toward the nucleosome. As such, the HSS domain improves the productive association of the ATPase domain at SHL2, which in turn enhances remodeling. The HSS domain could also optimize catalysis by weakening the DNA-histone interactions at the edge of the nucleosome. Through interactions with additional subunits and the linker DNA, the HSS may assist sensing the length of the linker or a preferred DNA sequence and therefore bias the remodeling reaction toward specific outcomes such as nucleosome spacing or positioning (Mueller-Planitz, 2013).

How do the conformational changes within the ATPase domain relate to previously reported structural changes in related enzymes? The catalytic domain of the distant relative Sulfolobus Sso1653 was crystallized with and without bound DNA. The two structures showed only minor differences well inside the ATPase core and therefore are unlikely to account for the increased exposure of peripheral arginines upon DNA binding. In conflict with the crystallographic data but in better agreement with the current results, a FRET study using the same Sulfolobus protein concluded that DNA binding leads to a major structural rearrangement between the two ATPase lobes (Mueller-Planitz, 2013).

Additional crystallographic evidence supports a high degree of flexibility between the two ATPase lobes. The ATPase lobes of relatives of ISWI crystallized in a multitude of very different orientations. Conformational changes between the two ATPase lobes may be functionally important for these enzymes, for example, for translocation on DNA or regulation of enzyme activity. Conceivably, multiple orientations of ISWI's ATPase lobes coexist in solution, accounting for the different enzyme species detected by ATPase experiments. DNA may preferentially stabilize a subset of these states, thereby aligning the composite catalytic site formed at the cleft between both lobes. As motifs of both ATPase lobes are thought to contact ATP, a proper alignment of the lobes might increase the affinity for ATP, explaining the biochemical data (Mueller-Planitz, 2013).

The increased exposure of peripheral arginines upon DNA binding also suggests that these regions undergo structural changes. Trypsin cleaved DNA-bound ISWI adjacent to a conserved acidic motif in the NTR. Despite a lack of sequence similarity, the NTR of Chd1 also contains a highly acidic motif, which was suggested to act as a pseudosubstrate and compete with DNA for binding to lobe 2. It has been proposed that, in excellent agreement with the current proteolytic results, DNA binding would force a structural rearrangement in Chd1 in which the NTR undocks from lobe 2 (Hauk, 2010). The NTRs of both enzymes may therefore fulfill similar roles and gate the entrance to the nucleic acid-binding site (Mueller-Planitz, 2013).

On the C-terminal side, trypsin cut the polypeptide chain within the 'brace' motif of lobe 2. The brace is in close contact with lobe 1 and is directly followed by a stretch of amino acids that folds back to form a bridge between both ATPase lobes. It is suggested that the brace or bridge may hold the ATPase lobes in a configuration that is not fully competent for ATP hydrolysis and that binding of nucleic acids relieves this inhibition. These results reinforce the notion that the ATPase domain represents an autonomous remodeling engine, which is optimized and modulated by the evolution of accessory domains and subunits (Mueller-Planitz, 2013).

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date revised: 25 August 2023

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