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).

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).

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date revised: 28 December 2011

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