The conserved chromatin remodeling and assembly factor CHD1 (chromodomains, helicase, DNA-binding domain) is present at active genes where it participates in histone turnover and recycling during transcription. In order to gain a more complete understanding of the mechanism of action of CHD1 during development, we created a novel genetic assay in Drosophila melanogaster to evaluate potential functional interactions between CHD1 and other chromatin factors. We found that overexpression of CHD1 results in defects in wing development and utilized this fully penetrant and reliable phenotype to conduct a small-scale RNAi-based candidate screen to identify genes that functionally interact with chd1 in vivo. Our results indicate that CHD1 may act in opposition to other remodeling factors, including INO80, and that the recruitment of CHD1 to active genes by RTF1 is conserved in flies.

CHD1 is a conserved chromatin remodeling factor that localizes to active genes and functions in nucleosome assembly and positioning as well as histone turnover. Mouse CHD1 is required for the maintenance of stem cell pluripotency while human CHD1 may function as a tumor suppressor. To investigate the action of CHD1 on higher order chromatin structure in differentiated cells, we examined the consequences of loss of CHD1 and over-expression of CHD1 on polytene chromosomes from salivary glands of third instar Drosophila melanogaster larvae. We observed that chromosome structure is sensitive to the amount of this remodeler. Loss of CHD1 resulted in alterations of chromosome structure and an increase in the heterochromatin protein HP1a, while over-expression of CHD1 disrupted higher order chromatin structure and caused a decrease in levels of HP1a. Over-expression of an ATPase inactive form of CHD1 did not result in severe chromosomal defects, suggesting that the ATPase activity is required for this in vivo phenotype. Interestingly, changes in CHD1 protein levels did not correlate with changes in the levels of the euchromatin mark H3K4me3 or elongating RNA Polymerase II. Thus, while CHD1 is localized to transcriptionally active regions of the genome, it can function to alter the levels of HP1a, perhaps through changes in methylation of H3K9.

Chd proteins are ATP-dependent chromatin remodeling enzymes implicated in biological functions from transcriptional elongation to control of pluripotency. Previous studies of the Chd1 subclass of these proteins have implicated them in diverse roles in gene expression including functions during initiation, elongation, and termination. Furthermore, some evidence has suggested a role for Chd1 in replication-independent histone exchange or assembly. Here, we examine roles of Chd1 in replication-independent dynamics of histone H3 in both Drosophila and yeast. We find evidence of a role for Chd1 in H3 dynamics in both organisms. Using genome-wide ChIP-on-chip analysis, we find that Chd1 influences histone turnover at the 5' and 3' ends of genes, accelerating H3 replacement at the 5' ends of genes while protecting the 3' ends of genes from excessive H3 turnover. Although consistent with a direct role for Chd1 in exchange, these results may indicate that Chd1 stabilizes nucleosomes perturbed by transcription. Curiously, we observe a strong effect of gene length on Chd1's effects on H3 turnover. Finally, we show that Chd1 also affects histone modification patterns over genes, likely as a consequence of its effects on histone replacement. Taken together, our results emphasize a role for Chd1 in histone replacement in both budding yeast and Drosophila melanogaster, and surprisingly they show that the major effects of Chd1 on turnover occur at the 3' ends of genes.

In this report we describe chd1 mutant alleles and show that the CHD1 chromatin-remodeling factor is important for wing development and fertility. While CHD1 colocalizes with elongating RNA polymerase II (Pol II) on polytene chromosomes, elongating Pol II can persist on chromatin in the absence of CHD1. These results clarify the roles of chromatin remodelers in transcription and provide novel insights into CHD1 function.

Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, we conducted an unbiased genetic screen to identify factors with which it interacts in vivo. We found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo.

Initiation by RNA polymerase II (Pol II) involves a host of enzymes, and the process of elongation appears similarly complex. Transcriptional elongation through chromatin requires the coordinated efforts of Pol II and its associated transcription factors: C-terminal domain kinases, elongation complexes, chromatin-modifying enzymes, chromatin remodeling factors, histone chaperones (nucleosome assembly factors), and histone variants. This review examines the following: (i) the consequences of the encounter between elongating Pol II and a nucleosome, and (ii) chromatin remodeling factors and nucleosome assembly factors that have recently been identified as important for the elongation stage of transcription.

Initiation by RNA polymerase II (Pol II) involves a host of enzymes, and the process of elongation appears similarly complex. Transcriptional elongation through chromatin requires the coordinated efforts of Pol II and its associated transcription factors: C-terminal domain kinases, elongation complexes, chromatin-modifying enzymes, chromatin remodeling factors, histone chaperones (nucleosome assembly factors), and histone variants. This review examines the following: (i) the consequences of the encounter between elongating Pol II and a nucleosome, and (ii) chromatin remodeling factors and nucleosome assembly factors that have recently been identified as important for the elongation stage of transcription.

Imitation SWI (ISWI) and other ATP-dependent chromatin-remodeling factors play key roles in transcription and other processes by altering the structure and positioning of nucleosomes. Recent studies have also implicated ISWI in the regulation of higher-order chromatin structure, but its role in this process remains poorly understood. To clarify the role of ISWI in vivo, we examined defects in chromosome structure and gene expression resulting from the loss of Iswi function in Drosophila. Consistent with a broad role in transcriptional regulation, the expression of a large number of genes is altered in Iswi mutant larvae. The expression of a dominant-negative form of ISWI leads to dramatic alterations in higher-order chromatin structure, including the apparent decondensation of both mitotic and polytene chromosomes. The loss of ISWI function does not cause obvious defects in nucleosome assembly, but results in a significant reduction in the level of histone H1 associated with chromatin in vivo. These findings suggest that ISWI plays a global role in chromatin compaction in vivo by promoting the association of the linker histone H1 with chromatin.

The Drosophila trithorax group gene brahma (brm) encodes the ATPase subunit of a 2-MDa chromatin-remodeling complex. brm was identified in a screen for transcriptional activators of homeotic genes and subsequently shown to play a global role in transcription by RNA polymerase II. To gain insight into the targeting, function, and regulation of the BRM complex, we screened for mutations that genetically interact with a dominant-negative allele of brm (brm(K804R)). We first screened for dominant mutations that are lethal in combination with a brm(K804R) transgene under control of the brm promoter. In a distinct but related screen, we identified dominant mutations that modify eye defects resulting from expression of brm(K804R) in the eye-antennal imaginal disc. Mutations in three classes of genes were identified in our screens: genes encoding subunits of the BRM complex (brm, moira, and osa), other proteins directly involved in transcription (zerknullt and RpII140), and signaling molecules (Delta and vein). Expression of brm(K804R) in the adult sense organ precursor lineage causes phenotypes similar to those resulting from impaired Delta-Notch signaling. Our results suggest that signaling pathways may regulate the transcription of target genes by regulating the activity of the BRM complex.

The Drosophila trithorax group gene kismet (kis) was identified in a screen for extragenic suppressors of Polycomb (Pc) and subsequently shown to play important roles in both segmentation and the determination of body segment identities. One of the two major proteins encoded by kis (KIS-L) is related to members of the SWI2/SNF2 and CHD families of ATP-dependent chromatin-remodeling factors. To clarify the role of KIS-L in gene expression, we examined its distribution on larval salivary gland polytene chromosomes. KIS-L is associated with virtually all sites of transcriptionally active chromatin in a pattern that largely overlaps that of RNA Polymerase II (Pol II). The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of KIS-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These data suggest that KIS-L facilitates an early step in transcriptional elongation by Pol II.

De novo chromatin assembly into regularly spaced nucleosomal arrays is essential for eukaryotic genome maintenance and inheritance. The Anti-Silencing Function 1 protein (ASF1) has been shown to be a histone chaperone, participating in DNA-replication-coupled nucleosome assembly. We show that mutations in the Drosophila asf1 gene derepress silencing at heterochromatin and that the ASF1 protein has a cell cycle-specific nuclear and cytoplasmic localization. Furthermore, using both genetic and biochemical methods, we demonstrate that ASF1 interacts with the Brahma (SWI/SNF) chromatin-remodelling complex. These findings suggest that ASF1 plays a crucial role in both chromatin assembly and SWI/SNF-mediated chromatin remodelling.

Drosophila brahma (brm) encodes the ATPase subunit of a 2 MDa complex that is related to yeast SWI/SNF and other chromatin‐remodeling complexes. BRM was identified as a transcriptional activator of Hox genes required for the specification of body segment identities. To clarify the role of the BRM complex in the transcription of other genes, we examined its distribution on larval salivary gland polytene chromosomes. The BRM complex is associated with nearly all transcriptionally active chromatin in a pattern that is generally non‐overlapping with that of Polycomb, a repressor of Hox gene transcription. Reduction of BRM function dramatically reduces the association of RNA polymerase II with salivary gland chromosomes. A few genes, such as induced heat shock loci, are not associated with the BRM complex; transcription of these genes is not compromised by loss of BRM function. The distribution of the BRM complex thus correlates with a dependence on BRM for gene activity. These data suggest that the chromatin remodeling activity of the BRM complex plays a general role in facilitating transcription by RNA polymerase II.

The homeotic genes controlling segment identity in Drosophila are repressed by the Polycomb group of genes (PcG) and are activated by genes of the trithorax group (trxG), An F-1 screen for dominant enhancers of Polycomb yielded a point mutation in the heat shock cognate gene, hsc4, along with mutations corresponding to several known PcG loci. The new mutation is a more potent enhancer of Polycomb phenotypes than an apparent null allele of hsc4 is, although even the null allele occasionally displays homeotic phenotypes associated with the PcG, Previous biochemical results had suggested that HSC4 might interact with BRAHMA, a trxG member. Further analyses now show that there is no physical or genetic interaction between HSC4 and the Brahma complex. HSC4 might be needed for the proper folding of a component of the Polycomb repression complex, or it may be a functional member of that complex.

The Drosophila trithorax group gene brahma ( brm ) encodes the ATPase subunit of a SWI/SNF-like chromatin-remodeling complex. A key question about chromatin-remodeling complexes is how they interact with DNA, particularly in the large genomes of higher eukaryotes. Here, we report the characterization of BAP111, a BRM-associated protein that contains a high mobility group (HMG) domain predicted to bind distorted or bent DNA. The presence of an HMG domain in BAP111 suggests that it may modulate interactions between the BRM complex and chromatin. BAP111 is an abundant nuclear protein that is present in all cells throughout development. By using gel filtration chromatography and immunoprecipitation assays, we found that the majority of BAP111 protein in embryos is associated with the BRM complex. Furthermore, heterozygosity for BAP111 enhanced the phenotypes resulting from a partial loss of brm function. These data demonstrate that the BAP111 subunit is important for BRM complex function in vivo .

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, we generated and characterized null and dominant-negative ISWI mutations. We found that 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.

complexes function in vitro to disrupt chromatin strucErythroid Krüppel-like factor (EKLF) is necessary for ture and facilitate factor binding (Côté et al., 1994; Imbalstage-specific expression of the human b-globin gene. zano et al., 1994; Kwon et al., 1994). Multiple mammalian We show that EKLF requires a SWI/SNF–related chroSWI/SNF complexes (BAF complexes) have been identimatin remodeling complex, EKLF coactivator-remodfied and shown to be diverse in subunit composition eling complex 1 (E-RC1), to generate a DNase I hyper(Wang et al., 1996a, 1996b, 1998). The mammalian SWI/ sensitive, transcriptionally active b-globin promoter SNF (BAF) complexes contain either BRG1 or human on chromatin templates in vitro. E-RC1 contains BRG1, brahma (hbrm). These ATPases are homologous to the BAF170, BAF155, and INI1 (BAF47) homologs of yeast yeast protein SWI2/SNF2 (Khavari et al., 1993; Muchardt SWI/SNF subunits, as well as a subunit unique to and Yaniv, 1993). BAF155 (BRG1-associated factor, 155 higher eukaryotes, BAF57, which is critical for chromakDa) and BAF170 are both homologs of yeast SWI3. tin remodeling and transcription with EKLF. E-RC1 Interestingly, subunits unique to SWI/SNF–like comdisplays functional selectivity toward transcription plexes of higher eukaryotes are now being identified. factors, since it cannot activate expression of chromaFor example, BAF57 is found in mammalian complexes tin-assembled HIV-1 templates with the E box–binding (Wang et al., 1998), and its counterpart, BAP111, was protein TFE-3. Thus, a member of the SWI/SNF family identified in the Drosophila SWI/SNF–related complex acts directly in transcriptional activation and may reg(Papoulas et al., 1998). These subunits both contain an ulate subsets of genes by selectively interacting with HMG-like domain, and no such subunits are found in specific DNA-binding proteins. the yeast complexes.