Polycomb and Trithorax Group Genes in Drosophila.

Polycomb group (PcG) and Trithorax group (TrxG) genes encode important regulators of development and differentiation in metazoans. These two groups of genes were discovered in Drosophila by their opposing effects on homeotic gene (Hox) expression. PcG genes collectively behave as genetic repressors of Hox genes, while the TrxG genes are necessary for HOX gene expression or function. Biochemical studies showed that many PcG proteins are present in two protein complexes, Polycomb repressive complexes 1 and 2, which repress transcription via chromatin modifications. TrxG proteins activate transcription via a variety of mechanisms. Here we summarize the large body of genetic and biochemical experiments in Drosophila on these two important groups of genes.

A novel approach for studying histone H1 function in vivo.

In this report, we investigate the mechanisms that regulate Drosophila histone H1 expression and its association with chromatin in vivo. We show that histone H1 is subject to negative autoregulation and exploit this result to examine the effects of mutations of the main phosphorylation site of histone H1.

Transcriptional regulation by trithorax-group proteins.

The trithorax group of genes (trxG) was identified in mutational screens that examined developmental phenotypes and suppression of Polycomb mutant phenotypes. The protein products of these genes are primarily involved in gene activation, although some can also have repressive effects. There is no central function for these proteins. Some move nucleosomes about on the genome in an ATP-dependent manner, some covalently modify histones such as methylating lysine 4 of histone H3, and some directly interact with the transcription machinery or are a part of that machinery. It is interesting to consider why these specific members of large families of functionally related proteins have strong developmental phenotypes.

A histone timer for zygotic genome activation.

Histone H1 variants play key roles in the regulation of higher-order chromatin structure and have been implicated in numerous developmental processes. In this issue of Developmental Cell, Pérez-Montero et al. (2013) present evidence that the Drosophila histone H1 variant dBigH1 prevents premature activation of the zygotic genome during early embryogenesis.

The trithorax group proteins Kismet and ASH1 promote H3K36 dimethylation to counteract Polycomb group repression in Drosophila.

Members of the Polycomb group of repressors and trithorax group of activators maintain heritable states of transcription by modifying nucleosomal histones or remodeling chromatin. Although tremendous progress has been made toward defining the biochemical activities of Polycomb and trithorax group proteins, much remains to be learned about how they interact with each other and the general transcription machinery to maintain on or off states of gene expression. The trithorax group protein Kismet (KIS) is related to the SWI/SNF and CHD families of chromatin remodeling factors. KIS promotes transcription elongation, facilitates the binding of the trithorax group histone methyltransferases ASH1 and TRX to active genes, and counteracts repressive methylation of histone H3 on lysine 27 (H3K27) by Polycomb group proteins. Here, we sought to clarify the mechanism of action of KIS and how it interacts with ASH1 to antagonize H3K27 methylation in Drosophila. We present evidence that KIS promotes transcription elongation and counteracts Polycomb group repression via distinct mechanisms. A chemical inhibitor of transcription elongation, DRB, had no effect on ASH1 recruitment or H3K27 methylation. Conversely, loss of ASH1 function had no effect on transcription elongation. Mutations in kis cause a global reduction in the di- and tri-methylation of histone H3 on lysine 36 (H3K36) - modifications that antagonize H3K27 methylation in vitro. Furthermore, loss of ASH1 significantly decreases H3K36 dimethylation, providing further evidence that ASH1 is an H3K36 dimethylase in vivo. These and other findings suggest that KIS antagonizes Polycomb group repression by facilitating ASH1-dependent H3K36 dimethylation.

The Drosophila MI-2 chromatin-remodeling factor regulates higher-order chromatin structure and cohesin dynamics in vivo.

dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells.

Drosophila ISWI regulates the association of histone H1 with interphase chromosomes in vivo.

Although tremendous progress has been made toward identifying factors that regulate nucleosome structure and positioning, the mechanisms that regulate higher-order chromatin structure remain poorly understood. Recent studies suggest that the ISWI chromatin-remodeling factor plays a key role in this process by promoting the assembly of chromatin containing histone H1. To test this hypothesis, we investigated the function of H1 in Drosophila. The association of H1 with salivary gland polytene chromosomes is regulated by a dynamic, ATP-dependent process. Reducing cellular ATP levels triggers the dissociation of H1 from polytene chromosomes and causes chromosome defects similar to those resulting from the loss of ISWI function. H1 knockdown causes even more severe defects in chromosome structure and a reduction in nucleosome repeat length, presumably due to the failure to incorporate H1 during replication-dependent chromatin assembly. Our findings suggest that ISWI regulates higher-order chromatin structure by modulating the interaction of H1 with interphase chromosomes.

Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II.

Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L) is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II). Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, we characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin. By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation-a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin.

Genetic identification of a network of factors that functionally interact with the nucleosome remodeling ATPase ISWI.

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.

ISWI regulates higher-order chromatin structure and histone H1 assembly in vivo.

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.

Genetic screens for enhancers of brahma reveal functional interactions between the BRM chromatin-remodeling complex and the delta-notch signal transduction pathway in Drosophila.

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 protein Kismet facilitates an early step in transcriptional elongation by RNA Polymerase II.

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.

Multiple roles for ISWI in transcription, chromosome organization and DNA replication.

ISWI functions as the ATPase subunit of multiple chromatin-remodeling complexes. These complexes use the energy of ATP hydrolysis to slide nucleosomes and increase chromatin fluidity, thereby modulating the access of transcription factors and other regulatory proteins to DNA. Here we discuss recent progress toward understanding the biological functions of ISWI, with an emphasis on its roles in transcription, chromosome organization and DNA replication.

Histone chaperone ASF1 cooperates with the Brahma chromatin-remodelling machinery.

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.

The Drosophila BRM complex facilitates global transcription by RNA polymerase II.

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.

Modulation of ISWI function by site-specific histone acetylation.

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). Here we show that 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 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.