Unknown,Transcriptomics,Genomics,Proteomics

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Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context (ChIP-chip)

ABSTRACT: The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at a subset of sites. However, the consensus sequence motif of entry sites (M-bM-^@M-^\MSL recognition elementM-bM-^@M-^] or MRE) is only slightly enriched on the X (~2 fold), and only a fraction of them is utilized by the MSL complex. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the NHGRI modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells, which contain MSL complex, and female Kc cells, which lack the complex, we find that the presence of active chromatin modifications, together with an elevated local GC content in surrounding sequence, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites, and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our finding serves as a model to understand how chromatin and local sequence features are involved in the selection of functional protein binding sites in the genome. The key Drosophila female sex determinant protein, SXL, represses dosage compensation by inhibiting MSL2 translation. Loss of SXL results in the expression, stabilization, and targetting of the MSL complex in female cells. Therefore, depletion of SXL by RNA interference (RNAi) in female Kc cells will lead to a MSL2-dependent increase in transcription from the female X chromosomes, consistent with the induction of dosage compensation. In this experiment, we generated ChIP-chip profiles of H4K16 acetylation (H4K16ac) in Kc cells of control (GFP) and Sxl RNAi. For ChIP, we used Upstate (now Millipore) anti-H4K16ac antibody, catalog # 07-329, lot #JBC1355376.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Joshua Ho

PROVIDER: E-GEOD-34857 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at a subset of sites. However, the consensus sequence motif of entry sites (“MSL recognition element” or MRE) is only slightly enriched on the X (~2 fold), and only a fraction of them is utilized by the MSL complex. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the NHGRI modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells, which contain MSL complex, and female Kc cells, which lack the complex, we find that the presence of active chromatin modifications, together with an elevated local GC content in surrounding sequence, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites, and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our finding serves as a model to understand how chromatin and local sequence features are involved in the selection of functional protein binding sites in the genome. The key Drosophila female sex determinant protein, SXL, represses dosage compensation by inhibiting MSL2 translation. Loss of SXL results in the expression, stabilization, and targetting of the MSL complex in female cells. Therefore, depletion of SXL by RNA interference (RNAi) in female Kc cells will lead to a MSL2-dependent increase in transcription from the female X chromosomes, consistent with the induction of dosage compensation. In this experiment, we generated gene expression profiles of Kc cells of control (GFP), Sxl RNAi and Sxl-Msl2 RNAi experiments.

Project description:Active genes on the X chromosome of Drosophila males are upregulated by the Male-specific lethal (MSL) complex containing five MSL proteins and two non-coding roX RNAs. To probe the targeting mechanism, we have solved the structure of the MSL3 chromodomain, designed point mutations in key residues that disrupt putative methyl-lysine recognition, and tested their effect on full length MSL3 function. Transgenic males expressing these site-directed point mutants or MSL3short, a naturally occurring MSL3 form lacking the chromodomain, are unhealthy and developmentally delayed. Genomewide analyses of the binding patterns of these mutants support a two-step model: the first step is chromodomain-independent association with “chromatin entry sites” carrying GA-rich MSL recognition elements (MREs). The second step involves spreading from entry sites to the majority of active genes on the X. Either deleting or introducing point mutations in the MSL3 chromodomain disrupts this second step. In vitro studies demonstrate that chromodomain mutants have diminished interaction with recombinant nucleosomes methylated at H3K36. We propose that MSL spreading depends, to a large extent, on the integrity of the MSL3 chromodomain to interact with lysine-methylated nucleosomes. Chromatin immunoprecipitation using TAP-tag was performed as described previously (Alekseyenko et al., 2006). The immunoprecipitated material was eluted from the beads by adding 10 ?L (100 U) of AcTEV protease into 450 ?L of TEV buffer followed by incubation at 25°C for 1 h. To reverse the cross-links, samples were brought to 0.3M NaCl and 1% SDS and incubated at 65°C for 12 h. The samples were then extracted with phenol/chloroform/isoamyl alcohol followed by chloroform, and precipitated by ethanol in the presence of glycogen. The resulting precipitated DNA and the input DNA were amplified using a DNA linker as described (Strutt et al., 1997). LM-PCR (25 cycles) was performed using Platinum Pfu (Invitrogen). The resulting DNA was labeled and hybridized to arrays by NimbleGen Systems, Inc. Two-channel tiling arrays containing 388,000 probes were designed based on FlyBase 3.2. Chromosomes X and 19.6Mb of 2L were covered. The ChIP sample of interest was hybridized on one channel and genomic DNA was used as the reference on the other channel. Each ChIP-chip experiment was performed in duplicate.

Project description:Full title: Complex patterns of genome accessibility discriminate sites of PcG repression, H4K16 acetylation and replication initiation Histone modifications have been proposed to regulate gene expression in part by modulating DNA accessibility and higher-order chromatin structure. However, there is limited direct evidence to support structural differences between euchromatic and heterochromatic fibers in the nucleus. To ask how histone modifications relate to chromatin compaction, we measured DNA accessibility throughout the genome by combining M.SssI methylase footprinting with methylated DNA immunoprecipitation (MeDIP-footprint). In the Drosophila genome, we find that accessibility to DNA methylase is variable in a manner that relates to the differential distribution of active and repressive histone modifications. Active promoters are highly permissive to M.SssI activity, yet inactive chromosomal domains decorated with H3 lysine 27 trimethylation are least accessible providing in vivo evidence for Polycomb-mediated chromatin compaction. Conversely, DNA accessibility is increased at active chromosomal regions marked with H4 lysine 16 acetylation and at the dosage-compensated male X chromosome suggesting that Drosophila transcriptional dosage compensation is facilitated by more permissive chromatin structure. Interestingly early replicating chromosomal regions and sites of replication initiation show also higher accessibility linking temporal and spatial control of genome duplication to the structural organization of chromatin. In conclusion, using a novel protocol we generated a comprehensive view of DNA accessibility and uncover different levels of chromatin organization, which are delineated by distinct patterns of posttranslational histone modifications and replication. Keywords: cell type comparison, ChIP-chip, MeDIP-footprint, RNA-seq, ChIP-seq MeDIP-footprint and ChIP-chip: ChIP-chip was performed for H3K4me3, H3K36me2, H3K36me3, H3K27me3, and H3K9me2 in Kc cells. We measured DNA accessibility throughout the genome by combining M.SssI methylase footprinting with methylated DNA immunoprecipitation (MeDIP-footprint) in Kc and S2 cells. RNA-seq: cDNA from RNA from Drosophila Kc cells was sequenced using Illumina deep sequencing. Reads were mapped and the abundance of all transcripts was determined. ChIP-seq: PSC ChIP from Drosophila Kc cells was sequenced using Illumina deep sequencing in three lanes. Reads were mapped and the binding profile of PSC was determined.

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Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context (ChIP-chip)

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