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New partners in regulation of gene expression: the Enhancer of Trithorax and Polycomb Corto interacts with methylated Ribosomal Protein L12 via its chromodomain.

ABSTRACT: Chromodomains are found in many regulators of chromatin structure. Most of them recognize methylated histones. Here, we investigate the role of the Corto chromodomain. This Drosophila melanogaster Enhancer of Polycomb and Trithorax is involved in both silencing and activation of gene expression. Overexpression of Corto chromodomain (CortoCD) in transgenic flies show that this domain is critical for Corto function and behaves as a chromatin-targeting module. Mass spectrometry analysis of peptides pulled down by CortoCD from nuclear extracts reveals that they correspond to nuclear ribosomal proteins (RPs). Notably, CortoCD binds with high affinity RPL12 tri-methylated on lysine 3 (RPL12K3me3) as demonstrated by real-time interaction analyses. Co-localization of Corto and RPL12 with active epigenetic marks on polytene chromosomes suggests that they are involved in fine-tuning transcription of genes located in open chromatin. Hence, pseudo-ribosomal complexes composed of various RPs might participate in regulation of gene expression in connection with chromatin regulators. RNA-seq analysis of wing imaginal discs overexpressing either Corto or RPL12 show that most deregulated genes are shared by both factors. Interestingly, these common targets are enriched in RP genes suggesting that Corto and RPL12 are involved in dynamic coordination of ribosome biogenesis.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE38435 | GEO | 2012/09/05

SECONDARY ACCESSION(S): PRJNA167951

REPOSITORIES: GEO

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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.

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New partners in regulation of gene expression: the Enhancer of Trithorax and Polycomb Corto interacts with methylated Ribosomal Protein L12 via its chromodomain.

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