Dataset Information

Set2 and H3K36 regulate the Drosophila male X chromosome in a context-specific manner (WL3 brain)

ABSTRACT: Current models of MSL spreading in the context of Drosophila dosage compensation (DC) focus on interactions of MSL3 (male-specific lethal 3) with histone marks; especially Set2-dependent H3 lysine-36 trimethylation (H3K36me3). However, previous studies investigating the role of H3K36me3 in DC do not fully account for: other targets of Set2, redundancy between canonical H3.2 and replication-dependent H3.3, and X chromosome effects that are not sex-specific.To differentiate amongst these possibilities and to assess whether Set2 and/or H3K36 is essential for DC, we use RNA-Seq in WL3 brain (male and female) and L1 larvae (mixed sex) to compare Set2, H3.2K36R, H3.3K36R, and combined H3K36R mutant genotypes. In brains, we observe heterogeneous regulation of chrX genes by these factors in both males and females. These effects are not clearly linked to MSL3 binding or H4K16ac occupancy, but in some groups of genes, are clearly stronger in males. At L1, we also observe heterogeneous gene regulation distinct from an H4K16R control. Simultaneous mutation of both H3.2K36 and H3.3K36 results in an increase in expression in genes most strongly reduced in H4K16R mutants. Overall, the data are inconsistent with the prevailing model wherein H3K36me3 is essential for spreading the MSL complex to genes along the male X. Rather, we propose that Set2 and H3K36 support DC indirectly, via processes that are utilized by MSL, but common to both sexes.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE270799 | GEO | 2024/10/14

REPOSITORIES: GEO

ACCESS DATA

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

Similar Datasets

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.