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Accession IconSRP078939

Dynamic Control of X-Chromosome Conformation and Repression by a Histone H4K20 Demethylase

Organism Icon Caenorhabditis elegans
Sample Icon No Downloadable Samples
Technology Badge IconIllumina HiSeq 2000

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Chromatin modification and higher-order chromosome structure play key roles in gene regulation, but their functional interplay in controlling gene expression is elusive. We discovered the machinery and mechanism underlying the dynamic enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during C. elegans dosage compensation and demonstrated H4K20me1''s pivotal role in regulating higher-order chromosome structure and X-chromosome-wide gene expression. Structure and activity of dosage-compensation-complex (DCC) subunit DPY-21 defined a novel Jumonji demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Selective inactivation of demethylase activity eliminated H4K20me1 enrichment in somatic cells, elevated X-linked gene expression, reduced X-chromosome compaction, and disrupted X-chromosome conformation by diminishing formation of topologically-associating domains (TADs). Unexpectedly, DPY-21 also associates with autosomes of germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Our findings demonstrate the direct link between chromatin modification and higher-order chromosome structure in long-range regulation of gene expression. Overall design: The chromatin modification H4K20me1 becomes enriched on the dosage-compensated X chromosomes of C. elegans hermaphrodites. We explored the machinery and mechanism underlying the enrichment of H4K20me1 on X chromosomes and discovered H4K20me1''s pivotal role in regulating higher-order chromosome structure and X-chromosome gene expression. Through X-ray crystallography and biochemical assays, we found that the DPY-21 subunit of the C. elegans dosage compensation complex defines a new subfamily of Jumonji C (JmjC) histone demethylases that converts H4K20me2 to H4K20me1 and is widely conserved from worms to mammals. ChIP-seq and immunofluorescence studies showed that inactivation of JmjC activity in vivo by genome editing abrogated H4K20me1 enrichment on X. Hi-C studies showed loss of demethylase activity reduced X-chromosome compaction and disrupted X-chromosome topology by weakening TAD boundaries. RNA-seq studies showed that loss of demethylase activity elevated X-linked gene expression, demonstrating a key role for H4K20me1 in DC. Unexpectedly in germ cells, DPY-21 associates with autosomes, but not X chromosomes, in a DCC-independent manner to enrich H4K20me1 and facilitate chromosome compaction. Thus, DPY-21 is an adaptable chromatin regulator that is harnessed during development for distinct biological functions. In both somatic cells and germ cells, H4K20me1 enrichment modulates 3D chromosome architecture, demonstrating the direct impact of chromatin modification on establishing higher-order chromosome structure. To examine the genome-wide distribution of H4K20me1 in C. elegans at high resolution and determine the effect of dpy-21 demethylase mutations on H4K20me1 enrichment, we performed ChIP-seq in mixed-stage embryos from wild-type, dpy-21(y607 JmjC), and dpy-21(e428 null) mutant strains. We were able to compare directly the levels of H4K20me1 in wild-type and dpy-21 mutant embryos, because we used wide-type C. tropicalis embryos as a spike-in control to normalize the read enrichment. Two biological replicates were examined for each genotype. To assay the effect of H4K20me1 on the regulation of X-chromosome gene expression, we performed RNA sequencing (RNA-seq) on three different dpy-21(JmjC) demethylase mutant strains that prevented the conversion of H4K20me2 to H4K20me1 on X chromosomes and compared the expression of genes on X and autosomes with that in wild-type strains and strains with reduced activity of set-1, the methyltransferase that converts H4K20me to H4K20me1 and set-4, the methyltransferase that converts H4K20me1 to H4K20me2/me3. Mixed-stage embryos were assayed from 3 biological replicates of dpy-21(y607 JmjC), 3 replicates of dpy-21(y618 JmjC), 3 replicates of dpy-21(y622 JmjC), 7 replicates of dpy-21(e428 null), 7 replicates of the wild-type (N2) strain, 2 replicates of the wild-type (N2) strain grown on control RNAi bacteria (L4440), 3 replicates of set-1(RNAi), and 3 replicates of set-4(n4600). To assess the effect of H4K20me1 enrichment on X-chromosome topology, we performed genome-wide chromosome conformation capture (Hi-C) experiments on mixed-stage wild-type (N2) and dpy-21(y607 JmjC) mutant embryos by combining a previously published Hi-C protocol with in-nucleus ligation (Crane et al., 2015; Rao et al.,
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