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Mitotic post-translational modifications of histones promote chromatin compaction in vitro

ABSTRACT: As histones undergo major changes in their post-translational modifications during mitotic entry, we speculated that the spectrum of cell cycle-specific histone modifications might contribute to chromosome compaction during mitosis. To test this hypothesis, we isolated core histones from interphase and mitotic cells and reconstituted chromatin with them. We used mass spectrometry to show that key post-translational modifications remained intact during our isolation procedure.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Gallus Gallus (chicken)

TISSUE(S): Cell Culture

SUBMITTER: Juan Jose Bonfiglio

LAB HEAD: William Earnshaw

PROVIDER: PXD007116 | Pride | 2018-10-24

REPOSITORIES: Pride

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			Action	DRS
	140829_QX1_HistonesSILAC2fast.raw	Raw
	140829_QX1_HistonesSILAC2sens.raw	Raw
	140829_QX1_HistonesSILAC3fast.raw	Raw
	140829_QX1_HistonesSILAC3fast_141015073936.raw	Raw
	140829_QX1_HistonesSILAC3sens.raw	Raw

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Mitotic post-translational modifications of histones promote chromatin compaction <i>in vitro</i>.

Zhiteneva Alisa A Bonfiglio Juan Jose JJ Makarov Alexandr A Colby Thomas T Vagnarelli Paola P Schirmer Eric C EC Matic Ivan I Earnshaw William C WC

Open biology 20170901 9

How eukaryotic chromosomes are compacted during mitosis has been a leading question in cell biology since the nineteenth century. Non-histone proteins such as condensin complexes contribute to chromosome shaping, but appear not to be necessary for mitotic chromatin compaction. Histone modifications are known to affect chromatin structure. As histones undergo major changes in their post-translational modifications during mitotic entry, we speculated that the spectrum of cell-cycle-specific histon ...[more]

PMID: 28903997

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Genome-wide analysis of condensin binding sites in vertebrate genome

Project description:The condensin complex is well known to be essential for correct packaging and segregation of chromosomes during mitosis and meiosis in all eukaryotes. To date, the genome wide location and the nature of condensin binding sites has remained elusive in vertebrate systems. A detailed knowledge of condensin binding sites is essential to understand the as-yet enigmatic function of this complex and to define its greater role in chromosome architecture. To address this, we have used next generation sequencing to map condensin I in chicken DT40 cells. Unexpectedly, we found condensin I binds predominately to promoter sequences in mitotic cells. We also found a striking enrichment at both centromeres and telomeres, highlighting the importance of the complex in chromosome segregation. Taken together, the results show condensin I is largely absent from heterochromatic regions. These results reveal for the first time the condensin I binding sites in the vertebrate genome, and demonstrate the importance of the complex in genome segregation and suggest a novel function in gene regulation. Condensin associated DNA from chicken DT40 cells were affinity purified using streptavidin and deep sequenced by Illumina Hiseq2000

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Project description:Purpose: to identify how condensin I removal affects gene expression globally. Methods: Chicken DT40 CAP-H KO cells were treated with or without dox for 36 h. Total RNA samples were extracted and subjected to sequencing using an Illumina Hiseq2000 platform. Library preparation and Illumina sequencing were performed by Macrogen (South Korea). The sequence tags were spliced-mapped onto the chicken genome galGal4 using Tophat and Bowtie2 following quality test using FASTQC. Differential expression of genes was analyzed using the Bioconductor v2.3 package edgeR v3.2.3. Tag enrichment in NCBI RefSeq genes was calculated between dox-treated and untreated cells using edgeR exact test with tag-wise dispersion estimation. Results: We identified a total of 3798 genes to be differentially expressed in G1 condensin I-depleted chicken DT40 cells: 2495 down regulated and 1303 up regulated. Conclusions: Removal of CAP-H leads to significant misregulation of gene expression, suggesting a key role for condensin I in transcription during interphase. Total RNA profiles of CAP-H KO cells with or without Dox were generated by sequencing using Illumina Hiseq2000 platform.

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Two distinct modes for propagation of histone PTMs across the cell cycle

Project description:Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, it remains unknown how histone-based information is transmitted. Here we combine nascent chromatin capture (NCC) and triple-SILAC labelling to track histone modifications and histone variants during DNA replication and across the cell cycle. We show that post-translational modifications (PTMs) are transmitted with parental histones to newly replicated DNA. Di- and tri-methylation marks are diluted two-fold upon DNA replication, as a consequence of new histone deposition. Importantly, within one cell cycle all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9me3 and H3K27me3 are propagated by continuous modification of parental and new histones, because the establishment of these marks extends over several cell generations. Together, our results reveal how histone marks propagate and demonstrate that chromatin states oscillate within the cell cycle.

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