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Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold proteins in Drosophila S2 cells – Transmission of hallmarks of nuclear organization through mitosis

ABSTRACT: During open mitosis chromatin condenses to form chromosomes and decondenses post-mitotically to re-occupy their designated nuclear territory in a precisely lineage specific manner. This necessitates that features of nuclear architecture persist through mitosis. Here we present a proteomic study, which shows that the features of nuclear architecture in interphase nucleus are retained on the mitotic chromosome. Proteomic comparison was made between nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an identical biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS). Our study elucidates that a large graction of the NuMat proteins are retained in the MiCS and possibly play an important role in maintenance of cell lineage specific features of nuclear architecture during cell division and thereby serve as components of cellular memory.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: RAHUL SUREKA

LAB HEAD: Rakesh Kumar Mishra

PROVIDER: PXD008404 | Pride | 2018-07-17

REPOSITORIES: Pride

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Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold Proteins in <i>Drosophila</i> S2 Cells-Transmission of Hallmarks of Nuclear Organization Through Mitosis.

Sureka Rahul R Wadhwa Rashi R Thakur Suman S SS Pathak Rashmi U RU Mishra Rakesh K RK

Molecular & cellular proteomics : MCP 20180710 10

Chromatin condenses several folds to form mitotic chromosomes during cell division and decondenses post-mitotically to reoccupy their nuclear territory and regain their specific transcriptional profile in a precisely lineage specific manner. This necessitates that the features of nuclear architecture and DNA topology persist through mitosis. We compared the proteome of nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an equivalent biochemical fr ...[more]

PMID: 29991507

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Project description:Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that they could provide transcriptional memory through mitosis. To obtain the first genome-wide view of the dynamics of chromatin structure during mitosis, we compared the DNase sensitivity of interphase and mitotic chromatin at two stages of cellular maturation in a rapidly dividingmurine erythroblastmodel. Despite global chromosome condensation visible during mitosis at the microscopic level, the chromatin accessibility landscape is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility, whereas distal regulatory elements preferentially lose accessibility. Promoters with the highest degree of accessibility preservation in mitosis tend to also be accessible across many murine tissues in interphase. Transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but does not visibly influence mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis and are modulated at the level of individual genes and regulatory elements. Dnase-Seq data is integrated with Chip-seq [GSE36589, GSE30142] and RNA-seq to examine epigentic changes in mitosis. We performed DNase-seq on two cell lines, G1E and G1E-ER4, both on an asynchronus population, and on a sample of cells in mitosis; each of the 4 experiments in triplicate.

Project description:During mitosis, RNA polymerase and most transcription factors are excluded from the chromosomes and transcription ceases. The transcriptional re-activation of the genome, following mitosis, requires the re-setting of cell-type specific programs that were initially established during development. However, only about one-fifth of transcription factors are retained on chromosomes throughout mitosis and a subset of these have been shown to facilitate target gene reactivation during mitotic exit. How such “bookmarking” factors bind to chromatin in mitosis and re-activate transcription is central to the stability of transcriptional programs across multiple cell cycles. We compared a diverse set of transcription factors involved in liver differentiation and found different modes of mitotic chromosome binding. The pioneer transcription factor FoxA1, which is among the first to bind liver genes in development, exhibits virtually complete mitotic chromosome binding, whereas other liver factors bind with a range of efficiencies. Yet genome-wide analysis shows that only about 15% of the FoxA1 interphase target sites are bound in mitosis; the latter include sites at genes for maintaining cell differentiation. FoxA1 mutants that perturb specific and nonspecific DNA binding reveal a significant contribution of nonspecific binding events in mitotic chromatin. Such nonspecific binding appears to spread from interphase FoxA1 targets and may serve as storage sites. The hierarchy of specific binding, nonspecific binding, partial chromatin binding, and failure to bind mitotic chromosomes reflects the temporal sequence of the factors’ developmental roles in gene activation. Gene expression was assessed in asynchronously cycling cells in order to determine whether FoxA1 is disposed to bind to high- or low-expressed genes during mitosis. Heatmap analysis shows that FoxA1's mitotic targets are among the highest-expressed genes in asynchronous cells. Total RNA was collected from three different plates with asynchronous HUH7 cells using the RNeasy mini kit (Qiagen Valencia CA). Expression microarrays were performed with a Human Gene 1.OST array (Affymetrix) at the UPenn Microarray Core Facility and assessed using Partek.

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