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RNA-seq and in situ Hi-C of primary mouse B cell progenitor populations after activation with LPS

ABSTRACT: During cellular differentiation, chromosome conformation is intricately remodelled to support the lineage-specific transcriptional programs required for initiating and maintaining lineage identity. When these changes occur in relation to cell cycle, division and time is unclear although it has been proposed they would occur during DNA synthesis 5 or after mitosis. Here we elucidate the chromosome conformational changes in B lymphocytes as they differentiate and massively expand from a naïve, quiescent state into antibody secreting plasma cells. Unexpectedly, we find dramatic gene-regulatory chromosome reorganization in late G1 phase prior to the first division, and that this configuration is remarkably stable as the cells massively 10 and rapidly clonally expansion. A second wave of conformational change occurs as cells terminally differentiate into plasma cells, coincident with increased time in G1 phase. In this study we stimulated naive B cells with LPS and harvested activated B cells or plasmablasts at different time points after stimulation. RNA-seq was used to profile gene expression and in situ Hi-C was used to profile DNA-DNA interactions at each time point.

ORGANISM(S): Mus musculus

PROVIDER: GSE147497 | GEO | 2020/12/08

REPOSITORIES: GEO

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Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs. We queried the yeast genome for gene expression after cells were treated with 200 mM hydroxyurea during S phase. Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 30 and 60 min after the 1 h exposure to HU. These samples are referred to as G1, HU 1h, R30, and R60, respectively. The strains from which the samples were collected are indicated before the time point, e.g. mec1_G1 or MEC1_R30. Stranded mRNA libraries were prepared according to manufacturer's suggestion and sequenced on Illumina MiSeq with paired-end reads of 75 bp.

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RNA-seq and in situ Hi-C of primary mouse B cell progenitor populations after activation with LPS

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