Dynamics of gene expression and chromatin marking during cell state transition

Abstract

SummaryWe have monitored the transcriptomic and epigenomic status of cells at twelve time-points during the transdifferentiation of human pre-B cells into macrophages. Using this data, we have investigated some fundamental questions regarding the role of chromatin in gene expression. We have found that, over time, genes are characterized by a limited number of chromatin states (combinations of histone modifications), and that, consistently, chromatin changes over genes tend to occur in a coordinated manner. We have observed strong association between these changes and gene expression only at the time of initial gene activation. Activation is preceded by H3K4me1 and H3K4me2, and followed in a precise order by most other histone modifications. Further changes in gene expression, comparable or even stronger than those at initial activation, occur without associated changes in histone modifications. The data generated here constitutes, thus, a unique resource to investigate transcriptomic and epigenomic dynamics during a differentiation process.

Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

“…This suggests that the impact of the deletion of these two lncRNAs on the process is likely due the disruption of a possible enhancer activity of the genomic sequence. Consistent with this hypothesis, both loci are enriched in H3K27ac, mark associated to active enhancers (34), and this enrichment is enhanced upon induction of the process (Supplementary Figure S11) (16).…”

“…The selection of target genes was based on RNA-seq data sampled at 12 time points (0h, 3h, 6h, 9h, 12h, 18h, 24h, 36h, 48h, 72h, 120h, 168h) during transdifferentiation of human BLaER1 cells to macrophages (16). The RNAseq data was quantified with GRAPE-nf (https://github.com/guigolab/grape-nf).…”

“…To identify coding and non-coding genes that may drive the BLaER1 transdifferentiation process, we analyzed available RNA-seq data at 12 time points along the seven days the process lasts, in two biological replicates (16). We identified 488 lncRNAs and 3,627 pc-genes with minimum expression values higher than 1 FPKM in at least one time point as well as expression changes higher than 2fold for lncRNAs and 4-fold for pc-genes (see Methods).…”

“…With the goal of discovering pc-genes and lncRNAs that are essential for the transition from B-cell to macrophage, and taking advantage of available RNA-Seq data produced along the transdifferentiation process (16), we have used the DECKO system with a combined library of paired guide RNAs (pgRNAS), targeting simultaneously 166 lncRNAs and 874 pc-genes upregulated along the transdifferentiation process. Towards that end, we have extended the CRISPETa bioinformatics pipeline (17) to design optimal pairs of sgRNAs for deletion of genomic regions including both pcgenes and lncRNAs.…”

Paired Guide RNA CRISPR-Cas9 Screening for Protein-Coding Genes and lncRNAs Involved in Transdifferentiation of Human B-Cells to Macrophages

“…With the goal of discovering pc-genes and lncRNAs essential for the transition from B-cell to macrophage, and taking advantage of available RNA-Seq data produced along the transdifferentiation process [15], we have used the DECKO system with a combined library of paired guide RNAs (pgRNAS), targeting simultaneously 166 lncRNAs and 874 pc-genes upregulated along the transdifferentiation process. Towards that end, we have extended the CRISPETa bioinformatics pipeline [16] to design optimal pairs of sgRNAs for deletion of genomic regions including both pc-genes and lncRNAs.…”