Biology subjects

Kakani, P.

Publications and source records attributed to Kakani, P..

3 recordsLinked to original sources

bioRxiv2024

Targeted H3K9 acetylation at lncSox1 promoter by cell type-specific epigenome editing promotes intermediate progenitor proliferation in developing mouse cortex

The distribution and level of epigenetic (chromatin) marks have implications for differential regulatory effects at specific gene loci. Herein, we applied a protocol which combines in vivo electroporation and a CRISPR-dead (d)Cas9 system to probe and edit a specific chromatin mark in the epigenome of intermediate progenitor cells (IPCs) in developing mouse cortex. We found that the promoter of lncSox1, a long non-coding gene, is a key genomic locus for H3K9 acetylation (H3K9ac) during IPC amplification. CRISPR-dCas9-mediated addition of H3K9ac at lncSox1 promoter resulted in lncSox1 upregulation, with attendant increase in IPC pool and augmented neurogenesis. Thus, we have identified dynamic regulation of lncSox1 as a major downstream target of H3 acetylation, and as part of an epigenetic mechanism involved in IPC proliferation during neocortex expansion. This finding is a proof-of-concept that our epigenome editing-based method can be used for manipulating specific epigenetic effectors to determine their (neuro)biological significance. MOTIVATIONThe abundance of basal progenitors is critical for cortical neurogenesis during brain development. There is growing interest in identifying how specific epigenetic factors regulate the genesis and expansion of basal progenitor cell sub-populations, including intermediate progenitor cells. We established a protocol that allowed us to identify the involvement of a long non-coding RNA (lncSox1) in regulating the proliferation of intermediate progenitor cells under the influence of H3K9 acetylation (H3K9ac). By enhancing H3K9ac at the promoter region of lncSox1 using a CRISPR-dCas9-mediated gene-editing tool, we were able to determine that lncSox1 upregulation is a downstream effect of H3K9ac acetylation and is necessary for intermediate progenitor pool amplification during cortical development. Highlights- Identification of H3 acetylation-dependent expression of ncRNAs in developing cortex. - Establishment of cell Cre/LoxP and CRISPR-dCas9-dependent H3K9ac epigenome editing. - CRISPR-dCas9-mediated addition of H3K9ac at lncSox1 promoter resulted in lncSox1 upregulation. - H3K9 acetylation at lncSox1 promoter enhances proliferation of TBR2-expressing IPCs. - Targeted epigenome editing revealed lncSox1 as a key regulator of cortical development.

neuroscience↗

bioRxiv2024

PRMT5-mediated histone methylation regulates alternative splicing via MECP2-PTBP1 to promote EMT in breast cancer hypoxia

Tumor hypoxia induced alterations in the epigenetic landscape and alternative splicing influence cellular adaptations. PRMT5 is a type II protein arginine methyltransferase that regulates several tumorigenic events in many cancer types. However, the regulation of PRMT5 and its direct implication on aberrant alternative splicing under hypoxia remains unexplored. In this study, we observed hypoxia induced upregulation of PRMT5 via the CCCTC binding factor, CTCF. Further, PRMT5-mediated symmetric arginine dimethylation H4R3me2s and H3R8me2s directly regulated the alternative splicing of Transcription Factor 3 (TCF3). Under hypoxia, PRMT5-mediated histone dimethylation at the intronic conserved region (ICR) present between TCF3 exon 18a and exon 18b recruits DNA methyltransferase 3A (DNMT3A), resulting in DNA methylation. DNA methylation at the TCF3-ICR is recognized and bound by Methyl CpG binding protein (MECP2) resulting in RNA-Pol II pausing, promoting the recruitment of the negative splicing factor PTBP1 to the splicing locus of TCF3 mRNA. PTBP1 promotes the exclusion of exon 18a which results in the production of the pro-invasive TCF3-18B (E47) isoform which promotes EMT and invasion of breast cancer cells under hypoxia. Collectively, our results indicate PRMT5-mediated symmetric arginine dimethylation of histones regulates alternative splicing of TCF3 gene thereby enhancing EMT and invasion in breast cancer hypoxia.

cancer biology↗

bioRxiv2023

Hypoxia-induced CTCF mediates alternative splicing via coupling chromatin looping and RNA Pol II pause to promote EMT in breast cancer

Cancer cells experiencing hypoxic stress employ Epithelial-Mesenchymal Transition (EMT) to undergo metastasis through rewiring chromatin landscape, epigenetics, and importantly alternative splicing. Here, we investigated the role of CTCF, chromatin, epigenetic and alternative splicing modulator under hypoxia to promote EMT. Our result shows that hypoxia-induced epigenetic changes upregulate CTCF expression in breast cancer. We delineate that CTCF is a direct target of HIF1 and the hypoxia-induced HIF1-CTCF axis functionally contributes to promoting EMT in breast cancer cells. Finally, our work uncovers COL5A1, an EMT gene, as a direct target of CTCF. We demonstrated that hypoxia-mediated CTCF enrichment on COL5A1 promoter regulates expression as well as alternative splicing events to promote EMT. Here, we put forward an intricate mechanism of alternative splicing where CTCF-mediated promoter-exon upstream looping regulates DNA de-methylation and CTCF-mediated RNA Pol II pausing at COL5A1 exon 64A. Our global analysis of CTCF-ChIP-seq data reveals that the hypoxia-induced differential CTCF occupancy possibly regulates gene expression and alternative splicing events of many genes that are enriched in the EMT pathway, cancer cell motility and invasion, angiogenesis, and stemness under hypoxia, similarly to the proposed model. Finally, we employed the epigenetic modulator dCas9-DNMT3A system to specifically disrupt HIF1 or CTCF binding under hypoxia and hence the HIF1-CTCF-COL5A1exon64A axis that alleviates the EMT potential of breast cancer cells that may represent a novel therapeutic target in breast cancer. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/539689v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e15c15org.highwire.dtl.DTLVardef@1649a71org.highwire.dtl.DTLVardef@1d77481org.highwire.dtl.DTLVardef@21f148_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

molecular biology↗