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Effects of CRISPR/Cas9-mediated deletion of Safb1 and Safb2 genes on the mature miRNA transcriptome

ABSTRACT: MicroRNAs (miRNAs) are 20-25 nucleotides (nt) RNAs that are predicted to post-transcriptionally silence expression of >60% of all protein coding genes in mammals. As such, they form an additional layer of gene regulation that controls most if not all biological processes. MiRNAs are generated from primary transcripts containing single or multiple clustered stem-loop structures that are thought to be recognized and cleaved by the DGCR8/DROSHA Microprocessor complex as independent entities. Contrasting this view, we find that the primary miR-15a stem-loop within the bicistronic miR-15a-16-1 cluster is a poor Microprocessor substrate and is consequently not processed on its own, but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted primary miR-15 cleavage. Small RNA sequencing further revealed that deletion of Safb1 and 2 genes reduces not only miR-15a levels, but also affects other clustered miRNAs, among them miR-15b, miR-181b and miR-92a. We therefore postulate a model in which SAFB2 enables processing of suboptimal substrates in a subset of clustered pri-miRNA transcripts.

ORGANISM(S): Homo sapiens

PROVIDER: GSE141098 | GEO | 2019/11/28

REPOSITORIES: GEO

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Project description:The scaffold attachment factors SAFB1 and SAFB2 are paralogs, which are involved in cell cycle regulation, apoptosis, differentiation, and stress response. They have been shown to function as estrogen receptor co-repressors, and there is evidence for a role in breast tumorigenesis. To identify their endogenous target genes in MCF-7 breast cancer cells, we utilized gene expression array analysis, which was set up in a two-by-four design, with vehicle and estrogen treatment, and control, SAFB1, SAFB2, and SAFB1/SAFB2 siRNA as variables. Using custom chips containing 1.5 kb upstream regulatory region, we identified 541 SAFB1/SAFB2 binding sites in promoters of known genes, with significant enrichment on chromosome 1 and 6. Gene expression analysis revealed that the majority of target genes were induced in the absence of SAFB1 or SAFB2, and less were repressed. In contrast to SAFB2, which shared most of its target genes with SAFB1, SAFB1 had many unique target genes, most of them involved in regulation of the immune system. A subsequent analysis of the estrogen treatment group revealed that twelve percent of estrogen-regulated genes were dependent on SAFB1, with the majority being estrogen-repressed genes. These were primarily genes involved in apoptosis, such as BBC3, NEDD9, and OPG. Thus, this study confirms SAFB1/SAFB2âs primary role as co-repressors, and also uncovers a previously unknown role for SAFB1 in regulation of immune genes, and in estrogen-mediated repression of genes. To identify transcripts which were altered upon decrease of SAFB1 or SAFB2 expression we performed gene expression array analysis in MCF-7 cells. A secondary goal was to identify ERÎ±-target genes for which estrogen regulation was dependent upon SAFB expression. We also added an experimental group in which we co-transfected siRNAs targeting both SAFB1 and SAFB2, which allowed us to address the question of interactions between the two paralogs. We therefore set up an experiment reflecting a 2x4 design, with a total of eight experimental groups.

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Effects of CRISPR/Cas9-mediated deletion of Safb1 and Safb2 genes on the mature miRNA transcriptome

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