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Effect of SAFB1/SAFB2 knockou or ERH knockout on the miRNA transcriptome of murine Baf3 cells

ABSTRACT: MicroRNAs are small non-coding RNAs that mediate post-transcriptional silencing of most mammalian genes. They are generated in a multi-step process initiated by the Microprocessor, a protein complex composed of DROSHA and DGCR8. Recent studies have described the phenomenon of “cluster assistance”, in which a prototypic primary miRNA hairpin can license the Microprocessor-mediated processing of a clustered suboptimal hairpin in cis. Mechanistic analyses led to the identification of two critical factors for this process, SAFB2 (scaffold attachment factor B2) and ERH (enhancer of rudimentary homolog), but it remains unclear to which extent these factors contribute to and are required for cluster assistance. Here, we have used a previously established dual fluorescence reporter system (doi: 10.1016/j.molcel.2020.05.011) to generate loss-of-function clones for SAFB2 and its closely related family member SAFB1 as well as for ERH.Our data indicate that loss of SAFB1/2 and of ERH results in overlapping, but not identical defects in primary miRNA biogenesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE311272 | GEO | 2025/12/05

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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Effect of SAFB1/SAFB2 knockou or ERH knockout on the miRNA transcriptome of murine Baf3 cells

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