MEG3 Chromatin isolation by RNA purification-DNA sequencing (ChIRP-Seq) in wild type Mouse emryonic fibroblasts (MEFs) and Beta actin Knock out MEFS

In the eukaryotic cell, ß-actin plays essential nuclear functions. Actin directly affects genome organization and regulates promoter-enhancer interactions as part of the BAF (SWI/SNF) chromatin remodeling complex. It also binds to the hyperphosphorylated carboxy-terminal domain (CTD) of RNA polymerases II, and depletion of ß-actin leads to differential gene expression that impacts key cellular processes such as differentiation. In this study, we investigated whether ß-actin controls the level of long non-coding RNAs (lncRNAs). Using a combination of bulk RNA-seq and qPCR analyses on total RNA isolated from WT mouse embryonic fibroblasts (MEFs), ß-actin heterozygous (HET) MEFs, and ß-actin KO MEFs. Our findings demonstrate that the expression of several lncRNAs is directly affected by ß-actin depletion. One of these lncRNAs, Meg3, shows increased expression in a dosage-dependent manner. Using ChIRP-seq, we observed alterations in Meg3 genomic association and Meg3 enrichment at or near enhancer sites following ß-actin depletion. These changes were accompanied by increased levels of H3K27 acetylation. At these sites, specific Meg3 enrichment disrupts promoter-enhancer interactions, as revealed by the Activity by Contact (ABC) model, through a sponging effect. Consequently, this leads to the repression of genes involved in metabolic biosynthetic pathways for chondroitin, heparan, dermatan sulfate, and phospholipases, impacting their synthesis. We propose that Meg3 potentially impairs local genome organization (or DNA looping) and negatively affects gene expression through its chromatin sponging effect that interferes with promoter-enhancer interactions. Overall design: Chromatin isolation by RNA purification of MEG3 in WT MEFs and KO MEFs, replicates were used using odd and even probes such that WTO1 is replicate one of WT MEFs odd MEG3 probes, WTO2 is replicate two of WT MEFs odd MEG3 probes, WTE1 is replicate one of WT MEFs Even MEG3 probes WTE2 is replicate two of WT MEFs Even MEG3 probes, KOO1 is replicate one of KO MEFs odd MEG3 probes, KOO2 is replicate two of KO MEFs odd MEG3 probes, KOE1 is replicate one of KO MEFs Even MEG3 probes and KOE2 is replicate two of KO MEFs Even MEG3 probes.