ABSTRACT: Memory B cells (MBCs) are long-lived and rapidly respond to cognate antigen with production of high-affinity, generally, class-switched antibodies. Here, we have developed an integrative analysis of differentially expressed (DE) mRNAs, miRNAs, lncRNAs, chromatin profiles and cis-regulatory elements to determine how gene expression intersects with epigenetic regulatory elements in human MBCs. Using a multiparameter cell sorting approach, we isolated CD27-IgD+NBCs, CD27+IgD+unswMBCs, CD27+IgG+swMBCs and CD27+IgA+swMBCs as well as total CD27-NBCs and CD27+MBCs from peripheral blood of healthy human subjects of different age, sex and ethnic background. Total RNA was used to construct RNA-Seq libraries and subjected to next generation sequencing for in-depth analysis of B cell transcriptome, including mRNAs, miRNAs and lncRNAs, as well as Ig heavy chain (H) V(D)J, and Ig light chain (L) gene transcripts. Differential expression analysis identified shared mRNA, miRNA and lncRNA profiles that distinguished CD27+IgG+swMBCs and CD27+IgA+swMBCs from CD27-IgD+NBCs, and stratified CD27+IgD+unswMBCs between NBCs and swMBCs. Further, targeted integration using Ingenuity Pathway Analysis (IPA) outlined distinct signaling pathways in human MBCs, while the activity of TFs was inferred from global changes in transcriptional activation. ATAC-Seq was integrated with RNA-Seq to correlate DE RNAs with chromatin accessibility, thereby uncovering distinct profiles of cis-regulatory elements in human MBCs. A deep downregulation of MIR181 was concomitant with upregulation of this microRNA target genes, which accounted for 11 percent of the DE mRNAs in swMBCs, indicating an important role for MIR181 in MBC differentiation and/or maintenance. Further, lncRNA MIAT, a sponge of MIR181, was upregulated in MBCs and inversely correlated with MIR181a and MIR181b expression. This along with chromatin accessibility contributes downregulation of MIR181 and promotes MBC-specific gene expression. Overall, our findings provide evidence for overlapping layers of regulation, including chromatin remodeling, cis-regulatory elements and distinct sets of miRNAs and lncRNAs, which integrate to dictate gene expression profiles and activation pathways characteristic of human MBCs.
