Integrative analysis of coding, non-coding transcriptome and chromatin accessibility reveals a distinct gene expression and epigenetic landscape in human memory B cells

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.记忆B细胞（Memory B cells, MBCs）是一类长寿命、可快速响应同源抗原并产生高亲和力、通常为类别转换型抗体的免疫细胞。本研究围绕人类记忆B细胞中基因表达与表观遗传调控元件的互作机制，整合分析了差异表达（differentially expressed, DE）的mRNA、miRNA、lncRNA、染色质图谱及顺式调控元件。研究采用多参数细胞分选策略，从不同年龄、性别及种族背景的健康人类外周血样本中，分离得到CD27-IgD+初始B细胞（naive B cells, NBCs）、CD27+IgD+未类别转换记忆B细胞（unswMBCs）、CD27+IgG+类别转换记忆B细胞（swMBCs）、CD27+IgA+类别转换记忆B细胞（swMBCs），以及总CD27阴性初始B细胞（CD27-NBCs）与总CD27阳性记忆B细胞（CD27+MBCs）。我们提取总RNA构建RNA测序（RNA-Seq）文库，并开展下一代测序，以深度解析B细胞转录组，涵盖mRNA、miRNA、lncRNA，以及免疫球蛋白重链（Ig heavy chain, H）V(D)J与免疫球蛋白轻链（Ig light chain, L）基因转录本。差异表达分析筛选得到共享的mRNA、miRNA及lncRNA表达谱，可有效区分CD27+IgG+swMBCs、CD27+IgA+swMBCs与CD27-IgD+NBCs，并能将CD27+IgD+unswMBCs在NBCs与swMBCs之间进行分层。进一步通过Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）开展靶向整合分析，勾勒出人类记忆B细胞中独特的信号通路；同时基于转录激活的全局变化，推断转录因子（transcription factors, TFs）的调控活性。我们将ATAC测序（ATAC-Seq）数据与RNA-Seq数据整合，以关联差异表达RNA与染色质开放状态，进而揭示人类记忆B细胞中顺式调控元件的独特图谱。研究发现，MIR181的显著下调与其靶基因的上调同步发生，这类靶基因占类别转换记忆B细胞中差异表达mRNA的11%，提示MIR181在记忆B细胞分化及/或维持过程中发挥关键作用。此外，作为MIR181海绵分子的长链非编码RNA MIAT在记忆B细胞中呈上调表达，且与MIR181a、MIR181b的表达呈负相关。这一现象结合染色质开放状态调控，共同促成了MIR181的下调，并促进记忆B细胞特异性基因的表达。综上，本研究结果证实了多层调控网络的协同作用，包括染色质重塑、顺式调控元件以及特定的miRNA与lncRNA群体，这些调控机制共同塑造了人类记忆B细胞特有的基因表达谱与激活通路。