RNA Sequencing Data-Driven Dissection of Human Plasma Cell Differentiation Reveals New Potential Transcription Regulators

Background: Plasma cells play an important role in the adaptive immune system through a continuous production of antibodies. Several hematological disorders, including some autoimmune diseases, multiple myeloma and plasmacytoma result from the malignant transformation of antibody-secreting cells. A clear understanding of the molecular processes underlying human plasma cell differentiation (PCD) and the long-term survival of ASCs will provide greater insight into such pathologies.

Methods: We performed RNA-seq analysis to obtain a global transcriptomic map of human PCD including memory B cells, preplasmablasts, plasmablasts and plasma cells. We implemented a DESeq2-based workflow to identify genes with temporal expression pattern during PCD. We used Reactome pathway enrichment analysis to identify key pathways of PCD. We next focused our analysis on the identification of new potential transcription factors and epigenetic enzymes involved in PCD.

Results: Our results reveal 8419 genes classified into four temporal gene expression patterns that we refer to one-step-up or one-step-down (mRNA level transitions from low to high or high to low, respectively, in two consecutive differentiation stages) and two-step-up-down (i.e. impulse-up) or two-step-down-up (i.e. impulse-down) (mRNA level transitions from low to high and back down or from high to low and back up, respectively, in a series of differentiation stages).

Consistent with known biology, genes upregulated during PCD were mainly involved in protein modification and metabolism. Interestingly, a stringent Reactome pathway enrichment analysis of these expression pattern highlights pathways largely unknown in PCD, including the heme biosynthesis and the glutathione conjugaison pathways.

To begin to understand thenature of the regulatory processes during human PCD, we focused on transcription factor (TF) and epigenetic enzyme (EE) genes. Collectively, we identified 617 TF/EE temporally regulated genes during PCD. 123 TF/EE genes fall into the one-step-up groups. 237 TF/EE genes are included in one-step-down groups, 49 in the impulse-up groups, 36 in the impulse-down groups.

Among these TFs/EEs we further analyze those exhibiting a one-step-up or impulse-up pattern, because they are highly expressed in one or more PCD stages. Among the one-step-up TFs/EEs are those encoding well-known proteins such as IRF4, BLIMP1 and XBP1. Interestingly we found many other new transcription factors with unknown function and a potential importance in PCD. New TFs with the most consistent and marked overexpression are BATF2, BHLHA15, IRF2, ZSCAN20, MIXL1, MAF and STAT1. Furthermore, our analysis identified EE genes consistently upregulated during PCD including histone methyltransferases (PRDM1, PRDM15, PRMT7, SETDB2, SMYD2 and SMYD4), de-novo DNA methylation enzyme (DNMT3B), DNA methylation readers (MBD1 and ZBTB38), DNA methylation editors/erasers (IDH1, IDH2, TET1, ALKBH1, ALKBH3 and MGMT) and histone phosphorylation editor (EYA2 and EYA3). Additionally, our results reveal several interesting TFs/EEs genes among the genes included in the impulse-up group, including AICDA, MYB, BATF3, FOXM1, ARNTL2, SUV39H2, WHSC1, MYBL2, TP53, EZH2, SUV39H1 and PRMT1.

Finally, we experimentally demonstrated that EZH2 inhibition, the catalytic component of polycomb repressive complex 2, affects the survival and the proliferation of plasma cell differentiation cell subpopulations, and significantly accelerates plasma cell differentiation.

Conclusion: This analysis thus identifies a discrete set of genes that function together to program plasma cell differentiation. Many of these genes have not yet been implicated in plasma cell biology. Together, the RNA-Seq analysis of temporal stages of PCD helped to identify coexpressed gene sets with associated up- /down- regulated TF/EE genes that could represent regulatory nodes for plasma cell differentiation.