Histone Lysine Methyltransferase MMSET : An Essential Regulator of Human Erythropoiesis.

Abstract

Erythropoiesis is the process by which hematopoietic stem cells proliferate, differentiate, and eventually form mature erythrocytes. Steady-state erythropoiesis is the result of the coordinated efforts of transcription factors and epigenetic regulators during differentiation. Histone methyltransferases (HMTs) are critical regulators that drive activation and repression of histone marks during erythroid lineage progression. MMSET (multiple myeloma SET domain) protein is aH3K36 methyltransferase (HKMT) required for normal hematopoietic development and essential for B cell differentiation and mature B cell function. Knockout of MMSET induces Wolf-Hirschhorn syndrome, where lymphocytes show abnormal functions, including a deficiency in antibody production. Studies have highlighted that MMSET acts as an oncogenic driver, classically associated with solid tumours, with a special focus on Multiple myeloma. However, the biological role and function of MMSET in human erythropoiesis is untouched and needs to be explored to address its role in the erythroid cascade. To this end, we examined the role of MMSET in human erythropoiesis by studying the genotypic and phenotypic modulatory effects observed upon MMSET knockdown (KD) in erythroid cells. Erythroid cells were generated from human G-CSF mobilized peripheral blood, hematopoietic stem and progenitor cells (HSPCs) as described by (Palii et al.,2011). Cultured cells were transduced with a lentiviral vector (pLKO.1) expressing MMSET andKD was observed after 72 hours of induction. 70-80% KD of MMSET in HSPCs resulted in reduced BFU-Es and CFU-Es in the semi-solid assay (Methocult TM) as compared to the control (pLKO.1 carrying scrambled sequence) (Fig. A), which was co-validated by FACS on days 2 and 4 (Phase I). Taken together, these results indicate that MMSET is required for early erythroid differentiation. Further, to access the role of MMSET during terminal stages of erythroid differentiation, lentiviral-mediated KD of late erythroblasts (days 8-12) (phase II) was done. MMSET KD erythroblasts displayed a block in terminally differentiating (Polychromatic and Orthochromatic) erythroblasts with an increase in Proerythroblasts and Basophilic erythroblasts. Thus, our results indicate that MMSET KD at later stages is associated with disordered terminal erythropoiesis. H3K36 methylation marks are involved in the regulation of DNA replication, recombination, and repair. Specifically, these marks keep DNA replication in check; hence, we noted that loss of MMSET correlates with increased transition of G2M phase cells from S phase cells with respect to control. In lieu of the above context, we noted increased proliferation (via Click-It Edu) in both phases (I and II). Studies have shown that MMSET is associated with DNA damage and repair (Evans et al., 2016). DNA damage and fragmentation mitigates apoptosis as a result of the signalling cascade downstream of effector caspases. We, therefore, observed a significant increase in apoptotic cells (via Annexin/PI staining) in both early and late erythroblasts upon KD of MMSET. To further gain insight, we carried out whole transcriptome analysis upon KD of MMSET and observed differentially expressed erythroid genes at three time points (days 4, 8, and 12) which revealed that GATA1, ALAS2, TAL1, KLF1, and FECH were significantly altered at the specified days (Fig. B). We further observed that erythrocyte development and differentiation, followed by regulation of the G2M transition of the cell cycle, apoptosis (programmed cell death), and cell proliferation, were the most enriched processes. Thus, in the present study, we systematically characterised erythroid impairment developing upon MMSET KD, leading to global downregulation of erythroid-specific genes, which are key players in normal erythroid development. An in-depth assessment of the MMSET -mediated erythroid gene regulatory network needs elucidation for further validation of MMSET as a direct player or mediator in human erythroid development. Our findings uncovered a previously unknown role for MMSET in human erythropoiesis.