Histone Lysine Methyltransferase (NSD2) : A Critical Regulator of Human Erythropoiesis
Authors: Katiyar Shobhita , Shah Arunim , Rai Bhuvnesh , Rahman Khaliqur , Tripathy Naresh , Nityanand Soniya , Yadav Sanjeev , Kashyap Rajesh , Chaturvedi Chandra Prakash 
Published: December 2024

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. NSD2 (nuclear receptor binding SET domain protein 2) is a H3K36 methyltransferase (HKMT) required for normal hematopoietic development and essential for B cell differentiation and mature B cell function. Knockout of NSD2 induces Wolf-Hirschhorn syndrome, where lymphocytes show abnormal functions, including a deficiency in antibody production. Studies have highlighted that NSD2 acts as an oncogenic driver, classically associated with solid tumours, with a special focus on Multiple myeloma. However, the biological role and function of NSD2 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 NSD2 in human erythropoiesis by studying the genotypic and phenotypic modulatory effects observed upon NSD2 knockdown (KD) in erythroid cells. Erythroid cells were obtained from human G-CSF mobilized peripheral blood containing hematopoietic stem and progenitor cells (HSPCs) as described by (Palii et al.,2011). Cultured cells were transduced with a lentiviral vector (pLKO.1) expressing NSD2, KD was observed after 72 hours of induction. 70-80% KD of NSD2 in HSPCs resulted in reduced BFU-Es and CFU-Es in the semi-solid assay (Methocult ) as compared to the control (pLKO.1 carrying scrambled sequence) which was co validated by FACS on days 2 and 7 . Taken together, these results indicate that NSD2 is required for early erythroid differentiation. Further, to access the role of NSD2 during terminal stages of erythroid differentiation, lentiviral-mediated KD of late erythroblasts (day 12) was done. NSD2 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 NSD2 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 NSD2 correlates with decreased transition of G2M phase cells from S phase cells with respect to control. In lieu of the above context, we noted decreased proliferation (Click-It Edu) at the three time points. Studies have shown that NSD2 is associated with DNA damage and repair. 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 ( Annexin/PI staining) at days 2,7 and 12 upon KD of NSD2.
To further gain insight, we carried out whole transcriptome analysis upon KD of NSD2 and observed differentially expressed erythroid genes (using heatmap2 software). RT-qPCR analysis revealed that GATA1, ALAS2, TAL1, KLF1, and FECH were significantly altered at the three time points. We further observed that erythrocyte development and differentiation, followed by regulation of cell cycle, apoptosis (programmed cell death), and cell proliferation, were the most enriched processes. Furthermore, KEGG pathway analysis revealed that Robo4 and VEGF crosstalk was highly enriched at three days upon knockdown of NSD2. VEGF has been shown to play key role in angiogenesis and regulation of erythropoiesis in embryonic stem cells. Few studies have highlighted VEGF being direct interacting partner of NSD2, therefore NSD2 might be involved as a direct or indirect player in regulating erythropoiesis.
Thus, in the present study, we systematically characterised erythroid impairment developing upon NSD2 KD, leading to global downregulation of erythroid-specific genes, which are key players in normal erythroid development. An in-depth assessment of the NSD2 -mediated erythroid gene regulatory network needs elucidation for further validation of NSD2 as a direct player or mediator in human erythroid development. Our findings uncovered a previously unknown role for NSD2 in human erythropoiesis.