Transcriptional analysis of direct NSD2 target genes in t(4;14) multiple myeloma reveals H3K36me2-dependent regulation and H3K27me3 antagonism [CUT&TAG]

This study aimed to identify the direct transcriptional targets of the H3K36me2 methyltransferase NSD2 in t(4;14) multiple myeloma (MM), where its aberrant expression drives oncogenesis, and to elucidate the molecular mechanisms underlying its regulatory role. To achieve this with precise temporal control and minimize cellular adaptation artifacts, we established an inducible dTAG-mediated acute NSD2 degradation system in the KMS-11 t(4;14) MM cell line. Using this model, we performed time-resolved SLAM-seq to measure newly synthesized mRNA levels at 7, 14, and 21 days post-NSD2 degradation, enabling the identification of primary transcriptional targets. Further SLAM-seq datasets were generated under conditions of NSD2 depletion followed by reconstitution with either wild-type NSD2 (NSD2WT) or a catalytically inactive mutant (NSD2Y1179A) to evaluate the requirement for SET domain activity. The contribution of H3K27me3 antagonism to NSD2-mediated gene regulation was investigated by performing SLAM-seq on NSD2-depleted cells treated with distinct PRC2 inhibitors (EPZ-6438 and MAK-658). Complementing the transcriptomic data, we performed CUTTAG to map the genomic distribution of the H3K27me3 histone mark following NSD2 depletion relative to control conditions. Analysis of these datasets revealed 308 primary NSD2 transcriptional targets, predominantly downregulated upon NSD2 loss and enriched for functionally relevant transcription factors. Reconstitution experiments demonstrated that NSD2's transcriptional effects are almost entirely dependent on its SET domain catalytic activity. Furthermore, PRC2 inhibition partially rescued the downregulation of approximately half of the NSD2 target genes, highlighting a significant role for H3K27me3 antagonism. Importantly, CUTTAG analysis showed that the compensatory increase in H3K27me3 upon NSD2 loss occurred primarily across genome-wide intergenic regions, rather than directly at the promoters or gene bodies of NSD2 target genes. Collectively, these deposited transcriptomic (SLAM-seq) and epigenomic (H3K27me3 CUTTAG) data support a model where NSD2 regulates target gene transcription via H3K36me2 deposition, which, in part, functions by antagonizing H3K27me3 deposition at distal regulatory elements to foster a permissive chromatin environment, providing a valuable resource for understanding NSD2 function in t(4;14) MM and Wolf-Hirschhorn syndrome.