Chromatin regulation of transcriptional enhancers and cell fate by the Sotos syndrome gene NSD1

Abstract

Nuclear receptor-binding SET-domain protein 1 (NSD1), a methyltransferase that catalyzes H3K36me2, is essential for mammalian development and is frequently dysregulated in diseases, including Sotos syndrome. Despite the impacts of H3K36me2 on H3K27me3 and DNA methylation, the direct role of NSD1 in transcriptional regulation remains largely unknown. Here, we show that NSD1 and H3K36me2 are enriched at cis-regulatory elements, particularly enhancers. NSD1 enhancer association is conferred by a tandem quadruple PHD (qPHD)-PWWP module, which recognizes p300-catalyzed H3K18ac. By combining acute NSD1 depletion with time-resolved epigenomic and nascent transcriptomic analyses, we demonstrate that NSD1 promotes enhancer-dependent gene transcription by facilitating RNA polymerase II (RNA Pol II) pause release. Notably, NSD1 can act as a transcriptional coactivator independent of its catalytic activity. Moreover, NSD1 enables the activation of developmental transcriptional programs associated with Sotos syndrome pathophysiology and controls embryonic stem cell (ESC) multilineage differentiation. Collectively, we have identified NSD1 as an enhancer-acting transcriptional coactivator that contributes to cell fate transition and Sotos syndrome development.

Keywords: H3K36 methylation; NSD1; Pol II pause release; Sotos syndrome; chromatin; enhancers; gene expression; histone methylation; reader domain; stem cells.

Figure 1.. NSD1 catalyzes the majority of H3K36me2 in mESCs

(A) Schematic showing the NSD1-dTAG alleles and targeted proteolysis. (B) Immunofluorescence in NSD1-dTAG mESCs. Scale bar representing 20 μm applies to all panels. In (B) and (C), NT, no treatment. (C and D) Western blots of whole-cell extracts from indicated mESCs. Actin, Tubulin and H3 used as loading controls. See also Figure S1.

Figure 2.. NSD1 and H3K36me2 are enriched at active enhancers

(A) Genome browser view of CUT&RUN profiles in NSD1-dTAG mESCs. Black bars represent active enhancers. In (A), (B) and (D), 72h dTAG-13 treated cells were used as control; NT, no treatment. (B) Heatmap showing enrichment of chromatin features at all NSD1-occupied promoter and promoter-distal regions. (C) Upper: genomic distribution of NSD1. Lower: relative enrichment of NSD1/H3K36me2 at distinct genomic regions. (D) Average profile of NSD1 and H3K36me2 at active enhancers (n = 15986) and super-enhancers (n = 705). Inset showing percentage occupied by NSD1. (E) Immunofluorescence of pluripotency markers KLF4 (naive) and OCT6 (primed). Scale bar representing 20 μm applies to all panels. (F) Overlap of NSD1 peaks in ESCs vs. EpiSCs. (G) Top three de novo motifs enriched at ESC and EpiSC unique NSD1 peaks (target coverage >10%; ranked by P values). Heatmap shows relative expression. (H) Average profile showing H3K27ac occupancy in ESCs and EpiSCs at indicated categories of NSD1 peaks. (I) Genome browser view of CUT&RUN profiles in NSD1-dTAG ESCs vs. EpiSCs. See also Figure S2.

Figure 3.. The tandem qPHD-PWWP module is essential for NSD1 chromatin association and enhancer recruitment

(A) Schematic of NSD1 (NSD1.1S) series. FL, full-length. PP, qPHD-PWWP. PC, PHD-C5HCH. C, C-terminal half. NLS, nuclear localization sequence. (B) Western blots of whole-cell extracts from indicated mESCs. In (B) and (J), Tubulin and H3 used as loading controls. (C) Western blots of sequential salt extraction fractions. ARID1A used as control. CNL, cytoplasmic and nuclear-leaked fraction. (D) Genome browser view showing occupancy of NSD1 C series. (E) Heatmap showing occupancy of NSD1 C series at all NSD1 C peaks (n = 2036). (F and G) Western blot analysis of nucleosome pull-down assay using recombinant nucleosomes and mESC nuclear extracts. H3 used as input control for bait nucleosomes. (H) Heatmap showing occupancy of indicated proteins and histone PTMs. (I) Average profile showing H3K18ac occupancy in ESCs and EpiSCs at indicated categories of NSD1 peaks. (J) Western blots of whole-cell extracts from indicated mESCs. (K) ChIP-qPCR analysis of relative enrichment of NSD1 C Sotos syndrome mutants at enhancers. Negative control regions (chr2 and chr6) and input were used for normalization; data represent mean ± SD from n=3 technical replicates. See also Figure S3.

Figure 4.. Impact of acute NSD1 degradation on H3K36 modifications and associated chromatin features

(A) Western blots of whole-cell extracts. Tubulin and H3 used as loading controls. (B) Genome browser view showing NSD1 and H3K36me2 occupancy in NSD1-dTAG mESCs. (C-L) Average profiles of indicated CUT&RUN and ATAC-seq signals in NSD1-dTAG mESCs at their respective peaks or all active genes (RPKM > 1) as indicated. Nsd1 KO2 cells used as control. (M) Scatterplots showing genome-wide correlation of H3K27me3 occupancy in NSD1-dTAG and Nsd1 KO2 mESCs. Gain of H3K27me3 was highlighted. (N) Mass spectrometry analysis of global DNA methylation. Data represent mean ± SD from n=3 independent culture. (O) Scatterplots showing genome-wide correlation of CpG methylation levels in NSD1-dTAG, WT and Nsd1 KO2 mESCs. Loss of CpG methylation was highlighted. See also Figure S4.

Figure 5.. Acute depletion of NSD1 leads to reduced gene transcription independent of its catalytic activity

(A) Schematic of SLAM-seq analysis of newly synthesized RNA following NSD1 degradation. (B) MA plots showing differential gene expression analysis in NSD1-dTAG mESCs. In (B), (G), (H), and (L), red dots represent genes significantly deregulated (q<0.05); Number of genes up- or down-regulated and percentage among all genes with detectable expression are shown; n=3 independent treatments. (C) Overlap of genes down-regulated by 6h of dTAG-13 treatment as in (B), genes bound by NSD1 at enhancers and/or TSS, and all super-enhancer proximal genes. (D) Western blots of whole-cell extracts from indicated mESCs. In (D) and (J), actin and H3 used as loading controls. (E) Magnitude of transcriptional downregulation upon dTAG-13 treatment. (F) Number of genes significantly down-regulated (q<0.05) upon dTAG-13 treatment. (G) and (H) MA plots showing differential gene expression analysis in indicated mESCs. (I) RT-qPCR of HBG1 expression in HEK293T cells expressing different combinations of dCas9 fusions. GAPDH used as internal reference. Data represent mean ± SD from n=3 technical replicates. DY, D1399Y. NQ, N1751Q. (J) Western blots of whole-cell extracts from SETD2-dTAG mESCs. (K) Average profiles of SETD2 and H3K36me3 CUT&RUN signal at active genes (RPKM > 1) in SETD2-dTAG mESCs. (L) MA plots showing differential gene expression analysis in SETD2-dTAG mESCs. See also Figure S5.

Figure 6.. NSD1 promotes enhancer activity and Pol II promoter pause release

(A) Magnitude of transcriptional downregulation (SLAM-seq) upon dTAG-13 treatment. (B) Average profile of Pol II S5P occupancy at active enhancers (n = 15986) and super-enhancers (n = 705) in NSD1-dTAG mESCs. (C) Genome browser view showing reduction of Pol II S5P occupancy at the Klf4 super-enhancer upon NSD1 degradation. (D) Average profile of total Pol II occupancy at TSS of active genes (RPKM > 1) in NSD1-dTAG mESCs. In (D), (F), and (H-K), TSS, transcription start site. (E) Empirical cumulative distribution function (ECDF) plot of Pol II pausing index following NSD1 loss. P values calculated using two-tailed t-test. (F) Average profile of Pol II S2P occupancy across active gene body and after TES. In (F-K), TES, transcript end site. (G) Western blots of whole-cell extracts from NSD1-dTAG mESCs. (H-K) Average profile showing occupancy of indicated factors (ChIP-seq) in NSD1-dTAG mESCs.. See also Figure S6.

Figure 7.. NSD1 facilitates activation of developmental transcriptional programs associated with Sotos syndrome pathogenesis

(A) Heatmap of RT-qPCR analysis of EB differentiation. In (A), (G), (I), (K) and (O), Rpl7 used as internal reference; Data represent mean from n=3 technical replicates. Color represents relative expression normalized to d0 in nontreated (NT) cells. Red bar indicates genes associated with cardiomyocyte differentiation. (B) Heatmap of differential gene expression by RNA-seq (|log2FC| > 2, q < 0.05). n = 3 independent differentiation. (C) Percentage of dTAG-13 down-regulated genes at EB day 6 (n = 164) that are up-regulated, down-regulated or stable during EB differentiation. (D) Gene ontology (GO) analysis of dTAG-13 down-regulated genes at EB day 6. (E) Percentage of cardiomyocytes in EBs by flow cytometric analysis. In (E) and (F), data represent mean ± SD from n=3 independent differentiation. In (E), (F), (H), (J), and (L), P values calculated using two-tailed t-test, ** P < 0.01, *** P < 0.001. (F) Percentage of contracting EBs. (G) Heatmap of RT-qPCR analysis of forebrain organoid differentiation using NSD1-dTAG EpiSCs. Color represents relative expression normalized to EpiSCs. (H) Boxplots showing read densities of indicated chromatin features at de novo enhancers (EBd6 vs. ESC) (n = 81) associated with EB d6 dTAG-13 down genes. (I) Heatmap of RT-qPCR analysis of EB differentiation using indicated mESCs. In (I) and (K), color represents relative expression normalized to d0 of Nsd1 KO2 cells. PP, qPHD-PWWP. (J) Percentage of contracting EBs. In (J) and (L), data represent mean ± SD from n=2 independent differentiation. (K) Heatmap of RT-qPCR analysis of EB differentiation using Nsd1 KO2 mESCs expressing NSD1 C with Sotos syndrome missense mutations. (L) Percentage of contracting EBs. (M) Western blots of whole-cell extracts from mESCs. H3 used as loading control. (N) Scatterplots showing genome-wide correlation of H3K36me2 occupancy. (O) Heatmap of RT-qPCR analysis of EB differentiation using indicated mESCs. Color represents relative expression normalized to d0 of WT E14 cells. See also Figure S6.