W. as a repressor. SAMD1 tethers L3MBTL3 to chromatin and interacts with the KDM1A histone demethylase CPI-0610 carboxylic acid complex to modulate H3K4me2 and H3K4me3 levels at CGIs, thereby providing a mechanism for SAMD1-mediated transcriptional repression. The absence of SAMD1 impairs ES cell differentiation processes, leading to misregulation of key biological pathways. Together, our work establishes SAMD1 as a newly identified chromatin regulator acting at unmethylated CGIs. INTRODUCTION Vertebrate CpG islands (CGIs) are specific genomic regions characterized by the accumulation of CpG dinucleotides. They are commonly found at gene promoters and play important roles in gene regulation (= 8733). The heatmaps of the KO, CGI position, and DNA methylation (MeDIP-seq) are shown in comparison. (H) Venn diagram showing the overlap of SAMD1 peaks (blue) with all CGIs (green) and methylated CGIs (red). (I) Top enriched motif at SAMD1-bound versus unbound CGIs is usually obtained by HOMER. DAPI, 4,6-diamidino-2-phenylindole. To verify our in vitro findings, we decided the cellular localization and genomic binding loci of SAMD1 in vivo. SAMD1 is usually expressed to comparable levels in different mouse organs, IGFBP2 with the strongest expression shown in mouse ES cells (fig. S2A). Thus, we used mouse ES cells as the model system for further investigations. We generated SAMD1 knockout (KO) cells (fig. S2B), which proliferated normally without any obvious phenotype (fig. S2, C and D). Using a custom-made antibody, we found that endogenous SAMD1 is usually predominantly nuclear localized, with a substantial proportion associated with chromatin (Fig. 1, D and E), supporting a potential chromatin-related function. Subsequently, using chromatin immunoprecipitation sequencing (ChIP-seq), we identified 8733 significant peaks and discovered that they strongly ( 90%) overlap with nonmethylated CGIs but not with methylated CGIs (Fig. 1, F to H). The ChIP-seq signal was absent in SAMD1 KO cells, demonstrating the specificity of the antibody (Fig. 1, F and G). SAMD1 is usually highly enriched at some CGIs such as those of the genes, while it shows only a subtle or no binding to other CGIs, suggesting preferential binding to certain CGIs (Fig. 1F). Comparing the sequences CPI-0610 carboxylic acid of the SAMD1-bound versus the unbound CGIs, a GCGC-containing motif is usually enriched (Fig. 1I), consistent with the motif identified by the in vitro PBM (Fig. 1C). SAMD1s WH domain name interacts with CPI-0610 carboxylic acid the minor and major groove of DNA To address the molecular details CPI-0610 carboxylic acid of the conversation of SAMD1 with DNA, we solved the crystal structure of the SAMD1-WH domain name in complex with 5-GCGC-3Ccontaining double-stranded DNA (dsDNA) at a resolution of 1 1.78 ? (Table 1). Unlike a typical WH domain name that contains three strands and two wing-like loops (named W1 and W2) ((?)45.93, 45.93, 132.6469.34, 69.34, 181.8966.43, 182.84, 66.9767.75, 67.75, 293.39??, , ()90.00, 90.00, 120.0090.00, 90.00, 120.0090.00, 93.32, 90.0090.00, 90.00, 120.00Resolution (?)50.00C1.78/ 0.01. Four biological replicates were performed. (D) Occupancy of up- and down-regulated genes with SAMD1. (E) Promoter profile of SAMD1 at up- and down-regulated genes. (F) Gene set enrichment analysis (GSEA) of the top 100 SAMD1-bound genes, using the RNA-seq data. (G) Expression level of SAMD1-bound up- and down-regulated genes in comparison to all SAMD1-bound genes. The whisker-box plots represent the lower quartile, median, and upper quartile of the data with 5 and 95% whiskers. (H) Promoter profiles of H3K4me3 and H2K27me3 at up- and down-regulated genes. NES, Normalized Enrichment Score. To gather information about the functional role of SAMD1 at those genes, we performed RNA sequencing (RNA-seq) in SAMD1 KO versus WT ES cells. This experiment identified 524 significantly ( 0.01) down-regulated and 257 up-regulated genes (Fig. 3C). Further investigation showed that this up-regulated but not the down-regulated genes are strongly occupied by SAMD1 (Fig. 3, D and E), suggesting that direct targets of SAMD1 become derepressed upon SAMD1 deletion. We confirmed via gene set enrichment analysis (GSEA) that this 100 genes with the highest levels of SAMD1 are, on average, significantly up-regulated.

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