The NSD family histone methyltransferases, including NSD1, NSD2 and NSD3, play crucial roles in chromatin regulation and are implicated in oncogenesis 1 , 2 . NSD enzymes exhibit an auto-inhibitory state that is relieved by nucleosome engagement, allowing for H3K36 di-methylation catalysis 3 – 7 . However, the molecular basis underlying this mechanism is largely unknown. Here, we have solved the cryo-EM structures of NSD2 and NSD3 bound to mononucleosomes at atomic resolution. We find that NSD2/3 mononucleosome engagement causes DNA near the linker region to unwrap, which facilitates insertion of their catalytic core in-between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between the nucleosome and NSD2/3 precisely define the enzymes’ position on the nucleosome, explaining the methylation specificity for H3K36. Further, NSD-nucleosome intermolecular contacts are altered by several recurrent cancer-associated NSD2/3 mutations. NSDs harboring these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes cancer cell proliferation and xenograft tumor growth. Together, our research provides molecular insights into the nucleosome-based recognition and modification mechanisms of NSD2 and NSD3, which should uncover strategies for therapeutic targeting of the NSD family of proteins.

CpG islands (CGIs) are key regulatory DNA elements at most promoters, but how they influence the chromatin status and transcription remains elusive. Here, we identify and characterize SAMD1 (SAM domain-containing protein 1) as an unmethylated CGI-binding protein. SAMD1 has an atypical winged-helix domain that directly recognizes unmethylated CpG-containing DNA via simultaneous interactions with both the major and the minor groove. The SAM domain interacts with L3MBTL3, but it can also homopolymerize into a closed pentameric ring. At a genome-wide level, SAMD1 localizes to H3K4me3-decorated CGIs, where it acts as a repressor. SAMD1 tethers L3MBTL3 to chromatin and interacts with the KDM1A histone demethylase 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.

The lysine acetyltransferase KAT6A (MOZ, MYST3) belongs to the MYST family of chromatin regulators, facilitating histone acetylation. Dysregulation of KAT6A has been implicated in developmental syndromes and the onset of acute myeloid leukemia (AML). Previous work suggests that KAT6A is recruited to its genomic targets by a combinatorial function of histone binding PHD fingers, transcription factors and chromatin binding interaction partners. Here, we demonstrate that a winged helix (WH) domain at the very N-terminus of KAT6A specifically interacts with unmethylated CpG motifs. This DNA binding function leads to the association of KAT6A with unmethylated CpG islands (CGIs) genome-wide. Mutation of the essential amino acids for DNA binding completely abrogates the enrichment of KAT6A at CGIs. In contrast, deletion of a second WH domain or the histone tail binding PHD fingers only subtly influences the binding of KAT6A to CGIs. Overexpression of a KAT6A WH1 mutant has a dominant negative effect on H3K9 histone acetylation, which is comparable to the effects upon overexpression of a KAT6A HAT domain mutant. Taken together, our work revealed a previously unrecognized chromatin recruitment mechanism of KAT6A, offering a new perspective on the role of KAT6A in gene regulation and human diseases.

SignificanceThe polymerase-associated factor 1 (PAF1) complex is a general transcription elongation factor of RNA polymerase II, which not only regulates various stages of the transcription cycle but also broadly influences gene expression through modulating chromatin structure and/or recruiting other transcription-related factors. This study presents a high-resolution crystal structure of the core region of the Paf1-Ctr9-Cdc73 ternary complex, which not only greatly facilitates our understanding of the overall architecture of the Paf1 complex but also provides a structure-based platform for understanding the molecular mechanism underlying the role of the Paf1 complex in regulating gene expression and sheds light toward deciphering the impact of its mutational spectrum on human diseases.

The fatty acid synthase type II (FAS-II) multienzyme system is the main target of drugs to inhibit mycolic acid synthesis in mycobacterium. Meromycolate extension acyl carrier protein (AcpM) serves as the carrier of fatty acyl chain shuttling among the individual FAS-II components during the progression of fatty acid elongation. In this paper, MSMEG_5634 in Mycobacterium smegmatis was determined to be a helix-grip structure protein with a deep hydrophobic pocket, preferring to form a complex with acyl-AcpM containing a fatty acyl chain at the C36-52 length, which is the medium product of FAS-II. MSMEG_5634 interacted with FAS-II components and presented relative accumulation at the cellular pole. By forming the MSMEG_5634/acyl-AcpM complex, which is free from FAS-II, MSMEG_5634 could transport acyl-AcpM away from FAS-II. Deletion of the MSMEG_5634 gene in M. smegmatis resulted in a mutant with decreased sensitivity to isoniazid and triclosan, two inhibitors of the FAS-II system. The isoniazid and triclosan sensitivity of this mutant could be restored by the ectopic expression of MSMEG_5634 or Rv0910, the MSMEG_5634 homologous protein in Mycobacterium tuberculosis H37Rv. These results suggest that MSMEG_5634 and its homologous proteins, forming a novel acyl-AcpM-binding protein family in mycobacterium, confer intrinsic sensitivity to FAS-II inhibitors.