Chromatin inheritance entails de novo nucleosome assembly after DNA replication by chromatin assembly factor-1 (CAF-1). Yet direct knowledge about CAF-1’s histone binding mode and nucleosome assembly process is lacking. In this work, we report the crystal structure of human CAF-1 in the absence of histones and the cryo–electron microscopy structure of CAF-1 in complex with histones H3 and H4. One histone H3-H4 heterodimer is bound by one CAF-1 complex mainly through the p60 subunit and the acidic domain of the p150 subunit. We also observed a dimeric CAF-1–H3-H4 supercomplex in which two H3-H4 heterodimers are poised for tetramer assembly and discovered that CAF-1 facilitates right-handed DNA wrapping of H3-H4 tetramers. These findings signify the involvement of DNA in H3-H4 tetramer formation and suggest a right-handed nucleosome precursor in chromatin replication.

The SLX1–SLX4 structure-specific endonuclease complex is involved in processing diverse DNA damage intermediates, including resolution of Holliday junctions, collapse of stalled replication forks and removal of DNA flaps. The nuclease subunit SLX1 is inactive on its own, but become activated upon binding to SLX4 via its conserved C-terminal domain (CCD). Yet, how the SLX1–SLX4 complex recognizes specific DNA structure and chooses cleavage sites remains unknown. Here we show, through a combination of structural, biochemical and computational analyses, that the SAP domain of SLX4 is critical for efficient and accurate processing of 5′-flap DNA. It binds the minor groove of DNA about one turn away from the flap junction, and the 5′-flap is implicated in binding the core domain of SLX1. This binding mode accounts for specific recognition of 5′-flap DNA and specification of cleavage site by the SLX1–SLX4 complex.

PHF8-promoted non-histone methylation switch plays a critical role in replication stress response and genome integrity.

Chromosomal translocations of MLL1 (Mixed Lineage Leukemia 1) yield oncogenic chimeric proteins containing the N-terminal portion of MLL1 fused with distinct partners. The MLL1–AF10 fusion causes leukemia through recruiting the H3K79 histone methyltransferase DOT1L via AF10’s octapeptide and leucine zipper (OM-LZ) motifs. Yet, the precise interaction sites in DOT1L, detailed interaction modes between AF10 and DOT1L, and the functional configuration of MLL1–AF10 in leukeomogenesis remain unknown. Through a combined approach of structural and functional analyses, we found that the LZ domain of AF10 interacts with the coiled-coil domains of DOT1L through a conserved binding mode and discovered that the C-terminal end of the LZ domain and the OM domain of AF10 mediate the formation of a DOT1L–AF10 octamer via tetramerization of the binary complex. We reveal that the oligomerization ability of the DOT1L–AF10 complex is essential for MLL1–AF10’s leukemogenic function. These findings provide insights into the molecular basis of pathogenesis by MLL1 rearrangements.

TRIM71 is an RNA-binding protein with ubiquitin ligase activity. Numerous functions of mammalian TRIM71, including cell cycle regulation, embryonic stem cell (ESC) self-renewal, and reprogramming of pluripotent stem cells, are related to its RNA-binding property. We previously reported that a long noncoding RNA (lncRNA) Trincr1 interacts with mouse TRIM71 (mTRIM71) to repress FGF/ERK pathway in mouse ESCs (mESCs). Herein, we identify an RNA motif specifically recognized by mTRIM71 from Trincr1 RNA, and solve the crystal structure of the NHL domain of mTRIM71 complexed with the RNA motif. Similar to the zebrafish TRIM71, mTRIM71 binds to a stem-loop structured RNA fragment of Trincr1, and an adenosine base at the loop region is crucial for the mTRIM71 interaction. We map similar hairpin RNAs preferably bound by TRIM71 in the mRNA UTRs of the cell-cycle related genes regulated by TRIM71. Furthermore, we identify key residues of mTRIM71, conserved among mammalian TRIM71 proteins, required for the RNA-binding property. Single-site mutations of these residues significantly impair the binding of TRIM71 to hairpin RNAs in vitro and to mRNAs of Cdkn1a/p21 and Rbl2/p130 in mESCs. Furthermore, congenital hydrocephalus (CH) specific mutation of mTRIM71 impair its binding to the RNA targets as well. These results reveal molecular mechanism behind the recognition of RNA by mammalian TRIM71 and provide insights into TRIM71 related diseases.