The mixed lineage leukaemia (MLL) family of proteins (including MLL1–MLL4, SET1A and SET1B) specifically methylate histone 3 Lys4, and have pivotal roles in the transcriptional regulation of genes involved in haematopoiesis and development. The methyltransferase activity of MLL1, by itself severely compromised, is stimulated by the three conserved factors WDR5, RBBP5 and ASH2L, which are shared by all MLL family complexes. However, the molecular mechanism of how these factors regulate the activity of MLL proteins still remains poorly understood. Here we show that a minimized human RBBP5–ASH2L heterodimer is the structural unit that interacts with and activates all MLL family histone methyltransferases. Our structural, biochemical and computational analyses reveal a two-step activation mechanism of MLL family proteins. These findings provide unprecedented insights into the common theme and functional plasticity in complex assembly and activity regulation of MLL family methyltransferases, and also suggest a universal regulation mechanism for most histone methyltransferases.

Telomeres are nucleoprotein complexes that play essential roles in protecting chromosome ends. Mammalian telomeres consist of repetitive DNA sequences bound by the shelterin complex. In this complex, the POT1-TPP1 heterodimer binds to single-stranded telomeric DNAs, while TRF1 and TRF2-RAP1 interact with double-stranded telomeric DNAs. TIN2, the linchpin of this complex, simultaneously interacts with TRF1, TRF2, and TPP1 to mediate the stable assembly of the shelterin complex. However, the molecular mechanism by which TIN2 interacts with these proteins to orchestrate telomere protection remains poorly understood. Here, we report the crystal structure of the N-terminal domain of TIN2 in complex with TIN2-binding motifs from TPP1 and TRF2, revealing how TIN2 interacts cooperatively with TPP1 and TRF2. Unexpectedly, TIN2 contains a telomeric repeat factor homology (TRFH)-like domain that functions as a protein-protein interaction platform. Structure-based mutagenesis analyses suggest that TIN2 plays an important role in maintaining the stable shelterin complex required for proper telomere end protection.

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Summary Telomeres employ TRF2 to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), thereby repressing ATM-dependent DNA damage checkpoint responses. How TRF2 prevents MRN activation at dysfunctional telomeres is unclear. Here, we show that the phosphorylation status of NBS1 determines the repair pathway choice of dysfunctional telomeres. The crystal structure of the TRF2-NBS1 complex at 3.0 Å resolution shows that the NBS1 429YQLSP433 motif interacts specifically with the TRF2TRFH domain. Phosphorylation of NBS1 serine 432 by CDK2 in S/G2 dissociates NBS1 from TRF2, promoting TRF2-Apollo/SNM1B complex formation and the protection of leading-strand telomeres. Classical-NHEJ mediated repair of telomeres lacking TRF2 requires phosphorylated NBS1S432 to activate ATM, while interaction of de-phosphorylated NBS1S432 with TRF2 promotes alternative-NHEJ repair of telomeres lacking POT1-TPP1. Our work advances understanding how the TRF2TRFH domain orchestrates telomere end protection, and reveals how the phosphorylation status of the NBS1S432 dictates repair pathway choice of dysfunctional telomeres.

The Type III-E RNA-targeting effector complex (gRAMP/Cas7-11) is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here we use cryo-electron microscopy snapshots of Craspase to explain its target RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5′ region of the guide RNA displaces a gating loop in gRAMP, which triggers an extensive conformational relay that allosterically aligns the protease catalytic dyad and opens an amino acid sidechain-binding pocket. We further define Csx30 as the endogenous protein substrate that is site-specifically proteolyzed by RNA-activated Craspase. This protease activity is switched off by target RNA cleavage by gRAMP, and is not activated by RNA targets containing a matching protospacer flanking sequence. We thus conclude that Craspase is a target RNA-activated protease with self-regulatory capacity.

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Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about 2/5 the size of Cas9 but shares similar domain organization. The associated ωRNA plays the combined role of crRNA and tracrRNA to guide dsDNA cleavage. Here we report a 2.78 Å cryo-EM structure of IscB-ωRNA bound to dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 RNPs. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9, and contacts the RNA/DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.