How the naked mole-rat resists senescence: a constraints-based theory

Here, I present a theory describing how the stabilization of constraints imposed on chromatin dynamics by the naked mole-rat's histone H1.0 protein—which in terminally differentiated cells constrains the accessibility of the nucleosome core particle for histone-modifying enzymes and chromatin remodeling factors—explains its resistance to both senescence and cancer. Further, this theory predicts that a mutant house mouse displaying such stabilization will be similarly resistant to both senescence and cancer. A proof-of-concept computational analysis is presented and two predictions for the direct testing of the theory are provided. These experiments comprise, as test subjects, mutant naked mole-rats synthesizing a house mouse (Mus musculus)-like histone H1.0, and mutant house mice synthesizing a naked mole-rat-like histone H1.0. The predictions are that the constraints on chromatin dynamics embodied by the respective mutant histone H1.0 proteins will negate the otherwise significant resistance to both senescence and cancer of the naked mole-rats and, conversely, confer such resistance to the house mice. A verification of these predictions will imply that constraints on chromatin dynamics embodied by naked mole-rat-like histone H1.0 proteins may confer significant resistance to both senescence and age-related cancer to otherwise senescence-prone and/or cancer-susceptible multicellular species, including humans.

Mechanism for DPY30 and ASH2L intrinsically disordered regions to modulate the MLL/SET1 activity on chromatin

Recent cryo-EM structures show the highly dynamic nature of the MLL1-NCP (nucleosome core particle) interaction. Functional implication and regulation of such dynamics remain unclear. Here we show that DPY30 and the intrinsically disordered regions (IDRs) of ASH2L work together in restricting the rotational dynamics of the MLL1 complex on the NCP. We show that DPY30 binding to ASH2L leads to stabilization and integration of ASH2L IDRs into the MLL1 complex and establishes new ASH2L-NCP contacts. The significance of ASH2L-DPY30 interactions is demonstrated by requirement of both ASH2L IDRs and DPY30 for dramatic increase of processivity and activity of the MLL1 complex. This DPY30 and ASH2L-IDR dependent regulation is NCP-specific and applies to all members of the MLL/SET1 family of enzymes. We further show that DPY30 is causal for de novo establishment of H3K4me3 in ESCs. Our study provides a paradigm of how H3K4me3 is regulated on chromatin and how H3K4me3 heterogeneity can be modulated by ASH2L IDR interacting proteins.

MLL1 Methyltransferase Activity is Regulated by Distinct Nucleosome Binding Modes

Here we solve the single particle cryoEM structure for the MLL1 complex with nucleosome core particle (NCP) carrying histone H3 lysine 4 to methionine mutation. The MLL1 complex displays significant rotational dynamics on the NCP, a feature distinct from the yeast SET1 complex. We identified two major binding modes of the MLL1 complex on the NCP. Both binding modes anchor on the NCP through ASH2L, but they differ drastically with regard to where the MLL1 SET domain and RbBP5 bind. We show that one of the binding modes is catalytically inactive since disrupting interactions unique to this binding mode does not affect overall MLL1 activity in an NCP-specific manner. Interestingly, the inactive binding mode is in a configuration similar to that of the ySET1-NCP complex, which is intrinsically inactive on an unmodified NCP. The high rotational dynamics of the MLL1 complex as well as distinction between MLL and yeast SET1 complexes may reflect the necessity for loci-specific regulation of H3K4 methylation states in higher eukaryotes.

Nucleosome-CHD4 chromatin remodeler structure maps human disease mutations

Chromatin remodeling plays important roles in gene regulation during development, differentiation and in disease. The chromatin remodeling enzyme CHD4 is a component of the NuRD and ChAHP complexes that are involved in gene repression. Here, we report the cryo-electron microscopy (cryo-EM) structure of Homo sapiens CHD4 engaged with a nucleosome core particle in the presence of the non-hydrolysable ATP analogue AMP-PNP at an overall resolution of 3.1 Å. The ATPase motor of CHD4 binds and distorts nucleosomal DNA at superhelical location (SHL) +2, supporting the ‘twist defect’ model of chromatin remodeling. CHD4 does not induce unwrapping of terminal DNA, in contrast to its homologue Chd1, which functions in gene activation. Our structure also maps CHD4 mutations that are associated with human cancer or the intellectual disability disorder Sifrim-Hitz-Weiss syndrome.

A methyl-TROSY approach for NMR studies of high-molecular-weight DNA with application to the nucleosome core particle

The development of methyl-transverse relaxation-optimized spectroscopy (methyl-TROSY)–based NMR methods, in concert with robust strategies for incorporation of methyl-group probes of structure and dynamics into the protein of interest, has facilitated quantitative studies of high-molecular-weight protein complexes. Here we develop a one-pot in vitro reaction for producing NMR quantities of methyl-labeled DNA at the C5 and N6 positions of cytosine (5mC) and adenine (6mA) nucleobases, respectively, enabling the study of high-molecular-weight DNA molecules using TROSY approaches originally developed for protein applications. Our biosynthetic strategy exploits the large number of naturally available methyltransferases to specifically methylate DNA at a desired number of sites that serve as probes of structure and dynamics. We illustrate the methodology with studies of the 153-base pair Widom DNA molecule that is simultaneously methyl-labeled at five sites, showing that high-quality13C-1H spectra can be recorded on 100 μM samples in a few minutes. NMR spin relaxation studies of labeled methyl groups in both DNA and the H2B histone protein component of the 200-kDa nucleosome core particle (NCP) establish that methyl groups at 5mC and 6mA positions are, in general, more rigid than Ile, Leu, and Val methyl probes in protein side chains. Studies focusing on histone H2B of NCPs wrapped with either wild-type DNA or DNA methylated at all 26 CpG sites highlight the utility of NMR in investigating the structural dynamics of the NCP and how its histone core is affected through DNA methylation, an important regulator of transcription.

The human telomeric nucleosome displays distinct structural and dynamic properties

Telomeres protect the ends of our chromosomes and are key to maintaining genomic integrity during cell division and differentiation. However, our knowledge of telomeric chromatin and nucleosome structure at the molecular level is limited. Here, we aimed to define the structure, dynamics as well as properties in solution of the human telomeric nucleosome. We first determined the 2.2 Å crystal structure of a human telomeric nucleosome core particle (NCP) containing 145 bp DNA, which revealed the same helical path for the DNA as well as symmetric stretching in both halves of the NCP as that of the 145 bp ‘601’ NCP. In solution, the telomeric nucleosome exhibited a less stable and a markedly more dynamic structure compared to NCPs containing DNA positioning sequences. These observations provide molecular insights into how telomeric DNA forms nucleosomes and chromatin and advance our understanding of the unique biological role of telomeres.

Phase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle reveals differential flexibility of the DNA ends

The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one (‘left’) 601 DNA end is well ordered whereas the other (‘right’) end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.