POT1 and TPP1 are part of the shelterin complex and are essential for telomere length regulation and maintenance. Naturally occurring mutations of the telomeric POT1–TPP1 complex are implicated in familial glioma, melanoma and chronic lymphocytic leukaemia. Here we report the atomic structure of the interacting portion of the human telomeric POT1–TPP1 complex and suggest how several of these mutations contribute to malignant cancer. The POT1 C-terminus (POT1C) forms a bilobal structure consisting of an OB-fold and a holiday junction resolvase domain. TPP1 consists of several loops and helices involved in extensive interactions with POT1C. Biochemical data shows that several of the cancer-associated mutations, partially disrupt the POT1–TPP1 complex, which affects its ability to bind telomeric DNA efficiently. A defective POT1–TPP1 complex leads to longer and fragile telomeres, which in turn promotes genomic instability and cancer.

RNase P protein subunits Rpp29, POP1, and Rpp21 interact with histone H3.3 upstream of nucleosome deposition, suggesting that a variant of this enzyme regulates H3.3 function. Rpp29 knockdown increases H3.3 chromatin incorporation, suggesting that it represses H3.3 nucleosome deposition, which has important implications for epigenetic regulation.

The incorporation of the histone H3 variant, H3.3, into chromatin by the H3.3-specific chaperone DAXX and the ATP-dependent chromatin remodeling factor ATRX is a critical mechanism for silencing repetitive DNA. DAXX and ATRX are also components of promyelocytic nuclear bodies (PML-NBs), which have been identified as sites of H3.3 chromatin assembly. Here, we use a transgene array that can be visualized in single living cells to investigate the mechanisms that recruit PML-NB proteins (i.e. PML, DAXX, ATRX, and SUMO-1, SUMO-2 and SUMO-3) to heterochromatin and their functions in H3.3 chromatin assembly. We show that DAXX and PML are recruited to the array through distinct SUMOylation-dependent mechanisms. Additionally, PML is recruited during S phase and its depletion increases H3.3 deposition. Since this effect is abrogated when PML and DAXX are co-depleted, it is likely that PML represses DAXX-mediated H3.3 chromatin assembly. Taken together, these results suggest that, at heterochromatin, PML-NBs coordinate H3.3 chromatin assembly with DNA replication, which has important implications for understanding how transcriptional silencing is established and maintained.

The regulators of mitochondrial cell death in cancer have remained elusive, hampering the development of new therapies. Here, we showed that protein isoforms of mitochondrial fission factor (MFF1 and MFF2), a molecule that controls mitochondrial size and shape, that is, mitochondrial dynamics, were overexpressed in patients with non-small cell lung cancer and formed homo-and heterodimeric complexes with the voltage-dependent anion channel-1 (VDAC1), a key regulator of mitochondrial outer membrane permeability. MFF inserted into the interior hole of the VDAC1 ring using Arg225, Arg236, and Gln241 as key contact sites. A cell-permeable MFF Ser223-Leu243 D-enantiomeric peptidomimetic disrupted the MFF-VDAC1 complex, acutely depolarized mitochondria, and triggered cell death in heterogeneous tumor types, including drug-resistant melanoma, but had no effect on normal cells. In preclinical models, treatment with the MFF peptidomimetic was well-tolerated and demonstrated anticancer activity in patientderived xenografts, primary breast and lung adenocarcinoma 3D organoids, and glioblastoma neurospheres. These data identify the MFF-VDAC1 complex as a novel regulator of mitochondrial cell death and an actionable therapeutic target in cancer.Significance: These findings describe mitochondrial fission regulation using a peptidomimetic agent that disturbs the MFF-VDAC complex and displays anticancer activity in multiple tumor models.

The human CST (Ctc1, Stn1 and Ten1) complex binds the telomeric overhang and regulates telomere length by promoting C-strand replication and inhibiting telomerase-dependent G-strand synthesis. Structural and biochemical studies on the human Stn1 and Ten1 complex revealed its mechanism of assembly and nucleic acid binding. However, little is known about the structural organization of the multi-domain Ctc1 protein and how each of these domains contribute to telomere length regulation. Here, we report the structure of a central domain of human Ctc1. The structure reveals a canonical OB-fold with the two identified disease mutations (R840W and V871M) contributing to the fold of the protein. In vitro assays suggest that although this domain is not contributing directly to Ctc1’s substrate binding properties, it affects full-length Ctc1 localization to telomeres and Stn1-Ten1 binding. Moreover, functional assays show that deletion of the entire OB-fold domain leads to significant increase in telomere length, frequency of internal single G-strands and fragile telomeres. Our findings demonstrate that a previously unknown OB-fold domain contributes to efficient Ctc1 telomere localization and chromosome end maintenance.

The histone H3 variant H3.3 is a highly conserved and dynamic regulator of chromatin organization. Therefore, fully elucidating its nucleosome incorporation mechanisms is essential to understanding its functions in epigenetic inheritance. We previously identified the RNase P protein subunit, Rpp29, as a repressor of H3.3 chromatin assembly. Here, we use a biochemical assay to show that Rpp29 interacts with H3.3 through a sequence element in its own N terminus, and we identify a novel interaction with histone H2B at an adjacent site. The fact that archaeal Rpp29 does not include this N-terminal region suggests that it evolved to regulate eukaryote-specific functions. Oncogenic H3.3 mutations alter the H3.3-Rpp29 interaction, which suggests that they could dysregulate Rpp29 function in chromatin assembly. We also used KNS42 cells, an H3.3(G34V) pediatric high-grade glioma cell line, to show that Rpp29 1) represses H3.3 incorporation into transcriptionally active protein-coding, rRNA, and tRNA genes; 2) represses mRNA, protein expression, and antisense RNA; and 3) represses euchromatic post-translational modifications (PTMs) and promotes heterochromatic PTM deposition ( histone H3 Lys-9 trimethylation (H3K9me3) and H3.1/2/3K27me3). Notably, we also found that K27me2 is increased and K36me1 decreased on H3.3(G34V), which suggests that Gly-34 mutations dysregulate Lys-27 and Lys-36 methylation in The fact that Rpp29 represses H3.3 chromatin assembly and sense and antisense RNA and promotes H3K9me3 and H3K27me3 suggests that Rpp29 regulates H3.3-mediated epigenetic mechanisms by processing a transcribed signal that recruits H3.3 to its incorporation sites.

<p>Mutant peptides derived from MFF1 peptide #8 used in this study</p>

<p>Morphology of MFF targeting in tumor cells</p>