Mutations in the X-linked MECP2 gene cause the progressive neurodevelopmental disorder Rett syndrome. Pathogenic missense mutation hotspots exist in the protein's Methyl DNA binding Domain (MBD), and the Nuclear receptor Co-Repressor (NCoR) Interaction Domain (NID), indicating these regions as critical for MeCP2 function. The NID binds to a co-repressor complex allowing transcriptional repression at target genes. A putative RNA Binding Domain (RBD) was identified that overlaps with the NID, yet the role that RNA interaction plays in MeCP2 function remains underexplored. Using cell-based and in vitro molecular assays, we validated RNA interaction at the NID/RBD of MeCP2 both to a dsRNA probe in vitro and to the lncRNA NEAT1_2 in cells. As expected, this region did not appear to affect MeCP2-chromatin interactions; however, we found that RNA-RBD interaction precludes MeCP2-NCoR binding in cells. Taken together, we find that RNA interaction at this non-canonical RNA binding domain regulates important MeCP2-protein interactions and therefore may be a key part of the pathophysiology of Rett syndrome.

Histone H4K16 acetylation (H4K16ac) is a key epigenetic mark essential for chromatin structure and DNA repair, which is substantially reduced in the accelerated aging disorder Hutchinson-Gilford progeria syndrome (HGPS). The specific enzymes governing H4K16ac homeostasis, particularly the deacetylase responsible for its loss in HGPS, remain poorly defined. Here, we sought to identify the enzymes regulating H4K16ac and determine if their inhibition could rescue HGPS-associated cellular defects. Using systematic siRNA screening in HeLa and U2OS cells, we confirmed that KAT8/MOF is the principal H4K16 acetyltransferase. Surprisingly, we identified HDAC2 as the dominant class I histone deacetylase for H4K16ac; knockdown of the highly homologous HDAC1 had no effect. While SIRT1 knockdown also increased H4K16ac, its contribution was minimal in HGPS vascular smooth muscle cells (VSMCs) compared to HDAC2. Crucially, selective pharmacological inhibition of HDAC2, but not SIRT1, robustly restored H4K16ac levels in HGPS VSMCs. This restoration led to a significant rescue of premature aging phenotypes, including improvements in nuclear morphology, preservation of proliferative capacity (Ki67) at late passages, and a significant reduction in cellular senescence. The effects of HDAC2 inhibition on cellular senescence and nuclear morphology suggests that HDAC2-mediated histone deacetylation contributes directly to the pathological features of HGPS, extending the functional impact of HDAC2 inhibition beyond DNA repair defects to fundamental aspects of cellular aging. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45, MOP-133570, PJT-178364

Background Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disorder driven by the accumulation of progerin, leading to severe vascular pathology. While epigenetic alterations are implicated, the spatiotemporal reorganization of the higher-order chromatin and its functional impact on vascular smooth muscle cell (VSMC) transcription remain poorly defined. Results Through an integrated multi-omics approach combining in situ high-throughput chromosome conformation capture (Hi-C) and Cleavage Under Targets and Tagmentation (CUT&Tag) profiling of CTCF, SMC1A, H3K27me3, H3K27ac, and H3K36me3 with transcriptomic analyses from control and HGPS iPSC-derived VSMCs, we reveal that global topologically associating domain (TAD) architecture remains largely intact in HGPS. However, the internal chromatin states of TADs undergo dynamic, passage-specific remodeling, characterized by a progressive accumulation of broad H3K27me3-repressed domains. This is accompanied by a loss of A/B compartment segregation, as confirmed by DNA-FISH, indicating a collapse of higher-order chromatin organization in late passage. Crucially, we uncover widespread rewiring of enhancer-promoter (E-P) loops, which is linked to the dysregulation of genes critical for vascular development, extracellular matrix organization, and atherosclerosis. Conclusions Our study demonstrates that spatiotemporal redistribution of repressive histone marks and reorganization of E-P interactions within a structurally resilient TAD framework underpin widespread transcriptional dysregulation in HGPS vascular pathogenesis. This uncovers a critical dissociation between higher-order chromatin architecture and histone modification landscape, providing a mechanistic basis for the failure of vascular homeostasis in progeria. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45

Hutchinson-Gilford Progeria syndrome (HGPS) is a lethal premature aging disorder caused by a de novo heterozygous mutation that leads to the accumulation of a splicing isoform of Lamin A termed progerin. Progerin expression deregulates the organization of the nuclear lamina and the epigenetic landscape. Progerin has also been observed to accumulate at low levels during normal aging in cardiovascular cells of adults that do not carry genetic mutations linked with HGPS. Therefore, the molecular mechanisms that lead to vascular dysfunction in HGPS may also play a role in vascular aging-associated diseases, such as myocardial infarction and stroke. Here, we show that HGPS patient-derived vascular smooth muscle cells (VSMCs) recapitulate HGPS molecular hallmarks. Transcriptional profiling revealed cardiovascular disease remodeling and reactive oxidative stress response activation in HGPS VSMCs. Proteomic analyses identified abnormal acetylation programs in HGPS VSMC replication fork complexes, resulting in reduced H4K16 acetylation. Analysis of acetylation kinetics revealed both upregulation of K16 deacetylation and downregulation of K16 acetylation. This correlates with abnormal accumulation of error-prone nonhomologous end joining (NHEJ) repair proteins on newly replicated chromatin. The knockdown of the histone acetyltransferase MOF recapitulates preferential engagement of NHEJ repair activity in control VSMCs. Additionally, we find that primary donor-derived coronary artery vascular smooth muscle cells from aged individuals show similar defects to HGPS VSMCs, including loss of H4K16 acetylation. Altogether, we provide insight into the molecular mechanisms underlying vascular complications associated with HGPS patients and normative aging.

Mutations in methyl-CpG binding protein 2 (MeCP2), such as the T158M, P152R, R294X, and R306C mutations, are responsible for most Rett syndrome (RTT) cases. These mutations often result in altered protein expression that appears to correlate with changes in the nuclear size; however, the molecular details of these observations are poorly understood. Using a C2C12 cellular system expressing human MeCP2-E1 isoform as well as mouse models expressing these mutations, we show that T158M and P152R result in a decrease in MeCP2 protein, whereas R306C has a milder variation, and R294X resulted in an overall 2.5 to 3 fold increase. We also explored the potential involvement of the MeCP2 PEST domains in the proteasome-mediated regulation of MeCP2. Finally, we used the R294X mutant to gain further insight into the controversial competition between MeCP2 and histone H1 in the chromatin context. Interestingly, in R294X, MeCP2 E1 and E2 isoforms were differently affected, where the E1 isoform contributes to much of the overall protein increase observed, while E2 decreases by half. The modes of MeCP2 regulation, thus, appear to be differently regulated in the two isoforms.

In higher eukaryotic cells, a string of nucleosomes, where long genomic DNA is wrapped around core histones, are rather irregularly folded into a number of condensed chromatin domains, which have been revealed by super-resolution imaging and Hi-C technologies. Inside these domains, nucleosomes fluctuate and locally behave like a liquid. The behavior of chromatin may be highly related to DNA transaction activities such as transcription and repair, which are often upregulated in cancer cells. To investigate chromatin behavior in cancer cells and compare those of cancer and non-cancer cells, we focused on oncogenic- HRAS (Gly12Val)-transformed mouse fibroblasts CIRAS-3 cells and their parental 10T1/2 cells. CIRAS-3 cells are tumorigenic and highly metastatic. First, we found that HRAS-induced transformation altered not only chromosome structure, but also nuclear morphology in the cell. Using single-nucleosome imaging/tracking in live cells, we demonstrated that nucleosomes are locally more constrained in CIRAS-3 cells than in 10T1/2 cells. Consistently, heterochromatin marked with H3K27me3 was upregulated in CIRAS-3 cells. Finally, Hi-C analysis showed enriched interactions of the B-B compartment in CIRAS-3 cells, which likely represents transcriptionally inactive chromatin. Increased heterochromatin may play an important role in cell migration, as they have been reported to increase during metastasis. Our study also suggests that single-nucleosome imaging provides new insights into how local chromatin is structured in living cells.

AbstractA defining feature of constitutive heterochromatin compartments is the heterochromatin protein-1 (HP1) family, whose members display fast internal mobility and rapid exchange with the surrounding nucleoplasm. Here, we describe a paradoxical state for the lysine methyltransferase KMT5C characterized by rapid internal diffusion but minimal nucleoplasmic exchange. This retentive behavior is conferred by sparse sequence features that constitute two modules tethered by an intrinsically disordered linker. While both modules harbor variant HP1 interaction motifs, the first comprises adjacent sequences that increase affinity using avidity. The second motif increases HP1 effective concentration to further enhance affinity in a context-dependent manner, which is evident using distinct heterochromatin recruitment strategies and heterologous linkers with defined conformational ensembles. Despite the linker sequence being highly divergent, it is under evolutionary constraint for functional length, suggesting conformational buffering can support cooperativity between modules across distant orthologs. Overall, we show that KMT5C has evolved a robust tethering strategy that uses minimal sequence determinants to harness highly dynamic HP1 proteins for retention within heterochromatin compartments.

PDF file - 46KB, Table S2: Effect of different fixative agents on BAP1 localization in cells after DNA damage. U2OS cells were left untreated or exposed to IR (2 Gy) or UV light for 2 min (60 J/min). Cells were allowed to recover for 1 hr before fixation as outlined in the above table and processed for immunostaining as described in the material and methods. Results are summarized in the above the table. Methanol, Met. Paraformaldehyde, PFA. Acetone, Acet.

PDF file - 46KB, Figure S2: ChIP analysis demonstrates enrichment of GFP-BAP1 WT but not GFP-BAP1-ΔUCH across reporter locus compared to IgG control. (A) U2OS cells containing the stably integrated reporter were co-transfected with GFP alone, GFP-BAP1 WT or GFP-BAP1-ΔUCH and Fok1 constructs for 24 hr. Cells were then fixed and subjected to chromatin immunoprecipitation (ChIP) using GFP or IgG antibodies. ch7 represents negative control locus on chromosome 7. Data are from two independent experiments. SEM represent standard error of the mean. (B) Knock down of BAP1 in cells using BAP1 specific shRNA. U2OS cells were transfected with control (Ctr) or one of two different BAP1 shRNA (BAP1 shRNA 1 and BAP1 shRNA2) for 48 hr. Nuclear extracts were prepared and immunoblotting was performed using the indicated antibodies. (C) Knockdown of ASXL1 in U2OS cells. U2OS cells were transfected with control or a ASXL1 shRNA for 48 h. Nuclear extracts were prepared and immunoblotted using ASXL1 and BAP1 antibodies. Actin was used as a loading control. (D) Association of BAP1 and ASXL1 is not altered in response to DNA damage. U2OS cells were left untreated or exposed to radiation (6 Gy) and allowed to recover for 2 hr. Nuclear extracts were prepared and immunoprecipitation was conducted with BAP1 or ASXL1 antibodies. Flow through (FT) and elute (El) and 20% of the input was immunoblotted as indicated.

PDF file - 70KB, Figure S1: The specificity of the BAP1 antibody used in this study. (A) Immunostaining of U2OS cells transfected with control (Ctr) or BAP1 specific shRNA (BAP1 shRNA). Cells were transfected with the indicated shRNA for 48 hr and immunostained with BAP1 antibody and counterstained with DAPI to reveal DNA. (B) Transfection of the FokI nuclease fusion construct into U2OS cells containing the stably integrated reporter leads to local accumulation of damage response proteins γH2AX and GFP-BAP1. 24 hours after co-transfection of GFP-BAP1 and Fok1 constructs, cells were immunostained with γH2AX antibody and counterstained with DAPI to reveal DNA. GFP-BAP1 enriches at the sites of DSBs. Arrows indicate the enrichment of proteins at sites of DSBs. Bars, 5 microm.

Transmission electron microscopy (TEM) has been essential in defining the structural organization of the cell due to its ability to image cell structures at molecular resolution. However, the absence of colour has made it very difficult to compare the distributions and relationships of two or more types of biomolecules simultaneously if they lack clear morphological distinctions. Furthermore, single-channel information limits functional analysis, particularly in the nucleoplasm, where fibrillar material could be chromatin, ribonucleic acid or protein. Where specific stains exist to discriminate among these molecules, they cannot be combined because conventional TEM is a single-channel technology. A potential path around this barrier is through electron spectroscopic imaging (ESI). ESI can map the distributions of chemical elements within an ultrathin section. Here, we present methods to stain specific molecules with elements that ESI can visualize to enable multichannel electron microscopy.

PDF file - 35KB, Figure S4. Immunoblot analysis of PARP1/2 expression. (A) U2OS cells were transfected with control (Ctr) or PARP1 siRNA for 48 hr. Nuclear extracts were prepared and immunoblotting was performed using the indicated antibodies. (B) Knockdown of PARP2 in U2OS cells. U2OS cells were transfected with control or PARP2 siRNA for 48 h. Nuclear extracts were prepared and immunoblotted using PARP2 antibody. Actin was used as a loading control. (C) PR-DUB complex does not bind PAR in vitro. SDS-PAGE of 100 ng purified GST-BAP1, GST-ASXL1 or BAP1/ASXL1 blotted onto a nitrocellulose membrane and incubated in 250 nM 32P-labeled PAR in TBS-T. As a control, histones and BSA were included as a positive and a negative control respectively.

Zinc finger (ZNF) motifs are some of the most frequently occurring domains in the human genome. It was only recently that ZNF proteins emerged as key regulators of genome integrity in mammalian cells. In this study, we report a new role for the Krüppel-type ZNF-containing protein ZNF432 as a novel poly(ADP-ribose) (PAR) reader that regulates the DNA damage response. We show that ZNF432 is recruited to DNA lesions via DNA- and PAR-dependent mechanisms. Remarkably, ZNF432 stimulates PARP-1 activity in vitro and in cellulo. Knockdown of ZNF432 inhibits phospho-DNA-PKcs and increases RAD51 foci formation following irradiation. Moreover, purified ZNF432 preferentially binds single-stranded DNA and impairs EXO1-mediated DNA resection. Consequently, the loss of ZNF432 in a cellular system leads to resistance to PARP inhibitors while its overexpression results in sensitivity. Taken together, our results support the emerging concept that ZNF-containing proteins can modulate PARylation, which can be embodied by the pivotal role of ZNF432 to finely balance the outcome of PARPi response by regulating homologous recombination.

Methyl CpG Binding Protein 2 (MeCP2) is a vertebrate DNA Reader that, through protein-protein interactions, participates in several molecular processes, such as regulation of transcription, chromatin organization, mRNA splicing, and miRNA biogenesis. However, the mechanism(s) that regulate these causative interactions in differing cellular contexts is/are unclear. Recently, a novel RNA Binding Domain (RBD) was identified in MeCP2, which lacks homology to well known “canonical” RBDs. Interestingly, the MeCP2 RBD represents a hotspot of missense mutations which cause the neurodevelopmental disorder known as Rett Syndrome, suggesting functional significance to this domain. Characterizing the molecular interactions with this novel, non-canonical RNA binding domain will help close the knowledge gap of how MeCP2 participates in myriad processes, and will contribute to the development of tailored therapies of MeCP2-related disorders, through understanding the downstream effects of MeCP2-RNA interaction. Here, Wild Type and RBD mutant MeCP2 proteins were expressed in human cells and in vitro. Biochemical methodologies including enzymatic chromatin fractionation as well as phase separation, in addition to immunoprecipitation and molecular docking were used to begin defining the type of RNA interaction occurring at the RBD of MeCP2, and to explore the biological implications of this interaction. I have shown that MeCP2-RNA and not MeCP2-chromatin interaction is mediated by its RNA Binding Domain, and I validated binding to a candidate RNA target, the lncRNA NEAT1L, in human cells. I then began to map where MeCP2 binds on the NEAT1L transcript, to postulate the structure- or sequence-specificity of MeCP2 for RNA. Finally, Co-immunoprecpitation data indicate that MeCP2-RNA interaction occurring at the RBD, directly and indirectly impacts MeCP2-protein interaction with two of its important transcriptional regulation complex partners, respectively. The elusive functional behaviour of MeCP2 has burdened researchers and families of children with MeCP2-related disorders world-wide for decades. I suggest that RNA interaction of the non-canonical RBD of MeCP2 may be one of the missing links to understanding and overcoming its related pathologies. The data from this proposal also represents a step towards understanding these emerging non-canonical RNA binding domains as well as a framework for determining RNA-based MeCP2 targets.

Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggest only modest compaction differences (∼2-10×) between the active nuclear compartment (ANC) and inactive nuclear compartment (INC). Here, we present maps of nuclear landscapes with true-to-scale DNA densities, ranging from <5 to >300 Mbp/μm3. Maps are generated from individual human and mouse cell nuclei with single-molecule localization microscopy at ∼20 nm lateral and ∼100 nm axial optical resolution and are supplemented by electron spectroscopic imaging. Microinjection of fluorescent nanobeads with sizes corresponding to macromolecular assemblies for transcription into nuclei of living cells demonstrates their localization and movements within the ANC and exclusion from the INC.

Poly(ADP-ribosylation) (PARylation) by poly(ADP-ribose) polymerases (PARPs) is a highly regulated process that consists of the covalent addition of polymers of ADP-ribose (PAR) through post-translational modifications of substrate proteins or non-covalent interactions with PAR via PAR binding domains and motifs, thereby reprogramming their functions. This modification is particularly known for its central role in the maintenance of genomic stability. However, how genomic integrity is controlled by an intricate interplay of covalent PARylation and non-covalent PAR binding remains largely unknown. Of importance, PARylation has caught recent attention for providing a mechanistic basis of synthetic lethality involving PARP inhibitors (PARPi), most notably in homologous recombination (HR)-deficient breast and ovarian tumors. The molecular mechanisms responsible for the anti-cancer effect of PARPi are thought to implicate both catalytic inhibition and trapping of PARP enzymes on DNA. However, the relative contribution of each on tumor-specific cytotoxicity is still unclear. It is paramount to understand these PAR-dependent mechanisms, given that resistance to PARPi is a challenge in the clinic. Deciphering the complex interplay between covalent PARylation and non-covalent PAR binding and defining how PARP trapping and non-trapping events contribute to PARPi anti-tumour activity is essential for developing improved therapeutic strategies. With this perspective, we review the current understanding of PARylation biology in the context of the DNA damage response (DDR) and the mechanisms underlying PARPi activity and resistance.

PDF file - 41KB, Figure S3. Time-lapse microscopy of GFP-BAP1 after laser micro-irradiation in: (A) U2OS cells treated with DMSO or ATM specific inhibitor (ATMi)( 10 uM) for 1 hr. (B) ATM deficient cells (EBS) or ATM deficient cells reconstituted with functional ATM (YZ5). Data are average of two independent experiments (n=15). Error bars represent stand error of the SEM. (C) Inhibition of ATM activity in cells. Immunostaining of U2OS cells treated with DMSO or ATM inhibitor (ATMi) (10 uM) for 1 hr. Cells were then irradiated with 2 Gy, allowed to recover for 30 min and immunostained with gammaH2AX antibody (as an example of ATM substrate) and counterstained with DAPI to reveal DNA. Bars, 5 microm.

PDF file - 47KB, Figure S6. BRCA1 siRNA efficiently knocked down BRCA1 in cells. (A) U2OS cells were transfected with control (Ctr) siRNA or BRCA1 siRNA for 48 hr. Nuclear extracts were prepared from these cells, and BRCA1 expression was determined by immunoblotting using BRCA1 specific antibody. As a loading control, membranes were probed for actin. (B) The specificity of the BRCA1 antibody used in this study. Immunostaining of U2OS cells transfected with control (Ctr) or BRCA1 specific siRNA (BRCA1 siRNA). Cells were transfected with the indicated siRNA for 48 hr. Cells were then irradiated with 2 Gy, allowed to recover for 1 hr and immunostained with BRCA1 antibody and counterstained with DAPI to reveal DNA. (C) Immunostaining of BMI1 and 53BP1 in response to DNA damage. U2OS were transfected with control (Ctr shRNA) or BAP1 specific shRNA (BAP1 shRNA) for 48 hr. Cell were then irradiated with 2 Gy of IR and left to recover for 30 min. Cells were immunostained with BMI1 and 53BP1 antibodies and counterstained with DAPI to reveal DNA. Bars, 5 microm.

PDF file - 39KB, Figure S5. Effect of MG132 on BAP1 recruitment to sites of DNA damage. (A) U2OS cells expressing GFP-BAP1 were either treated with DMSO or MG132 (1 uM) for 1 hr. Cells were monitored after micro-irradiation using time-lapse microscopy. The integrated intensity in the micro-irradiated areas was determined (n = 15) and the percentage of maximum value was plotted versus time. SEM of at least two independent experiments. (B) Flow cytometer profiles of the experiment done in figure 6A and B.

Cells assemble compartments around DNA double-strand breaks (DSBs). The assembly of this compartment is dependent on the phosphorylation of histone H2AX, the binding of MDC1 to phosphorylated H2AX, and the assembly of downstream signaling and repair components. The decision on whether to use homologous recombination or nonhomologous end-joining repair depends on competition between 53BP1 and BRCA1. A major point of control appears to be DNA replication and associated changes in the epigenetic state. This includes dilution of histone H4 dimethylation and an increase in acetylation of lysine residues on H2A and H4 that impair 53BP1 binding. In this article, we examined more closely the spatial relationship between 53BP1 and BRCA1 within the cell cycle. We find that 53BP1 can associate with early S-phase replicated chromatin and that the relative concentration of BRCA1 in DSB-associated compartments correlates with increased BRCA1 nuclear abundance as cells progress into and through S phase. In most cases during S phase, both BRCA1 and 53BP1 are recruited to these compartments. This occurs for both IR-induced DSBs and breaks targeted to an integrated LacO array through a LacI-Fok1-mCherry fusion protein. Having established that the array system replicates this heterogeneity, we further examined the spatial relationship between DNA repair components. This enabled us to precisely locate the DNA containing the break and map other proteins relative to that DNA. We find evidence for at least three subcompartments. The damaged DNA, single-stranded DNA generated from end resection of the array, and nuclease CtIP all localized to the center of the compartment. BRCA1 and 53BP1 largely occupied discrete regions of the focus. One of BRCA1 or 53BP1 overlaps with the array, while the other is more peripherally located. The array-overlapping protein occupied a larger volume than the array, CtIP, or single-stranded DNA (ssDNA). Rad51 often occupied a much larger volume than the array itself and was sometimes observed to be depleted in the array volume where the ssDNA exclusively localizes. These results highlight the complexity of molecular compartmentalization within DSB repair compartments.