Condensed Chromatin Behaves like a Solid on the Mesoscale In Vitro and in Living Cells.

The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.

Chromatin organization and behavior in HRAS-transformed mouse fibroblasts.

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.

ZNF432 stimulates PARylation and inhibits DNA resection to balance PARPi sensitivity and resistance.

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.

The Interchromatin Compartment Participates in the Structural and Functional Organization of the Cell Nucleus.

This article focuses on the role of the interchromatin compartment (IC) in shaping nuclear landscapes. The IC is connected with nuclear pore complexes (NPCs) and harbors splicing speckles and nuclear bodies. It is postulated that the IC provides routes for imported transcription factors to target sites, for export routes of mRNA as ribonucleoproteins toward NPCs, as well as for the intranuclear passage of regulatory RNAs from sites of transcription to remote functional sites (IC hypothesis). IC channels are lined by less-compacted euchromatin, called the perichromatin region (PR). The PR and IC together form the active nuclear compartment (ANC). The ANC is co-aligned with the inactive nuclear compartment (INC), comprising more compacted heterochromatin. It is postulated that the INC is accessible for individual transcription factors, but inaccessible for larger macromolecular aggregates (limited accessibility hypothesis). This functional nuclear organization depends on still unexplored movements of genes and regulatory sequences between the two compartments.

Domain analysis of PNKP-XRCC1 interactions: Influence of genetic variants of XRCC1.

Polynucleotide kinase/phosphatase (PNKP) and X-ray repair cross-complementing 1 (XRCC1) are key proteins in the single-strand DNA break repair pathway. Phosphorylated XRCC1 stimulates PNKP by binding to its forkhead-associated (FHA) domain, whereas nonphosphorylated XRCC1 stimulates PNKP by interacting with the PNKP catalytic domain. Here, we have further studied the interactions between these two proteins, including two variants of XRCC1 (R194W and R280H) arising from single-nucleotide polymorphisms (SNPs) that have been associated with elevated cancer risk in some reports. We observed that interaction of the PNKP FHA domain with phosphorylated XRCC1 extends beyond the immediate, well-characterized phosphorylated region of XRCC1 (residues 515-526). We also found that an XRCC1 fragment, comprising residues 166-436, binds tightly to PNKP and DNA and efficiently activates PNKP's kinase activity. However, interaction of either of the SNP-derived variants of this fragment with PNKP was considerably weaker, and their stimulation of PNKP was severely reduced, although they still could bind DNA effectively. Laser microirradiation revealed reduced recruitment of PNKP to damaged DNA in cells expressing either XRCC1 variant compared with PNKP recruitment in cells expressing WT XRCC1 even though WT and variant XRCC1s were equally efficient at localizing to the damaged DNA. These findings suggest that the elevated risk of cancer associated with these XRCC1 SNPs reported in some studies may be due in part to the reduced ability of these XRCC1 variants to recruit PNKP to damaged DNA.

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment.

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.

Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation.

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair.

In the current model of DNA SSBR, PARP1 is regarded as the sensor of single-strand breaks (SSBs). However, biochemical studies have implicated LIG3 as another possible SSB sensor. Using a laser micro-irradiation protocol that predominantly generates SSBs, we were able to demonstrate that PARP1 is dispensable for the accumulation of different single-strand break repair (SSBR) proteins at sites of DNA damage in live cells. Furthermore, we show in live cells for the first time that LIG3 plays a role in mediating the accumulation of the SSBR proteins XRCC1 and PNKP at sites of DNA damage. Importantly, the accumulation of LIG3 at sites of DNA damage did not require the BRCT domain-mediated interaction with XRCC1. We were able to show that the N-terminal ZnF domain of LIG3 plays a key role in the enzyme's SSB sensing function. Finally, we provide cellular evidence that LIG3 and not PARP1 acts as the sensor for DNA damage caused by the topoisomerase I inhibitor, irinotecan. Our results support the existence of a second damage-sensing mechanism in SSBR involving the detection of nicks in the genome by LIG3.

RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites.

In response to DNA damage, cells initiate complex signalling cascades leading to growth arrest and DNA repair. The recruitment of 53BP1 to damaged sites requires the activation of the ubiquitination cascade controlled by the E3 ubiquitin ligases RNF8 and RNF168, and methylation of histone H4 on lysine 20. However, molecular events that regulate the accessibility of methylated histones, to allow the recruitment of 53BP1 to DNA breaks, are unclear. Here, we show that like 53BP1, the JMJD2A (also known as KDM4A) tandem tudor domain binds dimethylated histone H4K20; however, JMJD2A is degraded by the proteasome following the DNA damage in an RNF8-dependent manner. We demonstrate that JMJD2A is ubiquitinated by RNF8 and RNF168. Moreover, ectopic expression of JMJD2A abrogates 53BP1 recruitment to DNA damage sites, indicating a role in antagonizing 53BP1 for methylated histone marks. The combined knockdown of JMJD2A and JMJD2B significantly rescued the ability of RNF8- and RNF168-deficient cells to form 53BP1 foci. We propose that the RNF8-dependent degradation of JMJD2A regulates DNA repair by controlling the recruitment of 53BP1 at DNA damage sites.

The differential mobilization of histones H3.1 and H3.3 by herpes simplex virus 1 relates histone dynamics to the assembly of viral chromatin.

During lytic infections, HSV-1 genomes are assembled into unstable nucleosomes. The histones required for HSV-1 chromatin assembly, however, are in the cellular chromatin. We have shown that linker (H1) and core (H2B and H4) histones are mobilized during HSV-1 infection, and proposed that the mobilized histones are available for assembly into viral chromatin. However, the actual relevance of histone mobilization remained unknown. We now show that canonical H3.1 and variant H3.3 are also mobilized during HSV-1 infection. Mobilization required no HSV-1 protein expression, although immediate early or early proteins enhanced it. We used the previously known differential association of H3.3 and H3.1 with HSV-1 DNA to test the relevance of histone mobilization. H3.3 binds to HSV-1 genomes first, whereas H3.1 only binds after HSV-1 DNA replication initiates. Consistently, H3.3 and H3.1 were differentially mobilized. H3.1 mobilization decreased with HSV-1 DNA replication, whereas H3.3 mobilization was largely unaffected by it. These results support a model in which previously mobilized H3.1 is immobilized by assembly into viral chromatin during HSV-1 DNA replication, whereas H3.3 is mobilized and assembled into HSV-1 chromatin throughout infection. The differential mobilizations of H3.3 and H3.1 are consistent with their differential assembly into viral chromatin. These data therefore relate nuclear histone dynamics to the composition of viral chromatin and provide the first evidence that histone mobilization relates to viral chromatin assembly.

Impaired in vivo binding of MeCP2 to chromatin in the absence of its DNA methyl-binding domain.

MeCP2 is a methyl-CpG-binding protein that is a main component of brain chromatin in vertebrates. In vitro studies have determined that in addition to its specific methyl-CpG-binding domain (MBD) MeCP2 also has several chromatin association domains. However, the specific interactions of MeCP2 with methylated or non-methylated chromatin regions and the structural characteristics of the resulting DNA associations in vivo remain poorly understood. We analysed the role of the MBD in MeCP2-chromatin associations in vivo using an MeCP2 mutant Rett syndrome mouse model (Mecp2(tm1.1Jae)) in which exon 3 deletion results in an N-terminal truncation of the protein, including most of the MBD. Our results show that in mutant mice, the truncated form of MeCP2 (ΔMeCP2) is expressed in different regions of the brain and liver, albeit at 50% of its wild-type (wt) counterpart. In contrast to the punctate nuclear distribution characteristic of wt MeCP2, ΔMeCP2 exhibits both diffuse nuclear localization and a substantial retention in the cytoplasm, suggesting a dysfunction of nuclear transport. In mutant brain tissue, neuronal nuclei are smaller, and ΔMeCP2 chromatin is digested faster by nucleases, producing a characteristic nuclease-resistant dinucleosome. Although a fraction of ΔMeCP2 is found associated with nucleosomes, its interaction with chromatin is transient and weak. Thus, our results unequivocally demonstrate that in vivo the MBD of MeCP2 together with its adjacent region in the N-terminal domain are critical for the proper interaction of the protein with chromatin, which cannot be replaced by any other of its protein domains.

Kdm4b histone demethylase is a DNA damage response protein and confers a survival advantage following γ-irradiation.

DNA damage evokes a complex and highly coordinated DNA damage response (DDR) that is integral to the suppression of genomic instability. Double-strand breaks (DSBs) are considered the most deleterious form damage. Evidence suggests that trimethylation of histone H3 lysine 9 (H3K9me3) presents a barrier to DSB repair. Also, global levels of histone methylation are clinically predictive for several tumor types. Therefore, demethylation of H3K9 may be an important step in the repair of DSBs. The KDM4 subfamily of demethylases removes H3K9 tri- and dimethylation and contributes to the regulation of cellular differentiation and proliferation; mutation or aberrant expression of KDM4 proteins has been identified in several human tumors. We hypothesize that members of the KDM4 subfamily may be components of the DDR. We found that Kdm4b-enhanced GFP (EGFP) and KDM4D-EGFP were recruited rapidly to DNA damage induced by laser micro-irradiation. Focusing on the clinically relevant Kdm4b, we found that recruitment was dependent on poly(ADP-ribose) polymerase 1 activity as well as Kdm4b demethylase activity. The Kdm4 proteins did not measurably accumulate at γ-irradiation-induced γH2AX foci. Nevertheless, increased levels of Kdm4b were associated with decreased numbers of γH2AX foci 6 h after irradiation as well as increased cell survival. Finally, we found that levels of H3K9me2 and H3K9me3 were decreased at early time points after 2 gray of γ-irradiation. Taken together, these data demonstrate that Kdm4b is a DDR protein and that overexpression of Kdm4b may contribute to the failure of anti-cancer therapy that relies on the induction of DNA damage.

A small molecule inhibitor of polycomb repressive complex 1 inhibits ubiquitin signaling at DNA double-strand breaks.

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.

CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage.

Polycomb group (PcG) proteins are involved in epigenetic silencing where they function as major determinants of cell identity, stem cell pluripotency and the epigenetic gene silencing involved in cancer development. Recently numerous PcG proteins, including CBX4, have been shown to accumulate at sites of DNA damage. However, it remains unclear whether or not CBX4 or its E3 sumo ligase activity is directly involved in the DNA damage response (DDR). Here we define a novel role for CBX4 as an early DDR protein that mediates SUMO conjugation at sites of DNA lesions. DNA damage stimulates sumoylation of BMI1 by CBX4 at lysine 88, which is required for the accumulation of BMI1 at DNA damage sites. Moreover, we establish that CBX4 recruitment to the sites of laser micro-irradiation-induced DNA damage requires PARP activity but does not require H2AX, RNF8, BMI1 nor PI-3-related kinases. The importance of CBX4 in the DDR was confirmed by the depletion of CBX4, which resulted in decreased cellular resistance to ionizing radiation. Our results reveal a direct role for CBX4 in the DDR pathway.

PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks.

After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood.

MeCP2 binds to nucleosome free (linker DNA) regions and to H3K9/H3K27 methylated nucleosomes in the brain.

Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2'-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me(2) and H3K27me(3) in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications.

Progeria-based vascular model identifies networks associated with cardiovascular aging and disease.

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.

The oncogenic potential of Jumonji D2 (JMJD2/KDM4) histone demethylase overexpression.

The Jumonji D2 proteins (JMJD2/KDM4) function to demethylate di- and trimethylated (me2/3) histone 3 lysine 9 (H3K9me2/3) and H3K36me3. Knockout mouse models for Kdm4b and Kdm4d have not resulted in gross abnormalities, while mouse models for Kdm4a and Kdm4c have not been reported. However, the KDM4 subfamily of demethylases are overexpressed in several tumor types. Overexpression of KDM4 proteins alters transcription and chromatin remodeling, driving cellular proliferation, anchorage-independent growth, invasion, and migration. Increased proliferation occurs through KDM4-mediated modification of cell cycle timing, as well as through increased numbers of replication forks. Recent evidence also suggests that KDM4C overexpression contributes to the maintenance of a pluripotent state. Together these data suggest that overexpression of KDM4 proteins induces numerous oncogenic effects.

Depletion of nuclear actin is a key mediator of quiescence in epithelial cells.

Functional differentiation is orchestrated by precise growth-regulatory controls conveyed by the tissue microenvironment. Cues from laminin 111 (LN1) lower transcription and suppress mammary epithelial cell growth in culture, but how LN1 induces quiescence is unknown. Recent literature points to involvement of nuclear ß-actin in transcriptional regulation. Here, we show that quiescence induced by growth factor withdrawal, or LN1 addition, rapidly decreases nuclear ß-actin. LN1, but not other extracellular matrix (ECM) molecules, decreases the levels of nuclear ß-actin and destabilizes RNA polymerase (RNA Pol) II and III binding to transcription sites, leading to a dramatic drop in transcription and DNA synthesis. Constitutive overexpression of globular ß-actin in the nucleus reverses the effect of LN1 on transcription and RNA Pol II association and prevents the cells from becoming quiescent in the presence of LN1. The physiological relevance of our findings was verified by identifying a clear spatial separation of LN1 and ß-actin in developing mammary end buds. These data indicate a novel role for nuclear ß-actin in growth arrest of epithelial cells and underscore the importance of the integrity of the basement membrane in homeostasis.