Homologous recombination (HR) is essential for genome integrity. Recombination proteins participate in tolerating DNA lesions that interfere with DNA replication, but can also generate toxic ...recombination intermediates and genetic instability when they are not properly regulated. Here, we have studied the role of the recombination proteins Rad51 and Rad52 at replication forks and replicative DNA lesions. We show that Rad52 loads Rad51 onto unperturbed replication forks, where they facilitate replication of alkylated DNA by non‐repair functions. The recruitment of Rad52 and Rad51 to chromatin during DNA replication is a prerequisite for the repair of the non‐DSB DNA lesions, presumably single‐stranded DNA gaps, which are generated during the replication of alkylated DNA. We also show that the repair of these lesions requires CDK1 and is not coupled to the fork but rather restricted to G2/M by the replicative checkpoint. We propose a new scenario for HR where Rad52 and Rad51 are recruited to the fork to promote DNA damage tolerance by distinct and cell cycle‐regulated replicative and repair functions.
The homologous recombination factors Rad51 and Rad52 load onto unperturbed replication forks, to facilitate eventual lesion bypass as well as post‐replicative single‐strand gap repair.
Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group ...proteins. The antisense noncoding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the polycomb repressive complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function, and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between noncoding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a mechanism by which noncoding RNA participates directly in epigenetic transcriptional repression.
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► CBX7 and its interacting ncRNA ANRIL are upregulated in prostate cancer tissues ► CBX7 chromodomain binds H3K27me3 and ANRIL with overlapping sites ► Both interactions are required for the INK4b/ARF/INK4a locus repression by CBX7 ► Disruption of either interaction results in premature growth arrest
The Polycomb Group (PcG) of chromatin modifiers regulates pluripotency and differentiation. Mammalian genomes encode multiple homologs of the Polycomb repressive complex 1 (PRC1) components, ...including five orthologs of the Drosophila Polycomb protein (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8). We have identified Cbx7 as the primary Polycomb ortholog of PRC1 complexes in embryonic stem cells (ESCs). The expression of Cbx7 is downregulated during ESC differentiation, preceding the upregulation of Cbx2, Cbx4, and Cbx8, which are directly repressed by Cbx7. Ectopic expression of Cbx7 inhibits differentiation and X chromosome inactivation and enhances ESC self-renewal. Conversely, Cbx7 knockdown induces differentiation and derepresses lineage-specific markers. In a functional screen, we identified the miR-125 and miR-181 families as regulators of Cbx7 that are induced during ESC differentiation. Ectopic expression of these miRNAs accelerates ESC differentiation via regulation of Cbx7. These observations establish a critical role for Cbx7 and its regulatory miRNAs in determining pluripotency.
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► Cbx7 is the primary Pc ortholog of the PRC1 complex in pluripotent cells ► Cbx7 repress its homologs to regulate PRC1 composition during ESC differentiation ► Cbx7 promotes a stem-cell-like state by repressing differentiation ► miR-181 and miR-125 regulate Cbx7 expression during ESC differentiation
Topoisomerase 2 (TOP2) DNA transactions proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer ...TOP2 drugs. How genotoxic TOP2 DNA-protein cross-links are resolved is unclear. We found that the SUMO (small ubiquitin-related modifier) ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a “split-SIM” SUMO2 engagement platform. These findings uncover a ZATT-TDP2–catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc.
RNA polymerase II (RNAPII) transcription elongation is a highly regulated process that greatly influences mRNA levels as well as pre-mRNA splicing. Despite many studies in vitro, how chromatin ...modulates RNAPII elongation in vivo is still unclear. Here, we show that a decrease in the level of available canonical histones leads to more accessible chromatin with decreased levels of canonical histones and variants H2A.X and H2A.Z and increased levels of H3.3. With this altered chromatin structure, the RNAPII elongation rate increases, and the kinetics of pre-mRNA splicing is delayed with respect to RNAPII elongation. Consistent with the kinetic model of cotranscriptional splicing, the rapid RNAPII elongation induced by histone depletion promotes the skipping of variable exons in theCD44gene. Indeed, a slowly elongating mutant of RNAPII was able to rescue this defect, indicating that the defective splicing induced by histone depletion is a direct consequence of the increased elongation rate. In addition, genome-wide analysis evidenced that histone reduction promotes widespread alterations in pre-mRNA processing, including intron retention and changes in alternative splicing. Our data demonstrate that premRNA splicing may be regulated by chromatin structure through the modulation of the RNAPII elongation rate.
Neurogenesis in developing and adult mammalian brain is a tightly regulated process that relies on neural stem cell (NSC) activity. There is increasing evidence that mitochondrial metabolism affects ...NSC homeostasis and differentiation but the precise role of mitochondrial function in the neurogenic process requires further investigation. Here, we have analyzed how mitochondrial complex I (MCI) dysfunction affects NSC viability, proliferation and differentiation, as well as survival of the neural progeny. We have generated a conditional knockout model (hGFAP-NDUFS2 mice) in which expression of the NDUFS2 protein, essential for MCI function, is suppressed in cells expressing the Cre recombinase under the human glial fibrillary acidic protein promoter, active in mouse radial glial cells (RGCs) and in neural stem cells (NSCs) that reside in adult neurogenic niches. In this model we observed that survival of central NSC population does not appear to be severely affected by MCI dysfunction. However, perinatal brain development was markedly inhibited and
knockout mice died before the tenth postnatal day. In addition,
studies of subventricular zone NSCs showed that active neural progenitors require a functional MCI to produce ATP and to proliferate.
differentiation of neural precursors into neurons and oligodendrocytes was also profoundly affected. These data indicate the need of a correct MCI function and oxidative phosphorylation for glia-like NSC proliferation, differentiation and subsequent oligodendrocyte or neuronal maturation.
Acute oxygen (O
2
) sensing is essential for adaptation of organisms to hypoxic environments or medical conditions with restricted exchange of gases in the lung. The main acute O
2
-sensing organ is ...the carotid body (CB), which contains neurosecretory chemoreceptor (glomus) cells innervated by sensory fibers whose activation by hypoxia elicits hyperventilation and increased cardiac output. Glomus cells have mitochondria with specialized metabolic and electron transport chain (ETC) properties. Reduced mitochondrial complex (MC) IV activity by hypoxia leads to production of signaling molecules (NADH and reactive O
2
species) in MCI and MCIII that modulate membrane ion channel activity. We studied mice with conditional genetic ablation of MCIII that disrupts the ETC in the CB and other catecholaminergic tissues. Glomus cells survived MCIII dysfunction but showed selective abolition of responsiveness to hypoxia (increased Ca
2+
and transmitter release) with normal responses to other stimuli. Mitochondrial hypoxic NADH and reactive O
2
species signals were also suppressed. MCIII-deficient mice exhibited strong inhibition of the hypoxic ventilatory response and altered acclimatization to sustained hypoxia. These data indicate that a functional ETC, with coupling between MCI and MCIV, is required for acute O
2
sensing. O
2
regulation of breathing results from the integrated action of mitochondrial ETC complexes in arterial chemoreceptors.
Upon telomerase inactivation, telomeres gradually shorten with each cell division until cells enter replicative senescence. In Saccharomyces cerevisiae, the kinases Mec1/ATR and Tel1/ATM protect the ...genome during pre-senescence by preventing telomere-telomere fusions (T-TFs) and the subsequent genetic instability associated with fusion-bridge-breakage cycles. Here we report that T-TFs in mec1Δ tel1Δ cells can be suppressed by reducing the pool of available histones. This protection associates neither with changes in bulk telomere length nor with major changes in the structure of subtelomeric chromatin. We show that the absence of Mec1 and Tel1 strongly augments double-strand break (DSB) repair by non-homologous end joining (NHEJ), which might contribute to the high frequency of T-TFs in mec1Δ tel1Δ cells. However, histone depletion does not prevent telomere fusions by inhibiting NHEJ, which is actually increased in histone-depleted cells. Rather, histone depletion protects telomeres from fusions by homologous recombination (HR), even though HR is proficient in maintaining the proliferative state of pre-senescent mec1Δ tel1Δ cells. Therefore, HR during pre-senescence not only helps stalled replication forks but also prevents T-TFs by a mechanism that, in contrast to the previous one, is promoted by a reduction in the histone pool and can occur in the absence of Rad51. Our results further suggest that the Mec1-dependent depletion of histones that occurs during pre-senescence in cells without telomerase (tlc1Δ) prevents T-TFs by favoring the processing of unprotected telomeres by Rad51-independent HR.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Vaccines against SARS-CoV-2 have been shown to be safe and effective but their protective efficacy against infection in the brain is yet unclear. Here, in the susceptible transgenic K18-hACE2 mouse ...model of severe coronavirus disease 2019 (COVID-19), we report a spatiotemporal description of SARS-CoV-2 infection and replication through the brain. SARS-CoV-2 brain replication occurs primarily in neurons, leading to neuronal loss, signs of glial activation and vascular damage in mice infected with SARS-CoV-2. One or two doses of a modified vaccinia virus Ankara (MVA) vector expressing the SARS-CoV-2 spike (S) protein (MVA-CoV2-S) conferred full protection against SARS-CoV-2 cerebral infection, preventing virus replication in all areas of the brain and its associated damage. This protection was maintained even after SARS-CoV-2 reinfection. These findings further support the use of MVA-CoV2-S as a promising vaccine candidate against SARS-CoV-2/COVID-19.
O2 is essential for aerobic life, and the classic view is that it diffuses freely across the plasma membrane. However, measurements of O2 permeability of lipid bilayers have indicated that it is much ...lower than previously thought, and therefore, the existence of membrane O2 channels has been suggested. We hypothesized that, besides its role as a water channel, aquaporin-1 (AQP-1) could also work as an O2 transporter, because this transmembrane protein appears to be CO2-permeable and is highly expressed in cells with rapid O2 turnover (erythrocytes and microvessel endothelium). Here we show that in mammalian cells overexpressing AQP-1 and exposed to hypoxia, the loss of cytosolic O2, as well as stabilization of the O2-dependent hypoxia-inducible transcription factor and expression of its target genes, is accelerated. In normoxic endothelial cells, knocking down AQP-1 produces induction of hypoxia-inducible genes. Moreover, lung AQP-1 is markedly up-regulated in animals exposed to hypoxia. These data suggest that AQP-1 has O2 permeability and thus could facilitate O2 diffusion across the cell membrane.
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