XRCC1 Prevents Replication Fork Instability during Misincorporation of the DNA Demethylation Bases 5-Hydroxymethyl-2′-Deoxycytidine and 5-Hydroxymethyl-2′-Deoxyuridine

Whilst avoidance of chemical modifications of DNA bases is essential to maintain genome stability, during evolution eukaryotic cells have evolved a chemically reversible modification of the cytosine base. These dynamic methylation and demethylation reactions on carbon-5 of cytosine regulate several cellular and developmental processes such as embryonic stem cell pluripotency, cell identity, differentiation or tumourgenesis. Whereas these physiological processes are well characterized, very little is known about the toxicity of these cytosine analogues when they incorporate during replication. Here, we report a role of the base excision repair factor XRCC1 in protecting replication fork upon incorporation of 5-hydroxymethyl-2′-deoxycytosine (5hmC) and its deamination product 5-hydroxymethyl-2′-deoxyuridine (5hmU) during DNA synthesis. In the absence of XRCC1, 5hmC exposure leads to increased genomic instability, replication fork impairment and cell lethality. Moreover, the 5hmC deamination product 5hmU recapitulated the genomic instability phenotypes observed by 5hmC exposure, suggesting that 5hmU accounts for the observed by 5hmC exposure. Remarkably, 5hmC-dependent genomic instability and replication fork impairment seen in Xrcc1−/− cells were exacerbated by the trapping of Parp1 on chromatin, indicating that XRCC1 maintains replication fork stability during processing of 5hmC and 5hmU by the base excision repair pathway. Our findings uncover natural epigenetic DNA bases 5hmC and 5hmU as genotoxic nucleosides that threaten replication dynamics and genome integrity in the absence of XRCC1.

The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis

ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis. Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

HP1β Chromo Shadow Domain facilitates H2A ubiquitination for BRCA1 recruitment at DNA double-strand breaks

Efficient DNA double strand break (DSB) repair by homologous recombination (HR), as orchestrated by histone and non-histone proteins, is critical to genome stability, replication, transcription, and cancer avoidance. Here we report that Heterochromatin Protein1 beta (HP1β) acts as a key component of the HR DNA resection step by regulating BRCA1 enrichment at DNA damage sites, a function largely dependent on the HP1β chromo shadow domain (CSD). HP1β itself is enriched at DSBs within gene-rich regions through a CSD interaction with Chromatin Assembly Factor 1 (CAF1) and HP1β depletion impairs subsequent BRCA1 enrichment. An added interaction of the HP1β CSD with the Polycomb Repressor Complex 1 ubiquitinase component RING1A facilitates BRCA1 recruitment by increasing H2A lysine 118-119 ubiquitination, a marker for BRCA1 recruitment. Our findings reveal that HP1β interactions, mediated through its CSD with RING1A, promote H2A ubiquitination and facilitate BRCA1 recruitment at DNA damage sites, a critical step in DSB repair by the HR pathway. These collective results unveil how HP1β is recruited to DSBs in gene-rich regions and how HP1β subsequently promotes BRCA1 recruitment to further HR DNA damage repair by stimulating CtIP-dependent resection.

Molecular and Cellular Functions of the Linker Histone H1.2

Linker histone H1.2, which belongs to the linker histone family H1, plays a crucial role in the maintenance of the stable higher-order structures of chromatin and nucleosomes. As a critical part of chromatin structure, H1.2 has an important function in regulating chromatin dynamics and participates in multiple other cellular processes as well. Recent work has also shown that linker histone H1.2 regulates the transcription levels of certain target genes and affects different processes as well, such as cancer cell growth and migration, DNA duplication and DNA repair. The present work briefly summarizes the current knowledge of linker histone H1.2 modifications. Further, we also discuss the roles of linker histone H1.2 in the maintenance of genome stability, apoptosis, cell cycle regulation, and its association with disease.

Chromatin profiling reveals heterogeneity in clinical isolates of the human pathogen Aspergillus fumigatus

Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.

Chicken blastoderms and primordial germ cells possess a higher expression of DNA repair genes and lower expression of apoptosis genes to preserve their genome stability

DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. When the cell fails to repair DNA damage, apoptosis is initiated. Although several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding of those gene expression is not well-understood in chicken tissues. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. PGCs are also undifferentiated progenitor cells that later differentiate into male and female germ cells. CEFs are differentiated and abundant somatic cells. Through WTS analysis, we identified that the DNA repair pathway genes were expressed more highly in blastoderms and high in PGCs than CEFs. Besides, the apoptosis pathway genes were expressed low in blastoderms and PGCs than CEFs. We have also examined the WTS-based expression profiling of candidate pluripotency regulating genes due to the conserved properties of blastoderms and PGCs. In the results, a limited number of pluripotency genes, especially the core transcriptional network, were detected higher in both blastoderms and PGCs than CEFs. Next, we treated the CEFs, blastoderm cells, and PGCs with hydrogen peroxide (H2O2) for 1 h to induce DNA damage. Then, the H2O2 treated cells were incubated in fresh media for 3–12 h to observe DNA repair. Subsequent analyses in treated cells found that blastoderm cells and PGCs were more likely to undergo apoptosis along with the loss of pluripotency and less likely to undergo DNA repair, contrasting with CEFs. These properties of blastoderms and PGCs should be necessary to preserve genome stability during the development of early embryos and germ cells, respectively.

Mandatory coupling of zygotic transcription to DNA replication in early Drosophila embryos

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. We hypothesized that collision-avoidance mechanisms safeguard the onset of zygotic transcription in these cycles. To explore this hypothesis, we used real-time imaging of transcriptional events at the onset of each interphase. Endogenously tagged RNA polymerase II (RNAPII) abruptly formed clusters before nascent transcripts accumulated, indicating recruitment prior to transcriptional engagement. Injection of inhibitors of DNA replication prevented RNAPII clustering, blocked formation of foci of the pioneer factor Zelda, and largely prevented expression of transcription reporters. Knockdown of Zelda or the histone acetyltransferase CBP prevented RNAPII cluster formation except at the replication-dependent (RD) histone gene locus. We suggest a model in which the passage of replication forks allows Zelda and a distinct pathway at the RD histone locus to reconfigure chromatin to nucleate RNAPII clustering and promote transcriptional initiation. The replication dependency of these events defers initiation of transcription and ensures that RNA polymerases transcribe behind advancing replication forks. The resulting coordination of transcription and replication explains how early embryos circumvent collisions and promote genome stability.

Retinoblastoma protein regulates carcinogen susceptibility at heterochromatic cancer driver loci

Carcinogenic insult, such as UV light exposure, creates DNA lesions that evolve into mutations if left unrepaired. These resulting mutations can contribute to carcinogenesis and drive malignant phenotypes. Susceptibility to carcinogens (i.e., the propensity to form a carcinogen-induced DNA lesion) is regulated by both genetic and epigenetic factors. Importantly, carcinogen susceptibility is a critical contributor to cancer mutagenesis. It is known that mutations can be prevented by tumor suppressor regulation of DNA damage response pathways; however, their roles carcinogen susceptibility have not yet been reported. In this study, we reveal that the retinoblastoma (RB1) tumor suppressor regulates UV susceptibility across broad regions of the genome. In particular, centromere and telomere-proximal regions exhibit significant increases in UV lesion susceptibility when RB1 is deleted. Several cancer-related genes are located within genomic regions of increased susceptibility, including telomerase reverse transcriptase, TERT, thereby accelerating mutagenic potential in cancers with RB1 pathway alterations. These findings reveal novel genome stability mechanisms of a tumor suppressor and uncover new pathways to accumulate mutations during cancer evolution.

Telomere Shortening and Its Association with Cell Dysfunction in Lung Diseases

Telomeres are localized at the end of chromosomes to provide genome stability; however, the telomere length tends to be shortened with each cell division inducing a progressive telomere shortening (TS). In addition to age, other factors, such as exposure to pollutants, diet, stress, and disruptions in the shelterin protein complex or genes associated with telomerase induce TS. This phenomenon favors cellular senescence and genotoxic stress, which increases the risk of the development and progression of lung diseases such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, SARS-CoV-2 infection, and lung cancer. In an infectious environment, immune cells that exhibit TS are associated with severe lymphopenia and death, whereas in a noninfectious context, naïve T cells that exhibit TS are related to cancer progression and enhanced inflammatory processes. In this review, we discuss how TS modifies the function of the immune system cells, making them inefficient in maintaining homeostasis in the lung. Finally, we discuss the advances in drug and gene therapy for lung diseases where TS could be used as a target for future treatments.