Twenty years ago, histone lysine demethylases (KDMs) were discovered. Since their discovery, they have been increasingly studied and shown to be important across species, development, and diseases. Considerable advances have been made toward understanding their (1) enzymology, (2) role as critical components of biological complexes, (3) role in normal cellular processes and functions, (4) implications in pathological conditions, and (5) therapeutic potential. This Review covers these key relationships related to the KDM field with the awareness that numerous laboratories have contributed to this field. The current knowledge coupled with future insights will shape our understanding about cell function, development, and disease onset and progression, which will allow for novel biomarkers to be identified and for optimal therapeutic options to be developed for KDM-related diseases in the years ahead.

DNA damage promotes mutations that fuel cancer, ageing and neurodegenerative diseases1-3, but surprisingly, the causes and types of damage remain largely unknown. There are three identified mechanisms that damage DNA during transcription: collision of RNA polymerase (RNAP) with the DNA-replication machinery head-on and co-directionally4-6, and R-loop-induced DNA breakage7-10. Here we identify novel DNA damage reaction intermediates11,12 and uncover a fourth transcription-related source of DNA damage: endogenous DNA damage at sites of terminated transcripts. We engineered proteins to capture single-stranded DNA (ssDNA) ends with 3' polarity in bacterial and human cells. In Escherichia coli, spontaneous 3'-ssDNA-end foci were unexpectedly frequent, at one or more per cell division, and arose via two identifiable pathways, both of which were dependent on DNA replication. A pathway associated with double-strand breaks was suppressed by overexpression of replicative DNA polymerase (pol) III, suggesting competition between pol III and DNA damage-promoting proteins. Mapping of recurrent 3'-ssDNA-ends identified distinct 3'-ssDNA-end-hotspots, mostly unrelated to double-strand breaks, next to the 5'-CCTTTTTT transcription-terminator-like sequence. These 3'-ssDNA-termini coincide with RNA 3'-termini identified by DirectRNA sequencing13 or simultaneous 5' and 3' end RNA sequencing (SEnd-seq)14 and were prevented by a mutant RNAP that reads through terminators. Our findings reveal that transcription termination or pausing can promote DNA damage and subsequent genomic instability.

Histones are integral components of eukaryotic chromatin that have a pivotal role in the organization and function of the genome. The dynamic regulation of chromatin involves the incorporation of histone variants, which can dramatically alter its structural and functional properties. Contrary to an earlier view that limited individual histone variants to specific genomic functions, new insights have revealed that histone variants exert multifaceted roles involving all aspects of genome function, from governing patterns of gene expression at precise genomic loci to participating in genome replication, repair and maintenance. This conceptual change has led to a new understanding of the intricate interplay between chromatin and DNA-dependent processes and how this connection translates into normal and abnormal cellular functions.

Elevated oxidative stress, which threatens genome stability, has been detected in almost all types of cancers. Cells employ various DNA repair pathways to cope with DNA damage induced by oxidative stress. Recently, a lot of studies have provided insights into DNA damage response upon oxidative stress, specifically in the context of transcriptionally active genomes. Here, we summarize recent studies to help understand how the transcription is regulated upon DNA double strand breaks (DSB) and how DNA repair pathways are selectively activated at the damage sites coupling with transcription. The role of RNA molecules, especially R-loops and RNA modifications during the DNA repair process, is critical for protecting genome stability. This review provides an update on how cells protect transcribed genome loci via transcription-coupled repair pathways.

DNA damage is a driver of cancer formation, leading to the impairment of repair mechanisms in cancer cells and rendering them susceptible to DNA-damaging therapeutic approaches. The concept of "synthetic lethality" in cancer clinics has emerged, particularly with the use of PARP inhibitors and the identification of DNA damage response (DDR) mutation biomarkers, emphasizing the significance of targeting DDR in cancer therapy. Novel approaches aimed at genome maintenance machinery are under development to further enhance the efficacy of cancer treatments. Natural compounds from traditional medicine, renowned for their anti-aging and anticarcinogenic properties, have garnered attention. Ginseng-derived compounds, in particular, exhibit anti-carcinogenic effects by suppressing reactive oxygen species (ROS) and protecting cells from DNA damage-induced carcinogenesis. However, the anticancer therapeutic effect of ginseng compounds has also been demonstrated by inducing DNA damage and blocking DDR. This review concentrates on the biphasic effects of ginseng compounds on DNA mutations-both inhibiting mutation accumulation and impairing DNA repair. Additionally, it explores other natural compounds targeting DDR directly, providing potential insights into enhancing cancer therapy efficacy.

The immune system plays a critical role in inflammation by initiating responses to infections or tissue damage. The nuclear factor-κB (NF-κB) pathway plays a key role in inflammation and innate immunity, as well as other cellular activities. Dysregulation of this well-choreographed pathway has been implicated in various diseases, including cancer. CARD11 is a key molecule in the BCL10-MALT1 complex, which is involved in transducing the signal downstream of the NF-κB pathway. This study aims to elucidate how CARD11 overexpression exacerbates the prognosis of colorectal cancer (CRC). To identify the cellular pathways influenced by CARD11, transcriptomic analysis in both CRC cell lines and patients was carried out on CARD11- overexpressed HCT-116 and HT-29 CRC cell lines alongside empty vector-transfected cell lines. Furthermore, a comparison of transcriptomic data from adenoma and carcinoma CRC patients with low- (CARD11-) and high-(CARD11+) CARD11 expression was carried out. Whole transcriptomics and bioinformatics analysis results indicate that CARD11 appears to play a key role in CRC progression. Absolute GSEA (absGSEA) on HCT-116 transcriptomics data revealed that CARD11 overexpression promotes cell growth and tissue remodeling and enhances immune response. Key genes co-expressed with CARD11, such as EP300, KDM5A, HIF1A, NFKBIZ, and DUSP1, were identified as mediators of these processes. In the HT-29 cell line, CARD11 overexpression activated pathways involved in chemotaxis and extracellular matrix (ECM) organization, marked by IL1RN, MDK, SPP1, and chemokines like CXCL1, CXCL3, and CCL22, which were shown to contribute to the more invasive stage of CRC. In patient samples, adenoma patients exhibited increased expression of genes associated with the tumor immune microenvironment, such as IL6ST, collagen family members, and CRC transition markers, such as GLI3 and PIEZO2, in CARD11+ adenoma patients. Carcinoma patients showed a dramatic increase in the expression of MAPK8IP2 in CARD11+ carcinoma patients alongside other cancer-related genes, including EMB, EPHB6, and CPEB4.

Epigenetic factors, including lysine-specific demethylases such as the KDM5 paralogs KDM5A and KDM5B have been implicated in cancer and the regulation of immune responses. Here, we performed a comprehensive multiomic study in cells lacking KDM5A or KDM5B to map changes in transcriptional regulation and chromatin organization. RNA-seq analysis revealed a significant decrease in the expression of Krüppel-associated box containing zinc finger (KRAB-ZNF) genes in KDM5A or KDM5B knockout cell lines, which was accompanied by changes ATAC-seq and H3K4me3 ChIP-seq. Pharmacological inhibition of KDM5A and KDM5B catalytic activity with a pan-KDM5 inhibitor, CPI-455, did not significantly change KRAB-ZNF expression, raising the possibility that regulation of KRAB-ZNF expression does not require KDM5A and KDM5B demethylase activity. KRAB-ZNF are recognized suppressors of the transcription of endogenous retroviruses (ERVs) and HAP1 cells with KDM5A or KDM5B gene inactivation showed elevated ERV expression, increased dsRNA levels and elevated levels of immune response genes. Acute degradation of KDM5A using a dTAG system in HAP1 cells led to increased ERV expression, demonstrating that de-repression of ERV genes occurs rapidly after loss of KDM5A. Co-immunoprecipitation of KDM5A revealed an interaction with the Nucleosome Remodeling and Deacetylase (NuRD) complex suggesting that KDM5A and NuRD may act together to regulate the expression of ERVs through KRAB-ZNFs. These findings reveal roles of KDM5A and KDM5B in modulating ERV expression and underscore the therapeutic potential of using degraders of KDM5A and KDM5B to modulate tumor immune responses.

Histone post-translational modifications (HPTMs), as one of the core mechanisms of epigenetic regulation, are garnering increasing attention due to their close association with the onset and progression of diseases and their potential as targeted therapeutic agents. Advances in high-throughput molecular tools and the abundance of bioinformatics data have led to the discovery of novel HPTMs which similarly affect gene expression, metabolism, and chromatin structure. Furthermore, a growing body of research has demonstrated that novel histone modifications also play crucial roles in the development and progression of various diseases, including various cancers, cardiovascular diseases, infectious diseases, psychiatric disorders, and reproductive system diseases. This review defines nine novel histone modifications: lactylation, citrullination, crotonylation, succinylation, SUMOylation, propionylation, butyrylation, 2-hydroxyisobutyrylation, and 2-hydroxybutyrylation. It comprehensively introduces the modification processes of these nine novel HPTMs, their roles in transcription, replication, DNA repair and recombination, metabolism, and chromatin structure, as well as their involvement in promoting the occurrence and development of various diseases and their clinical applications as therapeutic targets and potential biomarkers. Moreover, this review provides a detailed overview of novel HPTM inhibitors targeting various targets and their emerging strategies in the treatment of multiple diseases while offering insights into their future development prospects and challenges. Additionally, we briefly introduce novel epigenetic research techniques and their applications in the field of novel HPTM research.

The ubiquitin-proteasome system (UPS), which involves E3 ligases and deubiquitinates (DUBs), is critical for protein homeostasis. The epigenetic reader ZMYND8 (zinc finger MYND-type containing 8) has emerged as an oncoprotein, and its protein levels are elevated in various types of cancer, including breast cancer. However, the mechanism by which ZMYND8 protein levels are increased in cancer remains elusive. Although ZMYND8 has been reported to be regulated by the E3 ligase FBXW7, it is still unknown whether ZMYND8 could be modulated by DUBs. Here, we identified USP7 (ubiquitin carboxyl-terminal hydrolase 7) as a bona fide DUB for ZMYND8. Mechanically, USP7 directly binds to the PBP (PHD-BRD-PWWP) domain of ZMYND8 via its TRAF (tumor necrosis factor receptor-associated factor) domain and UBL (ubiquitin-like) domain and removes F-box and WD repeat domain containing 7 (FBXW7)-catalyzed poly-ubiquitin chains on lysine residue 1034 (K1034) within ZMYND8, thereby stabilizing ZMYND8 and stimulating the transcription of ZMYND8 target genes ZEB1 (zinc finger E-box binding homeobox 1) and VEGFA (Vascular Endothelial Growth Factor A). Consequently, USP7 enhances the capacity of breast cancer cells for migration and invasion through antagonizing FBXW7-mediated ZMYND8 degradation. Importantly, the protein levels of USP7 positively correlates with those of ZMYND8 in breast cancer tissues. These findings delineate an important layer of migration and invasion regulation by the USP7-ZMYND8 axis in breast cancer cells.

The genome is constantly exposed to DNA damage from endogenous and exogenous sources. Fine modulation of DNA repair, chromatin remodeling, and transcription factors is necessary for protecting genome integrity, but the precise mechanisms are still largely unclear. We found that after ionizing radiation (IR), global trimethylation of histone H3 at lysine 4 (H3K4me3) was decreased at an early (5 min) post-IR phase but increased at an intermediate (180 min) post-IR phase in both human and mouse hematopoietic cells. We demonstrated that PTIP, a component of the MLL histone methyltransferase complex, is required for H3K4me3 upregulation in the intermediate post-IR phase and promotes cell cycle arrest by epigenetically inducing a cell cycle inhibitor, PRDM1. In addition, we found that PTIP expression is specifically downregulated in acute myeloid leukemia patients. These findings collectively suggest that the PTIP-PRDM1 axis plays an essential role in proper DNA damage response and its deregulation contributes to leukemogenesis.

The maintenance of genome integrity by DNA damage response machineries is key to protect cells against pathological development. In cell nuclei, these genome maintenance machineries operate in the context of chromatin, where the DNA wraps around histone proteins. Here, we review recent findings illustrating how the chromatin substrate modulates genome maintenance mechanisms, focusing on the regulatory role of histone variants and post-translational modifications. In particular, we discuss how the pre-existing chromatin landscape impacts DNA damage formation and guides DNA repair pathway choice, and how DNA damage-induced chromatin alterations control DNA damage signaling and repair, and DNA damage segregation through cell divisions. We also highlight that pathological alterations of histone proteins may trigger genome instability by impairing chromosome segregation and DNA repair, thus defining new oncogenic mechanisms and opening up therapeutic options.

Cellular and molecular responses to DNA damage are highly orchestrated and dynamic, acting to preserve the maintenance and integrity of the genome. Histone proteins bind DNA and organize the genome into chromatin. Post-translational modifications of histones have been shown to play an essential role in orchestrating the chromatin response to DNA damage by regulating the DNA damage response pathway. Among the histone modifications that contribute to this intricate network, histone ADP-ribosylation (ADPr) is emerging as a pivotal component of chromatin-based DNA damage response (DDR) pathways. In this review, we survey how histone ADPr is regulated to promote the DDR and how it impacts chromatin and other histone marks. Recent advancements have revealed histone ADPr effects on chromatin structure and the regulation of DNA repair factor recruitment to DNA lesions. Additionally, we highlight advancements in technology that have enabled the identification and functional validation of histone ADPr in cells and in response to DNA damage. Given the involvement of DNA damage and epigenetic regulation in human diseases including cancer, these findings have clinical implications for histone ADPr, which are also discussed. Overall, this review covers the involvement of histone ADPr in the DDR and highlights potential future investigations aimed at identifying mechanisms governed by histone ADPr that participate in the DDR, human diseases, and their treatments.

DNA Double-strand breaks (DSBs) are harmful lesions and major sources of genomic instability. Studies have suggested that DSBs induce local transcriptional silencing that consequently promotes genomic stability. Several factors have been proposed to actively participate in this process, including ATM and Polycomb repressive complex 1 (PRC1). Here we found that disrupting PRC1 clustering disrupts DSB-induced gene silencing. Interactome analysis of PHC2, a PRC1 subunit that promotes the formation of the Polycomb body, found several nucleoporins that constitute the Nuclear Pore Complex (NPC). Similar to PHC2, depleting the nucleoporins also disrupted the DSB-induced gene silencing. We found that some of these nucleoporins, such as NUP107 and NUP43, which are members of the Y-complex of NPC, localize to DSB sites. These nucleoporin-enriched DSBs were distant from the nuclear periphery. The presence of nucleoporins and PHC2 at DSB regions were inter-dependent, suggesting that they act cooperatively in the DSB-induced gene silencing. We further found two structural components within NUP107 to be necessary for the transcriptional repression at DSBs: ATM/ATR-mediated phosphorylation at Serine37 residue within the N-terminal disordered tail, and the NUP133-binding surface at the C-terminus. These results provide a new functional interplay among nucleoporins, ATM and the Polycomb proteins in the DSB metabolism, and underscore their emerging roles in genome stability maintenance. *Hongseon Song, Yubin Bae, Sangin Kim, and Dante Deascanis contributed equally to this work.

Double-strand breaks (DSBs) are the most lethal form of DNA damage. Transcriptional activity at DSBs, as well as transcriptional repression around DSBs, are both required for efficient DNA repair. The chromatin landscape defines and coordinates these two opposing events. However, how the open and condensed chromatin architecture is regulated remains unclear. Here, we show that the GATAD2B-NuRD complex associates with DSBs in a transcription- and DNA:RNA hybrid-dependent manner, to promote histone deacetylation and chromatin condensation. This activity establishes a spatio-temporal boundary between open and closed chromatin, which is necessary for the correct termination of DNA end resection. The lack of the GATAD2B-NuRD complex leads to chromatin hyperrelaxation and extended DNA end resection, resulting in homologous recombination (HR) repair failure. Our results suggest that the GATAD2B-NuRD complex is a key coordinator of the dynamic interplay between transcription and the chromatin landscape, underscoring its biological significance in the RNA-dependent DNA damage response.

Breast cancer (BC) is the most frequent malignant cancer diagnosis and is a primary factor for cancer deaths in women. The clinical subtypes of BC include estrogen receptor (ER) positive, progesterone receptor (PR) positive, human epidermal growth factor receptor 2 (HER2) positive, and triple-negative BC (TNBC). Based on the stages and subtypes of BC, various treatment methods are available with variations in the rates of progression-free disease and overall survival of patients. However, the treatment of BC still faces challenges, particularly in terms of drug resistance and recurrence. The study of epigenetics has provided new ideas for treating BC. Targeting aberrant epigenetic factors with inhibitors represents a promising anticancer strategy. The KDM5 family includes four members, KDM5A, KDM5B, KDM5C, and KDMD, all of which are Jumonji C domain-containing histone H3K4me2/3 demethylases. KDM5 proteins have been extensively studied in BC, where they are involved in suppressing or promoting BC depending on their specific upstream and downstream pathways. Several KDM5 inhibitors have shown potent BC inhibitory activity in vitro and in vivo, but challenges still exist in developing KDM5 inhibitors. In this review, we introduce the subtypes of BC and their current therapeutic options, summarize KDM5 family context-specific functions in the pathobiology of BC, and discuss the outlook and pitfalls of KDM5 inhibitors in this disease.

Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis.

Histone modifications maintain genomic stability and orchestrate gene expression at the chromatin level. Benzo [a]pyrene (BaP) is the ubiquitous carcinogen widely spread in the environment, but the role and regulatory mechanism of histone modification in its toxic effects remain largely undefined. In this study, we found a dose-dependent reduction of histone H3 methylations at lysine4, lysine9, lysine27, lysine36 in HBE cells treated with BaP. We observed that inhibiting H3K27 and H3K36 methylation impaired cell proliferation, whereas the loss of H3K4, H3K9, H3K27, and H3K36 methylation led to increased genomic instability and delayed DNA repair. H3K36 mutation at both H3.1 and H3.3 exhibited the most significant impacts. In addition, we found that the expression of SET domain containing 2 (SETD2), the unique methyltransferase catalyzed H3K36me3, was downregulated by BaP dose-dependently in vitro and in vivo. Knockdown of SETD2 aggravated DNA damage of BaP exposure, which was consistent with the effects of H3K36 mutation. With the aid of chromatin immunoprecipitation (ChIP) -seq and RNA-seq, we found that H3K36me3 was responsible for transcriptional regulation of genes involved in pathways related to cell survival, lung cancer, metabolism and inflammation. The enhanced enrichment of H3K36me3 in genes (CYP1A1, ALDH1A3, ACOXL, WNT5A, WNT7A, RUNX2, IL1R2) was positively correlated with their expression levels, while the reduction of H3K36me3 distribution in genes (PPARGC1A, PDE4D, GAS1, RNF19A, KSR1) were in accordance with the downregulation of gene expression. Taken together, our findings emphasize the critical roles and mechanisms of histone lysine methylation in mediating cellular homeostasis during BaP exposure.

The SETD1B gene is instrumental in human intelligence and nerve development. Mutations in the SETD1B gene have been linked in recent studies to neurodevelopmental disorders, seizures, and language delay.

This study aimed to analyze the clinical manifestations and treatment of three patients suffering from mental retardation, epilepsy, and language delay resulting from a new mutation in the SETD1B gene. Three individuals with these symptoms were selected, and their clinical symptoms, gene test results, and treatment were analyzed. This article discusses the impact of the SETD1B gene mutation on patients and outlines the treatment approach. Among the three patients (two females and one male, aged 8, 4, and 1, respectively), all exhibited psychomotor retardation, attention deficit, and hyperactivity disorder, and two had epilepsy. Antiepileptic treatment with sodium tripolyvalproate halted the seizures in the affected child, although mental development remained somewhat delayed. Whole exome sequencing revealed new mutations in the SETD1B gene for all patients, specifically with c.5473C>T (p.Arg1825trp), c.4120C>T (p.Gln1374*, 593), c.14_15insC (p.His5Hisfs*33).

Possessing the SETD1B gene mutation may cause mental retardation accompanied by seizures and language delay. Although the exact mechanism is not fully understood, interventions such as drug therapy, rehabilitation training, and family support can assist patients in managing their symptoms and enhancing their quality of life. Furthermore, genetic testing supplies healthcare providers with more precise diagnostic and therapeutic guidance, informs families about genetic disease risks, and contributes to understanding disease pathogenesis and drug research and development.

Chemoresistance, a roadblock in the chemotherapy process, has been impeding its effective treatment. KDM5B, a member of the histone demethylase family, has been crucial in the emergence and growth of malignancies. More significantly, KDM5B has recently been linked closely to cancer's resistance to chemotherapy. In this review, we explain the biological properties of KDM5B, its function in the emergence and evolution of cancer treatment resistance, and our hopes for future drug resistance-busting combinations involving KDM5B and related targets or medications.

Eukaryotic nuclei are constantly being exposed to factors that break or chemically modify the DNA. Accurate repair of this DNA damage is crucial to prevent DNA mutations and maintain optimal cell function. To overcome the detrimental effects of DNA damage, a multitude of repair pathways has evolved. These pathways need to function properly within the different chromatin domains present in the nucleus. Each of these domains exhibit distinct molecular- and bio-physical characteristics that can influence the response to DNA damage. In particular, chromatin domains highly enriched for repetitive DNA sequences, such as nucleoli, centromeres and pericentromeric heterochromatin require tailored repair mechanisms to safeguard genome stability. Work from the past decades has led to the development of innovative imaging tools as well as inducible DNA damage techniques to gain new insights into the impact of these repetitive chromatin domains on the DNA repair process. Here we summarize these tools with a particular focus on Double-Strand Break (DSB) repair, and discuss the insights gained into our understanding of the influence of chromatin domains on DSB -dynamics and -repair pathway choice.

Tri-methylation of Histone 3 lysine 4 (H3K4) is an important epigenetic modification whose deposition and removal can affect the chromatin at structural and functional levels. KDM5A is one of the four known H3K4-specific demethylases. It is a part of the KDM5 family, which is characterized by a catalytic Jumonji domain capable of removing H3K4 di- and tri-methylation marks. KDM5A has been found to be involved in multiple cellular processes such as differentiation, metabolism, cell cycle, and transcription. Its link to various diseases, including cancer, makes KDM5A an important target for drug development. However, despite several studies outlining its significance in various pathways, our lack of understanding of its recruitment and function at the target sites on the chromatin presents a challenge in creating effective and targeted treatments. Therefore, it is essential to understand the recruitment mechanism of KDM5A to chromatin, and its activity therein, to comprehend how various roles of KDM5A are regulated. In this review, we discuss how KDM5A functions in a context-dependent manner on the chromatin, either directly through its structural domain, or through various interacting partners, to bring about a diverse range of functions.

The SWI/SNF family of ATP-dependent chromatin remodeling complexes is implicated in multiple DNA damage response mechanisms and frequently mutated in cancer. The BAF, PBAF and ncBAF complexes are three major types of SWI/SNF complexes that are functionally distinguished by their exclusive subunits. Accumulating evidence suggests that double-strand breaks (DSBs) in transcriptionally active DNA are preferentially repaired by a dedicated homologous recombination pathway. We show that different BAF, PBAF and ncBAF subunits promote homologous recombination and are rapidly recruited to DSBs in a transcription-dependent manner. The PBAF and ncBAF complexes promote RNA polymerase II eviction near DNA damage to rapidly initiate transcriptional silencing, while the BAF complex helps to maintain this transcriptional silencing. Furthermore, ARID1A-containing BAF complexes promote RNaseH1 and RAD52 recruitment to facilitate R-loop resolution and DNA repair. Our results highlight how multiple SWI/SNF complexes perform different functions to enable DNA repair in the context of actively transcribed genes.

Laser microirradiation coupled with live-cell fluorescence microscopy is a powerful technique that has been used widely in studying the recruitment and retention of proteins at sites of DNA damage. Results obtained from this technique can be found in published works by both seasoned and infrequent users of microscopy. However, like many other microscopy-based techniques, the presentation of data from laser microirradiation experiments is inconsistent; papers report a wide assortment of analytic techniques, not all of which result in accurate and/or appropriate representation of the data. In addition to the varied methods of analysis, experimental and analytical details are commonly under-reported. Consequently, publications reporting data from laser microirradiation coupled with fluorescence microscopy experiments need to be carefully and critically assessed by readers. Here, we undertake a systematic investigation of commonly reported corrections used in the analysis of laser microirradiation data. We validate the critical need to correct data for photobleaching and we identify key experimental parameters that must be accounted for when presenting data from laser microirradiation experiments. Furthermore, we propose a straightforward, four-step analytical protocol that can readily be applied across platforms and that aims to improve the quality of data reporting in the DNA damage field.

Cerebellar ataxia is often the first and irreversible outcome in the disease of ataxia-telangiectasia (A-T), as a consequence of selective cerebellar Purkinje neuronal degeneration. A-T is an autosomal recessive disorder resulting from the loss-of-function mutations of the ataxia-telangiectasia-mutated ATM gene. Over years of research, it now becomes clear that functional ATM-a serine/threonine kinase protein product of the ATM gene-plays critical roles in regulating both cellular DNA damage response and central carbon metabolic network in multiple subcellular locations. The key question arises is how cerebellar Purkinje neurons become selectively vulnerable when all other cell types in the brain are suffering from the very same defects in ATM function. This review intended to comprehensively elaborate the unexpected linkages between these two seemingly independent cellular functions and the regulatory roles of ATM involved, their integrated impacts on both physical and functional properties, hence the introduction of selective vulnerability to Purkinje neurons in the disease will be addressed.

Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1 to confer therapeutic resistance to ionizing radiation (IR).

We evaluated changes in the transcriptome and epigenome in a radioresistant mIDH1 patient-derived glioma cell culture (GCC) following treatment with an mIDH1-specific inhibitor, AGI-5198. We identified Zinc Finger MYND-Type Containing 8 (ZMYND8) as a potential target of mIDH1 reprogramming. We suppressed ZMYND8 expression by shRNA knockdown and genetic knockout (KO) in mIDH1 glioma cells and then assessed cellular viability to IR. We assessed the sensitivity of mIDH1 GCCS to pharmacologic inhibition of ZMYND8-interacting partners: HDAC, BRD4, and PARP.

Inhibition of mIDH1 leads to an upregulation of gene networks involved in replication stress. We found that the expression of ZMYND8, a regulator of DNA damage response, was decreased in three patient-derived mIDH1 GCCs after treatment with AGI-5198. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 KO exhibit significant phosphorylation of ATM and sustained γH2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells.

These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. See related commentary by Sachdev et al., p. 1648.

Farrerol, a natural flavanone, promotes homologous recombination (HR) repair to improve genome-editing efficiency, but the specific protein that farrerol directly targets to regulate HR repair and the underlying molecular mechanisms have not been determined. Here, we find that the deubiquitinase UCHL3 is the direct target of farrerol. Mechanistically, farrerol enhanced the deubiquitinase activity of UCHL3 to promote RAD51 deubiquitination, thereby improving HR repair. Importantly, we find that embryos of somatic cell nuclear transfer (SCNT) exhibited defective HR repair, increased genomic instability and aneuploidy, and that the farrerol treatment post nuclear transfer enhances HR repair, restores transcriptional and epigenetic network, and promotes SCNT embryo development. Ablating UCHL3 significantly attenuates farrerol-mediated stimulation in HR and SCNT embryo development. In summary, we identify farrerol as an activator of the deubiquitinase UCHL3, highlighted the importance of HR and epigenetic changes in SCNT reprogramming and provide a feasible method to promote SCNT efficiency.

Histone variants represent chromatin components that diversify the structure and function of the genome. The variants of H2A, primarily H2A.X, H2A.Z and macroH2A, are well-established participants in DNA damage response (DDR) pathways, which function to protect the integrity of the genome. Through their deposition, post-translational modifications and unique protein interaction networks, these variants guard DNA from endogenous threats including replication stress and genome fragility as well as from DNA lesions inflicted by exogenous sources. A growing body of work is now providing a clearer picture on the involvement and mechanistic basis of H2A variant contribution to genome integrity. Beyond their well-documented role in gene regulation, we review here how histone H2A variants promote genome stability and how alterations in these pathways contribute to human diseases including cancer.

Double-stranded breaks (DSBs) are toxic DNA damage and a serious threat to genomic integrity. Thus, all living organisms have evolved multiple mechanisms of DNA DSB repair, the two principal ones being classical-non homologous end joining (C-NHEJ), and homology dependent recombination (HDR). In mammals, C-NHEJ is the predominate DSB repair pathway, but how a cell chooses to repair a particular DSB by a certain pathway is still not mechanistically clear. To uncover novel regulators of DSB repair pathway choice, we performed a kinome-wide screen in a human cell line engineered to express a dominant-negative C-NHEJ factor. The intellectual basis for such a screen was our hypothesis that a C-NHEJ-crippled cell line might need to upregulate other DSB repair pathways, including HDR, in order to survive. This screen identified Bromodomain-containing Protein 3 (BRD3) as a protein whose expression was almost completely ablated specifically in a C-NHEJ-defective cell line. Subsequent experimentation demonstrated that BRD3 is a negative regulator of HDR as BRD3-null cell lines proved to be hyper-recombinogenic for gene conversion, sister chromatid exchanges and gene targeting. Mechanistically, BRD3 appears to be working at the level of Radiation Sensitive 51 (RAD51) recruitment. Overall, our results demonstrate that BRD3 is a novel regulator of human DSB repair pathway choice.

Alzheimer's disease (AD) is the most common neurodegenerative disease, which severely threatens the health of the elderly and causes significant economic and social burdens. The causes of AD are complex and include heritable but mostly aging-related factors. The primary aging hallmarks include genomic instability, telomere wear, epigenetic changes, and loss of protein stability, which play a dominant role in the aging process. Although AD is closely associated with the aging process, the underlying mechanisms involved in AD pathogenesis have not been well characterized. This review summarizes the available literature about primary aging hallmarks and their roles in AD pathogenesis. By analyzing published literature, we attempted to uncover the possible mechanisms of aberrant epigenetic markers with related enzymes, transcription factors, and loss of proteostasis in AD. In particular, the importance of oxidative stress-induced DNA methylation and DNA methylation-directed histone modifications and proteostasis are highlighted. A molecular network of gene regulatory elements that undergoes a dynamic change with age may underlie age-dependent AD pathogenesis, and can be used as a new drug target to treat AD.

Morphogenesis and organogenesis in the low organisms have been found to be modulated by a number of proteins, and one of such factor, deformed epidermal auto-regulatory factor-1 (DEAF-1) has been initially identified in Drosophila. The mammalian homologue of DEAF-1 and structurally related proteins have been identified, and they formed a family with over 20 members. The factors regulate gene expression through association with co-repressors, recognition of genomic marker, to exert histone modification by catalyze addition of some chemical groups to certain amino acid residues on histone and non-histone proteins, and degradation host proteins, so as to regulate cell cycle progression and execution of cell death. The formation of fused genes during chromosomal translocation, exemplified with myeloid transforming gene on chromosome 8 (MTG8)/eight-to-twenty one translocation (ETO) /ZMYND2, MTG receptor 1 (MTGR1)/ZMYND3, MTG on chromosome 16/MTGR2/ZMYND4 and BS69/ZMYND11 contributes to malignant transformation. Other anomaly like copy number variation (CNV) of BS69/ZMYND11 and promoter hyper methylation of BLU/ZMYND10 has been noted in malignancies. It has been reported that when fusing with Runt-related transcription factor 1 (RUNX1), the binding of MTG8/ZMYND2 with co-repressors is disturbed, and silencing of BLU/ZMYND10 abrogates its ability to inhibition of cell cycle and promotion of apoptotic death. Further characterization of the implication of ZMYND proteins in carcinogenesis would enhance understanding of the mechanisms of occurrence and early diagnosis of tumors, and effective antitumor efficacy.

Genome integrity is constantly challenged by endogenous and exogenous insults that cause DNA damage. To cope with these threats, cells have a surveillance mechanism, known as the DNA damage response (DDR), to repair any lesions. Although transcription has long been implicated in DNA repair, how transcriptional reprogramming is coordinated with the DDR is just beginning to be understood. In this review, we highlight recent advances in elucidating the molecular mechanisms underlying major transcriptional events, including RNA polymerase (Pol) II stalling and transcriptional silencing and recovery, which occur in response to DNA damage. Furthermore, we discuss how such transcriptional adaptation contributes to sensing and eliminating damaged DNA and how it can jeopardize genome integrity when it goes awry.

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.

In eukaryotic cells, DNA damage can occur at any time and at any chromatin locus, including loci at which active transcription is taking place. DNA double-strand breaks affect chromatin integrity and elicit a DNA damage response to facilitate repair of the DNA lesion. Actively transcribed genes near DNA lesions are transiently suppressed by crosstalk between DNA damage response factors and polycomb repressive complexes. Epigenetic modulation of the chromatin environment also contributes to efficient DNA damage response signaling and transcriptional repression. On the other hand, RNA transcripts produced in the G1 phase, as well as the active chromatin context of the lesion, appear to drive homologous recombination repair. Here, we discuss how the ISWI family of chromatin remodeling factors coordinates the DNA damage response and transcriptional repression, especially in transcriptionally active regions, highlighting the direct modulation of the epigenetic environment.

SWI/SNF (BAF) chromatin remodeling complexes regulate lineage-specific enhancer activity by promoting accessibility for diverse DNA-binding factors and chromatin regulators. Additionally, they are known to modulate the function of the epigenome through regulation of histone post-translational modifications and nucleosome composition, although the way SWI/SNF complexes govern the epigenome remains poorly understood. Here, we investigate the function of ARID1A, a subunit of certain mammalian SWI/SNF chromatin remodeling complexes associated with malignancies and benign diseases originating from the uterine endometrium.

Through genome-wide analysis of human endometriotic epithelial cells, we show that more than half of ARID1A binding sites are marked by the variant histone H3.3, including active regulatory elements such as super-enhancers. ARID1A knockdown leads to H3.3 depletion and gain of canonical H3.1/3.2 at ARID1A-bound active regulatory elements, and a concomitant redistribution of H3.3 toward genic elements. ARID1A interactions with the repressive chromatin remodeler CHD4 (NuRD) are associated with H3.3, and ARID1A is required for CHD4 recruitment to H3.3. ZMYND8 interacts with CHD4 to suppress a subset of ARID1A, CHD4, and ZMYND8 co-bound, H3.3+ H4K16ac+ super-enhancers near genes governing extracellular matrix, motility, adhesion, and epithelial-to-mesenchymal transition. Moreover, these gene expression alterations are observed in human endometriomas.

These studies demonstrate that ARID1A-containing BAF complexes are required for maintenance of the histone variant H3.3 at active regulatory elements, such as super-enhancers, and this function is required for the physiologically relevant activities of alternative chromatin remodelers.

Zinc Finger transcription factors are crucial in modulating various cellular processes, including differentiation. Chromatin reader Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8), an All-Trans Retinoic Acid (ATRA)-responsive gene, was previously shown to play a crucial role in promoting the expression of neuronal-lineage committed genes. Here, we report that ZMYND8 promotes neuronal differentiation by positively regulating canonical MAPT protein-coding gene isoform, a key player in the axonal development of neurons. Additionally, ZMYND8 modulates gene-isoform switching by epigenetically silencing key regulatory regions within the MAPT gene, thereby suppressing the expression of non-protein-coding isoforms such as MAPT213. Genetic deletion of ZMYND8 led to an increase in the MAPT213 that potentially suppressed the parental MAPT protein-coding transcript expression related to neuronal differentiation programs. In addition, ectopic expression of MAPT213 led to repression of MAPT protein-coding transcript. Similarly, ZMYND8-driven transcription regulation was also observed in other neuronal differentiation-promoting genes. Collectively our results elucidate a novel mechanism of ZMYND8-dependent transcription regulation of different neuronal lineage committing genes, including MAPT, to promote neural differentiation.

Much has been learned since the early 1960s about histone post-translational modifications (PTMs) and how they affect DNA-templated processes at the molecular level. This understanding has been bolstered in the past decade by the identification of new types of histone PTM, the advent of new genome-wide mapping approaches and methods to deposit or remove PTMs in a locally and temporally controlled manner. Now, with the availability of vast amounts of data across various biological systems, the functional role of PTMs in important processes (such as transcription, recombination, replication, DNA repair and the modulation of genomic architecture) is slowly emerging. This Review explores the contribution of histone PTMs to the regulation of genome function by discussing when these modifications play a causative (or instructive) role in DNA-templated processes and when they are deposited as a consequence of such processes, to reinforce and record the event. Important advances in the field showing that histone PTMs can exert both direct and indirect effects on genome function are also presented.

Over the course of a human lifespan, genome integrity erodes, leading to an increased abundance of several types of chromatin changes. The abundance of DNA lesions (chemical perturbations to nucleotides) increases with age, as does the number of genomic mutations and transcriptional disruptions caused by replication or transcription of those lesions, respectively. At the epigenetic level, precise DNA methylation patterns degrade, likely causing increasingly stochastic variations in gene expression. Similarly, the tight regulation of histone modifications begins to unravel. The genomic instability caused by these mechanisms allows transposon element reactivation and remobilization, further mutations, gene dysregulation, and cytoplasmic chromatin fragments. This cumulative genomic instability promotes cell signaling events that drive cell fate decisions and extracellular communications known to disrupt tissue homeostasis and regeneration. In this Review, we focus on age-related epigenetic changes and their interactions with age-related genomic changes that instigate these events.

ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene.

An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants.

ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the DrosophilaZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function.

We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.