Histone acylation at a glance.

An important mechanism of gene expression regulation is the epigenetic modification of histones. The cofactors and substrates for these modifications are often intermediary metabolites, and it is becoming increasingly clear that the metabolic and nutritional state of cells can influence these marks. These connections between the balance of metabolites, histone modifications and downstream transcriptional changes comprise a metabolic signaling program that can enable cells to adapt to changes in nutrient availability. Beyond acetylation, there is evidence now that histones can be modified by other acyl groups. In this Cell Science at a Glance article and the accompanying poster, we focus on these histone acylation modifications and provide an overview of the players that govern these acylations and their connections with metabolism.

ASXLs binding to the PHD2/3 fingers of MLL4 provides a mechanism for the recruitment of BAP1 to active enhancers.

The human methyltransferase and transcriptional coactivator MLL4 and its paralog MLL3 are frequently mutated in cancer. MLL4 and MLL3 monomethylate histone H3K4 and contain a set of uncharacterized PHD fingers. Here, we report a novel function of the PHD2 and PHD3 (PHD2/3) fingers of MLL4 and MLL3 that bind to ASXL2, a component of the Polycomb repressive H2AK119 deubiquitinase (PR-DUB) complex. The structure of MLL4 PHD2/3 in complex with the MLL-binding helix (MBH) of ASXL2 and mutational analyses reveal the molecular mechanism which is conserved in homologous ASXL1 and ASXL3. The native interaction of the Trithorax MLL3/4 complexes with the PR-DUB complex in vivo depends solely on MBH of ASXL1/2, coupling the two histone modifying activities. ChIP-seq analysis in embryonic stem cells demonstrates that MBH of ASXL1/2 is required for the deubiquitinase BAP1 recruitment to MLL4-bound active enhancers. Our findings suggest an ASXL1/2-dependent functional link between the MLL3/4 and PR-DUB complexes.

Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair.

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

[Epigenetic dysregulation and emerging treatment strategies in myelodysplastic syndromes].

The epigenome regulates transcription of target genes through DNA methylation- or histone methylation/acetylation/phosphorylation/ubiquitination-mediated alteration of genomic function or chromatin conformation. Recent genomic studies have shown that multiple genes encoding epigenetic regulators are frequently and recurrently mutated in MDS, suggesting that epigenetic dysregulation is significantly associated with the molecular pathogenesis and clinical features of MDS. In this review, we will present our recent findings together with others, focusing on physiological molecular functions of epigenetic regulators recurrently mutated in MDS and on functional correlation between dysregulated epigenomic regulators and molecular pathogenesis/clinical features of MDS.

[Clinicopathological characteristics of gangliogliomas with anaplastic morphology].

Objective: To investigate the clinical, radiological, and pathological features of anaplastic gangliogliomas (AGGs) and to determine whether these tumors represent a distinct entity. Methods: Consecutive 667 cases of ganglioglioma (GG) diagnosed at the Xuanwu Hospital, Capital Medical University, Beijing, China between January 2015 and July 2023 were screened. Among these cases, 9 pathologically confirmed AGG cases were identified. Their clinical, radiological, treatment, and outcome data were analyzed retrospectively. Most of the tumor samples were subject to next-generation sequencing, while a subset of them were subject to DNA methylation profiling. Results: Among the 9 patients, there were five males and four females, with a median age of 8 years. Epileptic seizures (5/9) were the most frequently presented symptom. Radiological examinations showed three types of radiological manifestations: four cases showed abnormal MRI signals with no significant mass effects and mild enhancement; two cases demonstrated a mixed solid-cystic density lesion with peritumoral edema, which showed significant heterogeneous enhancement and obvious mass effects, and one case displayed cystic cavity formation with nodules on MRI, which showed evident enhancements. All cases exhibited mutations that were predicted to activate the MAP kinase signaling pathway, including seven with BRAF p.V600E mutation and two with NF1 mutation. Five AGGs with mutations involving the MAP kinase signaling pathway also had concurrent mutations, including three with CDKN2A homozygous deletion, one with a TERT promoter mutation, one with a H3F3A mutation, and one with a PTEN mutation. Conclusions: AGG exhibits a distinct spectrum of pathology, genetic mutations and clinical behaviors, differing from GG. Given these characteristics suggest that AGG may be a distinct tumor type, further expansion of the case series is needed. Therefore, a comprehensive integration of clinical, histological, and molecular analyses is required to correctly diagnose AGG. It will also help guide treatments and prognostication.

Muscle-specific pyruvate kinase isoforms, PKM1 and PKM2, regulate mammalian SWI/SNF proteins and histone 3 phosphorylation during myoblast differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.

A Hypoxia-Epigenetics Axis Drives EMT in Pancreatic Cancer.

Epithelial-to-mesenchymal transition (EMT) is a classical cellular plasticity process induced by various cell-intrinsic and -extrinsic triggers. Although prominent factors, such as TGFß, mediate EMT via well-characterized pathways, alternative avenues are less well understood. Transcriptomic subtyping of pancreatic ductal adenocarcinoma (PDAC) has demonstrated that basal-like PDACs enrich a mesenchymal-like expression program, emphasizing the relevance of EMT in the disease. In this issue of Cancer Research, Brown and colleagues demonstrate the tight connection of EMT to hypoxia. Through a detailed mechanistic analysis, the authors deciphered that hypoxia-induced signals are integrated by the histone H3 lysine 36 di-methylation (H3K36me2) mark. On the one hand, hypoxia decreased activity of the H3K36me2 eraser KDM2A, while on the other hand promoting stabilization of the H3K36me2 writer NSD2. Hypoxia diminished the expression of a set of serine-threonine phosphatases, subsequently resulting in SRC kinase family-dependent activation of canonical MEK, ERK, and JNK signaling to impinge on NSD2 expression. In addition, reduced expression of the protein phosphatase PP2Cδ was linked to increased NSD2 protein expression. These discoveries illuminate the close relationship of hypoxia signaling to the epigenetic machinery and cellular plasticity processes. See related article by Brown et al., p. 1764.

Native and tagged CENP-A histones are functionally inequivalent.

BACKGROUND: Over the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function. RESULTS: We report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes. CONCLUSIONS: These data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant.

Activation of AMPK inhibits cervical cancer growth by hyperacetylation of H3K9 through PCAF.

BACKGROUND: Dysregulation in histone acetylation, a significant epigenetic alteration closely associated with major pathologies including cancer, promotes tumorigenesis, inactivating tumor-suppressor genes and activating oncogenic pathways. AMP-activated protein kinase (AMPK) is a cellular energy sensor that regulates a multitude of biological processes. Although a number of studies have identified the mechanisms by which AMPK regulates cancer growth, the underlying epigenetic mechanisms remain unknown. METHODS: The impact of metformin, an AMPK activator, on cervical cancer was evaluated through assessments of cell viability, tumor xenograft model, pan-acetylation analysis, and the role of the AMPK-PCAF-H3K9ac signaling pathway. Using label-free quantitative acetylproteomics and chromatin immunoprecipitation-sequencing (ChIP) technology, the activation of AMPK-induced H3K9 acetylation was further investigated. RESULTS: In this study, we found that metformin, acting as an AMPK agonist, activates AMPK, thereby inhibiting the proliferation of cervical cancer both in vitro and in vivo. Mechanistically, AMPK activation induces H3K9 acetylation at epigenetic level, leading to chromatin remodeling in cervical cancer. This also enhances the binding of H3K9ac to the promoter regions of multiple tumor suppressor genes, thereby promoting their transcriptional activation. Furthermore, the absence of PCAF renders AMPK activation incapable of inducing H3K9 acetylation. CONCLUSIONS: In conclusion, our findings demonstrate that AMPK mediates the inhibition of cervical cancer growth through PCAF-dependent H3K9 acetylation. This discovery not only facilitates the clinical application of metformin but also underscores the essential role of PCAF in AMPK activation-induced H3K9 hyperacetylation.

FBP1 orchestrates keratinocyte proliferation/differentiation and suppresses psoriasis through metabolic control of histone acetylation.

Keratinocyte proliferation and differentiation in epidermis are well-controlled and essential for reacting to stimuli such as ultraviolet light. Imbalance between proliferation and differentiation is a characteristic feature of major human skin diseases such as psoriasis and squamous cell carcinoma. However, the effect of keratinocyte metabolism on proliferation and differentiation remains largely elusive. We show here that the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) promotes differentiation while inhibits proliferation of keratinocyte and suppresses psoriasis development. FBP1 is identified among the most upregulated genes induced by UVB using transcriptome sequencing and is elevated especially in upper epidermis. Fbp1 heterozygous mice exhibit aberrant epidermis phenotypes with local hyperplasia and dedifferentiation. Loss of FBP1 promotes proliferation and inhibits differentiation of keratinocytes in vitro. Mechanistically, FBP1 loss facilitates glycolysis-mediated acetyl-CoA production, which increases histone H3 acetylation at lysine 9, resulting in enhanced transcription of proliferation genes. We further find that the expression of FBP1 is dramatically reduced in human psoriatic lesions and in skin of mouse imiquimod psoriasis model. Fbp1 deficiency in mice facilitates psoriasis-like skin lesions development through glycolysis and acetyl-CoA production. Collectively, our findings reveal a previously unrecognized role of FBP1 in epidermal homeostasis and provide evidence for FBP1 as a metabolic psoriasis suppressor.

Age-dependent regulation of ELP1 exon 20 splicing in Familial Dysautonomia by RNA Polymerase II kinetics and chromatin structure.

Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20.

Joint-specific regulation of homeobox D10 expression in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a systemic immune-mediated disease characterized by joint inflammation and destruction. The disease typically affects small joints in the hands and feet, later progressing to involve larger joints such as the knees, shoulders, and hips. While the reasons for these joint-specific differences are unclear, distinct epigenetic patterns associated with joint location have been reported. In this study, we evaluated the unique epigenetic landscapes of fibroblast-like synoviocytes (FLS) from hip and knee synovium in RA patients, focusing on the expression and regulation of Homeobox (HOX) transcription factors. These highly conserved genes play a critical role in embryonic development and are known to maintain distinct expression patterns in various adult tissues. We found that several HOX genes, especially HOXD10, were differentially expressed in knee FLS compared with hip FLS. Epigenetic differences in chromatin accessibility and histone marks were observed in HOXD10 promoter between knee and hip FLS. Histone modification, particularly histone acetylation, was identified as an important regulator of HOXD10 expression. To understand the mechanism of differential HOXD10 expression, we inhibited histone deacetylases (HDACs) with small molecules and siRNA. We found that HDAC1 blockade or deficiency normalized the joint-specific HOXD10 expression patterns. These observations suggest that epigenetic differences, specifically histone acetylation related to increased HDAC1 expression, play a crucial role in joint-specific HOXD10 expression. Understanding these mechanisms could provide insights into the regional aspects of RA and potentially lead to therapeutic strategies targeting specific patterns of joint involvement during the course of disease.

The PCNA-Pol δ complex couples lagging strand DNA synthesis to parental histone transfer for epigenetic inheritance.

Inheritance of epigenetic information is critical for maintaining cell identity. The transfer of parental histone H3-H4 tetramers, the primary carrier of epigenetic modifications on histone proteins, represents a crucial yet poorly understood step in the inheritance of epigenetic information. Here, we show the lagging strand DNA polymerase, Pol δ, interacts directly with H3-H4 and that the interaction between Pol δ and the sliding clamp PCNA regulates parental histone transfer to lagging strands, most likely independent of their roles in DNA synthesis. When combined, mutations at Pol δ and Mcm2 that compromise parental histone transfer result in a greater reduction in nucleosome occupancy at nascent chromatin than mutations in either alone. Last, PCNA contributes to nucleosome positioning on nascent chromatin. On the basis of these results, we suggest that the PCNA-Pol δ complex couples lagging strand DNA synthesis to parental H3-H4 transfer, facilitating epigenetic inheritance.

Gene regulatory landscape of cerebral cortex folding.

Folding of the cerebral cortex is a key aspect of mammalian brain development and evolution, and defects are linked to severe neurological disorders. Primary folding occurs in highly stereotyped patterns that are predefined in the cortical germinal zones by a transcriptomic protomap. The gene regulatory landscape governing the emergence of this folding protomap remains unknown. We characterized the spatiotemporal dynamics of gene expression and active epigenetic landscape (H3K27ac) across prospective folds and fissures in ferret. Our results show that the transcriptomic protomap begins to emerge at early embryonic stages, and it involves cell-fate signaling pathways. The H3K27ac landscape reveals developmental cell-fate restriction and engages known developmental regulators, including the transcription factor Cux2. Manipulating Cux2 expression in cortical progenitors changed their proliferation and the folding pattern in ferret, caused by selective transcriptional changes as revealed by single-cell RNA sequencing analyses. Our findings highlight the key relevance of epigenetic mechanisms in defining the patterns of cerebral cortex folding.

Cell surface CD55 traffics to the nucleus leading to cisplatin resistance and stemness by inducing PRC2 and H3K27 trimethylation on chromatin in ovarian cancer.

BACKGROUND: Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers. METHODS: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing. RESULTS: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2. CONCLUSIONS: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.

Discovery of NSD2 non-histone substrates and design of a super-substrate.

The human protein lysine methyltransferase NSD2 catalyzes dimethylation at H3K36. It has very important roles in development and disease but many mechanistic features and its full spectrum of substrate proteins are unclear. Using peptide SPOT array methylation assays, we investigate the substrate sequence specificity of NSD2 and discover strong readout of residues between G33 (-3) and P38 (+2) on H3K36. Unexpectedly, we observe that amino acid residues different from natural ones in H3K36 are preferred at some positions. Combining four preferred residues led to the development of a super-substrate which is methylated much faster by NSD2 at peptide and protein level. Molecular dynamics simulations demonstrate that this activity increase is caused by distinct hyperactive conformations of the enzyme-peptide complex. To investigate the substrate spectrum of NSD2, we conducted a proteome wide search for nuclear proteins matching the specificity profile and discovered 22 peptide substrates of NSD2. In protein methylation studies, we identify K1033 of ATRX and K819 of FANCM as NSD2 methylation sites and also demonstrate their methylation in human cells. Both these proteins have important roles in DNA repair strengthening the connection of NSD2 and H3K36 methylation to DNA repair.

Histone H1x in mouse ventral hippocampus associates with, but does not cause behavioral adaptations to stress.

Prior research has identified differential protein expression levels of linker histone H1x within the ventral hippocampus (vHipp) of stress-susceptible versus stress-resilient mice. These mice are behaviorally classified based on their divergent responses to chronic social stress. Here, we sought to determine whether elevated vHipp H1x protein levels directly contribute to these diverging behavioral adaptations to stress. First, we demonstrated that stress-susceptible mice uniquely express elevated vHipp H1x protein levels following chronic stress. Given that linker histones coordinate heterochromatin compaction, we hypothesize that elevated levels of H1x in the vHipp may impede pro-resilience transcriptional adaptations and prevent development of the resilient phenotype following social stress. To test this, 8-10-week-old male C57BL/6 J mice were randomly assigned to groups undergoing 10 days of chronic social defeat stress (CSDS) or single housing, respectively. Following CSDS, mice were classified as susceptible versus resilient based on their social interaction behaviors. We synthesized a viral overexpression (OE) vector for H1x and transduced all stressed and single housed mice with either H1x or control GFP within vHipp. Following viral delivery, we conducted social, anxiety-like, and memory-reliant behavior tests on distinct cohorts of mice. We found no behavioral adaptations following H1x OE compared to GFP controls in susceptible, resilient, or single housed mice. In sum, although we confirm elevated vHipp protein levels of H1x associate with susceptibility to social stress, we observe no significant behavioral consequence of H1x OE. Thus, we conclude elevated levels of H1x are associated with, but are not singularly sufficient to drive development of behavioral adaptations to stress.

Sex-specific transgenerational effects on murine thyroid gland imposed by ancestral exposure to neonicotinoid thiacloprid.

Neonicotinoids, a relatively new widely used class of insecticide is used in agriculture to control insect populations. We examined the capacity of ancestral exposure to the neonicotinoid thiacloprid (thia) to induce transgenerational effects on thyroid tissue. Pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5 to E15.5 using 0, 0.6, and 6 mg/kg/day doses. Thyroid paraffin sections were prepared for morphology analysis. We apply ELISA method to measure T4 and TSH levels, RT-qPCR for gene expression analysis, ChIP-qPCR techniques for sperm histone H3K4me3 analysis, and immunofluorescence microscopy and western blots for protein detection. We observed an alteration in the morphology of thyroids in both males and females in the F3 generation. We observed an increase in T4 hormone in F1 females and a significant T4 level decrease in F3 males. T4 changes in F1 females were associated with a TSH increase. We found that the amount of Iodothyronine Deiodinase 1 (DIO1) (an enzyme converting T4 to T3) was decreased in both F1 and F3 generations in female thyroids. GNAS protein which is important for thyroid function has increased in female thyroids. Gene expression analysis showed that the expression of genes encoding thyroid gland development, chromatin, biosynthesis and transport factors were affected in the thyroid gland in both sexes in F1 and F3. The analysis of sperm histone H3K4me3 showed that H3K4me3 occupancy at the Dio1 locus has decreased while Thyroglobulin (Tg) and Matrix Metallopeptidase 2 (Mmp2) genes have increased H3K4me3 occupancy in the sperm of F3 mice. Besides, DNA methylation analysis of our previously published datasets showed that, in the sperm of F1 and F3 thia-derived mice, several genes related to thyroid function show consistent alterations. Our data suggest that ancestral exposure to thiacloprid affects thyroid function not only in exposed but also in indirectly exposed F3 generation.

Heat-Stress Impacts on Developing Bovine Oocytes: Unraveling Epigenetic Changes, Oxidative Stress, and Developmental Resilience.

Extreme temperature during summer may lead to heat stress in cattle and compromise their productivity. It also poses detrimental impacts on the developmental capacity of bovine budding oocytes, which halt their fertility. To mitigate the adverse effects of heat stress, it is necessary to investigate the mechanisms through which it affects the developmental capacity of oocytes. The primary goal of this study was to investigate the impact of heat stress on the epigenetic modifications in bovine oocytes and embryos, as well as on oocyte developmental capacity, reactive oxygen species, mitochondrial membrane potential, apoptosis, transzonal projections, and gene expression levels. Our results showed that heat stress significantly reduced the expression levels of the epigenetic modifications from histone H1, histone H2A, histone H2B, histone H4, DNA methylation, and DNA hydroxymethylation at all stages of the oocyte and embryo. Similarly, heat stress significantly reduced cleavage rate, blastocyst rate, oocyte mitochondrial-membrane potential level, adenosine-triphosphate (ATP) level, mitochondrial DNA copy number, and transzonal projection level. It was also found that heat stress affected mitochondrial distribution in oocytes and significantly increased reactive oxygen species, apoptosis levels and mitochondrial autophagy levels. Our findings suggest that heat stress significantly impacts the expression levels of genes related to oocyte developmental ability, the cytoskeleton, mitochondrial function, and epigenetic modification, lowering their competence during the summer season.

Fusion of histone variants to Cas9 suppresses non-homologous end joining.

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.