Histone H2A Research Articles

Buforins: A Potential Antimicrobial Peptide Explored With Its Anticancer Efficacy‐A Review

ABSTRACTAntimicrobial peptides (AMPs), derived from numerous life forms, is being recognised as favourable contenders in today's era to overcome the multi drug resistance of cancer cells. Despite of their diverse structural orientations (β‐sheet, α‐helical, loop and extended peptides), they are immensely involved in immune defences and potentially involved for combating cancers and other types of infections via cellular membrane depolarization. Buforins (Bf), the histone H2A derived AMPs along with their analogues (like Bf‐I, Bf‐II and Bf‐IIb) demonstrated substantial anticancer efficacy despite of numerous challenges. They are quite effective in controlling the apoptosis in various cancer cell lines like breast, HeLa, ovarian, lung, liver and prostate cancers. Bf conjugated with bioconjugates were explored for enhancing the bioavailability, drug resistance and stability of these super giants in cancer therapy. The functionalized nanoparticles could possibly help to overwhelm the shortcomings of AMPs, towards cancer therapy. However, the success of in vivo approach may lead to the clinical translation of such therapeutics. In this review, we emphasized on the characteristic features, mechanisms of action, numerous anticancer approaches of Bf. Further discussion was continued with the challenges and their overcoming, advancement and future directions of Bf towards their success in chemotherapy.

JmjC catalysed histone H2a N-methyl arginine demethylation and C4-arginine hydroxylation reveals importance of sequence-reactivity relationships

2-Oxoglutarate (2OG) dependent Nε-methyl lysine demethylases (JmjC-KDMs) regulate eukaryotic transcription. We report studies showing that isolated forms of all human KDM4 and KDM5 JmjC enzymes catalyse demethylation of N-methylated Arg-3 of histone H2a. Unexpectedly, the results reveal that KDM4E and, less efficiently, KDM4D catalyse C-4 hydroxylation of Arg-20 of H2a on peptides, recombinant H2a, and calf histone extracts, including when the Arg-20 guanidino group is N-methylated. Combined with previous observations, our biochemical results highlight the importance of sequence context in determining the relative efficiencies of lysine and arginine demethylation reactions catalysed by KDM4s and KDM5s. At least in some cases changes in sequence can also enable a different JmjC reaction mode, such as C-4 arginine hydroxylation instead of demethylation. Further work is thus required to define the full scope of JmjC catalysed reactions in cells.

A tale of two serines: the effects of histone H2A mutations S122A and S129A on chromosome non-disjunction in Saccharomyces cerevisiae.

Near the C-terminus of histone H2A in the yeast S. cerevisiae, there are two serines (S122 and S129) that are targets of phosphorylation. The phosphorylation of Serine 129 in response to DNA damage is dependent on the Tel1 and Mec1 kinases. In S. pombe and S. cerevisiae, the phosphorylation of Serine 122 is dependent on the Bub1 kinase, and S. pombe strains with an alanine mutation of this serine have elevated levels of lagging chromosomes in mitosis. Strains that lack both Tel1 and Mec1 in S. cerevisiae have very elevated rates of non-disjunction. To clarify the functional importance of phosphorylation of serines 122 and 129 in H2A, we measured chromosome loss rates in single mutant strains and double mutant combinations. We also examined the interaction of mutations of BUB1, TEL1, and MEC1 in combination with mutations of serine 122 and 129 in H2A. We conclude that the phosphorylation state of S129 has no effect on chromosome disjunction whereas mutations that inactivate Bub1 or a S122A mutation in the histone H2A greatly elevate the rate of chromosome non-disjunction. Based on this analysis, we suggest that Bub1 exerts its primary effect on chromosome disjunction by phosphorylating S122 of histone H2A. However, Tel1, Mec1 and Bub1 also functionally redundant in a second pathway affecting chromosome disjunction that is at least partially independent of phosphorylation of S122 of H2A.

A Site-Specific Click Chemistry Approach to Di-Ubiquitylate H1 Variants Reveals Position-Dependent Stimulation of the DNA Repair Protein RNF168.

Ubiquitylation of histone H2A at lysines 13 and 15 by the E3 ligase RNF168 plays a key role in orchestrating DNA double-strand break (DSB) repair, which is often deregulated in cancer. RNF168 activity is triggered by DSB signaling cascades, reportedly through K63-linked poly-ubiquitylation of linker histone H1. However, direct experimental evidence of this mechanism has been elusive, primarily due to the lack of methods to specifically poly-ubiquitylate H1. Here, we developed a versatile click chemistry approach to covalently link multiple proteins in a site-specific, controlled, and stepwise manner. Applying this method, we synthesized H1 constructs bearing triazole-linked di-ubiquitin on four DNA repair-associated ubiquitylation hotspots (H1KxUb2, at K17, 46, 64 and 96). Integrated into nucleosome arrays, the H1KxUb2 variants stimulated H2A ubiquitylation by RNF168 in a position-dependent manner, with H1K17Ub2 showing the strongest RNF168 activation effect. Moreover, we show that di-ubiquitin binding is the driving force underlying RNF168 recruitment, introducing H1K17Ub2 into living U-2 OS cells. Together, our results support the hypothesis of poly-ubiquitylated H1 guiding RNF168 recruitment to DSB sites. Moreover, we demonstrate how the streamlined synthesis of H1KxUb2 variants enables mechanistic studies into RNF168 regulation, with potential implications for its inhibition in susceptible cancers.

HCV infection activates the proteasome via PA28γ acetylation and heptamerization to facilitate the degradation of RNF2, a catalytic component of polycomb repressive complex 1.

We previously reported that hepatitis C virus (HCV) infection or HCV core protein expression induces HOX gene expression by impairing histone H2A monoubiquitination via a proteasome-dependent reduction in the level of RNF2, a key catalytic component of polycomb repressive complex 1 (H. Kasai, K. Mochizuki, T. Tanaka, A. Yamashita, et al., J Virol 95:e01784-20, 2021, https://doi.org/10.1128/jvi.01784-20). In this study, we aimed to investigate the mechanism by which HCV infection accelerates RNF2 degradation. Yeast two-hybrid screening and an immunoprecipitation assay revealed that RNF2 is a PA28γ-binding protein. The proteasome activator PA28γ destabilized the RNF2 protein in a proteasome-dependent manner, since RNF2 degradation was impaired by PA28γ knockout or MG132 treatment. HCV infection or core protein expression reduced the levels of RNF2 and histone H2A K119 monoubiquitination and induced the expression of HOX genes in the presence of PA28γ, while PA28γ knockout reversed these changes. Treatment with a lysine acetyltransferase inhibitor inhibited the acetylation of PA28γ at K195 and the degradation of the RNF2 protein, while treatment with a lysine deacetylase inhibitor accelerated these events in a PA28γ-dependent manner. RNF2 protein degradation was increased by expression of the acetylation mimetic PA28γ mutant but not by expression of the acetylation-defective mutant or the proteasome activation-defective mutant. Furthermore, HCV infection or core protein expression facilitated the interaction between PA28γ and the lysine acetyltransferase CBP/p300 and then accelerated PA28γ acetylation and heptazmerization to promote RNF2 degradation. These data suggest that HCV infection accelerates the acetylation-dependent heptamerization of PA28γ to increase the proteasomal targeting of RNF2.IMPORTANCEHCV is a causative agent of HCV-related liver diseases, including hepatic steatosis, cirrhosis, and hepatocellular carcinoma. PA28γ, which, in heptameric form, activates the 20S core proteasome for the degradation of PA28γ-binding proteins, is responsible for HCV-related liver diseases. HCV core protein expression or HCV infection accelerates RNF2 degradation, leading to the induction of HOX gene expression via a decrease in the level of H2Aub on HOX gene promoters. However, the mechanism of RNF2 degradation in HCV-infected cells has not been clarified. The data presented in this study suggest that PA28γ acetylation and heptamerization are promoted by HCV infection or by core protein expression to activate the proteasome for the degradation of RNF2 and are responsible for HCV propagation. This study provides novel insights valuable for the development of therapies targeting both HCV propagation and HCV-related diseases.

Read-write mechanisms of H2A ubiquitination by Polycomb repressive complex 1.

Epigenetic inheritance of silent chromatin domains is fundamental to cellular memory during embryogenesis, but it must overcome the dilution of repressive histone modifications during DNA replication1. One such modification, histone H2A lysine 119 monoubiquitination (H2AK119Ub), needs to be re-established by the Polycomb repressive complex 1 (PRC1) E3 ligase to restore the silent Polycomb domain2,3. However, the exact mechanism behind this restoration remains unknown. Here, combining cryo-electron microscopy (cryo-EM) and functional approaches, we characterize the read-write mechanism of the non-canonical PRC1-containing RYBP (ncPRC1RYBP). This mechanism, which functions as a positive-feedback loop in epigenetic regulation4,5, emphasizes the pivotal role of ncPRC1RYBP in restoring H2AK119Ub. We observe an asymmetrical binding of ncPRC1RYBP to H2AK119Ub nucleosomes, guided in part by the N-terminal zinc-finger domain of RYBP binding to residual H2AK119Ub on nascent chromatin. This recognition positions the RING domains of RING1B and BMI1 on the unmodified nucleosome side, enabling recruitment of the E2 enzyme to ubiquitinate H2AK119 within the same nucleosome (intra-nucleosome read-write) or across nucleosomes (inter-nucleosome read-write). Collectively, our findings provide key structural and mechanistic insights into the dynamic interplay of epigenetic regulation, highlighting the significance of ncPRC1RYBP in H2AK119Ub restoration to sustain repressive chromatin domains.

Nucleosome flipping drives kinetic proofreading and processivity by SWR1

The yeast SWR1 complex catalyses the exchange of histone H2A–H2B dimers in nucleosomes, with Htz1–H2B dimers1, 2–3. Here we used single-molecule analysis to demonstrate two-step double exchange of the two H2A–H2B dimers in a canonical yeast nucleosome with Htz1–H2B dimers, and showed that double exchange can be processive without release of the nucleosome from the SWR1 complex. Further analysis showed that bound nucleosomes flip between two states, with each presenting a different face, and hence histone dimer, to SWR1. The bound dwell time is longer when an H2A–H2B dimer is presented for exchange than when presented with an Htz1–H2B dimer. A hexasome intermediate in the reaction is bound to the SWR1 complex in a single orientation with the ‘empty’ site presented for dimer insertion. Cryo-electron microscopy analysis revealed different populations of complexes showing nucleosomes caught ‘flipping’ between different conformations without release, each placing a different dimer into position for exchange, with the Swc2 subunit having a key role in this process. Together, the data reveal a processive mechanism for double dimer exchange that explains how SWR1 can ‘proofread’ the dimer identities within nucleosomes.

Seminal proteoforms from bulls with contrasting semen freezability: a story deciphered by top-down mass spectrometry.

Top-down proteomics was employed to construct proteoform atlas of sperm and seminal plasma (SP) from bulls with low (LF) and high (HF) semen freezability. Sperm and seminal proteins were fractionated by tandem size exclusion chromatography (< 30 kDa) and analyzed by reversed-phase liquid chromatography-tandem mass spectrometry. This approach enabled the identification of 299 SP (from 46 families) and 267 sperm proteoforms (from 139 families). Seventy proteoforms belonging to beta-defensin 10, c-type natriuretic peptide (NPPC), caltrin, seminal ribonuclease, osteopontin, and binder of sperm protein (BSP) 3 families were unique to HF bulls' SP. LF seminal proteins had unique 77 proteoforms, including caltrin, NPPC, osteopontin, BSP3, serpin family A member 5, and β-NGF families. Proteoform families of SP in HF and LF bulls were related to Ca2+ uptake, capacitation, acrosome reaction, sperm protection, fertilization and proteolytic processes. Thirty-three proteoforms of NPPC, caltrin, and cylicin-2 families were upregulated in HF sperm. Twenty-two proteoforms of caltrin, cylicin-2, ATP synthases, and malate dehydrogenase families were among those upregulated in LF sperm. Truncated and acetylated histone H2A and non-truncated and acetylated c-Myc binding protein were prevalent in LF sperm. Cylicin-2 proteoforms were observed in HF and LF sperm, and truncated glyceraldehyde-3-phosphate dehydrogenases, only in HF sperm. In silico analyses indicated the enrichment of mitochondrial metabolic pathways in HF sperm, including fatty acid metabolism and TCA cycle. Our study brings an unprecedented description of the bovine SP and sperm proteoforms. Post-translational processing appears to define the bio-properties of semen proteins and their associations with sperm cryoresistance.

Immunohistochemical Evaluation of p300, H2AacK5 and H3AcK27 in Odontogenic Cysts and Tumors.

The acetylation of histones H2A on lysine 5 (H2AacK5) and H3 on lysine 27 (H3AcK27) modulate several cellular mechanisms through the p300 enzyme in pathological lesions; however, their role in odontogenic lesions has not been addressed. This study aims to evaluate the immunoexpression of p300, H2AacK5, and H3AcK27 in samples of ameloblastoma (AMB) (n = 30), odontogenic keratocyst (OK) (n = 15), adenomatoid odontogenic tumor (AOT) (n = 10), odontogenic fibroma (OF) (n = 8), calcifying odontogenic cyst (COC) (n = 8), odontogenic myxoma (MIX) (n = 10), and ameloblastic fibroma (AF) (n = 06). The percentage of p300-positive cells was higher in AOT and decreased in COC, OK, AMB, AF, OF, and MIX. H2AacK5-positive cells were higher in AF and decreased in AOT, COC, OK, OF, AMB, and MIX, whereas H3acK27-positive cells were higher in AOT and decreased in COC, OK, AF, OF, AMB, and MIX. The expression of these proteins was higher in nonaggressive lesions in comparison to aggressive lesions. There was a positive correlation between p300 and H2AacK5, and H3acK27 in AMB, MIX, and OF, whereas there was a positive correlation between p300 and H2AacK5 in AOT and COC. The histone acetylation may be involved in the biological behavior of these lesions, which could be used to improve their diagnosis and treatment.

Dinochromosome Heterotermini with Telosomal Anchorages

Dinoflagellate birefringent chromosomes (BfCs) contain some of the largest known genomes, yet they lack typical nucleosomal micrococcal-nuclease protection patterns despite containing variant core histones. One BfC end interacts with extranuclear mitotic microtubules at the nuclear envelope (NE), which remains intact throughout the cell cycle. Ultrastructural studies, polarized light and fluorescence microscopy, and micrococcal nuclease-resistant profiles (MNRPs) revealed that NE-associated chromosome ends persisted post-mitosis. Histone H3K9me3 inhibition caused S-G2 delay in synchronous cells, without any effects at G1. Differential labeling and nuclear envelope swelling upon decompaction indicate an extension of the inner compartment into telosomal anchorages (TAs). Additionally, limited effects of low-concentration sirtinol on bulk BfCs, coupled with distinct mobility patterns in MNase-digested and psoralen-crosslinked nuclei observed on 2D gels, suggest that telomeric nucleosomes (TNs) are the primary histone structures. The absence of a nucleosomal ladder with cDNA probes, the presence of histone H2A and telomere-enriched H3.3 variants, along with the immuno-localization of H3 variants mainly at the NE further reinforce telomeric regions as the main nucleosomal domains. Cumulative biochemical and molecular analyses suggest that telomeric repeats constitute the major octameric MNRPs that provision chromosomal anchorage at the NE.

MLKL promotes extracellular trap releasing in Vibrio splendidus AJ01-challenged coelomocytes of sea cucumber Apostichopus japonicus

Extracellular traps (ETs) is a novel host-defense mechanism for the immobilization and killing of invading microorganisms, of which pseudokinase mixed lineage kinase-like protein (MLKL) is necessary for ET formation. Here, we aimed to investigate the effects of various stimulations (including classic ET inducers and pathogen) on ET formation in the coelomocytes of invertebrate sea cucumber Apostichopus japonicus. Our results show that stimulation of sea cucumber coelomocytes with phorbol 12-myristate 13-acetate (PMA), zymosan, Vibrio splendidus AJ01, and AJ01 flagella all led to the formation of typical ET fibers, which can be further digested by DNase I. This finding suggests that DNA is an essential component of coelomocyte ETs. Meanwhile, the signals of histones H2A and H2B transferred to extracellular spaces and colocalized with AJ01, which indicates that histones are also crucial components of coelomocyte ETs. Scanning electron microscopy observation and antimicrobial activity analysis successfully revealed that AJ01 was trapped and killed by ET fibers, and this finding implies the antimicrobial role of sea cucumber coelomocyte ETs. More importantly, silencing and inhibition of AjMLKL after AJ01 infection not only considerably depressed ET generation but also increased the survival rate of AJ01. All our results indicate that AjMLKL mediates sea cucumber ET formation and plays critical roles during pathogen infection.

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

Role and mechanism of KIAA1429 in regulating cellular ferroptosis and radioresistance in colorectal cancer.

Colorectal cancer (CRC) is one of the most common non-cutaneous malignancies, causing significant mortality and a substantial burden. This study aims to explore the role of KIAA1429 (also known as vir-like m6A methyltransferase associated [VIRMA]) protein in the radioresistance of CRC. CRC cells and a radioresistant cell line were cultured, and KIAA1429 expression was detected. After the down-regulation of KIAA1429, its effect on the radioresistance and ferroptosis of cancer cells was analyzed. The role of ferroptosis in radioresistance was verified. The binding relationship among long non-coding RNA endogenous Bornavirus-like nucleoprotein 3, pseudogene (lncRNA EBLN3P), microRNA (miR)-153-3p, and KIAA1429 was analyzed. KIAA1429 and lncRNA EBLN3P were highly expressed in CRC, while miR-153-3p was poorly expressed. KIAA1429 and lncRNA EBLN3P were further increased/decreased in the radioresistant cells. KIAA1429 knockdown decreased the survival rate of the radioresistant cell line after X-ray irradiation and increased gamma H2A histone family member X (γ-H2AX), ferroptosis, and oxidative stress. A ferroptosis inhibitor alleviated the inhibitory effect of KIAA1429 knockdown on radioresistance. KIAA1429-mediated m6A modification up-regulated lncRNA EBLN3P, and lncRNA EBLN3P increased KIAA1429 by competitively binding to miR-153-3p. miR-153-3p silencing or lncRNA EBLN3P overexpression attenuated the promotion of ferroptosis and the inhibition of radioresistance induced by KIAA1429 knockdown. Overall, KIAA1429-mediated m6A modification up-regulated lncRNA EBLN3P expression, and lncRNA EBLN3P increased KIAA1429 expression by competitively binding to miR-153-3p, thus reducing ferroptosis and increasing the radioresistance of CRC.

BMI-1 in Breast Cancer - Biological Role and Clinical Implications.

Breast cancer is the most frequent cancer among women. Despite extensive research in recent years the molecular basis of breast cancer development, growth and metastasis remains unclear. Numerous studies highlight the involvement of BMI-1 in tumorigenesis. BMI-1 protein is a key component of Polycomb Repressive Complex 1, which by ubiquitinylation of histone H2A, regulates expression of genes involved in various cellular processes including cell cycle, proliferation and programmed cell death. Overexpression of BMI-1 has been often associated with breast cancer development and progression. This review summarizes the current state of knowledge of BMI-1's role in breast cancer biology and its potential significance as prognostic marker and potential target of new anticancer therapy.

RPS19 and RPL5, the most commonly mutated genes in Diamond Blackfan anemia, impact DNA double-strand break repair.

Diamond Blackfan anemia (DBA) is caused by germline heterozygous loss-of-function pathogenic variants (PVs) in ribosomal protein (RP) genes, most commonly RPS19 and RPL5 . In addition to red cell aplasia, individuals with DBA are at increased risk of various cancers. Importantly, the mechanism(s) underlying cancer predisposition are poorly understood. We found that DBA patient-derived lymphoblastoid cells had persistent γ-H2AX foci following ionizing radiation (IR) treatment, suggesting DNA double-strand break (DSB) repair defects. RPS19- and RPL5-knocked down (KD) CD34+ cells had delayed repair of IR-induced DSBs, further implicating these RPs in DSB repair. Assessing the impact of RPS19- and RPL5-KD on specific DSB repair pathways, we found RPS19-KD decreased the efficiency of pathways requiring extensive end-resection, whereas RPL5-KD increased end-joining pathways. Additionally, RAD51 was reduced in RPS19- and RPL5-KD and RPS19- and RPL5-mutated DBA cells, whereas RPS19-deficient cells also had a reduction in PARP1 and BRCA2 proteins. RPS19-KD cells had an increase in nuclear RPA2 and a decrease in nuclear RAD51 foci post-IR, reflective of alterations in early, critical steps of homologous recombination. Notably, RPS19 and RPL5 interacted with poly(ADP)-ribose chains noncovalently, were recruited to DSBs in a poly(ADP)-ribose polymerase activity-dependent manner, and interacted with Ku70 and histone H2A. RPL5's recruitment, but not RPS19's, also required p53, suggesting that RPS19 and RPL5 directly participate in DSB repair via different pathways. We propose that defective DSB repair arising from haploinsufficiency of these RPs may underline the cancer predisposition in DBA.

Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally Defined K63-Polyubiquitylated Linker Histone H1.

The chemical synthesis of histones with homogeneous modifications is a powerful approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). In this study, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on H1 ubiquitylated chromatosomes with different ubiquitin chain lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and the chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub3 chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub3 di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally defined polyubiquitylated H1.0 by the CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.

Yeast Nat4 regulates DNA damage checkpoint signaling through its N-terminal acetyltransferase activity on histone H4.

The DNA damage response (DDR) constitutes a vital cellular process that safeguards genome integrity. This biological process involves substantial alterations in chromatin structure, commonly orchestrated by epigenetic enzymes. Here, we show that the epigenetic modifier N-terminal acetyltransferase 4 (Nat4), known to acetylate the alpha-amino group of serine 1 on histones H4 and H2A, is implicated in the response to DNA damage in S. cerevisiae. Initially, we demonstrate that yeast cells lacking Nat4 have an increased sensitivity to DNA damage and accumulate more DNA breaks than wild-type cells. Accordingly, upon DNA damage, NAT4 gene expression is elevated, and the enzyme is specifically recruited at double-strand breaks. Delving deeper into its effects on the DNA damage signaling cascade, nat4-deleted cells exhibit lower levels of the damage-induced modification H2AS129ph (γH2A), accompanied by diminished binding of the checkpoint control protein Rad9 surrounding the double-strand break. Consistently, Mec1 kinase recruitment at double-strand breaks, critical for H2AS129ph deposition and Rad9 retention, is significantly impaired in nat4Δ cells. Consequently, Mec1-dependent phosphorylation of downstream effector kinase Rad53, indicative of DNA damage checkpoint activation, is reduced. Importantly, we found that the effects of Nat4 in regulating the checkpoint signaling cascade are mediated by its N-terminal acetyltransferase activity targeted specifically towards histone H4. Overall, this study points towards a novel functional link between histone N-terminal acetyltransferase Nat4 and the DDR, associating a new histone-modifying activity in the maintenance of genome integrity.

Molecular basis of global promoter sensing and nucleosome capture by the SWR1 chromatin remodeler

The SWR1 chromatin remodeling complex is recruited to+1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. Here, we use cryoelectron microscopy (cryo-EM) to resolve the structural basis of the SWR1 interaction with free DNA, revealing a distinct open conformation of the Swr1 ATPase that enables sliding from accessible DNA to nucleosomes. A complete structural model of the SWR1-nucleosome complex illustrates critical roles for Swc2 and Swc3 subunits in oriented nucleosome engagement by SWR1. Moreover, an extended DNA-binding α helix within the Swc3 subunit enables sensing of nucleosome linker length and is essential for SWR1-promoter-specific recruitment and activity. The previously unresolved N-SWR1 subcomplex forms a flexible extended structure, enabling multivalent recognition of acetylated histone tails by reader domains to further direct SWR1 toward the+1 nucleosome. Altogether, our findings provide a generalizable mechanism for promoter-specific targeting of chromatin and transcription complexes.

UBTF mediates activation of L3MBTL2 to suppress NISCH expression through histone H2AK119 monoubiquitination modification in breast cancer.

Lethal(3)malignant brain tumor-like protein 2 (L3MBTL2) has been related to transcriptional inhibition and chromatin compaction. Nevertheless, the biological functions and mechanisms of L3MBTL2 are undefined in breast cancer (BRCA). Here, we revealed that L3MBTL2 is responsible for the decline of Nischarin (NISCH), a well-known tumor suppressor, in BRCA, and explored the detailed mechanism. Knockdown of L3MBTL2 reduced monoubiquitination of histone H2A at lysine-119 (H2AK119ub), leading to reduced binding to the NISCH promoter and increased expression of NISCH. Meanwhile, the knockdown of L3MBTL2 decreased proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of BRCA cells, and increased apoptosis, which were abated by NISCH knockdown. Nucleolar transcription factor 1 (UBTF) induced the transcription of L3MBTL2 in BRCA, and the suppressing effects of UBTF silencing on EMT in BRCA cells were also reversed by NISCH knockdown. Knockdown of UBTF slowed tumor progression and attenuated lung tumor infiltration, whereas simultaneous knockdown of NISCH accelerated EMT and increased tumor lung metastasis. Taken together, our results show that L3MBTL2, transcriptionally activated by UBTF, exerts oncogenic functions in BRCA, by catalyzing H2AK119Ub and reducing expression of NISCH.

Immunohistochemical expression of netosis markers (MPO, H2A, H2B) in placenta of RPL women in Basrah Province

Background. Recurrent Pregnancy Loss (RPL) is a multifactorial disorder significantly impacting women’s health globally. Emerging research has implicated Neutrophil Extracellular Traps (NETs) in the pathophysi­ology of RPL, suggesting an immunological underpinning to miscarriage occurrences. Aim. The study aim to evaluate the role and expression of NETosis markers (MPO, histones H2A, H2B) in women with RPL in comparison to pregnant women. Methods. This prospective cohort study, conducted from January 2022 to July 2023, included 30 participants (20 RPL patients and 10 controls) from Al Basrah Maternity and Child Hospital. Immunohistochemical technique were employed to detect Myeloperoxidase and histones in placental samples. Statistical analysis was performed using SPSS, with significance set at p&lt;90.05. Results. Immunohistochemical analysis of placental tissues indicated significant overexpression of Myelo­peroxidase (MPO), and histones H2A and H2B, in RPL patients compared to control, emphasizing the potential involvement of NETs in placental pathology associated with miscarriage. The expression levels of MPO were positive in 80% of RPL patients but undetected in control (p = 0.0001). Histone expressions also differed significantly, with H2A detected in 55% of RPL patients compared to none in control (p = 0.001) and H2B in 70% versus 30% (both p &lt;0.001).