Search Results (32)

Vitamin B12 (B12) is a strong antioxidant and a cofactor for methionine synthase supporting DNA/RNA/protein methylation. We previously demonstrated that oral high-dose B12 supplement mitigates diabetic cardiomyopathy in Akita diabetic mice expressing twice the normal levels of Elmo1 (Engulfment and cell motility 1). To assess how B12 prevents early kidney damage, we treated Elmo1^HH mice and diabetic Elmo1^HH Ins2^Akita/+ mice with or without B12 in drinking water starting at 8 weeks of age. At 16 weeks, markedly reduced mesangial expansion was detected in the B12-treated diabetic kidneys (22% of glomeruli affected vs. 70% in the untreated diabetic kidneys). RNAseq analysis of the kidneys revealed that B12 suppressed expression of genes for adaptive immune response, while it upregulated those for solute carrier transporters and antioxidant genes. Strikingly, B12 treatment suppressed activators of circadian rhythm, Clock and Bmal1, and upregulated repressors like Cry1/2, Per1-3 and Dbp, suggesting a shift in their rhythmicity. B12 also upregulated linker histone H1 variants, and enhanced chromatin stability and cell cycle regulation. In BU.MPT proximal tubular cells in culture, B12 shifted forward the circadian expression phase of Bmal1 and Per1. Taken together, B12 supplement effectively mitigates early development of diabetic nephropathy in diabetic mice, potentially involving regulation of circadian rhythm. Full article

Poly(ADP-ribose)polymerase 1 (PARP1) is an enzyme that interacts with chromatin during DNA repair and transcription processes; the molecular mechanisms of these processes remain to be determined. Previously, we have shown that PARP1 can bind to and reorganize nucleosomes using two modes of interaction with a mono-nucleosome, which are realized through PARP1 binding to the ends of linker DNA and to the nucleosome core. Here, it is shown that the latter mode of binding induces the reorganization of nucleosome structure and is more stable under the conditions of poly(ADP-ribosyl)ation (PARylation). The initial nucleosome structure is fully recovered after the dissociation of autoPARylated PARP1. The competition between PARP1 and linker histone H1.0 for binding to a nucleosome is mediated by the PARP1-H1.0 interaction and is affected by the length of linker DNA fragments. Longer linkers stabilize H1.0-nucleosome complexes, while shorter linkers facilitate displacement of H1.0 from the chromatosome by PARP1. PARylation removes both H1.0 and PARP1 from the complexes with nucleosomes. The data suggest that the H1.0 displacement from chromatin by PARP1 that is likely modulated by the density of nucleosomes might reduce chromatin compaction and facilitate access of PARP1-dependent DNA repair and transcription factors to nucleosome and inter-nucleosomal DNA. Full article

Background: Histone deacetylases (HDACs) are enzymes that deacetylate histone proteins, impacting the transcriptional repression and activation of cancer-associated genes such as P53 and Ras. The overexpression of HDACs in breast cancer (BC) underscores their significance as therapeutic targets for modulating gene expression through epigenetic regulation. Methods: In this study, a novel series of SAHA (suberoylanilide hydroxamic acid) analogs were designed using an in silico ligand-based strategy. These analogs were then synthesized and evaluated for their HDAC-inhibitory capacity as well as their antiproliferative capacity on breast cancer cells. These compounds retained an aliphatic LINKER, mimicking the natural substrate acetyl-lysine, while differing from the hydroxamic fragment present in SAHA. Results: The synthesized compounds exhibited HDAC inhibitory activity, suggesting potential for binding to these pharmacological targets. Compounds 5b, 6a, and 6b were identified as promising candidates in the evaluation on breast cancer cell lines MCF-7 and MDA-MB-231 at 72 h. Specifically, compound 6b, which contains an N-trifluoroacetyl group as a zinc-binding group (ZBG), demonstrated an IC₅₀ of 76.7 µM in the MDA-MB-231 cell line and 45.7 µM in the MCF-7 cell line. In the non-tumorigenic cell line, the compound exhibited an IC₅₀ of 154.6 µM. Conversely, SAHA exhibited an almost negligible safety margin with regard to its cytotoxic activity when compared to breast cancer cells and healthy cells (MCF-10A). This observation underscores the elevated toxicity exhibited by hydroxamic acid-derived molecules. Conclusions: The bioisosteric modification of ZBG by N-trifluoroacetyl in 6a and 6b demonstrated favorable cytotoxic activity, exhibiting a higher safety margin. This study underscores the challenge of identifying novel ZBGs to replace hydroxamic acid in the development of HDAC inhibitors, with the objective of enhancing their physicochemical and toxicological profile for utilization in BC treatment. Full article

Previously we discovered that among 15 DNA-binding plant secondary metabolites (PSMs) possessing anticancer activity, 11 compounds cause depletion of the chromatin-bound linker histones H1.2 and/or H1.4. Chromatin remodeling or multiH1 knocking-down is known to promote the upregulation of repetitive elements, ultimately triggering an interferon (IFN) response. Herein, using HeLa cells and applying fluorescent reporter assay with flow cytometry, immunofluorescence staining and quantitative RT-PCR, we studied effects of PSMs both evicting linker histones from chromatin and not influencing their location in nucleus. We found that (1) 8 PSMs, evicting linker histone H1.2 from chromatin, activated significantly the type I IFN signaling pathway and out of these compounds resveratrol, berberine, genistein, delphinidin, naringenin and curcumin also caused LINE1 expression. Fisetin and quercetin, which also induced linker histone H1.2 eviction from chromatin, significantly activated only type I IFN signaling, but not LINE1 expression; (2) curcumin, sanguinarine and kaempferol, causing significant depletion of the chromatin-bound linker histone H1.4 but not significantly influencing H1.2 presence in chromatin, activate type I IFN signaling less intensively without any changes in LINE1 expression; (3) four PSMs, which did not cause linker histone eviction, displayed neither IFN signaling activation nor enhancement of LINE1 expression. Thus, we have shown for the first time that chromatin destabilization observed by depletion of chromatin-bound linker histone H1.2 caused by anticancer DNA-binding PSMs is accompanied by enhancement of type I IFN signaling, and that LINE1 expression often impacts this activation. Full article

In eukaryotic nuclei, DNA is wrapped around an octamer of core histones to form nucleosomes. H1 binds to the linker DNA of nucleosome to form the chromatosome, the next structural unit of chromatin. Structural features on individual chromatosomes contribute to chromatin structure, but not fully characterized. In addition to canonical nucleosomes composed of two copies each of histones H2A, H2B, H3, and H4 (H3 nucleosomes), centromeres chromatin contain nucleosomes in which H3 is replaced with its analog CENP-A, changing structural properties of CENP-A nucleosomes. Nothing is known about the interaction of H1 with CENP-A nucleosomes. Here we filled this gap and characterized the interaction of H1 histone with both types of nucleosomes. H1 does bind both types of the nucleosomes forming more compact chromosome particles with elevated affinity to H3 nucleosomes. H1 binding significantly increases the stability of chromatosomes preventing their spontaneous dissociation. In addition to binding to the entry-exit position of the DNA arms identified earlier, H1 is capable of bridging of distant DNA segments. H1 binding leads to the assembly of mononucleosomes in aggregates, stabilized by internucleosome interactions as well as bridging of the DNA arms of chromatosomes. Contribution of these finding to the chromatin structure and functions are discussed. Full article

A thermodynamic analysis of the binary complex formation of the highly positively charged linker histone H1 and the highly negatively charged chaperone prothymosin α (ProTα) is detailed. ProTα and H1 have large opposite net charges (−44 and +53, respectively) and form complexes at physiological salt concentrations with high affinities. The data obtained for the binary complex formation are analyzed by a thermodynamic model that is based on counterion condensation modulated by hydration effects. The analysis demonstrates that the release of the counterions mainly bound to ProTα is the main driving force, and effects related to water release play no role within the limits of error. A strongly negative Δc_p (=−0.87 kJ/(K mol)) is found, which is due to the loss of conformational degrees of freedom. Full article

Histone H1.2 is a member of the linker histone family, which plays extensive and crucial roles not only in the regulation of chromatin dynamics, cell cycle, and cell apoptosis, but also in viral diseases and innate immunity response. Recently, it was discovered that H1.2 regulates interferon-β and inhibits influenza virus replication, whereas its role in other viral infections is poorly reported. Here, we first found the up-regulation of H1.2 during Encephalomyocarditis virus (EMCV) infection, implying that H1.2 was involved in EMCV infection. Overexpression of H1.2 inhibited EMCV proliferation, whereas knockdown of H1.2 showed a significant promotion of virus infection in HEK293T cells. Moreover, we demonstrated that overexpression of H1.2 remarkably enhanced the production of EMCV-induced type I interferon, which may be the crucial factor for H1.2 proliferation–inhibitory effects. We further found that H1.2 up-regulated the expression of the proteins of the MDA5 signaling pathway and interacted with MDA5 and IRF3 in EMCV infection. Further, we demonstrated that H1.2 facilitated EMCV-induced phosphorylation and nuclear translocation of IRF3. Briefly, our research uncovers the mechanism of H1.2 negatively regulating EMCV replication and provides new insight into antiviral targets for EMCV. Full article

Pericentric heterochromatin (PCH) forms spatio-temporarily distinct compartments and affects chromosome organization and stability. Albeit some of its components are known, an elucidation of its proteome and how it differs between tissues in vivo is lacking. Here, we find that PCH compartments are dynamically organized in a tissue-specific manner, possibly reflecting compositional differences. As the mouse brain and liver exhibit very different PCH architecture, we isolated native PCH fractions from these tissues, analyzed their protein compositions using quantitative mass spectrometry, and compared them to identify common and tissue-specific PCH proteins. In addition to heterochromatin-enriched proteins, the PCH proteome includes RNA/transcription and membrane-related proteins, which showed lower abundance than PCH-enriched proteins. Thus, we applied a cut-off of PCH-unspecific candidates based on their abundance and validated PCH-enriched proteins. Amongst the hits, MeCP2 was classified into brain PCH-enriched proteins, while linker histone H1 was not. We found that H1 and MeCP2 compete to bind to PCH and regulate PCH organization in opposite ways. Altogether, our workflow of unbiased PCH isolation, quantitative mass spectrometry, and validation-based analysis allowed the identification of proteins that are common and tissue-specifically enriched at PCH. Further investigation of selected hits revealed their opposing role in heterochromatin higher-order architecture in vivo. Full article

A series of novel quinazoline-4-(3H)-one derivatives were designed and synthesized as histone deacetylase 6 (HDAC6) inhibitors based on novel quinazoline-4-(3H)-one as the cap group and benzhydroxamic acid as the linker and metal-binding group. A total of 19 novel quinazoline-4-(3H)-one analogues (5a–5s) were obtained. The structures of the target compounds were characterized using ¹H-NMR, ¹³C-NMR, LC–MS, and elemental analyses. Characterized compounds were screened for inhibition against HDAC8 class I, HDAC4 class IIa, and HDAC6 class IIb. Among the compounds tested, 5b proved to be the most potent and selective inhibitor of HDAC6 with an IC₅₀ value 150 nM. Some of these compounds showed potent antiproliferative activity in several tumor cell lines (HCT116, MCF7, and B16). Amongst all the compounds tested for their anticancer effect against cancer cell lines, 5c emerged to be most active against the MCF-7 line with an IC₅₀ of 13.7 μM; it exhibited cell-cycle arrest in the G2 phase, as well as promoted apoptosis. Additionally, we noted a significant reduction in the colony-forming capability of cancer cells in the presence of 5c. At the intracellular level, selective inhibition of HDAC6 was enumerated by monitoring the acetylation of α-tubulin with a limited effect on acetyl-H3. Importantly, the obtained results suggested a potent effect of 5c at sub-micromolar concentrations as compared to the other molecules as HDAC6 inhibitors in vitro. Full article

Mitochondria are multifunctional, dynamic organelles important for stress response, cell longevity, ageing and death. Although the mitochondrion has its genome, nuclear-encoded proteins are essential in regulating mitochondria biogenesis, morphology, dynamics and function. Moreover, chromatin structure and epigenetic mechanisms govern the accessibility to DNA and control gene transcription, indirectly influencing nucleo-mitochondrial communications. Thus, they exert crucial functions in maintaining proper chromatin structure, cell morphology, gene expression, stress resistance and ageing. Here, we present our studies on the mtDNA copy number in Saccharomyces cerevisiae chromatin mutants and investigate the mitochondrial membrane potential throughout their lifespan. The mutants are arp4 (with a point mutation in the ARP4 gene, coding for actin-related protein 4—Arp4p), hho1Δ (lacking the HHO1 gene, coding for the linker histone H1), and the double mutant arp4 hho1Δ cells with the two mutations. Our findings showed that the three chromatin mutants acquired strain-specific changes in the mtDNA copy number. Furthermore, we detected the disrupted mitochondrial membrane potential in their chronological lifespan. In addition, the expression of nuclear genes responsible for regulating mitochondria biogenesis and turnover was changed. The most pronounced were the alterations found in the double mutant arp4 hho1Δ strain, which appeared as the only petite colony-forming mutant, unable to grow on respiratory substrates and with partial depletion of the mitochondrial genome. The results suggest that in the studied chromatin mutants, hho1Δ, arp4 and arp4 hho1Δ, the nucleus-mitochondria communication was disrupted, leading to impaired mitochondrial function and premature ageing phenotype in these mutants, especially in the double mutant. Full article

Epigenetic modifications are indispensable for regulating gene bodies and TE silencing. DECREASE IN DNA METHYLATION 1 (DDM1) is a chromatin remodeller involved in histone modifications and DNA methylation. Apart from maintaining the epigenome, DDM1 also maintains key plant traits such as flowering time and heterosis. The role of DDM1 in epigenetic regulation is best characterised in plants, especially arabidopsis, rice, maize and tomato. The epigenetic changes induced by DDM1 establish the stable inheritance of many plant traits for at least eight generations, yet DDM1 does not methylate protein-coding genes. The DDM1 TE silencing mechanism is distinct and has evolved independently of other silencing pathways. Unlike the RNA-directed DNA Methylation (RdDM) pathway, DDM1 does not depend on siRNAs to enforce the heterochromatic state of TEs. Here, we review DDM1 TE silencing activity in the RdDM and non-RdDM contexts. The DDM1 TE silencing machinery is strongly associated with the histone linker H1 and histone H2A.W. While the linker histone H1 excludes the RdDM factors from methylating the heterochromatin, the histone H2A.W variant prevents TE mobility. The DDM1-H2A.W strategy alone silences nearly all the mobile TEs in the arabidopsis genome. Thus, the DDM1-directed TE silencing essentially preserves heterochromatic features and abolishes mobile threats to genome stability. Full article

Linker histones (LH) are a critical component of chromatin in addition to the canonical histones (H2A, H2B, H3, and H4). In humans, 11 subtypes (7 somatic and 4 germinal) of linker histones have been identified, and their diverse cellular functions in chromatin structure, DNA replication, DNA repair, transcription, and apoptosis have been explored, especially for the somatic subtypes. Delineating the unique role of human linker histone (hLH) and their subtypes is highly tedious given their high homology and overlapping expression patterns. However, recent advancements in mass spectrometry combined with HPLC have helped in identifying the post-translational modifications (PTMs) found on the different LH subtypes. However, while a number of PTMs have been identified and their potential nuclear and non-nuclear functions explored in cellular processes, there are very few studies delineating the direct relevance of these PTMs in diseases. In addition, recent whole-genome sequencing of clinical samples from cancer patients and individuals afflicted with Rahman syndrome have identified high-frequency mutations and therefore broadened the perspective of the linker histone mutations in diseases. In this review, we compile the identified PTMs of hLH subtypes, current knowledge of the relevance of hLH PTMs in human diseases, and the correlation of PTMs coinciding with mutations mapped in diseases. Full article

The functioning of the eukaryotic cell genome is mediated by sophisticated protein-nucleic-acid complexes, whose minimal structural unit is the nucleosome. After the damage to genomic DNA, repair proteins need to gain access directly to the lesion; therefore, the initiation of the DNA damage response inevitably leads to local chromatin reorganisation. This review focuses on the possible involvement of PARP1, as well as proteins acting nucleosome compaction, linker histone H1 and non-histone chromatin protein HMGB1. The polymer of ADP-ribose is considered the main regulator during the development of the DNA damage response and in the course of assembly of the correct repair complex. Full article

Meiosis is a highly conserved specialized cell division process that generates haploid gametes. Many of its events are associated with dynamically regulated chromosomal structures and chromatin remodeling, which are mainly modulated by histone modifications. Histone H1 is a linker histone essential for packing the nucleosome into higher-order structures, and H1FOO (H1 histone family, member O, oocyte-specific) is a H1 variant whose expression pattern is restricted to growing oocytes and zygotes. To further explore the function of H1FOO, we generated mice lacking the H1foo gene by the CRISPR/Cas9 technique. Herein, we combine mouse genetics and cellular studies to show that H1foo-null mutants have no overt phenotype, with both males and females being fertile and presenting no gross defects in meiosis progression nor in synapsis dynamics. Accordingly, the histological sections show a normal development of gametes in both male and female mice. Considering the important role of oocyte constituents in enhancing mammalian somatic cell reprogramming, we analyzed iPSCs generation in H1foo mutant MEFs and observed no differences in the absence of H1FOO. Taken all together, in this work we present the first in vivo evidence of H1FOO dispensability for mouse fertility, clarifying the debate in the field surrounding its essentiality in meiosis. Full article

Yeast Hmo1 is a high mobility group B (HMGB) protein that participates in the transcription of ribosomal protein genes and rDNA, and also stimulates the activities of some ATP-dependent remodelers. Hmo1 binds both DNA and nucleosomes and has been proposed to be a functional yeast analog of mammalian linker histones. We used EMSA and single particle Förster resonance energy transfer (spFRET) microscopy to characterize the effects of Hmo1 on nucleosomes alone and with the histone chaperone FACT. Hmo1 induced a significant increase in the distance between the DNA gyres across the nucleosomal core, and also caused the separation of linker segments. This was opposite to the effect of the linker histone H1, which enhanced the proximity of linkers. Similar to Nhp6, another HMGB factor, Hmo1, was able to support large-scale, ATP-independent, reversible unfolding of nucleosomes by FACT in the spFRET assay and partially support FACT function in vivo. However, unlike Hmo1, Nhp6 alone does not affect nucleosome structure. These results suggest physiological roles for Hmo1 that are distinct from Nhp6 and possibly from other HMGB factors and linker histones, such as H1. Full article