Baf155 controls hematopoietic differentiation and regeneration through chromatin priming.

Chromatin priming promotes cell-type-specific gene expression, lineage differentiation, and development. The mechanism of chromatin priming has not been fully understood. Here, we report that mouse hematopoietic stem and progenitor cells (HSPCs) lacking the Baf155 subunit of the BAF (BRG1/BRM-associated factor) chromatin remodeling complex produce a significantly reduced number of mature blood cells, leading to a failure of hematopoietic regeneration upon transplantation and 5-fluorouracil (5-FU) injury. Baf155-deficient HSPCs generate particularly fewer neutrophils, B cells, and CD8+ T cells at homeostasis, supporting a more immune-suppressive tumor microenvironment and enhanced tumor growth. Single-nucleus multiomics analysis reveals that Baf155-deficient HSPCs fail to establish accessible chromatin in selected regions that are enriched for putative enhancers and binding motifs of hematopoietic lineage transcription factors. Our study provides a fundamental mechanistic understanding of the role of Baf155 in hematopoietic lineage chromatin priming and the functional consequences of Baf155 deficiency in regeneration and tumor immunity.

Emerging toolkits for decoding the co-occurrence of modified histones and chromatin proteins.

In eukaryotes, DNA is packaged into chromatin with the help of highly conserved histone proteins. Together with DNA-binding proteins, posttranslational modifications (PTMs) on these histones play crucial roles in regulating genome function, cell fate determination, inheritance of acquired traits, cellular states, and diseases. While most studies have focused on individual DNA-binding proteins, chromatin proteins, or histone PTMs in bulk cell populations, such chromatin features co-occur and potentially act cooperatively to accomplish specific functions in a given cell. This review discusses state-of-the-art techniques for the simultaneous profiling of multiple chromatin features in low-input samples and single cells, focusing on histone PTMs, DNA-binding, and chromatin proteins. We cover the origins of the currently available toolkits, compare and contrast their characteristic features, and discuss challenges and perspectives for future applications. Studying the co-occurrence of histone PTMs, DNA-binding proteins, and chromatin proteins in single cells will be central for a better understanding of the biological relevance of combinatorial chromatin features, their impact on genomic output, and cellular heterogeneity.

Impact of sperm DNA fragmentation in couples with unexplained recurrent pregnancy loss: A cross-sectional study.

OBJECTIVE: Recurrent pregnancy loss (RPL) has a multifactorial etiology, with a majority of cases remaining unexplained. To account for these unexplained cases, possible male factors are being explored. Conventional semen analysis lacks a qualitative assessment of sperms and information regarding sperm DNA integrity. Sperm DNA fragmentation (SDF) has diagnostic value in unexplained RPL, and it may account for a number of unexplained cases. Hence, we planned a study to explore and evaluate the impact of sperm DNA fragmentation in couples with unexplained recurrent pregnancy losses. STUDY DESIGN: Analytical cross-sectional study was conducted at a tertiary-level referral facility in India between August 2021 and July 2023. Participants (n = 70) were divided into two groups-male partners of couples with unexplained RPL (following spontaneous conceptions) (n = 35) and men with at least one previous live birth (spontaneous or following fertility treatments for female factor infertility such as ovulation induction or intrauterine insemination) as controls (n = 35). Neither of the two groups of couples recruited for this study had undergone ART as fertility treatment. Primary outcome assessed was mean DNA fragmentation index (DFI). Secondary outcomes included differences in semen parameters such as sperm concentration, progressive sperm motility and morphology, proportion of men with high (≥30%) and low DFI in the two groups, and the association between various semen parameters and DFI. RESULTS: Univariate logistic regression revealed that sperm DNA fragmentation was higher in men with unexplained RPL (30.0; IQR (interquartile range) 19.0, 46.0) as compared to controls (22.0; IQR 14.0, 30.0) although it was not statistically significant (OR, odds ratio, 1.02; 95% CI 1.0-1.1, p = 0.08). A higher proportion of men with unexplained RPL had DFI ≥30% compared to controls (54.2% vs. 25.7%; OR 3.43 (95% CI 1.2-9.4); p = 0.02). No statistically significant differences were observed in semen volume, sperm concentration, progressive motility, and morphology between the two groups. Sperm DNA fragmentation index also showed a weak but significant inverse relationship with sperm morphology (r = -0.336, p = 0.004). CONCLUSION: The current study did not show any significant difference in the mean sperm DNA fragmentation levels in male partners of couples with unexplained RPL compared to controls. However, a higher proportion of men with DFI ≥30% were observed in unexplained RPL population when compared to controls.

Chromatin and gene regulation in archaea.

The chromatinisation of DNA by nucleoid-associated proteins (NAPs) in archaea 'formats' the genome structure in profound ways, revealing both striking differences and analogies to eukaryotic chromatin. However, the extent to which archaeal NAPs actively regulate gene expression remains poorly understood. The dawn of quantitative chromatin mapping techniques and first NAP-specific occupancy profiles in different archaea promise a more accurate view. A picture emerges where in diverse archaea with very different NAP repertoires chromatin maintains access to regulatory motifs including the gene promoter independently of transcription activity. Our re-analysis of genome-wide occupancy data of the crenarchaeal NAP Cren7 shows that these chromatin-free regions are flanked by increased Cren7 binding across the transcription start site. While bacterial NAPs often form heterochromatin-like regions across islands with xenogeneic genes that are transcriptionally silenced, there is little evidence for similar structures in archaea and data from Haloferax show that the promoters of xenogeneic genes remain accessible. Local changes in chromatinisation causing wide-ranging effects on transcription restricted to one chromosomal interaction domain (CID) in Saccharolobus islandicus hint at a higher-order level of organisation between chromatin and transcription. The emerging challenge is to integrate results obtained at microscale and macroscale, reconciling molecular structure and function with dynamic genome-wide chromatin landscapes.

Novel insights on the positive correlation between sense and antisense pairs on gene expression.

A considerable proportion of the eukaryotic genome undergoes transcription, leading to the generation of noncoding RNA molecules that lack protein-coding information and are not subjected to translation. These noncoding RNAs (ncRNAs) are well recognized to have essential roles in several biological processes. Long noncoding RNAs (lncRNAs) represent the most extensive category of ncRNAs found in the human genome. Much research has focused on investigating the roles of cis-acting lncRNAs in the regulation of specific target gene expression. In the majority of instances, the regulation of sense gene expression by its corresponding antisense pair occurs in a negative (discordant) manner, resulting in the suppression of the target genes. The notion that a negative correlation exists between sense and antisense pairings is, however, not universally valid. In fact, several recent studies have reported a positive relationship between corresponding cis antisense pairs within plants, budding yeast, and mammalian cancer cells. The positive (concordant) correlation between anti-sense and sense transcripts leads to an increase in the level of the sense transcript within the same genomic loci. In addition, mechanisms such as altering chromatin structure, the formation of R loops, and the recruitment of transcription factors can either enhance transcription or stabilize sense transcripts through their antisense pairs. The primary objective of this work is to provide a comprehensive understanding of both aspects of antisense regulation, specifically focusing on the positive correlation between sense and antisense transcripts in the context of eukaryotic gene expression, including its implications towards cancer progression. This article is categorized under: RNA Processing > 3' End Processing Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Activated dormant stem cells recover spermatogenesis in chemoradiotherapy-induced infertility.

Male infertility is a recognized side effect of chemoradiotherapy. Extant spermatogonial stem cells (SSCs) may act as originators for any subsequent recovery. However, which type of SSCs, the mechanism by which they survive and resist toxicity, and how they act to restart spermatogenesis remain largely unknown. Here, we identify a small population of Set domain-containing protein 4 (Setd4)-expressing SSCs that occur in a relatively dormant state in the mouse seminiferous tubule. Extant beyond high-dose chemoradiotherapy, these cells then activate to recover spermatogenesis. Recovery fails when Setd4+ SSCs are deleted. Confirmed to be of fetal origin, these Setd4+ SSCs are shown to facilitate early testicular development and also contribute to steady-state spermatogenesis in adulthood. Upon activation, chromatin remodeling increases their genome-wide accessibility, enabling Notch1 and Aurora activation with corresponding silencing of p21 and p53. Here, Setd4+ SSCs are presented as the originators of both testicular development and spermatogenesis recovery in chemoradiotherapy-induced infertility.

Nucleosome remodeler exclusion by histone deacetylation enforces heterochromatic silencing and epigenetic inheritance.

Heterochromatin enforces transcriptional gene silencing and can be epigenetically inherited, but the underlying mechanisms remain unclear. Here, we show that histone deacetylation, a conserved feature of heterochromatin domains, blocks SWI/SNF subfamily remodelers involved in chromatin unraveling, thereby stabilizing modified nucleosomes that preserve gene silencing. Histone hyperacetylation, resulting from either the loss of histone deacetylase (HDAC) activity or the direct targeting of a histone acetyltransferase to heterochromatin, permits remodeler access, leading to silencing defects. The requirement for HDAC in heterochromatin silencing can be bypassed by impeding SWI/SNF activity. Highlighting the crucial role of remodelers, merely targeting SWI/SNF to heterochromatin, even in cells with functional HDAC, increases nucleosome turnover, causing defective gene silencing and compromised epigenetic inheritance. This study elucidates a fundamental mechanism whereby histone hypoacetylation, maintained by high HDAC levels in heterochromatic regions, ensures stable gene silencing and epigenetic inheritance, providing insights into genome regulatory mechanisms relevant to human diseases.

A replisome-associated histone H3-H4 chaperone required for epigenetic inheritance.

Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone-binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.

The fork protection complex promotes parental histone recycling and epigenetic memory.

The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.

Structural biology of shelterin and telomeric chromatin: the pieces and an unfinished puzzle.

The six-subunit shelterin complex binds to mammalian telomeres and protects them from triggering multiple DNA damage response pathways. The loss of this protective function by shelterin can have detrimental effects on cells. In this review, we first discuss structural studies of shelterin, detailing the contributions of each subunit and inter-subunit interactions in protecting chromosome ends. We then examine the influence of telomeric chromatin dynamics on the function of shelterin at telomeres. These studies provide valuable insights and underscore the challenges that future research must tackle to attain high-resolution structures of shelterin.

Evidence of innate training in bovine γδ T cells following subcutaneous BCG administration.

The Bacillus Calmette Guerin (BCG) vaccine has been shown to induce non-specific protection against diseases other than tuberculosis in vaccinated individuals, attributed to the induction of trained immunity. We have previously demonstrated that BCG administration induces innate immune training in mixed peripheral blood mononuclear cells and monocytes in calves. Gamma Delta (γδ) T cells are non-conventional T cells that exhibit innate and adaptive immune system features. They are in higher proportion in the peripheral blood of cattle than humans or rodents and play an essential role in bovine immune response to pathogens. In the current study, we determined if BCG administration induced innate immune training in bovine γδ T cells. A group of 16 pre-weaned Holstein calves (2-4 d age) were enrolled in the study and randomly assigned to vaccine and control groups (n=8/group). The vaccine group received two doses of 106 colony forming units (CFU) BCG Danish strain subcutaneously, separated by 2 weeks. The control group remained unvaccinated. Gamma delta T cells were purified from peripheral blood using magnetic cell sorting three weeks after receiving the 1st BCG dose. We observed functional changes in the γδ T cells from BCG-treated calves shown by increased IL-6 and TNF-α cytokine production in response to in vitro stimulation with Escherichia coli LPS and PAM3CSK4. ATAC-Seq analysis of 78,278 regions of open chromatin (peaks) revealed that γδ T cells from BCG-treated calves had an altered epigenetic status compared to cells from the control calves. Differentially accessible peaks (DAP) found near the promoters of innate immunity-related genes like Siglec14, Irf4, Ifna2, Lrrfip1, and Tnfrsf10d were 1 to 4-fold more accessible in cells from BCG-treated calves. MOTIF enrichment analysis of the sequences within DAPs, which explores transcription factor binding motifs (TFBM) upstream of regulatory elements, revealed TFBM for Eomes and IRF-5 were among the most enriched transcription factors. GO enrichment analysis of genes proximal to the DAPs showed enrichment of pathways such as regulation of IL-2 production, T-cell receptor signaling pathway, and other immune regulatory pathways. In conclusion, our study shows that subcutaneous BCG administration in pre-weaned calves can induce innate immune memory in the form of trained immunity in γδ T cells. This memory is associated with increased chromatin accessibility of innate immune response-related genes, thereby inducing a functional trained immune response evidenced by increased IL-6 and TNF-α cytokine production.

Loss of H3K9 trimethylation leads to premature aging.

Aging is the major risk factor for most human diseases and represents a major socio-economical challenge for modern societies. Despite its importance, the process of aging remains poorly understood. Epigenetic dysregulation has been proposed as a key driver of the aging process. Modifications in transcriptional networks and chromatin structure might be central to age-related functional decline. A prevalent feature described during aging is the overall reduction in heterochromatin, specifically marked by the loss of repressive histone modification, Histone 3 lysine 9 trimethylation (H3K9me3). However, the role of H3K9me3 in aging, especially in mammals, remains unclear. Here we show using a novel mouse strain, (TKOc), carrying a triple knockout of three methyltransferases responsible for H3K9me3 deposition, that the inducible loss of H3K9me3 in adulthood results in premature aging. TKOc mice exhibit reduced lifespan, lower body weight, increased frailty index, multi-organ degeneration, transcriptional changes with significant upregulation of transposable elements, and accelerated epigenetic age. Our data strongly supports the concept that the loss of epigenetic information directly drives the aging process. These findings reveal the importance of epigenetic regulation in aging and suggest that interventions targeting epigenetic modifications could potentially slow down or reverse age-related decline. Understanding the molecular mechanisms underlying the process of aging will be crucial for developing novel therapeutic strategies that can delay the onset of age-associated diseases and preserve human health at old age specially in rapidly aging societies.

Characterization of the Arabidopsis thaliana chromatin remodeler DEK3 for its interaction with histones and DNA.

Chromatin structure and dynamics regulate all DNA-templated processes, such as transcription, replication, and repair. Chromatin binding factors, chromatin architectural proteins, and nucleosome remodelers modulate chromatin structure and dynamics and, thereby, the various DNA-dependent processes. Arabidopsis thaliana DEK3, a member of the evolutionarily conserved DEK domain-containing chromatin architectural proteins, is an important factor for chromatin structure and function, involved in transcriptional programming to regulate flowering time and abiotic stress tolerance. AtDEK3 contains an uncharacterized N-terminal domain, a middle SAF domain (winged helix-like domain), and a C-terminal DEK domain, but their role in the interaction of AtDEK3 with histones and DNA remained poorly understood. Using biochemical and biophysical analyses, we provide a comprehensive in vitro characterization of the different AtDEK3 domains for their interaction with histone H3/H4 and DNA. AtDEK3 directly interacts with histone H3/H4 tetramers through its N-terminal domain and the C-terminal DEK domain in a 1:1 stoichiometry. Upon interaction with H3/H4, the unstructured N-terminal domain of AtDEK3 undergoes a conformational change and adopts an alpha-helical conformation. In addition, the in-solution envelope structures of the AtDEK3 domains and their complex with H3/H4 have been characterized. The SAF and DEK domains associate with double-stranded and four-way junction DNA. As DEK3 possesses a histone-interacting domain at the N- and the C-terminus and a DNA-binding domain in the middle and at the C-terminus, the protein might play a complex role as a chromatin remodeler.

Cancer, metastasis, and the epigenome.

Cancer is the second leading cause of death worldwide and disease burden is expected to increase globally throughout the next several decades, with the majority of cancer-related deaths occurring in metastatic disease. Cancers exhibit known hallmarks that endow them with increased survival and proliferative capacities, frequently as a result of de-stabilizing mutations. However, the genomic features that resolve metastatic clones from primary tumors are not yet well-characterized, as no mutational landscape has been identified as predictive of metastasis. Further, many cancers exhibit no known mutation signature. This suggests a larger role for non-mutational genome re-organization in promoting cancer evolution and dissemination. In this review, we highlight current critical needs for understanding cell state transitions and clonal selection advantages for metastatic cancer cells. We examine links between epigenetic states, genome structure, and misregulation of tumor suppressors and oncogenes, and discuss how recent technologies for understanding domain-scale regulation have been leveraged for a more complete picture of oncogenic and metastatic potential.

Bioengineered 3D ovarian model for long-term multiple development of preantral follicle: bridging the gap for poly(ε-caprolactone) (PCL)-based scaffold reproductive applications.

BACKGROUND: Assisted Reproductive Technologies (ARTs) have been validated in human and animal to solve reproductive problems such as infertility, aging, genetic selection/amplification and diseases. The persistent gap in ART biomedical applications lies in recapitulating the early stage of ovarian folliculogenesis, thus providing protocols to drive the large reserve of immature follicles towards the gonadotropin-dependent phase. Tissue engineering is becoming a concrete solution to potentially recapitulate ovarian structure, mostly relying on the use of autologous early follicles on natural or synthetic scaffolds. Based on these premises, the present study has been designed to validate the use of the ovarian bioinspired patterned electrospun fibrous scaffolds fabricated with poly(ε-caprolactone) (PCL) for multiple preantral (PA) follicle development. METHODS: PA follicles isolated from lamb ovaries were cultured on PCL scaffold adopting a validated single-follicle protocol (Ctrl) or simulating a multiple-follicle condition by reproducing an artificial ovary engrafted with 5 or 10 PA (AO5PA and AO10PA). The incubations were protracted for 14 and 18 days before assessing scaffold-based microenvironment suitability to assist in vitro folliculogenesis (ivF) and oogenesis at morphological and functional level. RESULTS: The ivF outcomes demonstrated that PCL-scaffolds generate an appropriate biomimetic ovarian microenvironment supporting the transition of multiple PA follicles towards early antral (EA) stage by supporting follicle growth and steroidogenic activation. PCL-multiple bioengineering ivF (AO10PA) performed in long term generated, in addition, the greatest percentage of highly specialized gametes by enhancing meiotic competence, large chromatin remodeling and parthenogenetic developmental competence. CONCLUSIONS: The study showcased the proof of concept for a next-generation ART use of PCL-patterned scaffold aimed to generate transplantable artificial ovary engrafted with autologous early-stage follicles or to advance ivF technologies holding a 3D bioinspired matrix promoting a physiological long-term multiple PA follicle protocol.

"To remember or forget: Insights into the mechanisms of epigenetic reprogramming and priming in early plant embryos".

Chromatin is dynamically modified throughout the plant life cycle to regulate gene expression in response to environmental and developmental cues. Although such epigenetic information can be inherited across generations in plants, chromatin features that regulate gene expression are typically reprogrammed during plant gametogenesis and directly after fertilization. Nevertheless, environmentally induced epigenetic marks on genes can be transmitted across generations. Moreover, epigenetic information installed on early embryonic chromatin can be stably inherited during subsequent growth and influence how plants respond to environmental conditions much later in development. Here, we review recent breakthroughs towards deciphering mechanisms underlying epigenetic reprogramming and transcriptional priming during early plant embryogenesis.

Current genomic deep learning models display decreased performance in cell type-specific accessible regions.

BACKGROUND: A number of deep learning models have been developed to predict epigenetic features such as chromatin accessibility from DNA sequence. Model evaluations commonly report performance genome-wide; however, cis regulatory elements (CREs), which play critical roles in gene regulation, make up only a small fraction of the genome. Furthermore, cell type-specific CREs contain a large proportion of complex disease heritability. RESULTS: We evaluate genomic deep learning models in chromatin accessibility regions with varying degrees of cell type specificity. We assess two modeling directions in the field: general purpose models trained across thousands of outputs (cell types and epigenetic marks) and models tailored to specific tissues and tasks. We find that the accuracy of genomic deep learning models, including two state-of-the-art general purpose models-Enformer and Sei-varies across the genome and is reduced in cell type-specific accessible regions. Using accessibility models trained on cell types from specific tissues, we find that increasing model capacity to learn cell type-specific regulatory syntax-through single-task learning or high capacity multi-task models-can improve performance in cell type-specific accessible regions. We also observe that improving reference sequence predictions does not consistently improve variant effect predictions, indicating that novel strategies are needed to improve performance on variants. CONCLUSIONS: Our results provide a new perspective on the performance of genomic deep learning models, showing that performance varies across the genome and is particularly reduced in cell type-specific accessible regions. We also identify strategies to maximize performance in cell type-specific accessible regions.

TERT upstream promoter methylation regulates TERT expression and acts as a therapeutic target in TERT promoter mutation-negative thyroid cancer.

BACKGROUND: DNA hypermethylation and hotspot mutations were frequently observed in the upstream and core promoter of telomerase reverse transcriptase (TERT), respectively, and they were associated with increased TERT expression and adverse clinical outcomes in thyroid cancer. In TERT promoter mutant cancer cells, the hypomethylated TERT mutant allele was active and the hypermethylated TERT wild-type allele was silenced. However, whether and how the upstream promoter methylation regulates TERT expression in TERT mutation-negative cells were largely unknown. METHODS: DNA demethylating agents 5-azacytidine and decitabine and a genomic locus-specific demethylation system based on dCas9-TET1 were used to assess the effects of TERT upstream promoter methylation on TERT expression, cell growth and apoptosis of thyroid cancer cells. Regulatory proteins binding to TERT promoter were identified by CRISPR affinity purification in situ of regulatory elements (CAPTURE) combined with mass spectrometry. The enrichments of selected regulatory proteins and histone modifications were evaluated by chromatin immunoprecipitation. RESULTS: The level of DNA methylation at TERT upstream promoter and expression of TERT were significantly decreased after treatment with 5-azacytidine or decitabine in TERT promoter wild-type thyroid cancer cells. Genomic locus-specific demethylation of TERT upstream promoter induced TERT downregulation, along with cell apoptosis and growth inhibition. Consistently, demethylating agents sharply inhibited the growth of thyroid cancer cells harboring hypermethylated TERT but had little effect on cells with TERT hypomethylation. Moreover, we identified that the chromatin remodeling protein CHD4 binds to methylated TERT upstream promoter and promotes its transcription by suppressing the enrichment of H3K9me3 and H3K27me3 at TERT promoter. CONCLUSIONS: This study uncovered the mechanism of promoter methylation mediated TERT activation in TERT promoter mutation-negative thyroid cancer cells and indicated TERT upstream promoter methylation as a therapeutic target for thyroid cancer.

Chromatin remodelers: a concise introduction for biophysicists.

Chromatin remodelers are molecular motors that act on nucleosomes: they move them along DNA or (dis-)assemble them. Despite the fact that they perform essential regulatory functions in cells-their deregulation can contribute to the development of cancers and lead to cell death-chromatin remodelers have only received meager attention in the biophysics community so far. In this short text, we attempt to present the key features of this interesting class of enzymes obtained with different experimental and theoretical methods, thereby providing a concise introduction for biophysicists to further stimulate interest in their properties.