Histone H3 lysine 4 trimethylation (H3K4me3) is abundant in mixed-lineage leukemia-rearranged (MLL-r) acute myeloid leukemia (AML) cells; however, the responsible enzymes and their roles remain unclear. This study aimed to identify the modifier responsible for high H3K4me3 modification in MLL-r leukemia and its downstream targets essential for the cell proliferation. Here, we performed a CRISPR-tiling screen against known H3K4 methylation modifiers in an MLL-r AML model. Disrupting the SETD1B catalytic SET domain caused depletion of FLT3-ITD or NrasG12D-expressing AML cells, and gene expression downregulation, particularly in the MYC pathway. SETD1B SET domain loss results in a significant decrease in H3K4me3 breadth. Exogenous MYC expression or disrupting H3K4 demethylase KDM5C significantly restored growth defects in SETD1B SET domain-mutant cells. These data indicated that SETD1B was required for H3K4me3 breadth and MYC expression. Thus, a thorough understanding of SETD1B-mediated H3K4me3 breadth is critical for developing markers and therapies for MYC-dependent leukemia subtypes.

Mitochondrial hormetic oxidative stress (mtHOS) is crucial in physiology and disease; however, its effects on epigenetic inheritance and organism fitness across generations remains elusive. Utilizing the C. elegans as a model, we elucidate that parental exposure to mtHOS not only elicits a lifespan extension in the exposed individuals but also confers this longevity advantage to the progeny through the transgenerational epigenetic inheritance (TEI) mechanism. This transgenerational transmission of lifespan prolongation depends on the activation of the UPRmt and the synergistic action of the transcription factors DAF-16/FOXO and SKN-1/Nrf2. Additionally, the H3K4me3 and H3K27me3 serve as epigenetic mediators, selectively marking and regulating the expression of genes associated with oxidative stress response and longevity determination. Our findings illuminate the mechanisms underlying the implementation and transmission of mtHOS, revealing a sophisticated interplay among oxidative stress response genes and chromatin remodeling that collectively enhances the progeny's adaptive resilience to future challenges.

Epigenetic programming governs cell fate determination during development through intricately controlling sequential gene activation and repression. Although H3K4me3 is widely recognized as a hallmark of gene activation, its role in modulating transcription output and timing within a continuously developing system remains poorly understood. In this study, we provide a detailed characterization of the epigenomic landscapes in developing male germ cells. We identified thousands of spermatid-specific broad H3K4me3 domains regulated by the SETD1B-RFX2 axis, representing a previously underappreciated form of H3K4me3. These domains, overlapping with H3K27ac-marked enhancers and promoters, play critical roles in orchestrating robust transcription and accurate temporal control of gene expression. Mechanistically, these broad H3K4me3 compete effectively with regular H3K4me3 for transcriptional machinery, thereby ensuring robust levels and precise timing of master gene expression in mouse spermiogenesis. Disruption of this mechanism compromises the accuracy of transcription dosage and timing, ultimately impairing spermiogenesis. Additionally, we unveil remarkable changes in the distribution of heterochromatin marks, including H3K27me3 and H3K9me2, during the mitosis-to-meiosis transition and completion of meiotic recombination, which closely correlates with gene silencing. This work underscores the highly orchestrated epigenetic regulation in spermatogenesis, highlighting the previously unrecognized role of Setd1b in the formation of broad H3K4me3 domains and transcriptional control, and provides an invaluable resource for future studies toward the elucidation of spermatogenesis.

Endogenous retroviruses (ERVs) shape human genome functionality and influence disease pathogenesis, including cancer. ERVK-7, a significant ERV, acts as an immune modulator and prognostic marker in lung adenocarcinoma (LUAD). Although ERVK-7 overexpression has been linked to the amplification of the 1q22 locus in approximately 10% of LUAD cases, it predominantly arises from alternative regulatory mechanisms. Our findings indicate that the canonical 5' long terminal repeat (LTR) of ERVK-7 is methylated and inactive, necessitating the use of alternative upstream promoters. We identified two novel transcripts, ERVK-7.long and ERVK-7.short, arising from distinct promoters located 2.8 kb and 13.8 kb upstream of the 5'LTR of ERVK-7, respectively. ERVK-7.long is predominantly overexpressed in LUAD. Through comprehensive epigenetic mapping and single-cell transcriptomics, we demonstrate that ERVK-7.long activation is predetermined by cell lineage, specifically in small airway epithelial cells (SAECs), where its promoter displays tumor-specific H3K4me3 modifications. Single-cell RNA sequencing further reveals a distinct enrichment of ERVK-7.long in LUAD tumor cells and alveolar type 2 epithelial cells, underscoring a cell-type-specific origin. Additionally, inflammatory signaling significantly influences ERVK-7 expression; TNF-α enhances ERVK-7.long, while interferon signaling preferentially augments ERVK-7.short by differential recruitment of NF-κB/RELA and IRF to their respective promoters. This differential regulation clarifies the elevated ERVK-7 expression in LUAD compared to lung squamous cell carcinoma (LUSC). Our study elucidates the complex regulatory mechanisms governing ERVK-7 in LUAD and proposes these transcripts as potential biomarkers and therapeutic targets, offering new avenues to improve patient outcomes.

The histone H3 lysine 4 (H3K4) methyltransferase KMT2D (also called MLL4) is one of the most frequently mutated epigenetic modifiers in many cancers, including medulloblastoma (MB). Notably, heterozygous KMT2D loss frequently occurs in MB and other cancers. However, its oncogenic role remains largely uncharacterized. Here, we show that heterozygous Kmt2d loss in murine cerebellar regions promotes MB genesis driven by heterozygous loss of the MB-suppressor gene Ptch via the upregulation of tumor-promoting programs (e.g., oxidative phosphorylation [OXPHOS]). Downregulation of the transcription-repressive tumor suppressor NCOR2 by heterozygous Kmt2d loss, along with Ptch+/--increased MYCN, upregulated tumor-promoting genes. Heterozygous Kmt2d loss substantially diminished enhancer marks (H3K4me1 and H3K27ac) and the H3K4me3 signature, including those for Ncor2. Combinatory pharmacological inhibition of the enhancer-decommissioning H3K4 demethylase LSD1 and OXPHOS significantly reduced the tumorigenicity of MB cells bearing heterozygous Kmt2d loss. Our findings suggest the molecular and epigenetic pathogenesis underlying the MB-promoting effect of heterozygous KMT2D loss.

FOXP3-expressing regulatory T (Treg) cells play a pivotal role in maintaining immune homeostasis and tolerance, with their activation being crucial for preventing various inflammatory responses. However, the mechanisms governing the epigenetic program in Treg cells during their dynamic activation remain unclear. In this study, we demonstrate that CXXC-finger protein 1 (CXXC1) interacts with the transcription factor FOXP3 and facilitates the regulation of target genes by modulating H3K4me3 deposition. Cxxc1 deletion in Treg cells leads to severe inflammatory disease and spontaneous T cell activation, with impaired immunosuppressive function. As a transcriptional regulator, CXXC1 promotes the expression of key Treg functional markers under steady-state conditions, which are essential for the maintenance of Treg cell homeostasis and their suppressive functions. Epigenetically, CXXC1 binds to the genomic regulatory regions of Treg program genes in mouse Treg cells, overlapping with FOXP3-binding sites. Given its critical role in Treg cell homeostasis, CXXC1 presents itself as a promising therapeutic target for autoimmune diseases.

The chromodomain helicase DNA binding protein 7 (CHD7) is a nucleosome repositioner implicated in multiple cellular processes, including neuronal differentiation. We identified CHD7 genome-wide binding sites that regulate neuronal differentiation in an otic stem cell line. We identified CHD7 enrichment at the Sox11 promoter and 3' untranslated region (UTR). Sox11 is a transcription factor essential for neuronal differentiation. CRISPRi of Sox11 promoter or 3'UTR displayed decreased neurite lengths and reduced neuronal marker expression TUBB3 expression. We showed that the Sox11 locus resides at TAD boundaries, and CTCF marks the 3'UTR. We propose that CHD7 modulates chromatin accessibility of the Sox11 promoter and CTCF-marked insulators in the 3'UTR to facilitate neuronal differentiation. CRISPRi of the insulator site alters 3D chromatin organization, affects gene expression and ultimately perturbs cellular processes. Our results implicate a general mechanism of CHD7 in facilitating neuronal differentiation and provide insight into CHD7 dysfunction in CHARGE syndrome, a congenital disorder associated with hearing loss.

Pluripotent stem cells (PSCs) possess the extraordinary capability to differentiate into a variety of cell types. This capability is tightly regulated by epigenetic mechanisms, particularly histone modifications. Moreover, the reprogramming of somatic or fate-committed cells into induced pluripotent stem cells (iPSCs) largely relies on these modifications, such as histone methylation and acetylation of histones. While extensive research has been conducted utilizing mouse models, the significance of histone modifications in human iPSCs is gaining increasing recognition. Recent studies underscore the importance of epigenetic regulators in both the reprogramming process and the regulation of cancer stem cells (CSCs), which are pivotal in tumor initiation and the development of treatment resistance. This review elucidates the dynamic alterations in histone modifications that impact reprogramming and emphasizes the necessity for a balance between activating and repressive marks. These epigenetic marks are influenced by enzymes such as DNA methyltransferases (DNMTs) and histone deacetylases (HDACs). Furthermore, this review explores therapeutic strategies aimed at targeting these epigenetic modifications to enhance treatment efficacy in cancer while advancing the understanding of pluripotency and reprogramming. Despite promising developments in the creation of inhibitors for histone-modifying enzymes, challenges such as selectivity and therapy resistance continue to pose significant hurdles. Therefore, future endeavors must prioritize biomarker-driven approaches and gene-editing technologies to optimize the efficacy of epigenetic therapies.

Intrinsic and extrinsic factors influence cancer cell identity throughout its lifespan. During tumor progression and metastasis formation, cancer cells are exposed to different environmental stimuli, resulting in a stepwise cellular reprogramming. Similar stepwise changes of cell identity have been shown as a major consequence of cancer treatment, as cells are exposed to extracellular stress that can result in the establishment of subpopulations exhibiting different epigenetic and transcriptional patterns, indicating a rapid adaptation mechanism of cellular identity by extrinsic stress factors. Both mechanisms, tumor progression-mediated changes and therapy response, rely on signaling pathways affecting the epigenetic and subsequent transcriptional landscape, which equip the cells with mechanisms for survival and tumor progression. These non-genetic alterations are propagated to the daughter cells, indicating a need for successful information propagation and transfer to the daughter generations, thereby allowing for a stepwise adaptation to environmental cues. However, the exact mechanisms how these cell identity changes are occurring, which context-specific mechanisms are behind and how this can be exploited for future therapeutic interventions is not yet fully understood and exploited. In this review, we discuss the current knowledge on cell identity maintenance mechanisms intra- and intergenerational in development and disease and how these mechanisms are altered in cancer. We will as well address how cancer treatment might target these properties.

Alcohol use disorder (AUD) contributes significantly to the global burden of disease. The susceptibility for AUD is mediated through an interaction of genetic risk factors and environmental influences. These gene × environment (G × E) interactions manifest as epigenetic regulations of gene expression, among other things. Previous research suggests an association between Ganglioside Induced Differentiation Associated Protein 1 (GDAP1) DNA methylation and AUD. Here, we investigate the epigenetic dysregulation of GDAP1 in AUD through comparing DNA methylation in whole blood and saliva, as well as H3K4-trimethylation (H3K4me3) in PBMC (Peripheral Blood Mononuclear Cell) samples of AUD patients and healthy control individuals. Additionally, the effect of abstinence-based therapy was investigated. AUD patients before treatment exhibit significantly lower promoter DNA methylation levels in whole blood and saliva, as well as lower H3K4me3 near the transcription start site. GDAP1 gene expression was not significantly altered. Following treatment, H3K4me3 was significantly increased in patients and no longer differed from control individuals. There was no significant effect of treatment on DNA methylation. We conclude that GDAP1 is epigenetically dysregulated in AUD patients, and is responsive to abstinence-based therapy at the level of H3K4me3. It should be investigated further to establish its potential as a diagnostic biomarker.

The study discloses the application of transformer-based deep learning models for the task of super-enhancers prediction in human tumor cell lines with a specific focus on sequence-only features within studied entities of super-enhancer and enhancer elements in the human genome. The proposed SE-prediction method included the GENA-LM application at handling long DNA sequences with the classification task, distinguishing super-enhancers from enhancers using H3K36me, H3K4me1, H3K4me3 and H3K27ac landscape datasets from HeLa, HEK293, H2171, Jurkat, K562, MM1S and U87 cell lines. The model was fine-tuned on relevant sequence data, allowing for the analysis of extended genomic sequences without the need for epigenetic markers as proposed in early approaches. The study achieved balanced accuracy metrics, surpassing previous models like SENet, particularly in HEK293 and K562 cell lines. Also, it was shown that super-enhancers frequently co-localize with epigenetic marks such as H3K4me3 and H3K27ac. Therefore, the attention mechanism of the model provided insights into the sequence features contributing to SE classification, indicating a correlation between sequence-only features and mentioned epigenetic landscapes. These findings support the potential transformer models use in further genomic sequence analysis for bioinformatics applications in enhancer/super-enhancer characterization and gene regulation studies.

Epigenetic modifications, including posttranslational modifications of histones, are closely linked to transcriptional regulation. Trimethylated H3 lysine 4 (H3K4me3) is one of the most studied histone modifications owing to its enrichment at the start sites of transcription and its association with gene expression and processes determining cell fate, development, and disease. In this review, we focus on recent studies that have yielded insights into how levels and patterns of H3K4me3 are regulated, how H3K4me3 contributes to the regulation of specific phases of transcription such as RNA polymerase II initiation, pause-release, heterogeneity, and consistency. The conclusion from these studies is that H3K4me3 by itself regulates gene expression and its precise regulation is essential for normal development and preventing disease.

Circular (circ)RNAs have emerged as crucial contributors to cancer progression. Nonetheless, the expression regulation, biological functions, and underlying mechanisms of circRNAs in mediating hepatocellular carcinoma (HCC) progression remain insufficiently elucidated.

We identified circUCK2(2,3) through circRNA sequencing, RT-PCR, and Sanger sequencing. CircUCK2(2,3) levels were measured in two independent HCC cohorts using quantitative real-time PCR (qRT-PCR). We explored the functions of circUCK2(2,3) using gain- and loss-of-function assays. Techniques such as RNA-sequencing, RNA immunoprecipitation (RIP), polysome fractionation, RNA pulldown, dual luciferase reporter assay, inhibitors of EGFR downstream signaling, CRISPR-Cas9, and medium transfer assays were employed to investigate the regulatory mechanisms and the protumoral activities of circUCK2(2,3). Additionally, in vitro cytotoxic assays and patient-derived xenograft (PDX) models assessed the effects of circUCK2(2,3) on the cytotoxic synergy of lenvatinib and EGFR inhibitors.

CircUCK2(2,3) is upregulated in HCC tissues and serves as an independent risk factor for poor recurrence-free survival. The expression of circUCK2(2,3) is independent on its host gene, UCK2, but is regulated by its upstream promoter and flanking inverted complementary sequences. Functionally, circUCK2(2,3) enhances HCC proliferation, migration, and invasion, both in vitro and in vivo. Mechanistically, by sponging miR-149-5p, circUCK2(2,3) increases CNIH4 levels, which in turn amplifies TGFα secretion, resulting in the activation of EGFR and downstream pAKT and pERK signaling pathways. Moreover, circUCK2(2,3) overexpression sensitizes HCC cells to EGFR inhibitors, and increases the synergistic cytotoxicity of combined lenvatinib and EGFR inhibitor treatment.

CircUCK2(2,3) regulates a novel oncogenic pathway, miR-149-5p-CNIH4-TGFα-EGFR, in HCC, presenting a viable therapeutic target and biomarker for the precision treatment of HCC.

T-cell acute lymphoblastic leukaemia (T-ALL) is a rare aggressive haematological malignancy characterised by the clonal expansion of immature T-cell precursors. It accounts for 15% of paediatric and 25% of adult ALL. T-ALL is associated with the overexpression of major transcription factors (TLX1/3, TAL1, HOXA) that drive specific transcriptional programmes and constitute the molecular classifying subgroups of T-ALL. Although the dysregulation of transcription factor oncogenes is frequently associated with chromosomal translocations in T-ALL, epigenetic dysregulation resulting in changes to post-translational modifications of histones has also been reported. This includes non-coding intergenic mutations that form oncogenic neo-enhancers. This review will focus on the known epigenetically activating intergenic mutations reported in T-ALL, and will discuss the wider implications of neo-enhancer mutations in cancer.

In early mammalian embryogenesis, a shift from non-canonical histone H3 lysine 4 trimethylation (H3K4me3) linked to transcriptional repression to canonical H3K4me3 indicating active promoters occurs during zygotic genome activation (ZGA). However, the mechanisms and roles of these H3K4me3 states in embryogenesis remain poorly understood. Our research reveals that the histone methyltransferase MLL2 is responsible for installing H3K4me3 (both non-canonical and canonical) in totipotent embryos, while a transition to SETD1A/B-deposited H3K4me3 occurs in pluripotent embryos. Interestingly, MLL2-mediated H3K4me3 operates independently of transcription, fostering a relaxed chromatin state conducive to totipotency rather than directly influencing transcription. Conversely, SETD1A/B-mediated H3K4me3, which depends on transcription, is crucial for facilitating expression of genes essential for pluripotency and pre-implantation development. Our findings highlight the role of the H3K4me3 transition, mediated by an MLL2-to-SETD1A/B relay mechanism, in the regulation of transition from totipotency to pluripotency during early embryogenesis.

The dynamics of three-dimensional (3D) genome organization are essential to transcriptional regulation. While enhancers regulate spatiotemporal gene expression, chromatin looping is a means for enhancer-promoter interactions yielding cell-type-specific gene expression. Further, non-canonical DNA secondary structures, such as G-quadruplexes (G4s), are related to increased gene expression. However, the role of G4s in promoter-distal regulatory elements, such as super-enhancers (SE), and in chromatin looping has remained elusive. Here we show that mature microRNA 9 (miR-9) is enriched at promoters and SE of genes that are inducible by transforming growth factor beta 1 (TGFB1) signaling. Moreover, we find that miR-9 is required for formation of G4s, promoter-super-enhancer looping and broad domains of the euchromatin histone mark H3K4me3 at TGFB1-responsive genes. Our study places miR-9 in the same functional context with G4s and promoter-enhancer interactions during 3D genome organization and transcriptional activation induced by TGFB1 signaling, a critical signaling pathway in cancer and fibrosis.

Zygotic genome activation (ZGA) is critical for early embryo development and is meticulously regulated by epigenetic modifications. H3K4me3 is a transcription-permissive histone mark preferentially found at promoters, but its distribution across genome features remains incompletely understood. In this study, we investigated the genome-wide enrichment of H3K4me3 during early embryo development and embryonic stem cells (ESCs) in both sheep and mice. We discovered that broad H3K4me3 domains were present in MII stage oocytes and were progressively diminished, while promoter H3K4me3 enrichment was increased and correlated with gene upregulation during ZGA in sheep. Additionally, we reported the dynamic distribution of H3K4me3 at the transposable elements (TEs) during early embryo development in both sheep and mice. Specifically, the H3K4me3 distribution of LINE1 and ERVL, two subsets of TEs, was associated with their expression during early embryo development in sheep. Furthermore, H3K4me3 enrichment in TEs was greatly increased during ZGA following Kdm5b knockdown, and the distribution of RNA polymerase II (Pol2) in TEs was also markedly increased in Kdm5b knockout ESCs in mice. These findings suggest that H3K4me3 plays important roles in regulating TE expression through interaction with RNA Pol2, providing valuable insights into the regulation of ZGA initiation and cell fate determination by H3K4me3.

KDM5 family proteins are best known for their demethylation of the promoter proximal chromatin mark H3K4me3. KDM5-regulated transcription is critical in the brain, with variants in the X-linked paralog KDM5C causing the intellectual disability (ID) disorder Claes-Jensen syndrome. Although the demethylase activity of KDM5C is known to be important for neuronal function, the contribution of non-enzymatic activities remain less characterized. We therefore used Drosophila to model the ID variant Kdm5 L854F , which disrupts a C5HC2 zinc finger adjacent to the enzymatic JmjC domain. Kdm5 L854F causes similar transcriptional changes in the brain to a demethylase dead strain, Kdm5 J1310C * , despite having little effect on enzymatic activity. KDM5 L854F is also distinct from KDM5 J1310C * in its reduced interactions with insulator proteins and enhancement of position effect variegation. Instead, the common transcriptional deficits likely result from both the JmjC and C5HC2 domains driving proper genomic organization through their activity in promoting proper loop architecture.

Family with sequence similarity 72 (FAM72) is a protein-coding gene family located on chromosome 1 in humans, uniquely featuring four paralogs: FAM72A, FAM72B, FAM72C, and FAM72D. While FAM72's presence as a gene pair with the SLIT-ROBO Rho GTPase-activating protein 2 (SRGAP2) is intriguing, its functional roles, particularly in neural stem cells, remain incompletely understood. This review explores the distinct characteristics of FAM72, shedding light on its expression patterns, potential roles in cell cycle regulation, stem cell renewal and implications in neurogenesis and tumorigenesis.

Periodontal disease is considered one of the diabetic complications with high morbidity and severity. Recent studies demonstrated the involvement of the epigenome on diabetic complications. Histone modifications change chromatin architecture and gene activation. Histone modifications have been reported to alter chromatin structure and regulate gene transcription. In this study, we investigated the impacts of H3 lysine 4 trimethylation (H3K4me3) and specific histone methyltransferases of H3K4 methylation, su(var)3-9, enhancer-of-zeste, and trithorax domain 1A (SETD1A) on periodontal tissue affected by the diabetic condition. We observed the increase in H3K4me3 and SETD1A in gingival tissue of diabetic rats compared with the normal rats. Cultured human fibroblasts (hGFs) confirmed a high glucose-induced increase in H3K4me3 and SETD1A. We further demonstrated that high glucose increased the gene expression of matrix metalloproteinase (MMP) 1 and MMP13, which were canceled by sinefungin, an SETD1A inhibitor. Our investigation suggests that diabetes triggers histone modifications in the gingival tissue, resulting in gingival inflammation. Histone modifications may play crucial roles in the development of periodontal disease in diabetes.

Inflammatory responses, leading to fibrosis and potential host rejection, significantly hinder the long-term success and widespread adoption of biomedical implants. The ability to control and investigated macrophage inflammatory responses at the implant-macrophage interface would be critical for reducing chronic inflammation and improving tissue integration. Nonetheless, the systematic investigation of how surface topography affects macrophage polarization is typically complicated by the restricted complexity of accessible nanostructures, difficulties in achieving exact control, and biased preselection of experimental parameters. In response to these problems, we developed a large-scale, high-content combinatorial biophysical cue (CBC) array for enabling high-throughput screening (HTS) of the effects of nanotopography on macrophage polarization and subsequent inflammatory processes. Our CBC array, created utilizing the dynamic laser interference lithography (DLIL) technology, contains over 1 million nanotopographies, ranging from nanolines and nanogrids to intricate hierarchical structures with dimensions ranging from 100 nm to several microns. Using machine learning (ML) based on the Gaussian process regression algorithm, we successfully identified certain topographical signals that either repress (pro-M2) or stimulate (pro-M1) macrophage polarization. The upscaling of these nanotopographies for further examination has shown mechanisms such as cytoskeletal remodeling and ROCK-dependent epigenetic activation to be critical to the mechanotransduction pathways regulating macrophage fate. Thus, we have also developed a platform combining advanced DLIL nanofabrication techniques, HTS, ML-driven prediction of nanobio interactions, and mechanotransduction pathway evaluation. In short, our developed platform technology not only improves our ability to investigate and understand nanotopography-regulated macrophage inflammatory responses but also holds great potential for guiding the design of nanostructured coatings for therapeutic biomaterials and biomedical implants.

Neuroblastoma is an embryonic cancer that contributes disproportionately to death in young children. Sequencing data have uncovered few recurrently mutated genes in this cancer, although epigenetic pathways have been implicated in disease pathogenesis. We used an expression-based computational screen that examined the impact of deubiquitinating enzymes on patient survival to identify potential new targets. We identified the histone H2B deubiquitinating enzyme USP44 as the enzyme with the greatest impact on survival in patients with neuroblastoma. High levels of USP44 significantly correlate with metastatic disease, unfavorable histology, advanced patient age, and MYCN amplification. The subset of patients with tumors expressing high levels of USP44 had significantly worse survival, including those with tumors lacking MYCN amplification. We showed experimentally that USP44 regulates neuroblastoma cell proliferation, migration, invasion, and neuronal development. Depletion of the histone H2B ubiquitin ligase subunit RNF20 resulted in similar findings, strongly implicating this histone mark as the target of USP44 activity in this disease. Integration of transcriptome and epigenome in analyses demonstrates a distinct set of genes that are regulated by USP44, including those in Hallmark MYC target genes in both murine embryonic fibroblasts and the SH-SY5Y neuroblastoma cell line. We conclude that USP44 is a novel epigenetic regulator that promotes aggressive features and may be a novel target in neuroblastoma. Implications: This study identifies a new genetic marker of aggressive neuroblastoma and identifies the mechanisms by which its overactivity contributes to the pathophysiology of this disease.

In acute promyelocytic leukemia (APL), the promyelocytic leukemia-retinoic acid receptor alpha (PML/RARα) fusion protein destroys PML nuclear bodies (NBs), leading to the formation of microspeckles. However, our understanding, largely learned from morphological observations, lacks insight into the mechanisms behind PML/RARα-mediated microspeckle formation and its role in APL leukemogenesis. This study presents evidence uncovering liquid-liquid phase separation (LLPS) as a key mechanism in the formation of PML/RARα-mediated microspeckles. This process is facilitated by the intrinsically disordered region containing a large portion of PML and a smaller segment of RARα. We demonstrate the coassembly of bromodomain-containing protein 4 (BRD4) within PML/RARα-mediated condensates, differing from wild-type PML-formed NBs. In the absence of PML/RARα, PML NBs and BRD4 puncta exist as two independent phases, but the presence of PML/RARα disrupts PML NBs and redistributes PML and BRD4 into a distinct phase, forming PML/RARα-assembled microspeckles. Genome-wide profiling reveals a PML/RARα-induced BRD4 redistribution across the genome, with preferential binding to super-enhancers and broad-promoters (SEBPs). Mechanistically, BRD4 is recruited by PML/RARα into nuclear condensates, facilitating BRD4 chromatin binding to exert transcriptional activation essential for APL survival. Perturbing LLPS through chemical inhibition (1, 6-hexanediol) significantly reduces chromatin co-occupancy of PML/RARα and BRD4, attenuating their target gene activation. Finally, a series of experimental validations in primary APL patient samples confirm that PML/RARα forms microspeckles through condensates, recruits BRD4 to coassemble condensates, and co-occupies SEBP regions. Our findings elucidate the biophysical, pathological, and transcriptional dynamics of PML/RARα-assembled microspeckles, underscoring the importance of BRD4 in mediating transcriptional activation that enables PML/RARα to initiate APL.

Histone 3 lysine 4 methylation (H3K4me) is a highly evolutionary conserved chromatin modification associated with active transcription, and its three methylation states-mono, di, and trimethylation-mark distinct regulatory elements. However, whether H3K4me plays functional roles in transcriptional regulation or is merely a by-product of histone methyltransferases recruited to actively transcribed loci is still under debate. Here, we outline the studies that have addressed this question in yeast, Drosophila, and mammalian systems. We review evidence from histone residue mutation, histone modifier manipulation, and epigenetic editing, focusing on the relative roles of H3K4me1 and H3K4me3. We conclude that H3K4me1 and H3K4me3 may have convergent functions in establishing open chromatin and promoting transcriptional activation during cell differentiation.

Biomolecular condensates play a significant role in chromatin activities, primarily by concentrating and compartmentalizing proteins and/or nucleic acids. However, their genomic landscapes and compositions remain largely unexplored due to a lack of dedicated computational tools for systematic identification in vivo. To address this, we develop CondSigDetector, a computational framework designed to detect condensate-like chromatin-associated protein co-occupancy signatures (CondSigs), to predict genomic loci and component proteins of distinct chromatin-associated biomolecular condensates. Applying this framework to mouse embryonic stem cells (mESC) and human K562 cells enable us to depict the high-resolution genomic landscape of chromatin-associated biomolecular condensates, and uncover both known and potentially unknown biomolecular condensates. Multi-omics analysis and experimental validation further verify the condensation properties of CondSigs. Additionally, our investigation sheds light on the impact of chromatin-associated biomolecular condensates on chromatin activities. Collectively, CondSigDetector provides an approach to decode the genomic landscape of chromatin-associated condensates, facilitating a deeper understanding of their biological functions and underlying mechanisms in cells.

Long terminal repeats (LTRs), which often contain promoter and enhancer sequences of intact endogenous retroviruses (ERVs), are known to be co-opted as cis-regulatory elements for fine-tuning host-coding gene expression. Since LTRs are mainly silenced by the deposition of repressive epigenetic marks, substantial activation of LTRs has been found in human cells after treatment with epigenetic inhibitors. Although the LTR12C family makes up the majority of ERVs activated by epigenetic inhibitors, how these epigenetically and transcriptionally activated LTR12C elements can regulate the host-coding gene expression remains unclear due to genome-wide alteration of transcriptional changes after epigenetic inhibitor treatments. Here, we specifically transactivated >600 LTR12C elements by using single guide RNA-based dCas9-SunTag-VP64, a site-specific targeting CRISPR activation (CRISPRa) system, with minimal off-target events. Interestingly, most of the transactivated LTR12C elements acquired the H3K27ac-marked enhancer feature, while only 20% were co-marked with promoter-associated H3K4me3 modifications. The enrichment of the H3K4me3 signal was intricately associated with downstream regions of LTR12C, such as internal regions of intact ERV9 or other types of retrotransposons. Here, we leverage an optimized CRISPRa system to identify two distinct epigenetic signatures that define LTR12C transcriptional activation, which modulate the expression of proximal protein-coding genes.

Female infertility due to declining oocyte quality with age remains a significant challenge for patients and physicians, despite extensive research efforts. Recent studies suggest that microRNAs (miRNAs), which respond to various stressors in the aging process, may provide a promising solution. With the approval of small RNA drugs for clinical use, miRNA-based treatment of oocyte aging appears to be a viable option. Through high-throughput sequencing, miR-425-5p was identified as the only miRNA elevated under natural aging and oxidative stress. Microinjection of inhibitors to inhibit miR-425-5p effectively improved compromised phenotypes of old oocytes in vitro. Further investigation revealed that Crebzf acts as a mediator of miR-425-5p's age-related functions in old oocytes. In vivo treatment with miR-425-5p antagomirs significantly improved impaired oocyte development in reproductively old females by targeting Crebzf. Single-cell RNA sequencing revealed that Crebzf plays a vital role in regulating mRNAs targeting histone H3, trimethylated lysine 4 (H3K4me3), a crucial marker for transcriptional silencing. Overexpression of miR-425-5p could hinder oocyte maturation by downregulating Crebzf expression and disrupting transcriptional regulation. Our findings provide new insights into the potential of miR-425-5p antagomirs as a treatment for female infertility and highlight an elegant mechanism by which miR-425-5p inhibition of Crebzf inhibits a developmental switch in GV oocytes by regulating a group of histone methyltransferase mRNAs.

The crosstalk between metabolism and epigenetics is an emerging field that is gaining importance in different areas such as cancer and aging, where changes in metabolism significantly impacts the cellular epigenome, in turn dictating changes in chromatin as an adaptive mechanism to bring back metabolic homeostasis. A key metabolic pathway influencing an organism's epigenetic state is one-carbon metabolism (OCM), which includes the folate and methionine cycles. Together, these cycles generate S-adenosylmethionine (SAM), the universal methyl donor essential for DNA and histone methylation. SAM serves as the sole methyl group donor for DNA and histone methyltransferases, making it a crucial metabolite for chromatin modifications. In this review, we will discuss how SAM and its byproduct, S-adenosylhomocysteine (SAH), along with the enzymes and cofactors involved in OCM, may function in the different cellular compartments, particularly in the nucleus, to directly regulate the epigenome in aging and cancer.

DNA hydroxymethylation (5hmC), the most abundant oxidative derivative of DNA methylation, is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in age-related diseases, its functional role in aging remains unknown. Here, using mouse liver and cerebellum as model organs, we show that 5hmC accumulates in gene bodies associated with tissue-specific function and restricts the magnitude of gene expression changes with age. Mechanistically, 5hmC decreases the binding of splicing associated factors and correlates with age-related alternative splicing events. We found that various age-related contexts, such as prolonged quiescence and senescence, drive the accumulation of 5hmC with age. We provide evidence that this age-related transcriptionally restrictive function is conserved in mouse and human tissues. Our findings reveal that 5hmC regulates tissue-specific function and may play a role in longevity.

The genomes and annotated genes of allotetraploid cotton Gossypium hirsutum have been extensively studied in recent years. However, the expression, regulation, and evolution of intergenic genes (ITGs) have not been completely deciphered. In this study, we identified a novel set of actively expressed ITGs in G. hirsutum cotton, through transcriptome profiling based on deep sequencing data, as well as chromatin immunoprecipitation, followed by sequencing (ChIP-seq) of histone modifications and how the ITGs evolved. Totals of 17,567 and 8249 ITGs were identified in G. hirsutum and Gossypium arboreum, respectively. The expression of ITGs in G. hirsutum was significantly higher than that in G. arboreum. Moreover, longer exons were observed in G. hirsutum ITGs. Notably, 42.3% of the ITGs from G. hirsutum were generated by the long terminal repeat (LTR) insertions, while their proportion in genic genes was 19.9%. The H3K27ac and H3K4me3 modification proportions and intensities of ITGs were equivalent to genic genes. The H3K4me1 modifications were lower in ITGs. Additionally, evolution analyses revealed that the ITGs from G. hirsutum were mainly produced around 6.6 and 1.6 million years ago (Mya), later than the pegged time for genic genes, which is 7.0 Mya. The characterization of ITGs helps to elucidate the evolution of cotton genomes and shed more light on their biological functions in the transcriptional regulation of eukaryotic genes, along with the roles of histone modifications in speciation and diversification.

The three major anatomic regions of the human kidney include the cortex, medulla and papilla, with different functions and vulnerabilities to kidney diseases. Epigenetic mechanisms underlying these anatomic structures are incompletely understood. Here, we performed chromatin conformation capture with Hi-C and histone modification H3K4me3/H3K27me3 Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing on the kidney cortex, medulla and papilla dissected from one individual donor. Nuclear suspensions were generated from each region and split subjected to paired Hi-C and CUT&RUN sequencing. We evaluated the quality of next-generation sequencing data, Hi-C chromatin contact matrices and CUT&RUN peak calling. H3K4me3 and H3K27me3 histone modifications represent active and repressive gene transcription, respectively, and differences in chromatin conformation between kidney regions can be analyzed with this dataset. All raw and processed data files are publicly available, allowing researchers to survey the epigenetic landscape across regional human kidney anatomy.

Comparative genomics has revealed the rapid expansion of multiple gene families involved in immunity. Members within each gene family often evolved distinct roles in immunity. However, less is known about the evolution of their epigenome and cis-regulation. Here we systematically profile the epigenome of the recently expanded murine Ly49 gene family that mainly encode either inhibitory or activating surface receptors on natural killer cells. We identify a set of cis-regulatory elements (CREs) for activating Ly49 genes. In addition, we show that in mice, inhibitory and activating Ly49 genes are regulated by two separate sets of proximal CREs, likely resulting from lineage-specific losses of CRE activity. Furthermore, we find that some Ly49 genes are cross-regulated by the CREs of other Ly49 genes, suggesting that the Ly49 family has begun to evolve a concerted cis-regulatory mechanism. Collectively, we demonstrate the different modes of cis-regulatory evolution for a rapidly expanding gene family.

Swd2/Cps35 is a common component of the COMPASS H3K4 methyltransferase and CPF transcription termination complex in Saccharomyces cerevisiae. The deletion of SWD2 is lethal, which results from transcription termination defects in snoRNA genes. This study isolated a yeast strain that showed significantly reduced protein level of Swd2 following epitope tagging at its N-terminus (9MYC-SWD2). The reduced level of Swd2 in the 9MYC-SWD2 strain was insufficient for the stability of the Set1 H3K4 methyltransferase, H3K4me3 and snoRNA termination, but the level was enough for viability and growth similar to the wildtype strain. In addition, we presented the genes differentially regulated by the essential protein Swd2 under optimal culture conditions for the first time. The expression of genes known to be decreased in the absence of Set1 and H3K4me3, including NAD biosynthetic process genes and histone genes, was decreased in the 9MYC-SWD2 strain, as expected. However, the effects of Swd2 on the ribosome biogenesis (RiBi) genes were opposite to those of Set1, suggesting that the expression of RiBi genes is regulated by more complex relationship between COMPASS and other Swd2-containing complexes. These data suggest that different concentrations of Swd2 are required for its roles in H3K4me3 and viability and that it may be either contributory or contrary to the transcriptional regulation of Set1/H3K4me3, depending on the gene group.

Cells of multicellular organisms generate heterogeneity in a controlled and transient fashion during embryogenesis, which can be reactivated in pathologies such as cancer. Although genomic heterogeneity is an important part of tumorigenesis, continuous generation of phenotypic heterogeneity is central for the adaptation of cancer cells to the challenges of tumorigenesis and response to therapy. Here I discuss the capacity of generating heterogeneity, hereafter called cell hetness, in cancer cells both as the activation of hetness oncogenes and inactivation of hetness tumor suppressor genes, which increase the generation of heterogeneity, ultimately producing an increase in adaptability and cell fitness. Transcriptomic high hetness states in therapy-tolerant cell states denote its importance in cancer resistance to therapy. The definition of the concept of hetness will allow the understanding of its origins, its control during embryogenesis, its loss of control in tumorigenesis and cancer therapeutics and its active targeting.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies, highlighting the urgent need to elucidate the underlying oncogenic mechanisms. VIRMA is a classic isoform of methyltransferases that participates in epigenetic transcriptomic modification in eukaryotic mRNAs. However, the exact roles of VIRMA in PDAC remain unclear. Here, we identified that VIRMA is highly expressed in PDAC, and histone modifications of the promoter may partly account for this dysregulation. Moreover, VIRMA is closely related to glycolysis and poor prognosis in PDAC. We further determined that STRA6 is a direct downstream target of VIRMA in PDAC by RNA sequencing (RNA-seq) and m6A sequencing (m6A-seq). VIRMA is involved in gene expression regulation via 3' UTR targeting of STRA6 mRNA. Furthermore, the m6A reader IGF2BP2 was shown to critically contribute to the stability of STRA6 mRNA. We describe the role of VIRMA in promoting signaling via the STRA6/STAT3 axis, which results in increased levels of HIF-1α, a key activator of glycolysis. In vivo and in vitro experiments reveal that the VIRMA-STRA6-STAT3-HIF-1α axis plays an instrumental role in glycolysis and tumor progression in PDAC. In conclusion, we demonstrate that VIRMA can increase glycolysis in PDAC by upregulating STRA6, a cell surface membrane protein that stimulates the STAT3 pathway, thereby activating HIF-1α and leading to pancreatic cancer malignancy. Overall, our data strongly suggest that the VIRMA-STRA6-STAT3-HIF-1α axis is a viable therapeutic target in PDAC.