Inactivation of α-thalassemia/mental retardation X-linked (ATRX) represents a defining molecular feature in large subsets of malignant glioma. ATRX deficiency gives rise to abnormal G-quadruplex (G4) DNA secondary structures, enhancing replication stress and genomic instability. Building on earlier work, we evaluated the extent to which pharmacological G4 stabilization selectively enhances DNA damage and cell death in ATRX-deficient preclinical glioma models.

Using the G4 stabilizer CX-5461, we treated patient-derived glioma stem cells (GSCs) in vitro and GSC flank and intracranial murine xenografts in vivo to evaluate efficacy as both a single agent and in combination with ionizing radiation (IR), the latter a central element of current treatment standards.

CX-5461 promoted dose-sensitive lethality in ATRX-deficient GSCs relative to ATRX-intact controls. Mechanistic studies revealed that CX-5461 disrupted histone variant H3.3 deposition, enhanced replication stress and DNA damage, activated p53-independent apoptosis, and induced G2/M arrest to a greater extent in ATRX-deficient GSCs than in ATRX-intact counterparts. These data were corroborated in vivo, where CX-5461/IR treatment profoundly delayed tumor growth and prolonged survival in mice bearing ATRX-deficient flank xenografts. Histopathological analyses revealed decreased proliferation, increased apoptosis, and significant G4 induction, replication stress, and DNA damage in CX-5461-treated tumors, both alone and in combination with IR. Finally, despite suboptimal blood-brain-barrier penetration, systemic CX-5461 treatment induced tangible pharmacodynamic effects in ATRX-deficient intracranial GSC models.

In totality, our work substantively demonstrates efficacy and defines mechanisms of action for G4 stabilization as a novel therapeutic strategy targeting ATRX-deficient malignant glioma, laying the groundwork for clinical translation.

Mitofusin 1 (MFN1) plays a crucial role in mitochondrial fusion and oocyte development. However, its function in preimplantation embryonic development and its potential involvement in epigenetic regulation remain poorly understood. In this study, it is shown that MFN1 interacts with PADI6, a key component of the cytoplasmic lattice in oocytes and early embryos. MFN1 deficiency in mice results in reduced PADI6 levels and decreased expression of translational machinery components, which suppress protein synthesis activity and lower histone H3.3 abundance. These disruptions lead to the failure of male pronucleus formation, aberrant zygotic genome activation, and impaired embryonic development. It is further demonstrated that the MFN1 activator S89 promotes H3.3 incorporation and rescues early development in maternally aged embryos with low MFN1 levels. Additionally, a positive correlation between MFN1 and H3.3 protein levels in early human embryos is observed. Together, these findings provide new insights into MFN1's role in regulating epigenetic reprogramming during preimplantation embryo development.

ATRX (alpha-thalassemia/mental retardation, X-linked), a chromatin remodeler, is one of the most commonly mutated genes in human cancer. The ATRX protein functions as a histone chaperone, facilitating the proper folding and assembly of histone proteins into nucleosome cores. Investigations into its molecular mechanisms have significantly advanced our understanding of its roles in diseases associated with chromosomal instability and defective DNA repair. In this comprehensive review, we delineate ATRX's critical function in maintaining heterochromatin integrity and genomic stability under physiological conditions. We further explore the pathogenesis of ATRX-deficient tumors and ATRX syndrome, systematically evaluate current therapeutic strategies for these conditions, and propose novel perspectives on potential targeted therapies for ATRX-mutated malignancies. This review provides useful resource for regarding the etiology and treatment of ATRX deficiency-related diseases.

Daxx and HIRA are key proteins in the host response to DNA virus infections. Daxx is involved in apoptosis, transcription regulation, and stress responses. During DNA virus infections, Daxx helps modulate the immune response and viral progression. Viruses like adenoviruses and herpesviruses can exploit Daxx to evade immune detection, either by targeting it for degradation or inhibiting its function. Daxx also interacts with chromatin to regulate transcription, which viruses can manipulate to enhance their own gene expression and replication. HIRA is a histone chaperone and reported to be essential for chromatin assembly and gene regulation. It plays a critical role in maintaining chromatin structure and modulating gene accessibility. During DNA virus infection, HIRA influences chromatin remodeling, affecting both viral and host DNA accessibility, which impacts viral replication and gene expression. Additionally, the histone variant H3.3 is crucial for maintaining active chromatin states. It is incorporated into chromatin independently of DNA replication and is associated with active gene regions. During viral infections, H3.3 dynamics can be altered, affecting viral genome accessibility and replication efficiency. Overall, Daxx and HIRA are integral to orchestrating viral infection programs, maintaining latency and/or persistence, and influencing virus-induced transformation by modulating chromatin dynamics and host immune responses, making them significant targets for therapeutic strategies once fully understood. Here, we summarize various DNA viruses and their crosstalk with Daxx and HIRA.

The alternative lengthening of telomeres (ALT) pathway is a telomerase-independent mechanism for immortalization in cancer cells and is commonly activated in low-grade and high-grade glioma, as well as osteosarcoma. The ALT pathway can be activated under various conditions and has often been shown to include mutational loss of ATRX. However, this is insufficient in isolation and so other cellular event must also be implicated. It has been shown that excessive accumulation of DNA:RNA hybrid structures (R-loops) and/or formation of DNA-protein crosslinks (DPCs) can be other important driving factors. The underlying cellular events leading to R-loop and DPC formation in ALT cancer cells to date remain unclear. Here, we demonstrate that excessive cellular reactive oxygen species (ROS) is an important causative factor in the evolution of ALT-telomere maintenance in ATRX-deficient glioma. We identified three sources of elevated ROS in ALT-positive gliomas: co-mutation of SETD2, downregulation of DRG2, and hypoxic tumour microenvironment. We demonstrate that elevated ROS leads to accumulation of R-loops and, crucially, resolution of R-loops by the enzyme RNase H1 prevents ALT pathway activity in cells exposed to elevated ROS. Further, we found a possible causal link between the formation of R-loops and the accumulation of DPCs, in particular, formation of TOP1 complexes covalently linked to DNA (Top1cc). We also demonstrate that elevation of ROS can trigger over-activity of the ALT pathway in osteosarcoma and glioma cell lines, resulting in excessive DNA damage and cell death. This work presents important mechanistic insights into the endogenous origin of excessive R-loops and DPCs in ALT-positive cancers, as well as highlighting potential novel therapeutic approaches in these difficult-to-treat cancer types.

A significant portion of human cancers utilize a recombination-based pathway, alternative lengthening of telomeres (ALT), to extend telomeres. To gain further insights into this pathway, we developed a high-throughput imaging-based screen named TAILS (telomeric ALT in situ localization screen) to identify genes that either promote or inhibit ALT activity. Screening over 1,000 genes implicated in DNA transactions, TAILS reveals both well-established and putative ALT modulators. Here, we present the validation of factors that promote ALT, such as the nucleosome-remodeling factor CHD4 and the chromatin reader SGF29, as well as factors that suppress ALT, including the RNA helicases DExD-box helicase 39A/B (DDX39A/B), the replication factor TIMELESS, and components of the chromatin assembly factor CAF1. Our data indicate that defects in histone deposition significantly contribute to ALT-associated phenotypes. Based on these findings, we demonstrate that pharmacological treatments can be employed to either exacerbate or suppress ALT-associated phenotypes.

Identifying novel targets for molecular radiosensitization is critical for improving the efficacy of colorectal cancer (CRC) radiotherapy. Alpha-thalassemia/mental retardation X-linked (ATRX), a member of the SWI/SNF-like chromatin remodeling protein family, functions in the maintenance of genomic integrity and the regulation of apoptosis and senescence. However, whether ATRX is directly involved in the radiosensitivity of CRC remains unclear. Our results showed that silencing ATRX increased the radiosensitivity of HCT116 CRC cells, which was further strengthened when p53 was depleted. To explore the potential mechanism, we focused on the impact of the ionizing radiation (IR)-induced DNA damage response (DDR), apoptosis, and senescence and the activation of the Daxx/MDM2/p53 pathway caused by ATRX loss. The results showed that IR induced DNA damage and G2/M arrest after depleting ATRX, especially in p53-depleted HCT116 cells, and inhibited ATM/Chk2 pathway activation, indicating that ATRX loss leads to failure of triggering the ATM/Chk2 pathway. Accordingly, ATRX loss promotes cell apoptosis and attenuates cell senescence. Interestingly, our results indicate that ATRX loss upregulates p53 function via the Daxx/MDM2 pathway to mediate radiosensitivity. Thus, ATRX may represent a novel radiosensitizing target for CRC, particularly p53-deficient CRC.

A significant portion of human cancers utilize a recombination-based pathway, Alternative Lengthening of Telomeres (ALT), to extend telomeres. To gain further insights into this pathway, we developed a high-throughput imaging-based screen named TAILS (Telomeric ALT In situ Localization Screen), to identify genes that either promote or inhibit ALT activity. Screening over 1000 genes implicated in DNA transactions, TAILS revealed both well-established and novel ALT modulators. We have identified new factors that promote ALT, such as the nucleosome-remodeling factor CHD4 and the chromatin reader SGF29, as well as factors that suppress ALT, including the RNA helicases DDX39A/B, the replication factor TIMELESS, and components of the chromatin assembly factor CAF1. Our data indicate that defects in histone deposition significantly contribute to ALT-associated phenotypes. Based on these findings, we demonstrate that pharmacological treatments can be employed to either exacerbate or suppress ALT-associated phenotypes.

Miscarriages cause a greater loss-of-life than cardiovascular diseases, but knowledge about environmentally induced miscarriages is limited. Cultured naïve pluripotent embryonic stem cells (ESC) differentiate into extra-embryonic endoderm/extraembryonic endoderm (XEN) or formative pluripotent ESC, during the period emulating maximal miscarriage of peri-implantation development. In previous reports using small marker sets, hyperosmotic sorbitol, or retinoic acid (RA) decreased naïve pluripotency and increased XEN by FACS quantitation.

Bulk and single cell (sc)RNAseq analyses of two cultured ESC lines was done, corroborated by qPCR. Transcriptomic responses were analyzed of cultured ESC stressed by Sorbitol, with Leukemia inhibitory factor (LIF + ; stemness growth factor), RA without LIF to control for XEN induction, and compared with normal differentiation (LIF - , ND).

Sorbitol and RA increase subpopulations of 2-cell embryo-like (2CEL) and XEN sub-lineages; primitive, parietal, and visceral endoderm (VE) cells and suppress formative pluripotency, imbalancing alternate lineage choices of initial naïve pluripotent cultured ESC compared with ND. Although bulk RNAseq and gene ontology (GO) group analyses suggest that stress induces anterior VE-head organizer and placental markers, scRNAseq reveals relatively few cells. But VE and placental markers/cells were in adjacent stressed cell clusters in the UMAP, like recent, normal UMAP of conceptuses. UMAPs show that dose-dependent stress overrides stemness to force premature lineage imbalance.

Hyperosmotic stress, and other toxicological stresses, like drugs with active ingredient RA, may cause premature, lineage imbalance, resulting in miscarriages or birth defects.

Canonical histone H3 and histone variant H3.3 are posttranslationally modified with the genomic distribution of these marks denoting different features and these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the amino acid H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent but exhibit a reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue within H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells.

The transcriptional regulator ATRX, a genetic factor, is associated with a range of disabilities, including intellectual, hematopoietic, skeletal, facial, and urogenital disabilities. ATRX mutations substantially contribute to the pathogenesis of ATRX syndrome and are frequently detected in gliomas and many other cancers. These mutations disrupt the organization, subcellular localization, and transcriptional activity of ATRX, leading to chromosomal instability and affecting interactions with key regulatory proteins such as DAXX, EZH2, and TERRA. ATRX also functions as a transcriptional regulator involved in the pathogenesis of neuronal disorders and various diseases. In conclusion, ATRX is a central protein whose abnormalities lead to multiple diseases.

ATRX limits the accumulation of human cytomegalovirus (HCMV) Immediate Early (IE) proteins at the start of productive, lytic infections, and thus is a part of the cell-intrinsic defenses against infecting viruses. ATRX is a chromatin remodeler and a component of a histone chaperone complex. Therefore, we hypothesized ATRX would inhibit the transcription of HCMV IE genes by increasing viral genome heterochromatinization and decreasing its accessibility. To test this hypothesis, we quantitated viral transcription and genome structure in cells replete with or depleted of ATRX. We found ATRX did indeed limit viral IE transcription, increase viral genome chromatinization, and decrease viral genome accessibility. The inhibitory effects of ATRX extended to Early (E) and Late (L) viral protein accumulation, viral DNA replication, and progeny virion output. However, we found the negative effects of ATRX on HCMV viral DNA replication were independent of its effects on viral IE and E protein accumulation but correlated with viral genome heterochromatinization. Interestingly, the increased number of viral genomes synthesized in ATRX-depleted cells were not efficiently packaged, indicating the ATRX-mediated restriction to HCMV viral DNA replication may benefit productive infection by increasing viral fitness. Our work mechanistically describes the antiviral function of ATRX and introduces a novel, pro-viral role for this protein, perhaps explaining why, unlike during infections with other herpesviruses, it is not directly targeted by a viral countermeasure in HCMV infected cells.

The histone variant H3.3 acts in coordination with histone posttranslational modifications and other chromatin features to facilitate appropriate transcription. Canonical histone H3 and histone variant H3.3 are post-translationally modified with the genomic distribution of these marks denoting different features and with more recent evidence suggesting that these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the residue H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent, albeit with reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue of H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells.

Acquiring a telomere maintenance mechanism is a hallmark of high-risk neuroblastoma and commonly occurs by expressing telomerase (TERT). Telomerase-negative neuroblastoma has long telomeres and utilizes the telomerase-independent alternative lengthening of telomeres (ALT) mechanism. Conversely, no discernable telomere maintenance mechanism is detected in a fraction of neuroblastoma with long telomeres. Here, we show, unlike most cancers, DNA of the TERT promoter is broadly hypomethylated in neuroblastoma. In telomerase-positive neuroblastoma cells, the hypomethylated DNA promoter is approximately 1.5 kb. The TERT locus shows active chromatin marks with low enrichment for the repressive mark, H3K27me3. MYCN, a commonly amplified oncogene in neuroblstoma, binds to the promoter and induces TERT expression. Strikingly, in neuroblastoma with long telomeres, the hypomethylated region spans the entire TERT locus, including multiple nearby genes with enrichment for the repressive H3K27me3 chromatin mark. Furthermore, subtelomeric regions showed enrichment of repressive chromatin marks in neuroblastomas with long telomeres relative to those with short telomeres. These repressive marks were even more evident at the genic loci, suggesting a telomere position effect (TPE). Inhibiting H3K27 methylation by three different EZH2 inhibitors induced the expression of TERT in cell lines with long telomeres and H3K27me3 marks in the promoter region. EZH2 inhibition facilitated MYCN binding to the TERT promoter in neuroblastoma cells with long telomeres. Taken together, these data suggest that epigenetic regulation of TERT expression differs in neuroblastoma depending on the telomere maintenance status, and H3K27 methylation is important in repressing TERT expression in neuroblastoma with long telomeres.

The epigenetic landscape of the TERT locus is unique in neuroblastoma. The DNA at the TERT locus, unlike other cancer cells and similar to normal cells, are hypomethylated in telomerase-positive neuroblastoma cells. The TERT locus is repressed by polycomb repressive complex-2 complex in neuroblastoma cells that have long telomeres and do not express TERT. Long telomeres in neuroblastoma cells are also associated with repressive chromatin states at the chromosomal termini, suggesting TPE.

Glioblastoma are highly malignant type of primary brain tumors. Treatment for glioblastoma multiforme (GBM) generally involves surgery combined with chemotherapy and radiotherapy. However, the development of tumoral chemo- and radioresistance induces complexities in clinical practice. Multiple signaling pathways are known to be involved in radiation-induced cell survival. However, the role of alpha-thalassemia X-linked mutant retardation syndrome (ATRX), a chromatin remodeling protein, in GBM radioresistance remains unclear.

In the present study, the ATRX mutation rate in patients with glioma was obtained from The Cancer Genome Atlas, while its expression analyzed using bioinformatics. Datasets were also obtained from the Gene Expression Omnibus, and ATRX expression levels following irradiation of GBM were determined. The effects of ATRX on radiosensitivity were investigated using a knockdown assays.

The present study demonstrated that the ATRX mutation rate in patients with GBM was significantly lower than that in patients with low-grade glioma, and that patients harboring an ATRX mutation exhibited a prolonged survival, compared with to those harboring the wild-type gene. Single-cell RNA sequencing demonstrated that ATRX counts increased 2 days after irradiation, with ATRX expression levels also increasing in U-251MG radioresistant cells. Moreover, the results of in vitro irradiation assays revealed that ATRX expression was increased in U-251MG cells, while ATRX knockdown was associated with increased levels of radiosensitivity.

High ATRX expression levels in primary GBM may contribute to high levels of radioresistance. Thus ATRX is a potential target for overcoming the radioresistance in GBM.

A majority of cancers (~85%) activate the enzyme telomerase to maintain telomere length over multiple rounds of cellular division. Telomerase-negative cancers activate a distinct, telomerase-independent mechanism of telomere maintenance termed alternative lengthening of telomeres (ALT). ALT uses homologous recombination to maintain telomere length and exhibits features of break-induced DNA replication. In malignant gliomas, the activation of either telomerase or ALT is nearly ubiquitous in pediatric and adult tumors, and the frequency with which these distinct telomere maintenance mechanisms (TMMs) is activated varies according to genetically defined glioma subtypes. In this review, we summarize the current state of the field of TMMs and their relevance to glioma biology and therapy. We review the genetic alterations and molecular mechanisms leading to telomerase activation or ALT induction in pediatric and adult gliomas. With this background, we review emerging evidence on strategies for targeting TMMs for glioma therapy. Finally, we comment on critical gaps and issues for moving the field forward to translate our improved understanding of glioma telomere maintenance into better therapeutic strategies for patients.

Alterations in the tumor suppressor ATRX are recurrently observed in mesenchymal neoplasms. ATRX has multiple epigenetic functions including heterochromatin formation and maintenance and regulation of transcription through modulation of chromatin accessibility. Here, we show in murine mesenchymal progenitor cells (MPCs) that Atrx deficiency aberrantly activated mesenchymal differentiation programs. This includes adipogenic pathways where ATRX loss induced expression of adipogenic transcription factors and enhanced adipogenic differentiation in response to differentiation stimuli. These changes are linked to loss of heterochromatin near mesenchymal lineage genes together with increased chromatin accessibility and gains of active chromatin marks. We additionally observed depletion of H3K9me3 at transposable elements, which are derepressed including near mesenchymal genes where they could serve as regulatory elements. Finally, we demonstrated that loss of ATRX in a mesenchymal malignancy, undifferentiated pleomorphic sarcoma, results in similar epigenetic disruption and de-repression of transposable elements. Together, our results reveal a role for ATRX in maintaining epigenetic states and transcriptional repression in mesenchymal progenitors and tumor cells and in preventing aberrant differentiation in the progenitor context.

ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome features genital and testicular abnormalities including atypical genitalia and small testes with few seminiferous tubules. Our mouse model recapitulated the testicular defects when Atrx was deleted in Sertoli cells (ScAtrxKO) which displayed G2/M arrest and apoptosis. Here, we investigated the mechanisms underlying these defects. In control mice, Sertoli cells contain a single novel "GATA4 PML nuclear body (NB)" that contained the transcription factor GATA4, ATRX, DAXX, HP1α, and PH3 and co-localized with the Y chromosome short arm (Yp). ScAtrxKO mice contain single giant GATA4 PML-NBs with frequent DNA double-strand breaks (DSBs) in G2/M-arrested apoptotic Sertoli cells. HP1α and PH3 were absent from giant GATA4 PML-NBs indicating a failure in heterochromatin formation and chromosome condensation. Our data suggest that ATRX protects a Yp region from DNA damage, thereby preventing Sertoli cell death. We discuss Y chromosome damage/decondensation as a mechanism for testicular failure.

Glioblastoma is currently considered the most common and, unfortunately, also the most aggressive primary brain tumor, with the highest morbidity and mortality rates. The average survival of patients diagnosed with glioblastoma is 14 months, and only 2% of patients survive 3 years after surgery. Based on our clinical experience and knowledge from extensive clinical studies, survival is mainly related to the molecular biological properties of glioblastoma, which are of interest to the general medical community. Our study examined a total of 71 retrospective studies published from 2016 through 2022 and available on PubMed that deal with mutations of selected genes in the pathophysiology of GBM. In conclusion, we can find other mutations within a given gene group that have different effects on the prognosis and quality of survival of a patient with glioblastoma. These mutations, together with the associated mutations of other genes, as well as intratumoral heterogeneity itself, offer enormous potential for further clinical research and possible application in therapeutic practice.

Nucleosome remodeling complexes and other regulatory factors work in concert to build a chromatin environment that directs the expression of a distinct set of genes in each cell using cis-regulatory elements (CREs), such as promoters and enhancers, that drive transcription of both mRNAs and CRE-associated non-coding RNAs (ncRNAs). Two classes of CRE-associated ncRNAs include upstream antisense RNAs (uaRNAs), which are transcribed divergently from a shared mRNA promoter, and enhancer RNAs (eRNAs), which are transcribed bidirectionally from active enhancers. The complicated network of CRE regulation by nucleosome remodelers remains only partially explored, with a focus on a select, limited number of remodelers. We endeavored to elucidate a remodeler-based regulatory network governing CRE-associated transcription (mRNA, eRNA, and uaRNA) in murine embryonic stem (ES) cells to test the hypothesis that many SNF2-family nucleosome remodelers collaborate to regulate the coding and non-coding transcriptome via alteration of underlying nucleosome architecture. Using depletion followed by transient transcriptome sequencing (TT-seq), we identified thousands of misregulated mRNAs and CRE-associated ncRNAs across the remodelers examined, identifying novel contributions by understudied remodelers in the regulation of coding and noncoding transcription. Our findings suggest that mRNA and eRNA transcription are coordinately co-regulated, while mRNA and uaRNAs sharing a common promoter are independently regulated. Subsequent mechanistic studies suggest that while remodelers SRCAP and CHD8 modulate transcription through classical mechanisms such as transcription factors and histone variants, a broad set of remodelers including SMARCAL1 indirectly contribute to transcriptional regulation through maintenance of genomic stability and proper Integrator complex localization. This study systematically examines the contribution of SNF2-remodelers to the CRE-associated transcriptome, identifying at least two classes for remodeler action.

Nucleosome, the building block of chromatin, plays pivotal roles in all DNA-related processes. While cryogenic-electron microscopy (cryo-EM) has significantly advanced our understanding of nucleosome structures, the emerging field of single-molecule force spectroscopy is illuminating their dynamic properties. This technique is crucial for revealing how nucleosome behavior is influenced by chaperones, remodelers, histone variants, and post-translational modifications, particularly in their folding and unfolding mechanisms under tension. Such insights are vital for deciphering the complex interplay in nucleosome assembly and structural regulation, highlighting the nucleosome's versatility in response to DNA activities. In this Perspective, we aim to consolidate the latest advancements in nucleosome dynamics, with a special focus on the revelations brought forth by single-molecule manipulation. Our objective is to highlight the insights gained from studying nucleosome dynamics through this innovative approach, emphasizing the transformative impact of single-molecule manipulation techniques in the field of chromatin research.

Malignant melanoma remains the most lethal form of skin cancer, exhibiting poor prognosis after forming distant metastasis. Owing to their potential tumor-suppressive properties by regulating oncogenes and tumor suppressor genes, microRNAs are important player in melanoma development and progression. We defined the loss of miR-101-3p expression in melanoma cells compared with melanocytes and melanoblast-related cells as an early event in tumor development and aimed to understand the tumor suppressive role of miR-101-3p and its regulation of important cellular processes. Reexpression of miR-101-3p resulted in inhibition of proliferation, increase in DNA damage, and induction of apoptosis. We further determined the nuclear structure protein Lamin B1, which influences nuclear processes and heterochromatin structure, ATRX, CASP3, and PARP as an important direct target of miR-101-3p. RNA sequencing and differential gene expression analysis after miR-101-3p reexpression supported our findings and the importance of loss of mir-101-3p for melanoma progression. The validated functional effects are related to genomic instability, as recent studies suggest miRNAs plays a key role in mediating this cellular process. Therefore, we concluded that miR-101-3p reexpression increases the genomic instability, leading to irreversible DNA damage, which leads to apoptosis induction. Our findings suggest that the loss of miR-101-3p in melanoma serves as an early event in melanoma progression by influencing the genomic integrity to maintain the increased bioenergetic demand.

Aging is a significant risk factor for Alzheimer's disease (AD), although the precise mechanism and molecular basis of AD are not yet fully understood. Epigenetic mechanisms, such as DNA methylation and hydroxymethylation, mitochondrial DNA methylation, histone modifications, and non-coding RNAs (ncRNAs), play a role in regulating gene expression related to neuron plasticity and integrity, which are closely associated with learning and memory development. This review describes the impact of dynamic and reversible epigenetic modifications and factors on memory and plasticity throughout life, emphasizing their potential as target for therapeutic intervention in AD. Additionally, we present insight from postmortem and animal studies on abnormal epigenetics regulation in AD, as well as current strategies aiming at targeting these factors in the context of AD therapy.

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.

A G-quadruplex (G4) is a typical non-B DNA structure and involved in various DNA-templated events in eukaryotic genomes. PEG and PDS chemicals have been widely applied for promoting the folding of in vivo or in vitro G4s. However, how PEG and PDS preferentially affect a subset of G4 formation genome-wide is still largely unknown. We here conducted a BG4-based IP-seq in vitro under K++PEG or K++PDS conditions in the rice genome. We found that PEG-favored IP-G4s+ have distinct sequence features, distinct genomic distributions and distinct associations with TEGs, non-TEGs and subtypes of TEs compared to PDS-favored ones. Strikingly, PEG-specific IP-G4s+ are associated with euchromatin with less enrichment levels of DNA methylation but with more enriched active histone marks, while PDS-specific IP-G4s+ are associated with heterochromatin with higher enrichment levels of DNA methylation and repressive marks. Moreover, we found that genes with PEG-specific IP-G4s+ are more expressed than those with PDS-specific IP-G4s+, suggesting that PEG/PDS-specific IP-G4s+ alone or coordinating with epigenetic marks are involved in the regulation of the differential expression of related genes, therefore functioning in distinct biological processes. Thus, our study provides new insights into differential impacts of PEG and PDS on G4 formation, thereby advancing our understanding of G4 biology.

Despite the fact that important advances in research on neuroendocrine neoplasms (NENs) have been made, consistent data about their pathogenetic mechanism are still lacking. Furthermore, different primary sites may recognize different pathogenetic mechanisms.

This review analyzes the possible biological and molecular mechanisms that may lead to NEN onset and progression in different organs. Through extensive research of the literature, risk factors including hypercholesterolemia, inflammatory bowel disease, chronic atrophic gastritis are evaluated as potential pathogenetic mechanisms. Consistent evidence is available regarding sporadic gastric NENs and MEN1 related duodenopancreatic NENs precursor lesions, and genetic-epigenetic mutations may play a pivotal role in tumor development and bone metastases onset. In lung neuroendocrine tumors (NETs), diffuse proliferation of neuroendocrine cells on the bronchial wall (DIPNECH) has been proposed as a premalignant lesion, while in lung neuroendocrine carcinoma nicotine and smoke could be responsible for carcinogenic processes. Also, rare primary NENs such as thymic (T-NENs) and Merkel cell carcinoma (MCC) have been analyzed, finding different possible pathogenetic mechanisms.

New technologies in genomics and epigenomics are bringing new light to the pathogenetic landscape of NENs, but further studies are needed to improve both prevention and treatment in these heterogeneous neoplasms.

Transformed cells must acquire specific characteristics to be malignant. Weinberg and Hanahan characterize these characteristics as cancer hallmarks. Though these features are independently driven, substantial signaling crosstalk in transformed cells efficiently promotes these feature acquisitions. Telomerase is an enzyme complex that maintains telomere length. However, its main component, Telomere reverse transcriptase (TERT), has been found to interact with various signaling molecules like cMYC, NF-kB, BRG1 and cooperate in transcription and metabolic reprogramming, acting as a strong proponent of malignant features such as cell death resistance, sustained proliferation, angiogenesis activation, and metastasis, among others. It allows cells to avoid replicative senescence and achieve endless replicative potential. This review summarizes both the canonical and noncanonical functions of TERT and discusses how they promote cancer hallmarks. Understanding the role of Telomerase in promoting cancer hallmarks provides vital insight into the underlying mechanism of cancer genesis and progression and telomerase intervention as a possible therapeutic target for cancer treatment. More investigation into the precise molecular mechanisms of telomerase-mediated impacts on cancer hallmarks will contribute to developing more focused and customized cancer treatment methods.

The Drosophila melanogaster dADD1 and dXNP proteins are orthologues of the ADD and SNF2 domains of the vertebrate ATRX (Alpha-Thalassemia with mental Retardation X-related) protein. ATRX plays a role in general molecular processes, such as regulating chromatin status and gene expression, while dADD1 and dXNP have similar functions in the Drosophila genome. Both ATRX and dADD1/dXNP interact with various protein partners and participate in various regulatory complexes. Disruption of ATRX expression in humans leads to the development of α-thalassemia and cancer, especially glioma. However, the mechanisms that allow ATRX to regulate various cellular processes are poorly understood. Studying the functioning of dADD1/dXNP in the Drosophila model may contribute to understanding the mechanisms underlying the multifunctional action of ATRX and its connection with various cellular processes. This review provides a brief overview of the currently available information in mammals and Drosophila regarding the roles of ATRX, dXNP, and dADD1. It discusses possible mechanisms of action of complexes involving these proteins.

ATRX is an ATP-dependent chromatin remodeling protein with essential roles in safeguarding genome integrity and modulating gene expression. Deficiencies in this protein cause ATR-X syndrome, a condition characterized by intellectual disability and an array of developmental abnormalities, including features of autism. Previous studies demonstrated that deleting ATRX in mouse forebrain excitatory neurons postnatally resulted in male-specific memory deficits, but no apparent autistic-like behaviours.

We generated mice with an earlier embryonic deletion of ATRX in forebrain excitatory neurons and characterized their behaviour using a series of memory and autistic-related paradigms.

We found that mutant mice displayed a broader spectrum of impairments, including fear memory, decreased anxiety-like behaviour, hyperactivity, as well as self-injurious and repetitive grooming. Sex-specific alterations were also observed, including male-specific aggression, sensory gating impairments, and decreased social memory.

Collectively, the findings indicate that early developmental abnormalities arising from ATRX deficiency in forebrain excitatory neurons contribute to the presentation of fear memory deficits as well as autistic-like behaviours.

Advancing the technologies for cellular reprogramming with high efficiency has significant impact on regenerative therapy, disease modeling, and drug discovery. Biophysical cues can tune the cell fate, yet the precise role of external physical forces during reprogramming remains elusive. Here the authors show that temporal cyclic-stretching of fibroblasts significantly enhances the efficiency of induced pluripotent stem cell (iPSC) production. Generated iPSCs are proven to express pluripotency markers and exhibit in vivo functionality. Bulk RNA-sequencing reveales that cyclic-stretching enhances biological characteristics required for pluripotency acquisition, including increased cell division and mesenchymal-epithelial transition. Of note, cyclic-stretching activates key mechanosensitive molecules (integrins, perinuclear actins, nesprin-2, and YAP), across the cytoskeletal-to-nuclear space. Furthermore, stretch-mediated cytoskeletal-nuclear mechano-coupling leads to altered epigenetic modifications, mainly downregulation in H3K9 methylation, and its global gene occupancy change, as revealed by genome-wide ChIP-sequencing and pharmacological inhibition tests. Single cell RNA-sequencing further identifies subcluster of mechano-responsive iPSCs and key epigenetic modifier in stretched cells. Collectively, cyclic-stretching activates iPSC reprogramming through mechanotransduction process and epigenetic changes accompanied by altered occupancy of mechanosensitive genes. This study highlights the strong link between external physical forces with subsequent mechanotransduction process and the epigenetic changes with expression of related genes in cellular reprogramming, holding substantial implications in the field of cell biology, tissue engineering, and regenerative medicine.

Approximately 15% of human cancers depend on the alternative lengthening of telomeres (ALT) pathway to maintain telomeres and proliferate. Telomeres that are elongated using ALT display unique features raising the exciting prospect of tailored cancer therapies. ALT-mediated telomere elongation shares several features with recombination-based DNA repair. Strikingly, cells that use the ALT pathway display abnormal levels of replication stress at telomeres and accumulate abundant extrachromosomal telomeric DNA. In this review, we examine recent findings that shed light on the ALT mechanisms and the strategies currently available to suppress this telomere elongation mechanism.

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. In addition, HIRA KO deregulates glucocorticoid- (GR) driven transcription of genes co-regulated by AR and GR, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity.

The aggregation of cancer cells provides a survival signal for disseminating cancer cells; however, the underlying molecular mechanisms have yet to be elucidated. Using qPCR gene arrays, this study investigated the changes in cancer-specific genes as well as genes regulating mitochondrial quality control, metabolism, and oxidative stress in response to aggregation and hypoxia in our progressive ovarian cancer models representing slow- and fast-developing ovarian cancer. Aggregation increased the expression of anti-apoptotic, stemness, epithelial-mesenchymal transition (EMT), angiogenic, mitophagic, and reactive oxygen species (ROS) scavenging genes and functions, and decreased proliferation, apoptosis, metabolism, and mitochondrial content genes and functions. The incorporation of stromal vascular cells (SVF) from obese mice into the spheroids increased DNA repair and telomere regulatory genes that may represent a link between obesity and ovarian cancer risk. While glucose had no effect, glutamine was essential for aggregation and supported proliferation of the spheroid. In contrast, low glucose and hypoxic culture conditions delayed adhesion and outgrowth capacity of the spheroids independent of their phenotype, decreased mitochondrial mass and polarity, and induced a shift of mitochondrial dynamics towards mitophagy. However, these conditions did not reduce the appearance of polarized mitochondria at adhesion sites, suggesting that adhesion signals that either reversed mitochondrial fragmentation or induced mitobiogenesis can override the impact of low glucose and oxygen levels. Thus, the plasticity of the spheroids' phenotype supports viability during dissemination, allows for the adaptation to changing conditions such as oxygen and nutrient availability. This may be critical for the development of an aggressive cancer phenotype and, therefore, could represent druggable targets for clinical interventions.

Chromatin is a crucial regulator of gene expression and tightly controls development across species. Mutations in only one copy of multiple histone genes were identified in children with developmental disorders characterized by microcephaly, but their mechanistic roles in development remain unclear. Here we focus on dominant mutations affecting histone H4 lysine 91. These H4K91 mutants form aberrant nuclear puncta at specific heterochromatin regions. Mechanistically, H4K91 mutants demonstrate enhanced binding to the histone variant H3.3, and ablation of H3.3 or the H3.3-specific chaperone DAXX diminishes the mutant localization to chromatin. Our functional studies demonstrate that H4K91 mutant expression increases chromatin accessibility, alters developmental gene expression through accelerating pro-neural differentiation, and causes reduced mouse brain size in vivo, reminiscent of the microcephaly phenotypes of patients. Together, our studies unveil a distinct molecular pathogenic mechanism from other known histone mutants, where H4K91 mutants misregulate cell fate during development through abnormal genomic localization.

Alterations in the tumor suppressor ATRX are recurrently observed in several cancer types including sarcomas, which are mesenchymal neoplasms. ATRX has multiple epigenetic functions including heterochromatin formation and maintenance and regulation of transcription through modulation of chromatin accessibility. Here, we show in murine mesenchymal progenitor cells (MPCs) that Atrx deficiency aberrantly activated mesenchymal differentiation programs. This includes adipogenic pathways where ATRX loss induced expression of adipogenic transcription factors (Pparγ and Cebpα) and enhanced adipogenic differentiation in response to differentiation stimuli. These changes are linked to loss of heterochromatin near mesenchymal lineage genes together with increased chromatin accessibility and gains of active chromatin marks at putative enhancer elements and promoters. Finally, we observed depletion of H3K9me3 at transposable elements, which are derepressed including near mesenchymal genes where they could serve as regulatory elements. Our results demonstrate that ATRX functions to buffer against differentiation in mesenchymal progenitor cells, which has implications for understanding ATRX loss of function in sarcomas.

Telomere maintenance is a hallmark of malignant cells and allows cancers to divide indefinitely. In some cancers, this is achieved through the alternative lengthening of telomeres (ALT) pathway. Whilst loss of ATRX is a near universal feature of ALT-cancers, it is insufficient in isolation. As such, other cellular events must be necessary - but the exact nature of the secondary events has remained elusive. Here, we report that trapping of proteins (such as TOP1, TOP2A and PARP1) on DNA leads to ALT induction in cells lacking ATRX. We demonstrate that protein-trapping chemotherapeutic agents, such as etoposide, camptothecin and talazoparib, induce ALT markers specifically in ATRX-null cells. Further, we show that treatment with G4-stabilising drugs cause an increase in trapped TOP2A levels which leads to ALT induction in ATRX-null cells. This process is MUS81-endonuclease and break-induced replication dependent, suggesting that protein trapping leads to replication fork stalling, with these forks being aberrantly processed in the absence of ATRX. Finally, we show ALT-positive cells harbour a higher load of genome-wide trapped proteins, such as TOP1, and knockdown of TOP1 reduced ALT activity. Taken together, these findings suggest that protein trapping is a fundamental driving force behind ALT-biology in ATRX-deficient malignancies.

Cultured naïve pluripotent ESC differentiate into first lineage, XEN or second lineage, formative pluripotency. Hyperosmotic stress (sorbitol), like retinoic acid, decreases naive pluripotency and increases XEN in two ESC lines, as reported by bulk and scRNAseq, analyzed by UMAP. Sorbitol overrides pluripotency in two ESC lines as reported by bulk and scRNAseq, analyzed by UMAP. UMAP analyzed the effects of 5 stimuli - three stressed (200-300mM sorbitol with leukemia inhibitory factor +LIF) and two unstressed (+LIF, normal stemness-NS and -LIF, normal differentiation-ND). Sorbitol and RA decrease naive pluripotency and increase subpopulations of 2-cell embryo-like and XEN sub-lineages; primitive, parietal, and visceral endoderm (VE). Between the naïve pluripotency and primitive endoderm clusters is a stress-induced cluster with transient intermediate cells with higher LIF receptor signaling, with increased Stat3, Klf4, and Tbx3 expression. Sorbitol, like RA, also suppresses formative pluripotency, increasing lineage imbalance. Although bulk RNAseq and gene ontology group analyses suggest that stress induces head organizer and placental markers, scRNAseq reveals few cells. But VE and placental markers/cells were in adjacent clusters, like recent reports. UMAPs show that dose-dependent stress overrides stemness to force premature lineage imbalance. Hyperosmotic stress induces lineage imbalance, and other toxicological stresses, like drugs with RA, may cause lineage imbalance, resulting in miscarriages or birth defects.

Integration of retroviruses into the host genome can impair the genomic and epigenomic integrity of the cell. As a defense mechanism, epigenetic modifications on the proviral DNA repress retroviral sequences in mouse embryonic stem cells (ESC). Here, we focus on the histone 3 variant H3.3, which is abundant in active transcription zones, as well as centromeres and heterochromatinized repeat elements, e.g., endogenous retroviruses (ERV).

To understand the involvement of H3.3 in the epigenetic silencing of retroviral sequences in ESC, we depleted the H3.3 genes in ESC and transduced the cells with GFP-labeled MLV pseudovirus. This led to altered retroviral repression and reduced Trim28 recruitment, which consequently led to a loss of heterochromatinization in proviral sequences. Interestingly, we show that H3.3 depletion has a differential effect depending on which of the two genes coding for H3.3, H3f3a or H3f3b, are knocked out. Depletion of H3f3a resulted in a transient upregulation of incoming retroviral expression and ERVs, while the depletion of H3f3b did not have the same effect and repression was maintained. However, the depletion of both genes resulted in a stable activation of the retroviral promoter. These findings suggest that H3.3 is important for regulating retroviral gene expression in mouse ESC and provide evidence for a distinct function of the two H3.3 genes in this regulation. Furthermore, we show that Trim28 is needed for depositing H3.3 in retroviral sequences, suggesting a functional interaction between Trim28 recruitment and H3.3 loading.

Identifying the molecular mechanisms by which H3.3 and Trim28 interact and regulate retroviral gene expression could provide a deeper understanding of the fundamental processes involved in retroviral silencing and the general regulation of gene expression, thus providing new answers to a central question of stem cell biology.

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. AR KO reduced levels of H3.3 at enhancers, indicating feedback mechanism. In addition, HIRA KO deregulates glucocorticoid-driven transcription, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity.

*H3.3 at enhancers promotes acetylation of H3K27Ac and retention of AR/BRD4 complex for transcription regulation*Knockout of H3.3 chaperone HIRA suppresses PC cells growth and deregulates androgen-induced transcription*H3.3/HIRA pathway regulates both AR and GR, suggesting a common HIRA/H3.3 mechanism of nuclear receptors function.

Adult pluripotent stem cell (aPSC) populations underlie whole-body regeneration in many distantly-related animal lineages, but how the underlying cellular and molecular mechanisms compare across species is unknown. Here, we apply single-cell RNA sequencing to profile transcriptional cell states of the acoel worm Hofstenia miamia during postembryonic development and regeneration. We identify cell types shared across stages and their associated gene expression dynamics during regeneration. Functional studies confirm that the aPSCs, also known as neoblasts, are the source of differentiated cells and reveal transcription factors needed for differentiation. Subclustering of neoblasts recovers transcriptionally distinct subpopulations, the majority of which are likely specialized to differentiated lineages. One neoblast subset, showing enriched expression of the histone variant H3.3, appears to lack specialization. Altogether, the cell states identified in this study facilitate comparisons to other species and enable future studies of stem cell fate potentials.