Repressive Marks Research Articles

Polymorphic transposable elements contribute to variation in recombination landscapes

Meiotic recombination is a prominent force shaping genome evolution, and understanding why recombination rates vary within and between species has remained a central, though challenging, question. Variation in recombination is widely thought to influence the efficacy of selection in purging transposable elements (TEs), prevalent selfish genetic elements, leading to widely observed negative correlations between TE abundance and recombination rates across taxa. However, accumulating evidence suggests that TEs could instead be the cause rather than the consequence of this relationship. To test this prediction, we formally investigated the influence of polymorphic, putatively active TEs on recombination rates. We developed and benchmarked an approach that uses PacBio long-read sequencing to efficiently, accurately, and cost-effectively identify crossovers (COs), a key recombination product, among large numbers of pooled recombinant individuals. By applying this approach to Drosophila strains with distinct TE insertion profiles, we found that polymorphic TEs, especially RNA-based TEs and TEs with local enrichment of repressive marks, reduce the occurrence of COs. Such an effect leads to different CO frequencies between homologous sequences with and without TEs, contributing to varying CO maps between individuals. The suppressive effect of TEs on CO is further supported by two orthogonal approaches-analyzing the distributions of COs in panels of recombinant inbred lines in relation to TE polymorphism and applying marker-assisted estimations of CO frequencies to isogenic strains with and without transgenically inserted TEs. Our investigations reveal how the constantly changing TE landscape can actively modify recombination, shaping genome evolution within and between species.

Activating antiviral immune responses potentiates immune checkpoint inhibition in glioblastoma models.

Viral mimicry refers to the activation of innate antiviral immune responses due to the induction of endogenous retroelements (REs). Viral mimicry augments antitumor immune responses and sensitizes solid tumors to immunotherapy. Here, we found that targeting what we believe to be a novel, master epigenetic regulator, Zinc Finger Protein 638 (ZNF638), induces viral mimicry in glioblastoma (GBM) preclinical models and potentiates immune checkpoint inhibition (ICI). ZNF638 recruits the HUSH complex, which precipitates repressive H3K9me3 marks on endogenous REs. In GBM, ZNF638 is associated with marked locoregional immunosuppressive transcriptional signatures, reduced endogenous RE expression, and poor immune cell infiltration. Targeting ZNF638 decreased H3K9 trimethylation, increased REs, and activated intracellular dsRNA signaling cascades. Furthermore, ZNF638 knockdown upregulated antiviral immune programs and significantly increased PD-L1 immune checkpoint expression in diverse GBM models. Importantly, targeting ZNF638 sensitized mice to ICI in syngeneic murine orthotopic models through innate IFN signaling. This response was recapitulated in recurrent GBM (rGBM) samples with radiographic responses to checkpoint inhibition with widely increased expression of dsRNA, PD-L1, and perivascular CD8 cell infiltration, suggesting that dsRNA signaling may mediate response to immunotherapy. Finally, low ZNF638 expression was a biomarker of clinical response to ICI and improved survival in patients with rGBM and patients with melanoma. Our findings suggest that ZNF638 could serve as a target to potentiate immunotherapy in gliomas.

Epigenetic regulation of reprogramming and pluripotency: insights from histone modifications and their implications for cancer stem cell therapies.

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.

CTCF-mediated insulation and chromatin environment modulate Car5b escape from X inactivation

BackgroundGenes that escape X-chromosome inactivation (XCI) in female somatic cells vary in number and levels of escape among mammalian species and tissues, potentially contributing to species- and tissue-specific sex differences. CTCF, a master chromatin conformation regulator, is enriched at escape regions and may play an important role in regulating escape, but the molecular mechanisms remain elusive.ResultsCTCF binding profiles and epigenetic features were systematically examined at escape genes (escapees) using mouse allelic systems with skewed XCI to distinguish the inactive X (Xi) and active X (Xa) chromosomes. We found that six constitutive and two facultative escapees are located inside 30-800 kb domains marked by convergent arrays of CTCF binding sites, consistent with the formation of chromatin loops. Facultative escapees show clear differences in CTCF binding depending on their XCI status in specific cell types/tissues. In addition, sets of strong and in some cases divergent CTCF binding sites located at the boundary between an escapee and its adjacent neighbors subject to XCI would also help insulate domains. Indeed, deletion but not inversion of a CTCF binding site at the boundary between the facultative escapee Car5b and its silent neighbor Siah1b results in a dramatic reduction of Car5b escape. This is associated with reduced CTCF and cohesin binding, which indicates loss of looping and insulation and is supported by 3C combined with Hi-C analysis. In addition, enrichment in the repressive mark H3K27me3 invades the Car5b domain in deleted cells, consistent with loss of expression from the Xi. In contrast, cells with an inversion of the CTCF binding site retain CTCF and cohesin binding, as well as looping, in line with persistence of escape. Interestingly, the levels of escape increase in cells with deletion of either Dxz4, which disrupts the Xi-specific compact 3D structure, or Firre, which results in lower H3K27me3 enrichment on the Xi, indicating that the structural and epigenetic features of the Xi constrain escape from XCI in wild type conditions.ConclusionsTaken together, our findings support the idea that escape from XCI in female somatic cells is modulated by both the topological insulation of domains via CTCF binding and the surrounding heterochromatin environment.

P53-mediated regulation of LINE1 retrotransposon-derived R-loops.

p53-mediated regulation of LINE1 retrotransposon-derived R-loops.

Argonaute CSR-1A promotes H3K9me3 maintenance to protect somatic development in offspring.

Parental stress can be encoded into altered epigenetic information to influence their offspring. Concurrently, it is vital for the preservation of a parent's epigenetic information, despite environmental challenges, to ensure accurate inheritance by the next generation. Nevertheless, the complexities of this process and the specific molecular mechanisms involved are not yet fully understood. Here we report that Argonaute CSR-1A potentiates the recovery of histone H3 lysine 9 trimethylation (H3K9me3) in spermatocyte to secure the developmental competence of male offspring. CSR-1A employs its repetitive RG motif to engage with putative histone 3 lysine 9 (H3K9) methyltransferases SET-25 and -32, and helps to restore repressive H3K9me3 chromatin marks following heat-stress, protecting the late development of somatic cells in the progeny. Finally, among the genes regulated by CSR-1A, we identified dim-1, at which decreased H3K9me3 persists in the progeny, and RNAi of dim-1 mitigates the somatic defects associated with csr-1a loss under stress. Thus, CSR-1A coordinates a paternal epigenetic program that shields development from the influences of the paternal environment. We speculate that, driven by both natural environmental stressors and the unique characteristics of spermatogenic chromatin, the emergence of multiple RG motif-featured and spermatogenesis-specific CSR-1A and small RNA serves as a protective strategy to safeguard against variability in the orchestration of inherited developmental programs from the paternal lineage.

Genome-wide CRISPR screening identifies PHF8 as an effective therapeutic target for KRAS- or BRAF-mutant colorectal cancers

BackgroundMutations in KRAS and BRAF genes are prevalent in colorectal cancer (CRC), which strikingly promote tumorigenesis and lead to poor response to a variety of treatments including immunotherapy by activating the MAPK/ERK pathway. Thus, there is an urgent need to discover effective therapeutic targets and strategies.MethodsCRISPR-Cas9 lentiviral knockout library was used to screen the suppressors of anti-PD1 immunotherapy. Bioinformatic analysis was used to analyze the correlation between PHF8 expression and immune indicators in CRC. In vitro and in vivo experiments were utilized to determine the effects of PHF8 on the immune indexes and malignant phenotypes of CRC cells. qRT-PCR, western blotting, immunohistochemical (IHC) staining, and chromatin immunoprecipitation (ChIP)-qPCR assays were used to determine the regulatory effects of PHF8 on PD-L1, KRAS, BRAF, and c-Myc and the regulatory effect c-Myc/miR-22-3p signaling axis on PHF8 expression in CRC cells.ResultsThis study identified histone lysine demethylase PHF8 as a negative regulator for the efficacy of anti-PD1 therapy and found that it was highly expressed in CRCs and strongly associated with poor patient survival. Functional studies showed that PHF8 played an oncogenic role in KRAS- or BRAF-mutant CRC cells, but not in wild-type ones. Mechanistically, PHF8 up-regulated the expression of PD-L1, KRAS, BRAF, and c-Myc by increasing the levels of transcriptional activation marks H3K4me3 and H3K27ac and decreasing the levels of transcriptional repression mark H3K9me2 within their promoter regions, promoting immune escape and tumor progression. Besides, our data also demonstrated that PHF8 was up-regulated by the c-Myc/miR-22-3p signaling axis to form a positive feedback loop. Targeting PHF8 substantially improved the efficacy of anti-PD1 therapy and inhibited the malignant phenotypes of KRAS- or BRAF-mutant CRC cells.ConclusionOur data demonstrate that PHF8 may be an effective therapeutic target for KRAS- or BRAF-mutant CRCs.

KDM6B preferentially promotes bone formation over resorption to facilitate postnatal bone mass accrual through CTHRC1-mediated PKCδ/MAPKs signaling.

Lysine demethylase 6B (KDM6B) plays a role in regulating osteoblast differentiation and fetal bone ossification. Nevertheless, its involvement in regulating postnatal bone homeostasis and bone mass accrual remains unclear. In this study, we generated mice lacking Kdm6b gene specifically in mesenchyme and osteoprogenitor cells using a conditional strategy. The adult mice of both mutant strains had decreased cancellous bone mass. The absence of Kdm6b in mesenchyme led to decreased numbers of osteoblasts and osteoclasts, increased marrow adipocytes, as well as repressed bone formation and resorption. Additionally, Kdm6b-deficient bone marrow stromal cells (BMSCs) displayed impaired osteogenic differentiation and exerted an inhibitory effect on osteoclastogenesis. RNA-seq combined with gene expression analysis uncovered downregulation of collagen triple helix repeat containing 1 (CTHRC1) and a lower receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) ratio in BMSCs of the mutant mice. Further mechanistic explorations demonstrated that KDM6B epigenetically upregulated CTHRC1 expression by removing the repressive H3K27me3 mark from its promoter, thereby triggering PKCδ/MAPKs signaling to facilitate osteoblast differentiation. CTHRC1 was able to mitigate the dysregulated osteogenic and adipogenic differentiation induced by Kdm6b deficiency. This study provides evidence that KDM6B regulates postnatal bone homeostasis through balancing osteoblast and osteoclast differentiation. Given its predominant promotion of osteoblastic bone formation over osteoclastic bone resorption, KDM6B tends to promote postnatal bone mass accrual.

De-novo DNA Methylation of Bivalent Promoters Induces Gene Activation through PRC2 Displacement.

Promoter DNA methylation is a key epigenetic mark, commonly associated with gene silencing. However, we noticed that a positive association between promoter DNA methylation and expression is surprisingly common in cancer. Here, we use hit-and-run CRISPR/dCas9 epigenome editing to evaluate how deposition of DNA methylation can regulate gene expression dependent on pre-existing chromatin environment. While the predominant effect of DNA methylation in non-bivalent promoters is gene repression, we show that in bivalent promoters this often leads to gene activation. We demonstrate that gain of DNA methylation leads to reduced MTF2 binding and eviction of H3K27me3, a repressive mark that guards bivalent genes against activation. Our cancer patient data analyses reveal that in cancer, this mechanism likely leads to activation of a large group of transcription factors regulating pluripotency, apoptosis, and senescence signalling. In conclusion, our study uncovers an activating role of DNA methylation in bivalent promoters, with broad implications for cancer and development.

Meioc-Piwil1 complexes regulate rRNA transcription for differentiation of spermatogonial stem cells.

Ribosome biogenesis is vital for sustaining stem cell properties, yet its regulatory mechanisms are obscure. Herein, we show unique properties of zebrafish meioc mutants in which spermatogonial stem cells (SSCs) do not differentiate or upregulate rRNAs. Meioc colocalized with Piwil1 in perinuclear germ granules, but Meioc depletion resulted in Piwil1 accumulation in nucleoli. Nucleolar Piwil1 interacted with 45S pre-rRNA. piwil1 +/- spermatogonia with reduced Piwil1 upregulated rRNAs, and piwil1 +/- ;meioc -/- spermatogonia recovered differentiation later than those in meioc -/- . Further, Piwil1 interacted with Setdb1 and HP1α, and meioc -/- spermatogonia exhibited high levels of H3K9me3 and methylated CpG in the 45S-rDNA region. These results indicate that zebrafish SSCs maintain low levels of rRNA transcription with repressive marks similar to Drosophila piRNA targets of RNA polymerase II, and that Meioc has a unique function on preventing localization of Piwil1 in nucleoli to upregulate rRNA transcripts and to promote SSC differentiation.

Multi-locus CRISPRi targeting with a single truncated guide RNA.

A critical goal in functional genomics is evaluating which non-coding elements contribute to gene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10 nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription factor binding at most sequence match target sites. This approach can be a valuable screening step for testing transcription factor binding motifs or other repeated genomic sequences and is easily implemented with existing tools.

Barriers Composed of tRNA Genes Can Complement the Benefits of a Ubiquitous Chromatin Opening Element to Enhance Transgene Expression.

Random integration of transgenes into host cell genomes often occurs in epigenetically unstable regions, leading to variable and unreliable transgene expression. To address this, biomanufacturing organizations frequently employ barrier elements, such as the widely-used ubiquitous chromatin opening element (UCOE). We have compared UCOE barrier activity against a barrier provided by tRNA genes. We demonstrate that the tRNA genes provide a more effective barrier than a UCOE in preventing transgene silencing in Chinese hamster ovary (CHO) cells. Nevertheless, the UCOE offers other benefits, increasing expression strongly, albeit transiently, and reducing production variability. Both the UCOE and tRNA genes counteract the repressive heterochromatin mark H3K9me3, but only the tRNA genes sustain euchromatic H3K27ac and recruitment of RNA polymerase II (Pol II) throughout long-term culture. A hybrid combining these distinct types of elements can provide benefits of both, enhancing expression in a more enduring manner. This synthetic hybrid offers potential for biomanufacturing applications.

Targeting the IKZF1/BCL-2 axis as a novel therapeutic strategy for treating acute T-cell lymphoblastic leukemia

ABSTRACT Objectives Acute T-cell lymphoblastic leukemia (T-ALL) is a severe hematologic malignancy with limited treatment options and poor long-term survival. This study explores the role of IKZF1 in regulating BCL-2 expression in T-ALL. Methods CUT&Tag and CUT&Run assays were employed to assess IKZF1 binding to the BCL-2 promoter. IKZF1 overexpression and knockdown experiments were performed in T-ALL cell lines. The effects of CX-4945 and venetoclax, alone and in combination, were evaluated in vitro and in vivo T-ALL models. Results CUT&Tag sequencing identified IKZF1 binding to the BCL-2 promoter, establishing it as a transcriptional repressor. Functional assays demonstrated that IKZF1 overexpression reduced BCL-2 mRNA levels and increased repressive histone marks at the BCL-2 promoter, while IKZF1 knockdown led to elevated BCL-2 expression. CX-4945, a CK2 inhibitor, could reduced BCL-2 levels in T-ALL cells. Notably, knockdown of IKZF1 partially rescued the CX-4945-induced repression of BCL-2. These results underscore the CK2-IKZF1 signaling axis as a key regulator of BCL-2 expression. In vitro, CX-4945 enhanced the cytotoxicity of venetoclax, with the combination showing significant synergistic effects and increased apoptosis in T-ALL cell lines. In vivo studies with cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models demonstrated that CX-4945 and venetoclax combined therapy provided superior therapeutic efficacy, reducing tumor burden and prolonging survival compared to single-agent treatments. Conclusions IKZF1 represses BCL-2 in T-ALL, and targeting the CK2-IKZF1 axis with CX-4945 and venetoclax offers a promising therapeutic strategy, showing enhanced efficacy and potential as a novel treatment approach for T-ALL.

Mineral Stress Drives Loss of Heterochromatin: An Early Harbinger of Vascular Inflammaging and Calcification.

Vascular calcification is a detrimental aging pathology markedly accelerated in patients with chronic kidney disease. PLA (prelamin A) is a biomarker of vascular smooth muscle cell aging that accelerates calcification however the mechanisms remain undefined. Vascular smooth muscle cells were transduced with PLA using an adenoviral vector and epigenetic modifications were monitored using immunofluorescence and targeted polymerase chain reaction array. Epigenetic findings were verified in vivo using immunohistochemistry in human vessels, in a mouse model of inducible prelamin A expression, and in a rat model of chronic kidney disease-induced calcification. Transcriptomic and chromatin immunoprecipitation followed by sequencing analyses were used to identify gene targets impacted by changes in the epigenetic landscape. Molecular tools and antibody arrays were used to monitor the effects of mineral dysregulation on heterochromatin, inflammation, aging, and calcification. Here, we report that depletion of the repressive heterochromatin marks, H3K9me3 (histone H3, lysine 9, trimethylation) and H3K27me3 (histone H3, lysine 27,trimethylation), is an early hallmark of vascular aging induced by both nuclear lamina dysfunction and dysregulated mineral metabolism, which act to modulate the expression of key epigenetic writers and erasers. Global analysis of H3K9me3 and H3K27me3 marks and pathway analysis revealed deregulation of insulin signaling and autophagy pathways as well as cross-talking DNA damage and NF-κB (nuclear factor κB) inflammatory pathways consistent with early activation of the senescence-associated secretory phenotype. Expression of PLA in vivo induced loss of heterochromatin and promoted inflammation and osteogenic differentiation which preceded aging indices, such as DNA damage and senescence. Vessels from children on dialysis and rats with chronic kidney disease showed prelamin A accumulation and accelerated loss of heterochromatin before the onset of calcification. Dysregulated mineral metabolism drives changes in the epigenetic landscape and nuclear lamina dysfunction that together promote early induction of inflammaging pathways priming the vasculature for downstream pathological change.

Marek's disease virus-encoded microRNA-M6-5p facilitates viral latent infection by targeting histone demethylase KDM2B.

Marek's disease virus (MDV), a highly contagious and oncogenic avian alphaherpesvirus, establishes a latent infection primarily in CD4+ T cells. Latent infections are necessary for both persistent lifelong MDV infection and viral tumorigenesis. MicroRNAs (miRNAs) play critical roles as post-transcriptional regulators of viral infections. However, the role of miRNAs in regulating MDV latency remains unclear. In this study, we found that an MDV-encoded miRNA, miR-M6-5p, inhibited viral lytic replication in vitro by functional screening and that infection with an MDV mutant lacking miR-M6-5p resulted in impaired MDV latency, proliferation, and tumor formation in vivo. Importantly, we identified lysine-specific demethylase 2b (KDM2B), an important epigenetic factor, as a target of miR-M6-5p. Furthermore, KDM2B knockdown increased the level of the transcriptionally repressive histone mark H3K27me3 on the key lytic gene pp38 promoter, accompanied by suppression of pp38 expression and reduced latent-to-lytic switch in MDV-latently infected cells, while treatment of cells with H3K27me3 inhibitors (GSK126 and Tazemetostat) markedly promoted the expression of pp38 and MDV reactivation from latency. Thus, miR-M6-5p facilitates MDV latency by epigenetically suppressing pp38 expression by targeting KDM2B. These findings unravel the mechanism by which a virus-encoded miRNA plays a critical role in the regulation of latent MDV infection.IMPORTANCESimilar to other herpesviruses, MDV can establish a lifelong latent infection in the host. During the latency, MDV integrates its genome into the host genome to maintain the viral genome, which is considered a prerequisite for tumor formation. Reactivation of the latent viral genome in response to intracellular and extracellular stimuli re-enters lytic replication, resulting in pathological recurrence and/or viral shedding. However, the regulatory mechanisms underlying MDV latency remain poorly understood. In the present study, we investigated the role of virus-encoded miRNAs in MDV latency. We found that miR-M6-5p facilitated MDV latency, proliferation, and tumor formation in vivo. Mechanistically, miR-M6-5p epigenetically suppressed the expression of the viral lytic gene pp38 by directly targeting the histone demethylase KDM2B. These findings will advance our understanding of the role of virus-encoded miRNA in the regulation of viral latency and will help guide the development of novel strategies for the effective control of MDV.

Regulation of epigenetics and chromosome structure by human ORC2.

The six subunit Origin Recognition Complex (ORC) is a DNA replication initiator that also promotes heterochromatinization in some species. A multi-omics study in a human cell line with mutations in three subunits of ORC, reveals that the subunits bind to DNA independent of each other rather than as part of a common six-subunit ORC. While DNA-bound ORC2 was seen to compact chromatin and attract repressive histone marks, the activation of chromatin and protection from repressive marks was seen at a large number of sites. The epigenetic changes regulate hundreds of genes, including some epigenetic regulators, adding an indirect mechanism by which ORC2 regulates epigenetics without local binding. DNA-bound ORC2 also prevents the acquisition of CTCF at focal sites in the genome to regulate chromatin loops. Thus, individual ORC subunits are major regulators, in both directions, of epigenetics, gene expression and chromosome structure, independent of the role of ORC in replication.

Dual ASXL1 and CSF3R mutations drive myeloid biased stem cell expansion and enhance neutrophil differentiation.

Dual ASXL1 and CSF3R mutations drive myeloid biased stem cell expansion and enhance neutrophil differentiation.

Structural basis for the inhibition of PRC2 by active transcription histone posttranslational modifications

Polycomb repressive complex 2 (PRC2) trimethylates histone H3 on K27 (H3K27me3) leading to gene silencing that is essential for embryonic development and maintenance of cell identity. PRC2 is regulated by protein cofactors and their crosstalk with histone modifications. Trimethylated histone H3 on K4 (H3K4me3) and K36 (H3K36me3) localize to sites of active transcription and inhibit PRC2 activity through unknown mechanisms. Using cryo-electron microscopy, we reveal that histone H3 tails containing H3K36me3 engage poorly with PRC2 and preclude its effective interaction with chromatin, while H3K4me3 binds to the allosteric site in the EED subunit, acting as an antagonist that competes with activators required for spreading of the H3K27me3 repressive mark. Thus, the location of the H3K4me3 and H3K36me3 modifications along the H3 tail allows them to target two requirements for efficient trimethylation of H3K27 by PRC2. We further show that the JARID2 cofactor modulates PRC2 activity in the presence of these histone modifications.

KDM6A facilitates Xist upregulation at the onset of X inactivation

BackgroundX chromosome inactivation (XCI) is a female-specific process in which one X chromosome is silenced to balance X-linked gene expression between the sexes. XCI is initiated in early development by upregulation of the lncRNA Xist on the future inactive X (Xi). A subset of X-linked genes escape silencing and thus have higher expression in females, suggesting female-specific functions. One of these genes is the highly conserved gene Kdm6a, which encodes a histone demethylase that removes methyl groups at H3K27 to facilitate gene expression. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in development and cancer.MethodsKdm6a was knocked out (KO) using CRISPR/Cas9 gene editing in hybrid female mouse embryonic stem (ES) cells derived either from a 129 × Mus castaneus (cast) cross or a BL6 x cast cross. In one of the lines a transcriptional stop signal inserted in Tsix results in completely skewed X silencing upon differentiation. The effects of both homozygous and heterozygous Kdm6a KO on Xist expression during the onset of XCI were measured by RT-PCR and RNA-FISH. Changes in gene expression and in H3K27me3 enrichment were investigated using allele-specific RNA-seq and Cut&Run, respectively. KDM6A binding to the Xist gene was characterized by Cut&Run.ResultsWe observed impaired upregulation of Xist and reduced coating of the Xi during early stages of differentiation in Kdm6a KO cells, both homozygous and heterozygous, suggesting a threshold effect of KDM6A. This was associated with aberrant overexpression of genes from the Xi after differentiation, indicating loss of X inactivation potency. Consistent with KDM6A having a direct role in Xist regulation, we found that the histone demethylase binds to the Xist promoter and KO cells show an increase in H3K27me3 at Xist, consistent with reduced expression.ConclusionsThese results reveal a novel female-specific role for the X-linked histone demethylase, KDM6A in the initiation of XCI through histone demethylase-dependent activation of Xist during early differentiation.Plain language summaryX chromosome inactivation is a female-specific mechanism that evolved to balance sex-linked gene dosage between females (XX) and males (XY) by silencing one X chromosome in females. X inactivation begins with the upregulation of the long noncoding RNA Xist on the future inactive X chromosome. While most genes become silenced on the inactive X chromosome some genes escape inactivation and thus have higher expression in females compared to males, suggesting that escape genes may have female-specific functions. One such gene encodes the histone demethylase KDM6A which function is to turn on gene expression by removing repressive histone modifications. In this study, we investigated the role of KDM6A in the regulation of Xist expression during the onset of X inactivation. We found that KDM6A binds to the Xist gene to remove repressive histone marks and facilitate its expression in early development. Indeed, depletion of KDM6A prevents upregulation of Xist due to abnormal persistence of repressive histone modifications. In turn, this results in aberrant overexpression of genes from the inactive X chromosome. Our findings point to a novel mechanism of Xist regulation during the initiation of X inactivation, which may lead to new avenues of treatment to alleviate congenital disorders such as Kabuki syndrome and sex-biased immune disorders where X-linked gene dosage is perturbed.

Suppression of the H3K27me3 demethylase disrupts diapause formation in mosquito Culex pipiens.

Suppression of the H3K27me3 demethylase disrupts diapause formation in mosquito Culex pipiens.