Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Sex-biased and parental allele-specific gene regulation by KDM6A
Abstract Background KDM6A is a demethylase encoded by a gene with female-biased expression due to escape from X inactivation. Its main role is to facilitate gene expression through removal of the repressive H3K27me3 mark, with evidence of some additional histone demethylase-independent functions. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in cancer. Methods Kdm6a was knocked out using CRISPR/Cas9 gene editing in F1 male and female mouse embryonic stem cells (ES) derived from reciprocal crosses between C57BL6 x Mus castaneus . Diploid and allelic RNA-seq analyses were done to compare gene expression between wild-type and Kdm6a knockout (KO) clones. The effects of Kdm6a KO on sex-biased gene expression were investigated by comparing gene expression between male and female ES cells. Changes in H3K27me3 enrichment and chromatin accessibility at promoter regions of genes with expression changes were characterized by ChIP-seq and ATAC-seq followed by diploid and allelic analyses. Results We report that Kdm6a KO in male and female embryonic stem (ES) cells derived from F1 hybrid mice cause extensive gene dysregulation, disruption of sex biases, and specific parental allele effects. Among the dysregulated genes are candidate genes that may explain abnormal developmental features of Kabuki syndrome caused by KDM6A mutations in human. Strikingly, Kdm6a knockouts result in a decrease in sex-biased expression and in preferential downregulation of the maternal alleles of a number of genes. Most promoters of dysregulated genes show concordant epigenetic changes including gain of H3K27me3 and loss of chromatin accessibility, but there was less concordance when considering allelic changes. Conclusions Our study reveals new sex-related roles of KDM6A in the regulation of developmental genes, the maintenance of sex-biased gene expression, and the differential expression of parental alleles.  more » « less
Award ID(s):
1751317
PAR ID:
10419586
Author(s) / Creator(s):
; ; ; ; ;
Date Published:
Journal Name:
Biology of Sex Differences
Volume:
13
Issue:
1
ISSN:
2042-6410
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ; ; ; et al (, Biology of Sex Differences)
    Abstract 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 lncRNAXiston 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 geneKdm6a, which encodes a histone demethylase that removes methyl groups at H3K27 to facilitate gene expression.KDM6Amutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in development and cancer. MethodsKdm6awas 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 xcastcross. In one of the lines a transcriptional stop signal inserted inTsixresults in completely skewed X silencing upon differentiation. The effects of both homozygous and heterozygousKdm6aKO onXistexpression 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 theXistgene was characterized by Cut&Run. ResultsWe observed impaired upregulation ofXistand reduced coating of the Xi during early stages of differentiation inKdm6aKO 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 inXistregulation, we found that the histone demethylase binds to theXistpromoter and KO cells show an increase in H3K27me3 atXist, 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 ofXistduring 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 RNAXiston 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 ofXistexpression during the onset of X inactivation. We found that KDM6A binds to theXistgene to remove repressive histone marks and facilitate its expression in early development. Indeed, depletion of KDM6A prevents upregulation ofXistdue 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 ofXistregulation 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. 
    more » « less
  2. ; ; ; ; ; ; ; (, bioRxiv)
    Abstract BackgroundMorphologic sex differences between males and females typically emerge after the primordial germ cell migration and gonad formation, although sex is determined at fertilization based on chromosome composition. A key debated sexual difference is the embryonic developmental rate, within vitroproduced male embryos often developing faster. However, the molecular mechanisms driving early embryonic sex differences remain unclear. ResultsTo investigate the transcriptional sex difference during early development,in vitroproduced bovine blastocysts were collected and sexed by PCR. A significant male-biased development was observed in expanded blastocysts. Ultra-low input RNA-seq analysis identified 837 DEGs, with 231 upregulated and 606 downregulated in males. Functional enrichment analysis revealed male-biased DEGs were associated with metabolic regulation, whereas female-biased DEGs were related to female gonad development, sex differentiation, inflammatory pathways, and TGF-beta signaling. Comparing X chromosome and autosome expression ratio, we found that female-biased DEGs contributed to the higher X-linked gene dosage, a phenomenon not observed in male embryos. Moreover, we identified the sex-biased transcription factors and RNA-bind proteins, including pluripotent factors such asSOX21andPRDM14, and splicing factorsFMR1andHNRNPH2. Additionally, we revealed 1,555 significantly sex-biased differential alternative splicing (AS), predominantly skipped exons, mapped to 906 genes, with 59 overlapping with DEGs enriched in metabolic and autophagy pathways. By incorporating novel isoforms from long reads sequencing, we identified 1,151 sex-biased differentially expressed isoforms (DEIs) associated with 1,017 genes. Functional analysis showed that female-biased DEIs were involved in the negative regulation of transcriptional activity, while male-biased DEIs were related to energy metabolism. Furthermore, we identified sex-biased differential exon usage inDENND1B, DIS3L2, DOCK11, IL1RAPL2,andZRSR2Y,indicating their sex-specific regulation in early embryo development. ConclusionThis study provided a comprehensive analysis of transcriptome differences between male and female bovine blastocysts, integrating sex-biased gene expression, alternative splicing, and isoform dynamics. Our findings indicate that enriched metabolism processes in male embryos may contribute to the faster developmental pace, providing insights into sex-specific regulatory mechanisms during early embryogenesis. Plain English summaryMale and female early embryos develop at different speeds, with male embryos often developing faster than female embryos. However, the reasons behind these early differences remain unclear. In this study, we examined gene activity in bovine embryos to uncover the biological factors regulating these early sex differences. We collected in vitro-produced bovine blastocysts, examined their sex, and confirmed that male embryos develop faster. By analyzing global gene activity, including alternative splicing, which allows one gene to code for multiple RNA isoforms and proteins, we found distinct gene expression profiles between male and female embryos. Male embryos showed higher activity in genes related to metabolism and cellular functions, while female embryos had increased activity in genes associated with female-specific gonad development and gene expression regulation. We also examined differences in how genes on the X chromosome were expressed. Female embryos had higher X-linked gene expression, which may contribute to sex-specific developmental regulation. Additionally, we identified sex-specific transcription factors and RNA-binding proteins that regulate early embryo development, some of which are known to control pluripotency and gene splicing. Overall, our study provides new insights into how gene activity shapes early sex differences, suggesting that enhanced metabolism in male embryos may be a key driver of their faster developmental rate. HighlightsMale embryos develop faster due to increased gene expression in metabolism pathwaysFemale embryos exhibit higher X-linked gene expression, suggesting X-dosage compensation plays a role in early developmentSex-biased alternative splicing events contribute to embryonic metabolism, autophagy, and transcriptional regulation in embryosSex-biased isoform diversity contributes to distinct developmental regulation in male and female embryosKey pluripotency factors (SOX21, PRDM14) and splicing regulators (FMR1, HNRNPH2) drive sex-specific gene expression 
    more » « less
  3. ; ; ; ; ; ; ; ; ; ; et al (, Molecular Biology and Evolution)
    Wittkopp, Patricia (Ed.)
    Abstract In Drosophila melanogaster and D. simulans head tissue, 60% of orthologous genes show evidence of sex-biased expression in at least one species. Of these, ∼39% (2,192) are conserved in direction. We hypothesize enrichment of open chromatin in the sex where we see expression bias and closed chromatin in the opposite sex. Male-biased orthologs are significantly enriched for H3K4me3 marks in males of both species (∼89% of male-biased orthologs vs. ∼76% of unbiased orthologs). Similarly, female-biased orthologs are significantly enriched for H3K4me3 marks in females of both species (∼90% of female-biased orthologs vs. ∼73% of unbiased orthologs). The sex-bias ratio in female-biased orthologs was similar in magnitude between the two species, regardless of the closed chromatin (H3K27me2me3) marks in males. However, in male-biased orthologs, the presence of H3K27me2me3 in both species significantly reduced the correlation between D. melanogaster sex-bias ratio and the D. simulans sex-bias ratio. Male-biased orthologs are enriched for evidence of positive selection in the D. melanogaster group. There are more male-biased genes than female-biased genes in both species. For orthologs with gains/losses of sex-bias between the two species, there is an excess of male-bias compared to female-bias, but there is no consistent pattern in the relationship between H3K4me3 or H3K27me2me3 chromatin marks and expression. These data suggest chromatin state is a component of the maintenance of sex-biased expression and divergence of sex-bias between species is reflected in the complexity of the chromatin status. 
    more » « less
  4. ; ; ; ; ; ; ; ; ; ; et al (, Cell & Bioscience)
    Abstract BackgroundMorphologic sex differences between males and females typically emerge after the primordial germ cell migration and gonad formation, although sex is determined at fertilization based on chromosome composition. A key debated sexual difference is the embryonic developmental rate, with in vitro produced male embryos often developing faster. However, the molecular mechanisms driving early embryonic sex differences remain unclear. ResultsTo investigate the transcriptional sex difference during early development, in vitro produced bovine blastocysts were collected and sexed by PCR. A significant male-biased development is consistently observed in expanded blastocysts. Ultra-low input RNA-seq analysis identified 837 DEGs, 1555 significantly sex-biased differential alternative splicing (AS), and 1151 differentially expressed isoforms (DEIs). Among all of the DEGs, there were 231 upregulated and 606 downregulated in males. Functional enrichment analysis revealed male-biased DEGs were associated with metabolic regulation, whereas female-biased DEGs were related to female gonad development, sex differentiation, inflammatory pathways, and TGF-beta signaling. Comparing X chromosome and autosome expression ratio, we found that female-biased DEGs contributed to the higher X-linked gene dosage, a phenomenon not observed in male embryos. Moreover, we identified the sex-biased transcription factors and RNA-bind proteins, including pluripotent factors such asSOX21andPRDM14, and splicing factorsFMR1andHNRNPH2. Additionally, we revealed that the significantly sex-biased differential AS were predominantly skipped exons, and they could be mapped to 906 genes, with 59 overlapping with DEGs enriched in metabolic and autophagy pathways. By incorporating novel isoforms from long reads sequencing, the sex-biased DEIs were associated with 1017 genes. Functional analysis showed that female-biased DEIs were involved in the negative regulation of transcriptional activity, while male-biased DEIs were related to energy metabolism. Furthermore, we identified sex-biased differential exon usage inDENND1B, DIS3L2, DOCK11, IL1RAPL2,andZRSR2Y,indicating their sex-specific regulation in early embryo development. ConclusionThis study provided a comprehensive analysis of transcriptome differences between male and female bovine blastocysts, integrating sex-biased gene expression, alternative splicing, and isoform dynamics. Our findings indicate that enriched metabolism processes in male embryos may contribute to the faster developmental pace, providing insights into sex-specific regulatory mechanisms during early embryogenesis. 
    more » « less
  5. ; ; ; (, Frontiers in Physiology)
    Sex differences in gene expression tend to increase with age across a variety of species, often coincident with the development of sexual dimorphism and maturational changes in hormone levels. However, because most transcriptome-wide characterizations of sexual divergence are framed as comparisons of sex-biased gene expression across ages, it can be difficult to determine the extent to which age-biased gene expression within each sex contributes to the emergence of sex-biased gene expression. Using RNAseq in the liver of the sexually dimorphic brown anole lizard ( Anolis sagrei ), we found that a pronounced increase in sex-biased gene expression with age was associated with a much greater degree of age-biased gene expression in males than in females. This pattern suggests that developmental changes in males, such as maturational increases in circulating testosterone, contribute disproportionately to the ontogenetic emergence of sex-biased gene expression. To test this hypothesis, we used four different experimental contrasts to independently characterize sets of genes whose expression differed as a function of castration and/or treatment with exogenous testosterone. We found that genes that were significantly male-biased in expression or upregulated as males matured tended to be upregulated by testosterone, whereas genes that were female-biased or downregulated as males matured tended to be downregulated by testosterone. Moreover, the first two principal components describing multivariate gene expression indicated that exogenous testosterone reversed many of the feminizing effects of castration on the liver transcriptome of maturing males. Collectively, our results suggest that developmental changes that occur in males contribute disproportionately to the emergence of sex-biased gene expression in the Anolis liver, and that many of these changes are orchestrated by androgens such as testosterone. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Text Encoded Conversion
  • XML
  • Save / Share this Record
  • Send to Email