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
Dai, Xiangyan; Pradhan, Ajay; Liu, Jiao; Liu, Ruolan; Zhai, Gang; Zhou, Linyan; Dai, Jiyan; Shao, Feng; Yuan, Zhiyong; Wang, Zhijian; et al(
, Biology of Sex Differences)
AbstractBackground
Sexually dimorphic mating behaviors differ between sexes and involve gonadal hormones and possibly sexually dimorphic gene expression in the brain. However, the associations among the brain, gonad, and sexual behavior in teleosts are still unclear. Here, we utilized germ cells-freetdrd12knockout (KO) zebrafish, and steroid synthesis enzymecyp17a1-deficient zebrafish to investigate the differences and interplays in the brain–gonad–behavior axis, and the molecular control of brain dimorphism and male mating behaviors.
Methods
Tdrd12+/−;cyp17a1+/−double heterozygous parents were crossed to obtaintdrd12−/−;cyp17a1+/+(tdrd12 KO),tdrd12+/+;cyp17a1−/−(cyp17a1 KO), andtdrd12−/−;cyp17a1−/−(double KO) homozygous progenies. Comparative analysis of mating behaviors were evaluated using Viewpoint zebrafish tracking software and sexual traits were thoroughly characterized based on anatomical and histological experiments in these KOs and wild types. The steroid hormone levels (testosterone, 11-ketotestosterone and 17β-estradiol) in the brains, gonads, and serum were measured using ELISA kits. To achieve a higher resolution view of the differences in region-specific expression patterns of the brain, the brains of these KOs, and control male and female fish were dissected into three regions: the forebrain, midbrain, and hindbrain for transcriptomic analysis.
Results
Qualitative analysis of mating behaviors demonstrated thattdrd12−/−fish behaved in the same manner as wild-type males to trigger oviposition behavior, whilecyp17a1−/−and double knockout (KO) fish did not exhibit these behaviors. Based on the observation of sex characteristics, mating behaviors and hormone levels in these mutants, we found that the maintenance of secondary sex characteristics and male mating behavior did not depend on the presence of germ cells; rather, they depended mainly on the 11-ketotestosterone and testosterone levels secreted into the brain–gonad regulatory axis. RNA-seq analysis of different brain regions revealed that the brain transcript profile oftdrd12−/−fish was similar to that of wild-type males, especially in the forebrain and midbrain. However, the brain transcript profiles ofcyp17a1−/−and double KO fish were distinct from those of wild-type males and were partially biased towards the expression pattern of the female brain. Our results revealed important candidate genes and signaling pathways, such as synaptic signaling/neurotransmission, MAPK signaling, and steroid hormone pathways, that shape brain dimorphism and modulate male mating behavior in zebrafish.
Conclusions
Our results provide comprehensive analyses and new insights regarding the endogenous interactions in the brain–gonad–behavior axis. Moreover, this study revealed the crucial candidate genes and neural signaling pathways of different brain regions that are involved in modulating brain dimorphism and male mating behavior in zebrafish, which would significantly light up the understanding the neuroendocrine and molecular mechanisms modulating brain dimorphism and male mating behavior in zebrafish and other teleost fish.
Abstract Sweet orange originated from the introgressive hybridizations of pummelo and mandarin resulting in a highly heterozygous genome. How alleles from the two species cooperate in shaping sweet orange phenotypes under distinct circumstances is unknown. Here, we assembled a chromosome-level phased diploid Valencia sweet orange (DVS) genome with over 99.999% base accuracy and 99.2% gene annotation BUSCO completeness. DVS enables allele-level studies for sweet orange and other hybrids between pummelo and mandarin. We first configured an allele-aware transcriptomic profiling pipeline and applied it to 740 sweet orange transcriptomes. On average, 32.5% of genes have a significantly biased allelic expression in the transcriptomes. Different cultivars, transgenic lineages, tissues, development stages, and disease status all impacted allelic expressions and resulted in diversified allelic expression patterns in sweet orange, but particularly citrus Huanglongbing (HLB) shifted the allelic expression of hundreds of genes in leaves and calyx abscission zones. In addition, we detected allelic structural mutations in an HLB-tolerant mutant (T19) and a more sensitive mutant (T78) through long-read sequencing. The irradiation-induced structural mutations mostly involved double-strand breaks, while most spontaneous structural mutations were transposon insertions. In the mutants, most genes with significant allelic expression ratio alterations (≥1.5-fold) were directly affected by those structural mutations. In T19, alleles located at a translocated segment terminal were upregulated, including CsDnaJ, CsHSP17.4B, and CsCEBPZ. Their upregulation is inferred to keep phloem protein homeostasis under the stress from HLB and enable subsequent stress responses observed in T19. DVS will advance allelic level studies in citrus.
Adhikari, Kiran; Son, Jae Hak; Rensink, Anna H.; Jaweria, Jaweria; Bopp, Daniel; Beukeboom, Leo W.; Meisel, Richard P.(
, Molecular Ecology)
Abstract
Sex determination, the developmental process by which sexually dimorphic phenotypes are established, evolves fast. Evolutionary turnover in a sex determination pathway may occur via selection on alleles that are genetically linked to a new master sex determining locus on a newly formed proto‐sex chromosome. Species with polygenic sex determination, in which master regulatory genes are found on multiple different proto‐sex chromosomes, are informative models to study the evolution of sex determination and sex chromosomes. House flies are such a model system, with male determining loci possible on all six chromosomes and a female‐determiner on one of the chromosomes as well. The two most common male‐determining proto‐Y chromosomes form latitudinal clines on multiple continents, suggesting that temperature variation is an important selection pressure responsible for maintaining polygenic sex determination in this species. Temperature‐dependent fitness effects could be manifested through temperature‐dependent gene expression differences across proto‐Y chromosome genotypes. These gene expression differences may be the result ofcisregulatory variants that affect the expression of genes on the proto‐sex chromosomes, ortranseffects of the proto‐Y chromosomes on genes elswhere in the genome. We used RNA‐seq to identify genes whose expression depends on proto‐Y chromosome genotype and temperature in adult male house flies. We found no evidence for ecologically meaningful temperature‐dependent expression differences of sex determining genes between male genotypes, but we were probably not sampling an appropriate developmental time‐point to identify such effects. In contrast, we identified many other genes whose expression depends on the interaction between proto‐Y chromosome genotype and temperature, including genes that encode proteins involved in reproduction, metabolism, lifespan, stress response, and immunity. Notably, genes with genotype‐by‐temperature interactions on expression were not enriched on the proto‐sex chromosomes. Moreover, there was no evidence that temperature‐dependent expression is driven by chromosome‐widecis‐regulatory divergence between the proto‐Y and proto‐X alleles. Therefore, if temperature‐dependent gene expression is responsible for differences in phenotypes and fitness of proto‐Y genotypes across house fly populations, these effects are driven by a small number of temperature‐dependent alleles on the proto‐Y chromosomes that may havetranseffects on the expression of genes on other chromosomes.
Richardson, Victorya; Engel, Nora; Kulathinal, Rob J.(
, Biology of Sex Differences)
AbstractBackground
Mammalian gonadal sex is determined by the presence or absence of a Y chromosome and the subsequent production of sex hormones contributes to secondary sexual differentiation. However, sex chromosome-linked genes encoding dosage-sensitive transcription and epigenetic factors are expressed well before gonad formation and have the potential to establish sex-biased expression that persists beyond the appearance of gonadal hormones. Here, we apply a comparative bioinformatics analysis on a pair of published single-cell datasets from mouse and human during very early embryogenesis—from two-cell to pre-implantation stages—to characterize sex-specific signals and to assess the degree of conservation among early acting sex-specific genes and pathways.
Results
Clustering and regression analyses of gene expression across samples reveal that sex initially plays a significant role in overall gene expression patterns at the earliest stages of embryogenesis which potentially may be the byproduct of signals from male and female gametes during fertilization. Although these transcriptional sex effects rapidly diminish, sex-biased genes appear to form sex-specific protein–protein interaction networks across pre-implantation stages in both mammals providing evidence that sex-biased expression of epigenetic enzymes may establish sex-specific patterns that persist beyond pre-implantation. Non-negative matrix factorization (NMF) on male and female transcriptomes generated clusters of genes with similar expression patterns across sex and developmental stages, including post-fertilization, epigenetic, and pre-implantation ontologies conserved between mouse and human. While the fraction of sex-differentially expressed genes (sexDEGs) in early embryonic stages is similar and functional ontologies are conserved, the genes involved are generally different in mouse and human.
Conclusions
This comparative study uncovers much earlier than expected sex-specific signals in mouse and human embryos that pre-date hormonal signaling from the gonads. These early signals are diverged with respect to orthologs yet conserved in terms of function with important implications in the use of genetic models for sex-specific disease.
Sex chromosome evolution results in the disparity in gene content between heterogametic sex chromosomes and creates the need for dosage compensation to counteract the effects of gene dose imbalance of sex chromosomes in males and females. It is not known at which stage of sex chromosome evolution dosage compensation would evolve. We used global gene expression profiling in male and female papayas to assess gene expression patterns of sex-linked genes on the papaya sex chromosomes. By analyzing expression ratios of sex-linked genes to autosomal genes and sex-linked genes in males relative to females, our results showed that dosage compensation was regulated on a gene-by-gene level rather than whole sex-linked region in papaya. Seven genes on the papaya X chromosome exhibited dosage compensation. We further compared gene expression ratios in the two evolutionary strata. Y alleles in the older evolutionary stratum showed reduced expression compared to X alleles, while Y alleles in the younger evolutionary stratum showed elevated expression compared to X alleles. Reduced expression of Y alleles in the older evolutionary stratum might be caused by accumulation of deleterious mutations in regulatory regions or transposable element-mediated methylation spreading. Most X-hemizygous genes exhibited either no or very low expression, suggesting that gene silencing might play a role in maintaining transcriptional balance between females and males.
Ma, Wenxiu, Fang, He, Pease, Nicolas, Filippova, Galina N., Disteche, Christine M., and Berletch, Joel B. Sex-biased and parental allele-specific gene regulation by KDM6A. Retrieved from https://par.nsf.gov/biblio/10419586. Biology of Sex Differences 13.1 Web. doi:10.1186/s13293-022-00452-0.
Ma, Wenxiu, Fang, He, Pease, Nicolas, Filippova, Galina N., Disteche, Christine M., & Berletch, Joel B. Sex-biased and parental allele-specific gene regulation by KDM6A. Biology of Sex Differences, 13 (1). Retrieved from https://par.nsf.gov/biblio/10419586. https://doi.org/10.1186/s13293-022-00452-0
Ma, Wenxiu, Fang, He, Pease, Nicolas, Filippova, Galina N., Disteche, Christine M., and Berletch, Joel B.
"Sex-biased and parental allele-specific gene regulation by KDM6A". Biology of Sex Differences 13 (1). Country unknown/Code not available. https://doi.org/10.1186/s13293-022-00452-0.https://par.nsf.gov/biblio/10419586.
@article{osti_10419586,
place = {Country unknown/Code not available},
title = {Sex-biased and parental allele-specific gene regulation by KDM6A},
url = {https://par.nsf.gov/biblio/10419586},
DOI = {10.1186/s13293-022-00452-0},
abstractNote = {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.},
journal = {Biology of Sex Differences},
volume = {13},
number = {1},
author = {Ma, Wenxiu and Fang, He and Pease, Nicolas and Filippova, Galina N. and Disteche, Christine M. and Berletch, Joel B.},
}
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