Exploring XIST Silencing and Repressor Factors on the X Chromosome in Early Human and Bovine Embryos

Research Background

X chromosome inactivation is a dosage compensation mechanism in mammals, aimed at balancing the expression of X-linked genes between females and males. The XIST gene locus can trigger transcriptional silencing of the additional X chromosome, making it the inactive X. However, how to protect one active X chromosome in each cell from inactivation by its own XIST gene locus remains not fully elucidated in all mammals. Previous studies on autosomal repeats suggest that genes that repress XIST on the active X may exist on the short arm of human chromosome 19.

To verify this hypothesis, researchers analyzed the transcription timing of candidate genes in human and bovine embryos derived from single-cell RNA sequencing data. The results confirmed that XIST is expressed from the future active X in both sexes, and the XIST locus is repressed in both sexes during the upregulation of pluripotency factors, i.e., between the 4-8 cell and morula stages in human and bovine embryos. These data support the roles of DNMT1, UHRF1, SAFB, and SAFB2 in XIST repression and exclude XACT and other 19p candidate genes, providing transcription timing for some genes never sequenced before in early human or bovine embryos.

Research Source

This study was conducted by Melis A. Aksit, Bo Yu, Bernard A. J. Roelen, and Barbara R. Migeon, affiliated with institutions including Johns Hopkins University and Utrecht University. The research findings were published in the European Journal of Human Genetics in 2022.

Research Process

The research team adopted the following steps to explore the mechanism of XIST silencing on the active X chromosome: a) Research subjects included human and bovine early embryos at different developmental stages. b) Single-cell RNA sequencing data analysis was performed, as well as quantitative RT-PCR (qRT-PCR) analysis of bovine embryos. c) XIST expression patterns and pluripotency factors (NANOG, POU5F1, and SOX2) expression were analyzed to explore the period of XIST repression. d) In vitro developed bovine embryo samples were used in the laboratory, and sex-specific embryo analysis materials were obtained by fertilization with X or Y sorted sperm.

Research Results

The study identified several candidate genes potentially involved in XIST repression, including DNMT1, UHRF1, SAFB/SAFB2, HNRNPM, and UBL5. Among them, DNMT1 and UHRF1 were expressed earliest and at the highest levels among the candidate genes, suggesting they might be important XIST repressors. Furthermore, the study verified similar XIST expression patterns in bovine embryos as in human embryos, strengthening the persuasiveness of these genes as key XIST repressors.

Research Conclusions

The study suggests that candidate genes such as DNMT1, UHRF1, SAFB, and SAFB2 may work together during early embryo development, specifically between the 4-8 cell and morula stages, to repress XIST on the future active X during the initial activation of pluripotency. This result provides important clues for future functional validation of candidate genes, further research on mammalian X chromosome inactivation mechanisms, and potential medical applications.

Research Highlights

This study uncovers the mechanism that silences the XIST gene on the future active X chromosome, which helps explain how XIST on the X chromosome balances expression between individuals. At the same time, this research opens up a new theoretical perspective, namely through key regulatory factors in selective activation or silencing of X chromosomes during early embryo development, thereby providing potential therapeutic methods for treating sex chromosome abnormalities.