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Silencing in order to survive

One of the copies of the X chromosome is inactivated in females during the early days of embryonic development

CAVALLINI JAMES/BSIP/ALAMY/LATINSTOCK The X and Y chromosomes, which are found in male cells, and the two copies of the X chromosome, which are typical of female cellsCAVALLINI JAMES/BSIP/ALAMY/LATINSTOCK

Five or six days after the sperm fertilizes the egg, a genetic phenomenon occurs to ensure that healthy female embryos develop: one of the two copies of the X chromosome is silenced. Although this process is important to the viability of female embryos, little is known about when this process begins, especially in human beings. In a study published in September in the journal Scientific Reports, the geneticists Lygia da Veiga Pereira and Maria Vibranovski, both from the Institute of Biosciences at the University of São Paulo (IB-USP), offered a new explanation about the beginning of this phenomenon. By analyzing RNA sequences in cells from human embryos available in a public database, they found that inactivation of the X chromosome occurs in the first days after fertilization, earlier than previously thought.

Human cells have 46 chromosomes, each of which contains 22 pairs of somatic chromosomes, which are the same in men and women, and one pair of sex chromosomes: females have two X chromosomes, and males have one X and one Y. Because they have a double dose of the genes present on the X chromosome, females could produce twice the proteins related to these genes than men. But this does not happen, because during embryonic development one of the two X chromosomes is silenced, preventing these genes from being overexpressed. “Inactivation of one copy of the X chromosome is an important epigenetic mechanism responsible for calibrating the activity of genes linked to these DNA sequences in women,” explains Pereira.

Over the past two decades, many hypotheses have been presented in an attempt to explain this phenomenon, but no conclusion has been reached on how and when it occurs. The most recent explanation was proposed in March 2016 by a group of Swedish researchers in a study published in the journal Cell. Unlike nearly all previous hypotheses, this group dismissed the existence of a mechanism which inactivates the X chromosome early on in embryonic development. They believed that genetic activity was aligned by reducing the levels of genetic expression in the two X chromosomes in female embryos.

The first clues about the process of inactivating the X chromosome were identified in the 1940s by the Canadian physician Murray Llewellyn Barr (1908–1995), who observed a small cluster of DNA in women along the inner face of the cell nucleus membrane which does not unfold during mitosis. In the 1960s, the work of the English geneticist Mary Lyon (1925–2014) showed that this structure was an inactivated X chromosome, and that this characteristic was unique to female mammalian cells, ensuring proper development of the embryo. In the following years, this process was seen to occur differently in other organisms; in female worms, gene expression from the two X chromosomes was reduced by half, while male flies doubled the expression of genes from their single X chromosome.

In mammals, inactivation of the X chromosome has mainly been studied in mice embryo cells, where this process occurs as soon as the blastocyst implants into the uterine wall and cells begin to differentiate. Today it is known that this inactivation can affect both the paternal and maternal X chromosomes, and that once the inactive X chromosome is defined its genes are no longer expressed, which also applies in the daughter cells. It had previously been assumed that the process in humans would be the same as that observed in mice. But in recent years, the discovery of human embryonic stem cells and the development of a new technique for DNA mapping which can sequence the complete genome from a single cell permitted the study of this phenomenon, and many groups consequently began to investigate the inactivation of the X chromosome.

The group led by Pereira and Vibranovski decided to pursue this line of research in 2013. Alongside the biologist Joana Carvalho Moreira de Mello, they began to work on sequencing RNA from isolated human embryonic cells. This strategy allows scientists to identify which genes are active, or to determine when they become active. “We wanted to analyze the activity of the genes linked to the X chromosome in embryos at different stages of development,” explains Vibranovski. However, that same year a group of Chinese researchers published an article analyzing the change in genetic expression in embryos, using the same experiments they had planned to conduct.

Unexpected shortcut
The Chinese group had done all the laboratory work: they obtained the human embryos, separated the cells, and extracted and sequenced the RNA, although they did not look at the inactivation of the X chromosome. They also provided all the information in genomic databases. “We consequently didn’t need to spend time on the experiments, only analyzing the finished data,” says Pereira, who also heads the Brazilian National Laboratory of Embryonic Stem Cells and is a lead researcher at the Center for Cell-Based Therapy at USP, one of FAPESP’s Research, Innovation and Dissemination Centers (RIDC). They interrupted the RNA sequencing and began to analyze the data using bioinformatics strategies, determining the sex of the embryos based on RNA data and evaluating the expression of genes on the X chromosome.

In their study, they monitored the dynamics of gene expression from the X chromosome at the beginning of human embryonic development. They confirmed that the XIST gene, which is responsible for initiating the inactivation, was expressed in female embryos from the eight-cell stage, and that its expression was stabilized by the blastocyst stage, six days after fertilization. The results suggest that the process of inactivating the X chromosome starts earlier in human embryos than in mice. “The adjustment of gene activity in humans occurs by inactivating one of the X chromosomes, as it does in mice,” explains Pereira. “The difference is that in humans, this process begins before the embryo attaches to the wall of the uterus, while in mice this occurs when the cells are beginning to specialize.”

In the assessment of geneticist Anamaria Camargo, of the Molecular Oncology Center at the Sírio-Libanês Hospital in São Paulo, this project is notable for its scientific rigor and careful analysis of the data. “The observed pattern of expression is compatible with the model of inactivating one of the X chromosomes, and refutes the model proposed by the Swedish researchers,” says Camargo, who did not participate in the study published in Scientific Reports. “It is a valuable contribution toward a better understanding of the mechanism for adjusting the activity of genes linked to the X chromosome in humans.”

According to Pereira, the next step is to study how the silenced chromosome is selected, in other words, whether the process is random. To do so, the researchers plan to analyze the process of inactivating the X chromosome soon after fertilization. They also intend to continue monitoring the stages following inactivation of the X chromosome to further investigate this mechanism.

1. Cell Therapy Center – CTC (No. 13/08135-2); Grant Mechanism Research, Innovation, and Dissemination Centers (RIDC); Principal Investigator Dimas Tadeu Covas (FMRP-USP); Investment R$25,560,734.64 (entire project).
2. The role of gametogenesis in the origin and evolution of new genes (No. 15/20844-4); Grant Mechanism Regular Research Grant – Junior Researcher; Principal Investigator Maria Dulcetti Vibranovski (IB-USP); Investment R$394,581.10.
3. In silico analysis of the epigenetic state of the X chromosome in human embryos in the preimplantation stage (No. 15/03610-0); Grant Mechanism Post-Doctoral Grant; Principal Investigator Maria Dulcetti Vibranovski (IB-USP); Scholarship Beneficiary Joana Carvalho Moreira de Mello; Investment R$233,872.74.

Scientific articles
MELLO, J. C. et al. Early X chromosome inactivation during human preimplantation development revealed by single-cell RNA-sequencing. Scientific Reports. Sept. 2017.
LANNER, F. et al. Single-cell RNA-Seq reveals lineage and X chromosome dynamics in human preimplantation embryos. Cell. V. 165, No. 4, p. 1012–26. May 2016.