When two is a crowd: Mapping the incomplete silencing of the X chromosome

The cells of female mammals have two X chromosomes, but if all the genes on each chromosome were active, the double-dose of proteins would spell disaster for the cell. To ensure that X chromosome genes are expressed in female cells at a similar level to that in cells of XY males, one of the two female X chromosomes is silenced in a process called X chromosome inactivation (XCI).

A decade ago, scientists discovered that although XCI neatly explains how the cell compensates for the twin X chromosomes, in reality it is often an incomplete task. Many genes somehow escape XCI and remain active on both copies of the X chromosome, and still others are inconsistently silenced — these may be active on only one X chromosome in some females, but active on both in others.

Previous studies of the phenomenon were limited to small numbers of samples, with no good way to investigate how XCI varies among different human tissues. However, the recent advent of single-cell transcriptomic methods, which measure the activity of all genes in individual cells, has made it possible to take a more thorough look at XCI. In addition, scientists now have access to a database that correlates gene expression measurements with genetic variation in dozens of tissues across the human body, known as the Genotype Tissue Expression Project (GTEx), led in part by scientists at the Broad Institute of MIT and Harvard.

GTEx researchers recently published a suite of papers from the project’s second phase, including an effort led by Daniel MacArthur, an institute member at the Broad, aimed at assessing the landscape of X-chromosome inactivation — and its incompleteness — across human tissues.

In Nature, MacArthur, first author Taru Tukiainen, and colleagues describe their survey of XCI. In their analysis, the team integrated more than 5,500 transcriptomes from 449 people spanning 29 tissues from GTEx and 940 single-cell transcriptomes, combined with data on the individuals’ DNA sequences.

The researchers found that most of the X chromosome genes in female cells were uniformly silenced across different tissues, suggesting a global process controlling this phenomenon. However, they also uncovered many instances of XCI variation between tissues, individuals, and cells. Nearly a quarter of the 186 X-chromosomal genes assessed in the study were incompletely inactivated and were expressed from both copies of the chromosome, and the activity of these so-called “escape genes” varied from one female to another. The team further determined that escape from X inactivation results in gene expression differences between males and females.

“These significant expression-biases suggest that escape from X inactivation can contribute to the many phenotypic differences between males and females, and possibly play a role in phenotypes seen in conditions marked by abnormal numbers of sex chromosomes,” said Tukiainen. Another notable finding was that nearly 6 percent of genes on the X chromosome escape XCI only in certain tissues (e.g., the gene KAL1 which escaped XCI in lung tissue only), illustrating the importance of including multiple tissue types in studies to uncover the full extent of XCI escape.

This study also demonstrated the power of single-cell approaches to yield insight into what appears to be a largely random phenomenon. The specific X chromosome to be inactivated — either that from the mother or that from the father — is randomly selected in cells during development, so that a normal female has the maternal X chromosome active in half of her cells and the paternal one in the other half. Distinguishing expression of genes from the active X or the inactivated X chromosome in bulk samples (e.g., a patch of tissue) is nearly impossible.

Tukiainen and colleagues overcame this challenge by complementing their GTEx analyses with single-cell RNA sequencing on nearly 1,000 single cells from four females, providing the means to assess XCI on the cellular level — a level of detail not previously observed in human samples. The results pinpoint new escape genes, as well as variability in the pattern of XCI both between females and between seemingly identical cells from the same individual.

“Our work, the first published application of single-cell RNA sequencing to study X inactivation in adult humans, shows how much we can learn by studying individual cells on a large scale and utilizing the rich treasure trove of data created by GTEx,” said Tukiainen.