Biology is never as straightforward as we learned in high school. Take sex-linked chromosomes as an example: we’re taught that biological males get XY chromosomes while biological females get two copies of X instead, and to compensate for this double dosage of X-linked genes, one of the X chromosomes is simply switched off in a process called X-chromosome inactivation (XCI).
But, of course, things are never really that black and white. While most of the X-linked genes are silenced on the inactive X chromosome, “a significant number of X-linked genes called escapees (~15-30% in human and 3-7% in mouse) escape silencing, leading to a higher expression in females than males,” says Dr. Christine Disteche, a University of Washington professor and member of the Cancer Consortium program in Basic Cancer Biology. Her lab in the Department of Laboratory Medicine and Pathology focuses on the molecular mechanisms that change the structure and function of sex chromosomes.
“The number and expression levels of genes that escape XCI vary among tissues, cell types, and individuals. These genes contribute to variable sex difference manifestation,” Dr. Disteche explains. “Understanding where, when and how escape from X inactivation is controlled will help delineate molecular mechanisms underlying sex dimorphisms in health and disease.”
This is a tricky research question for a few reasons. First, there are multiple redundant mechanisms involved in XCI, including transcriptional silencing facilitated by the non-coding RNA Xist and genome condensation and repression by heterochromatin markers.
Second, it’s technically challenging to identify which X chromosome a gene may be expressed from. Only recently have techniques been developed to identify whether a gene is coming from the inactive X chromosome (called the Xi) or the active X chromosome (the Xa). “Studies of these mechanisms require allele-specific analyses,” Dr. Disteche explains.
In a new publication in BMC Biology, Dr. Disteche and fellow UW Pathology faculty Drs. Xinxian Deng and He Fang looked into the role of chromatin factor CTCF (CCCTC-binding factor) in XCI escape.
CTCF is a master chromatin regulator that can function as a transcription factor and also plays a fascinating role in 3D genome architecture. Binding of CTCF to two of its binding sites in 3D proximity create a barrier to stall a protein complex called cohesin that mediates DNA loop extrusion, resulting in a loop of chromatin. One interesting facet of CTCF binding is that its motifs are directional, which can impact how these chromatin loops form.
CTCF binding sites have been found at the boundaries of many genes that can break free of X-chromosome silencing. Furthermore, past work has shown that artificial tethering of CTCF to sites it normally does not bind on the Xi can induce reactivation of Xi genes.
The Deng and Disteche teams identified several escapee genes in mouse cells that were flanked by CTCF binding motifs. They confirmed CTCF binding using ChIP-seq and CUT&RUN, two methods that locate binding of proteins. They then compared these sites with 3D chromosome contacts obtained through Hi-C, which maps long-range interactions of DNA strands by crosslinking them together, digesting the DNA into small fragments, and sequencing.
The gene that their team chose to study is Car5b, a mitochondrial carbonic anhydrase. In humans, CA5B isn't silenced and is expressed from the Xi in a majority of tissues. In contrast, in mice, Car5b is only a facultative escapee, i.e. it can elude X silencing in some cell types but not others. The question of the paper is: how does Car5b resist XCI, and is this potentially mediated by CTCF?
To answer this, the Disteche and Deng teams performed an elegant series of experiments where they either deleted CTCF binding sites around Car5b or inverted them to see if the presence or direction of CTCF binding on the Xi impacts Car5b expression.
“Deletion but not inversion of CTCF binding sites at the boundary between Car5b and its neighbor gene subject to X inactivation abolish escape by allowing the spreading of inactive chromatin features,” Dr. Disteche reports.
In other words, they think that Car5b is located on a DNA loop that CTCF makes on the Xi, and that this loop insulates Car5b from marks that would silence it. Deleting CTCF binding around the Car5b locus correlates with intrusion of repressive marks (like H3K27me3) into this domain and kills expression of Car5b. In contrast, merely switching the directionality of the CTCF-binding motifs doesn’t seem to impact the insulation provided by CTCF.
“While escape domains other than Car5b need to be evaluated to generalize this finding, this study provides insights into future studies that aim to manipulate gene expression levels of endogenous genes by disrupting chromatin architecture,” Dr. Disteche concludes. “Such manipulation of chromatin loop formation on each allele could help, for example, to reactivate normal alleles of X-linked genes or of tumor suppressor genes, or conversely, to suppress expression of oncogenes, thereby providing new avenues of gene therapy.”
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members He Fang, Steven Henikoff, Jay Shendure, and Christine Disteche, contributed to this research.
The spotlighted research was funded by the National Institutes of Health Common Fund 4D Nucleome, the National Institute of General Medical Sciences, and the Howard Hughes Medical Institute.
Fang H, Tronco AR, Bonora G, Nguyen T, Thakur J, Berletch JB, Filippova GN, Henikoff S, Shendure J, Noble WS, Duan Z, Disteche CM, Deng X. 2025. CTCF-mediated insulation and chromatin environment modulate Car5b escape from X inactivation. BMC Biology. doi: 10.1186/s12915-025-02137-7.
Hannah Lewis is a postdoctoral research fellow with Jim Boonyaratanakornkit’s group in the Vaccine and Infectious Disease Division (VIDD). She is developing screens to find rare B cells that produce protective antibodies against human herpesviruses. She obtained her PhD in molecular and cellular biology from the University of Washington.
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