**Abstract:** A collaborative study led by Professor Wei Jie and Assistant Professor Hai-Feng Wang from the School of Life Sciences at Tsinghua University, along with Professor Sundeep Kalantry from the University of Michigan Medical School, has elucidated the dynamic process of establishing and regulating the higher-order chromatin structure of the inactive X chromosome during early mouse embryonic development. The findings were published in the journal *Nature Genetics* on September 10, under the title "Stepwise de novo establishment of inactive X chromosome architecture in early development." In mammals, females (XX) and males (XY) exhibit sex chromosome dimorphism. To balance gene expression levels, one X chromosome in female cells undergoes transcriptional inactivation (XCI) during early embryonic development. Abnormalities in XCI can lead to severe developmental defects, multiple intellectual disability disorders, and even embryonic lethality. Therefore, understanding the mechanisms and regulation of XCI is of significant scientific and clinical importance. The study utilized high-sensitivity sisHi-C technology to systematically analyze the three-dimensional chromatin conformation of the X chromosome from the 1-cell stage to E9.5 extraembryonic tissues and E13.5 embryonic cells, covering the establishment and maintenance of imprinted and random XCI. The researchers observed that during the inactivation process, the topologically associating domains (TADs) and compartments of the X chromosome exhibited a weakening trend similar to that seen in mouse embryonic stem cells (mESCs) during differentiation. However, a unique structure called Xist-separated megadomains (X-megadomains) emerged in post-implantation extraembryonic tissues and was briefly present during the initial stages of random XCI in embryonic tissues. This structure is characterized by the Xist locus serving as a boundary. Further investigation revealed that the formation of X-megadomains is closely associated with the enhancer activity of the Xist Regulatory Region (XRR) and the binding of cohesin. In XEN cells, deletion of the XRR region, silencing of Xist expression, or specific induction of cohesin degradation disrupted the X-megadomains, while degradation of CTCF had a lesser impact. This led the researchers to propose a molecular mechanism for X-megadomain formation: the highly active enhancer signals in the XRR region promote the loading of cohesin, which then accumulates in nearby regions and forms X-megadomains through loop extrusion. The disruption of X-megadomains resulted in the abnormal activation of genes and transcriptional regulatory sequences near the Xist locus, suggesting that the enrichment of cohesin in the Xist and XRR regions isolates active DNA regions to prevent their transcriptional activity from spreading to surrounding silenced regions, thereby avoiding the abnormal activation of nearby silenced genes. The study also highlights the potential function of X-megadomains in maintaining the necessary gene activation and global transcriptional silencing on the inactive X chromosome. By isolating active genes through enhancer-mediated cohesin loading, cells can better regulate gene expression during XCI, ensuring proper developmental outcomes. The key contributors to this research include Dr. Zhenhai Du, doctoral students Liangjun Hu, Zhaoning Zou, and Meishuo Liu from Tsinghua University, and several other researchers from Tsinghua University, the University of Michigan, and Chongqing Medical University. The study was supported by various organizations, including the National Natural Science Foundation of China, the Ministry of Science and Technology of China, the Tsinghua-Peking Center for Life Sciences, the National Institutes of Health (NIH), and the China Postdoctoral Science Foundation. Professor Wei Jie is an HHMI International Research Scholar and a New Foundations Researcher, while Assistant Professor Hai-Feng Wang received funding from Tsinghua University's Dushih Special Program and the Benyuan Public Welfare Foundation. For more details, the full paper can be accessed at: https://www.nature.com/articles/s41588-024-01897-2 **Key Events, People, Locations, and Time Elements:** - **Event:** Publication of a study on the establishment and regulation of higher-order chromatin structure of the inactive X chromosome during early mouse embryonic development. - **People:** Professor Wei Jie, Assistant Professor Hai-Feng Wang (Tsinghua University); Professor Sundeep Kalantry (University of Michigan Medical School); Dr. Zhenhai Du, doctoral students Liangjun Hu, Zhaoning Zou, and Meishuo Liu (Tsinghua University); and other researchers from Tsinghua University, the University of Michigan, and Chongqing Medical University. - **Locations:** Tsinghua University (Beijing, China); University of Michigan Medical School (Ann Arbor, Michigan, USA); Chongqing Medical University (Chongqing, China). - **Time Elements:** The study was published on September 10, 2024, in *Nature Genetics*. **Scientific and Clinical Significance:** The research provides crucial insights into the mechanisms of XCI and the relationship between chromatin structure and gene transcription regulation. Understanding these processes can help in the diagnosis and treatment of developmental disorders and intellectual disabilities associated with XCI abnormalities. The findings also contribute to the broader field of epigenetics and chromatin biology, offering a deeper understanding of how chromatin structure influences gene expression during development.