A Novel cis Regulatory Element Regulates Human XIST in a CTCF-Dependent Manner

ABSTRACT

The long noncoding RNA XIST is the master regulator for the process of X chromosome inactivation (XCI) in mammalian females. Here, we report the existence of a hitherto-uncharacterized cis regulatory element (cRE) within the first exon of human XIST, which determines the transcriptional status of XIST during the initiation and maintenance phases of XCI. In the initiation phase, pluripotency factors bind to this cRE and keep XIST repressed. In the maintenance phase of XCI, the cRE is enriched for CTCF, which activates XIST transcription. By employing a CRISPR-dCas9-KRAB-based interference strategy, we demonstrate that binding of CTCF to the newly identified cRE is critical for regulating XIST in a YY1-dependent manner. Collectively, our study uncovers the combinatorial effect of multiple transcriptional regulators influencing XIST expression during the initiation and maintenance phases of XCI.

KEYWORDS:

• XIST
• cis regulatory element
• pluripotency factors
• YY1
• CTCF

ACKNOWLEDGMENTS

We thank Peter Andrews (University of Sheffield) for kindly gifting NT2/D1 cells, Ajay Labade and Madhu Kabra (IISER Pune) for providing help with the FISH experiments, and Mukul Rawat and Sneha Tripathi (IISER Pune) for helping with the imaging and analysis.

R.S. and S.G. conceived the project and designed experiments. R.S. performed all experiments except X chromosome paint (Fig. 1E) and dCas9-KRAB-based targeting (Fig. 5D to G), interpreted data, and wrote the manuscript. A.S. performed and analyzed dCas9-KRAB experiments in HEK293T cells. A.K. performed bioinformatics analysis and interpretations and also helped with acquisition of the RNA FISH images. K.S. performed X chromosome FISH. S.G. interpreted data, supervised the project, and wrote the manuscript. All authors read and approved the final manuscript.

Work was supported by the Centre of Excellence in Epigenetics program (Phase II) of the Department of Biotechnology (BT/COE/34/SP17426/2016), Government of India, and the JC Bose Fellowship (JCB/2019/000013) from the Science and Engineering Research Board, Government of India, to S.G. R.S. was supported by a fellowship from the Council of Scientific and Industrial Research, India. A.S. was supported by a fellowship from the University Grants Commission, India. A.K. was supported by the Wellcome Trust-DBT India Alliance Early Career Fellowship.

We declare no conflict of interest.