Periodic Reporting for period 2 - CisTune (Regulatory Logic, Thresholds and Epigenetic Memory: How cis-regulatory landscapes tune gene activity during mammalian development)
Reporting period: 2023-02-01 to 2024-07-31
The project CisTune aims at deciphering how mammalian genes decode information. A key prerequisite of multicellular life is the restriction of gene activity to defined cellular contexts. To this end, each gene must integrate information on space, time and cell type. How genes decode complex information remains largely unknown. CisTune uses the Xist locus as a model to study information processing by mammalian genes. Xist is active only in females, where it will inactivate one of the two X chromosomes to ensure dosage compensation for X-chromosomal genes between the sexes. It therefore senses the sex of the cell and the developmental context. The project combines a series of state-of-the-art experimental and computational approaches to dissect the complex quantitative interplay between multiple regulators and cis-regulatory elements in controlling Xist. CisTune will enhance our understanding of the basic mechanisms that underlie gene control. This knowledge will be helpful for therapeutic interventions and diagnostic applications. Moreover, Xist regulation and X-chromosome inactivation are thought to affect sex bias in disease susceptibility, suggesting that the project results might have implications for gender medicine.
To comprehensively identify factors involved in Xist regulation, we have performed a CRISPR screen to test all transcription factors for a role in Xist regulation. The identified factors were then extensively characterized with respect to their mode of action at the Xist locus. This analysis revealed two sets of Xist regulators. Early regulators are active when Xist is first upregulated and modulate the probability of Xist activation through the promoter-proximal region. Late regulators remain expressed after X-inactivation has been initiated, are important to maintain high Xist levels and control distal regulatory regions. A subset of early regulators are more abundant in female compared to male cells, suggesting a role in female-specific Xist expression. One of the identified factors is encoded on the X-chromosome, making it a prime candidate for X-dosage dependent Xist regulation. To assess the dosage-dependence of this regulatory interaction we have developed a new method, called CasTuner, that allows tuning endogenous gene expression homogenously across cells. With CasTuner we have assessed the dose-response relationship between Xist and known and newly identified X-linked Xist regulators. After data analysis with the use of a mathematical model, we could show that female specificity can be achieved through a combined action of two different X-linked regulators with distinct dose-response profiles.
How female-specific and monoallelic Xist expresion is ensured is a long-standing question in biology, since Xist is the master regulator that drives the essential process of X-chromosome inactivation in mammals. In CisTune we have identified central new regulators of the Xist locus. Moreover, we have developed and applied a new method for tuning endogenous gene expression, which might we useful to investigate quantitative effects also in other biological contexts. During the second half of the project we expect to develop and apply another methods, which allows high-throughput mapping of functional interactions between regulatory elements and transcription factors. We expect that this method will provide central new insights in how regulatory elements decode their input signals.