1. How is the mono-allelic and female-specific expression pattern of Xist ensured?
Each female cell in the early embryo randomly selects exactly one X-chromosome to up-regulate the long non-coding RNA Xist and undergo X-inactivation. To this end the cell has to quantitatively detect the dosage of X-linked genes to thus sense the number of X-chromosomes present in the cell and to ensure the female-specific expression of Xist. In addition, Xist up-regulation must be limited to exactly one out of two X-chromosomes, potentially through a combination of cis- and trans-acting, positive and negative feedback loops. To investigate the structure and function of the gene-regulatory network that ensures the mono-allelic and female-specific expression pattern of Xist, we are using an interdisciplinary approach that combines mathematical modeling with a series of quantitative experimental techniques, such as single-molecule RNA-FISH, single-cell RNA-Seq, allele-specific expression analysis and gene induction/repression using CRISPRa/i. We have recently found that in the minimal network required for female-specific and mono-allelic Xist up-regulation a trans-acting negative feedback must collaborate with a cis-acting positive feedback loop (Mutzel et al., BioRxiv, 2017).
2. Understand potential function of antisense transcription
An important regulator of Xist is its antisense transcript Tsix, another long non-coding RNA. We develop and analyze mathematical models of antisense transcription to understand, whether and under what conditions it can give rise to non-linear behavior such as switch-like responses. To validate our models, we interfere experimentally at different points of the antisense transcription unit and compare the system's response to model simulations. Moreover, we aim to identify transcription units with a similar architecture as the Xist/Tsix locus to understand whether the regulatory principles we have identfied are also employed by other cellular switches.
3. Finding unknown regulators of X-inactivation
Although several important regulators of X-inactivation have been identified in the past 20 years, several key players in the network still remain to be identified. To find new genes involved in X-inactivation, we perform Cripsr-based functional screens and also dissect the cis-regulatory landscape around the Xist locus in collaboration of the Ohler lab at the MDC.
4. How is X-inactivation integrated with stem cell differentiation?
X-inactivation is initiated upon the exit from the stem cell state, when the repression of Xist by pluripotency factors is released. However, in turn also the process of X-inactivation modulates the differentiation process since a double dose of X-linked genes inhibits differentiation, potentially to ensure a tight coordination of X-inactivation and development. To shed light into the underlying mechanisms we are using CRISPR screens to search for the X-linked genes mediating the effect and we are performing a systematic comparison of the signaling and gene-regulatory networks in cells with one or two X-chromosomes using genomics and phospho-proteomics in collaboration with the Blüthgen lab at the Charite Berlin.
5. What determines Xist-mediated gene silencing kinetics?
Although nearly all X-lines genes are silenced during X-inactivation, the kinetics of silencing varies widely between different genes. Based on chromosome-wide measurements of gene silencing, we aim at identifying features that determine the susceptibility for Xist-mediated silencing for a given gene, in collaboration with the Heard lab in Paris and the Marsico lab here at the MPIMG. In the long term this will help us to gain a better understanding of the mechanisms that mediate X-inactivation.