Determination of transcription factor cooperativity driving expression from the inactive X chromosome

Project Information

TAXi-ch

Grant agreement ID: 882771

DOI

10.3030/882771

Project closed

EC signature date 21 February 2020

Start date 1 March 2020

End date 29 August 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 162 806,40

EU contribution

€ 162 806,40

Coordinated by

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Germany

Periodic Reporting for period 1 - TAXi-ch (Determination of transcription factor cooperativity driving expression from the inactive X chromosome)

Reporting period: 2020-03-01 to 2022-02-28

Summary of the context and overall objectives of the project

In mammals, in order to compensate for the double dose of X-linked genes between XX females and XY males, female cells inactivate one of the X chromosomes during embryonic development. This process triggers chromosome-wide silencing of most genes on one X chromosome. Once the inactive X (Xi) chromosome has been established, its silent state is preserved during subsequent cell divisions. However, there is a small percentage of genes for which expression is detectable from the Xi chromosome (so-called escapees), albeit usually at lower levels than from the active X (Xa) chromosome in general. Among these genes, several have been associated with neurological, metabolic and autoimmune disorders, and considering the increased incidence of some of these pathologies in females, their proper regulation is likely to be important for female physiology. Indeed, for some escapee genes this has been directly shown (e.g. the implication of TLR7 escape from XCI in systemic lupus erythematosus).

Escapees have been identified in several species and tissues, yet, there is a lack of knowledge about their regulation and their expression dynamics during the silencing process. For instance, it is not known if genes expressed from the Xi chromosome are regulated by shared regulatory networks than their counterparts in the Xa chromosome or if the transcription factors (TFs) involved in these networks play the exact same roles. This is important in order to understand how escapee genes are expressed from the Xi, and whether this can ultimately help in detecting or treating associated disorders. In consequence, the TAXi-ch project was designed to shed light into the regulators of escapees and their interplay by:

1. Identifying TFs involved in the regulation of expression from the Xi chromosome and assessing their contribution.
2. Generating gene regulatory networks of X-linked genes.

TAXi-ch advances the current knowledge of gene regulation in the Xi chromosome by identifying candidate regulators that can be explored in the future, and by investigating the role and mechanism of action of one of them. Moreover, the fellow (Yuvia A. Pérez-Rico) improved professional skills that are relevant to become a competitive candidate for leadership positions in Europe.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Research results obtained for the defined objectives:

1. During the action, genomics data in clonal, F1 hybrid female mouse neural progenitor cells (NPCs) established in the hosting group were generated, to determine the transcriptional status of genes, DNA accessible regions and the presence of enhancers. As the NPC clones were derived from crosses of distant mouse species this enabled the bioinformatics analysis of the data in an allele-specific manner, to distinguish the signal originating from the Xi or Xa chromosomes. These data were used to identify enriched TF binding motifs in regulatory regions of the Xi chromosome. The motif corresponding to the architectural protein, CTCF, was detected in this analysis. As part of a collaboration in the group, genomics data to delineate CTCF binding in the X chromosomes were generated, as well as, cell lines to assess the impact of CTCF depletion in the expression of escapees. Notably, CTCF has been suggested to play a role in the process of escape since years and this work is contributing to identify its exact functions in this process.

2. The variability of expression from the Xi chromosome was also investigated using the genomics data of NPC clones. Although the transcriptional data used for this analysis were only derived from NPC samples, there was high variability in the number of escapees between clones and no strong correlation between the frequency of escape and level of expression was observed.

A manuscript summarizing some of the results obtained is currently being drafted, and we expect to submit this to a peer-reviewed journal in the next months.

Concomitant to the research activities, Dr Pérez-Rico improved her data analysis skills with the R and python programming languages and gained knowledge in machine learning methods. Importantly, she learnt to implement bioinformatic pipelines using a workflow manager, which increases analysis reproducibility, in line with the objectives of the Data Management Plan. In addition, Dr Pérez-Rico participated in training offered by EMBL administration and the Heidelberg International Professional (HIP) Women’s Forum to hone transferable skills, in particular, communication and leadership. These skills proved extremely useful during the supervision of a master student during one semester and the dissemination of results.

Project results were communicated both as poster talks in conferences with expert audiences and with the general public. Outreach activities included talks with high school students and professional women of the Heidelberg Area, participation as jury in science fairs and a pen pal program. These activities were broadly communicated in my Twitter account and with a blog entry (https://emblpenpals.wordpress.com/2020/05/16/how-to-fulfill-our-social-responsibility-as-scientists). The fellow is particularly grateful for having had the opportunity to interact with young students in developing countries (Mexico and Nigeria) that often lack access to outreach activities, as she experienced during her childhood.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

TAXi-ch contributed to the understanding of how gene expression is regulated from the Xi chromosome and the role of CTCF, known to act as activator or repressor depending on the chromatin context via the formation of DNA interactions. This project predicted additional TFs that may play a role in the regulation of escapee genes and they can be studied using the approach implemented for CTCF. As escape from XCI is related to sex-biased diseases, understanding the role of these TFs is important because this knowledge could be used as the basis for future therapies. Importantly, the multiomics dataset generated by this project will be further exploited by researchers of the hosting group that are developing projects with the same cell lines, and also by researchers working in the X chromosome inactivation field or generally interested in the transcriptional regulation in neuronal lineages. Moreover, the allele-specific workflows developed as part of this project are publicly available and should ease the analysis of samples with hybrid genetic background.

The management of this project had a profound impact in the career of Dr Pérez-Rico because she had the opportunity to hone molecular biology, data analysis and transferable skills that are essential in her future establishment as an independent leader outside academia. These skills improved her competence portfolio positioning her as a highly qualified computational biologist.

One of the lessons learnt by the general public during the COVID-19 pandemic was the significant differences in the immune response that can occur between females and males. Although this depends on multiple factors, the study of X-linked gene dosage is becoming increasingly attractive. Therefore, the tools and results generated by TAXi-ch are paving the way to deepen the understanding of escapees’ regulation and the consequences of their dysregulation in pathogenesis, and in the long-term, to contribute to the Sustainable Development Goal of “Good health and well-being”.

Model system and sequencing methods used to study the variability of escape from XCI and CTCF roles.