Periodic Reporting for period 2 - RANdOM (Systematic search of RegulAtory elements coNtrOlling autosomal Monoallelic expression)
Reporting period: 2020-01-01 to 2020-12-31
Although it is estimated that only 1-10% of the autosomes show such an expression pattern, the widespread of functional classes observed among the affected genes underlies diverse pathways and emphasizes the potential for disease consequent from the perturbation of RME and RIE regulation. Indeed, great focus has been given to the role of allelic-specific cis-regulatory genetic effects on disease, but these studies are insensitive to the random allelic effects of epigenetic nature, either because these are canceled out (e.g. RNA-Seq studies in bulk tissues), the technology is not (yet) sensitive enough to reliably call mitotically-stable epigenetic allelic biases (single-cell RNA-Seq), or are not high-throughput (e.g. FISH) and thus lack power. As a consequence, the impact of allelic imbalances of epigenetic nature on disease remains uncertain.
The overall objective of this action is to identify mechanisms of regulation of mitotically stable allelic imbalances in murine cells. Additionally, we will characterize the dynamics of allele-specific expression along the cell cycle progression and characterize for the first time the highly stable monoallelic expression states in hematopoietic stem cells in the mouse model, in vivo. These studies will consolidate the base knowledge in the field and set the ground for the introduction of the clonal parameter on studies of functional genetics.
The Gimelbrant lab thrives for the development of new methodologies and specialized variations of established ones. This strategy led to the development of the “screen-by-sequencing” methodology for the introduction of perturbations in clonal cell lines and detection of their effects in the expression of targeted genes, with allelic resolution. With this work, we have detected for the first time a mechanism for the maintenance of allele-specific epigenetic regulation in the mouse model. Part of this work has been deposited as a pre-print in the biorxiv repository, is under review by peers, and free access to the public will be granted as soon as it is published.
To further understand the mechanisms of allele-specific expression regulation, I have isolated the fractions of cells that are synchronized regarding the cell cycle stages in clonal cell lines, performed RNA-Seq, and analyzed the results with an optimized allele-specific expression pipeline developed at the outgoing laboratory. In collaboration with the Churchman laboratory (HMS, Boston), I have further fractionated the chromatin compartments of the cell-cycle sorted partitions of a clonal cell population and analyzed the allelic expression in the chromatin compartments. The results of this study show for the first time the dynamics of allele expression along the cell cycle progression.
Finally, we have set to study the stability of the allelic epigenetic marks in hematopoietic stem cells (HSC) in the mouse model, in vivo. By analyzing terminally differentiated cells that make up different hematopoietic compartments (B cells, T cells, Macrophages) and share the same hematopoietic stem cell ancestor, we are able to measure the extant allele-specific expression in the stem cell that has resisted multiple rounds of mitotic propagation and differentiation stages.
In order to understand the contribution of clonal effects in phenotypes, two questions need to be solved: 1) What is the clonal distribution of the cells that compose a tissue and its variability between organisms? 2) What is the extent of stable allelic imbalances of epigenetic nature in clonal populations and what are the mechanisms of maintenance of these marks? This study greatly contributes to the solution to the second question. For the first time, we have identified a mechanism that contributes to the maintenance of stable mitotic allelic expression biases. Remarkably, we were able to artificially disrupt this mechanism with a drug that is used in clinical settings, disclosing a possible important consequence of therapies: the disruption of homeostasis of tissues. These findings are a breakthrough in the field, consolidating the perception that clonal variability and structure should be taken to the clinical studies. These efforts of characterizing the nature of epigenetic allelic biases are being complemented by our analysis of the dynamics of allele-specific expression along the cell cycle. Furthermore, we are analyzing clonal systems in vivo. With the latter, we have conclusively determined, for the first time, the extant of stable epigenetic allelic-specific regulation that can be observed in differentiated cells derived from hematopoietic stem cells and will be able to contrast with the stable allelic biases in clones of differentiated cells. These studies will set the ground for the development of future projects, aiming at measuring the impact of the clonal background on the cellular phenotypes of disease variants in human cellular models.