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Establishment and maintenance of gene expression by heterochromatin factors

Periodic Reporting for period 4 - METACHROM (Establishment and maintenance of gene expression by heterochromatin factors)

Reporting period: 2021-12-01 to 2023-11-30

Metastable epialleles are alleles that are variably expressed in genetically identical individuals. These epialleles are established during early development by epigenetic modifications in a largely unknown manner. However it is clear that this process is influenced by stress and the environment. The epiallele’s state can subsequently be maintained throughout development and adult life. While chromatin marks associated with this metastability are known (DNA methylation and histone H3 lysine 9 tri-methylation), the targeting mechanisms and the mechanisms underlying inter-individual variations are unknown. Studying the mechanisms underlying establishment and maintenance of chromatin states is critical to understanding how the environment can shape the epigenome and how it can impact on diseases and aging. Intriguingly, all known examples of metastable epialleles are linked to the insertion of a transposable element in the vicinity and this insertion plays a major role. Since the control of transposable elements is key to maintain genome stability and gene expression homeostasis in development and throughout adulte life, understanding the mechanisms that control these transposable elements is fundamental and the focus of this study.
Our overall objectives are:
-to understand the nature and the function of the special heterochromatin that forms on telomeres and on transposable elements. We addressed the former in Gauchier et al, Science Adv. 2019. The latter is currently being addressed by two members of the team (Amandine Barral, PhD student and Mathilde Gauchier, post-doc).
-to understand the mechanistics of this heterochromatin. We have addressed this (Gohei Nishibushi) by performing the heterochromatin reconstitution experiments explained in aim2 of the proposal and are currently finalizing this part of the proposal.
-to uncover all the players involved in the metastable silencing of transposble elements in the mouse genome. We have identified a potentially crucial transposon defense mechanism
The long reaching implications of our work are in several area of fundamental research and on diseases: it is known that defective heterochromatin can lead to loss of cellular identity and cancer. Our work will aim at explaining some of the important mechanisms behind such deleterious phenomena.
Upon characterizing the nature of the chromatin that forms on telomeres, we realized that, depending on the cell type, the histone methyltransferase SETDB1 was responsible for local heterochromatinization. Counterintuitively, we found that this heterochromatin was permissive to transcription and a recombination based mode of telomere maintenance named ALT (Alternative lenghtening of telomeres), present in a subset of human cancers. We have described these results in 2019 in Science Advances (data presented at the EMBO telomere meeting in 2018, the Cold Spring Harbor Asia Temoere meeting in 2018). These unexpected results opened novel questions: the heterochromatin discovered at telomeres is better known to drive the silencing of some transposable elements in the same cells and therefore we embarked on trying to address the major differences found on SETDB1 controlled telomeres and on SETDB1 controlled transposable elements. We have developped a novel version of a locus specifc chromatin capture approach that allowed us to purify transposable elements of the Intracisternal A PArticle type (IAP). Surprisingly we found that the heterochromatin components at IAP are not much different from the heterochromatin components found at the telomeres with the exception of only a handful factors. We have focused our studies on these factors.
-some are known to be involved in the sensing of abnormal nucleic acid molecules and to trigger an innate immune response. We are currently working on the role of these factors in the control of transposable element chromatin: Mathilde Gauchier presented the first data on this at the Cold Spring Harbor Transposon Meeting in 2018, and I and her have presented our latest data on this at the CHSL Transposon meeting in Oct 2020 and at the MBSJ in October 2021. We are finalizing some experiments to be able to publish these data
-We also developped genome-wide approaches to identify all the chromosomal regions which harbor a heterochromatin which is similar to the one identified at telomeres. We have identified set of ~5,000 domains, which share the same chromatin features as the telomeres and are testing the function of this heterochromatin in the control of gene expression profiles and genome stability. We uncovered that these regions are actually critical locus control regions that are important for cellular identity. This work has been publihsed in Molecular Cell (17 Feb 2022 issue)
-We have also reconstituted a metastable transgene to which heterochromatin can be induced and destroyed very rapidly. While the system works and highlights some differences in the type of heterochromatin that can form on a gene, we have so far, failed to create a transgenic chromatin able of memory (hence original Aim3 has been cancelled)
-Our efforts to induce durable epigenetic changes in the genome by inducing diverse types of stress on stem cells have, for the moment, yielded no clear data: and we therefore decided not to pursue this at the moment.
-Our serendipitous discovery of the mechanisms responsible for t-loop formation at telomeric heterochromatin is the subject of a scientific article under review at Science.
In the course of characterizing the IAP purification described above, we realized that these transposable elements seem to be able to interact over long distances to other types of DNA repeats. This was an unexpected result and we decided to explore more this aspect and its biological significance. It could be that such unexpected interactions might explain the interindividual variations observed in metastable epialleles.
The discovery of a novel mode of t-loop formation, independent of the TRF2 shelterin protein, will have major implication in the way we think about chromosome protection.