The importance of transcription-associated R-loops for chromosome condensation – TRACC

We sought to develop protocols allowing an in-depth characterization of R-Loops in the genome of the fission yeast S. pombe. This meant the development of protocols to estimate precisely the amount of R-Loops in the genome of different mutants and/or in different conditions but also the mapping of R-Loop forming regions at high resolution. A comprehensive view of where and when R-Loops form in the genome is an essential pre-requisite for a good understanding of R-Loop functions. To determine the exact features that lead to R-Loop formation, such mapping must be strand-specific and have the greatest possible resolution. To achieve this goal, we have developed protocols to sequence the RNA moiety of the R-Loops. We have also looked for ways of varying the amount of R-Loops in the genome. We have also developed protocols to establish which proteins bind to R-Loops. In particular, we have tried to establish whether or not condensin can bind to R-Loops. Finally, we have developed protocols to visualize at the single molecule resolution in vitro transcribed R-Loops using AFM microscopy.

Our results confirm that transcription produces the chromatin structure that recruits condensin but exclude the possibility that this structure could be R-Loops. Instead, our results indicate that topological stress facilitates the binding of condensin. From our analysis of the structure of R-Loops at the single molecule resolution, we formulated a model to explain why some R-Loops can interfere with genome stability and contribute to human pathologies. Our work has also considerably improved the toolkit available to characterize the functions and position of R-Loops in the genome.

Our results open new avenues of research which we will now investigate: 1. Our work suggests that transcription-dependent topological stress facilitates the binding of condensin to chromatin. One would therefore expect that the amount of topological stress changes through the cell-cycle and increases particularly at mitotic entry when condensin binds to chromatin. This has yet to be demonstrated. 2. If this is indeed the case, then how is transcription-dependent topological stress regulated through the cell-cycle? Which factors regulate the formation of topological stress through the cell-cycle? Is it possible to follow the dynamics of nascent RNA transcription through the cell-cycle? 3. Is it possible to demonstrate that topological stress produces the single-stranded DNA that is recognized by condensin? In other words, is there a tight co-localization between topological stress, single-stranded DNA and condensin at the beginning of mitosis? 4. We have shown that in vitro transcribed R-Loops adopt a very specific conformation. Can we demonstrate that this conformation is at the origin of their toxicity in vivo?

Some of the results we have obtained in the course of this project have already been published in three peer-reviewed publications. Two more manuscripts are in preparation and will be submitted shortly. We have been invited to present our work orally at eight conferences, both national and international.

An ever-growing number of studies show that chromosome abnormalities can lead to premature aging or to serious diseases such as cancer and developmental disorders. To protect public health, it is therefore a major challenge to understand how these chromosomal alterations arise. I am interested in the molecular mechanisms that ensure the faithful transmission of chromosomes through generations, and thereby contribute to genome stability.
A key aspect of chromosome biology is the process of mitotic chromosome condensation, whereby chromatin becomes tightly packed and as a result, chromosomes individualize. Chromosome condensation is extensively conserved through evolution and is a hallmark of mitosis. When deficient, duplicated chromosomes cannot segregate properly and instead remain entangled. This often leads to broken or abnormal chromosomes. I have recently shown in the fission yeast Schizosaccharomyces pombe that even a small chromosome condensation defect is sufficient to induce chromosome breaks, in particular close to telomeres. These breaks trigger the irreparable loss of genetic information. As a clear illustration of the importance of chromosome condensation for cell survival, Rb-dependent chromosome condensation was recently shown to be tumour-suppressive. Chromosome condensation was first described in the late 1800s. Yet, its molecular mechanisms remain poorly understood.

A significant number of studies have shown that chromatin compaction and gene expression are mutually exclusive. Chromatin compaction can, at least locally, contribute to the reduction of gene expression. Reciprocally, a number of studies have suggested that transcription and/or the transcription machinery itself can prevent chromosome condensation, at least locally. Whether this antagonism, that can act locally to shape chromatin, can also act to structure the whole chromosome in mitosis is seldom addressed. I propose that the reorganization of the transcription machinery contributes to chromosome condensation upon mitotic entry. My preliminary data suggest that the local impairment of the machinery responsible for the 3’end maturation of RNAs contributes to chromosome condensation. This reorganization is promoted by a highly conserved protein complex. Our data indicate that one consequence of this reorganization is the generation of transient single-stranded DNA (ssDNA) in the genome. Our results indicate that this ssDNA helps the recruitment on chromatin of the Condensin complex, the main enforcer of chromosome condensation. These data establish for the first time an unsuspected link between the local impairment of the transcription machinery, the production of ssDNA and chromosome condensation. The overall aim of my proposal is to understand the molecular basis for this regulation.

Research tends to be divided in independent fields of investigation that rarely interact. In order to build a holistic view of biological phenomena, it is essential to built bridges between subjects. By bridging the gap between our understanding of transcriptional regulation and the mechanisms controlling chromosome condensation, the work carried out in this project will participate to this process and will contribute to building an integrated view of cell-cycle transitions at the level of the whole chromosome.