Periodic Reporting for period 2 - ChromoSilence (Chromosome-based mechanisms of transcriptional inactivation during mitosis)
Reporting period: 2023-04-01 to 2024-08-31
Cell division and transcriptional regulation are two essential processes for proper cellular function, yet these processes are strictly temporally separated. As cells reach mitosis, the process of dividing their chromosomes into two identical sets between two daughter cells, transcriptional activity is largely suppressed. This phenomenon, known as Mitotic Transcriptional Inactivation (MTI), ensures that the cellular machinery focuses on the precise segregation of chromosomes rather than the synthesis of RNA. However, the molecular mechanisms driving such drastic changes in the transcriptional state remain largely unknown.
ChromoSilence aims to uncover how transcriptional shutdown is coordinated with mitotic processes. By pioneering new methods to manipulate and observe MTI with unprecedented precision, this project seeks to elucidate how this transcriptional silencing is vital for accurate chromosome segregation and genome stability. Insights gained from this work could lead to novel approaches to maintaining genome integrity, which is essential for preventing diseases such as cancer. Additionally, understanding MTI's role in transcriptional program maintenance during development could have significant implications for developmental biology and regenerative medicine, potentially informing strategies to address developmental disorders and improve tissue repair therapies.
ChromoSilence aims to uncover how transcriptional shutdown is coordinated with mitotic processes. By pioneering new methods to manipulate and observe MTI with unprecedented precision, this project seeks to elucidate how this transcriptional silencing is vital for accurate chromosome segregation and genome stability. Insights gained from this work could lead to novel approaches to maintaining genome integrity, which is essential for preventing diseases such as cancer. Additionally, understanding MTI's role in transcriptional program maintenance during development could have significant implications for developmental biology and regenerative medicine, potentially informing strategies to address developmental disorders and improve tissue repair therapies.
In AIM 1, we aim to uncover the molecular players that drive MTI. We center our analysis on chromosome-based events, which we propose as key contributors to MTI regulation. We initially focused our analysis on classic chromosome assembly factors, long postulated to drive or contribute to mitotic transcriptional shutdown. In contrast with this dogma, we have found that perturbations in the critical chromosome structural factors do not perturb the kinetics of transcriptional shutdown. These findings propose that the change in the transcriptional status is not a byproduct of chromosome compaction but is instead driven by dedicated mechanisms. Following this novel notion, we showed that a poorly characterised helicase-like protein – Lodestar – promotes the removal of nascent transcripts upon mitotic entry (Carmo et al, EMBO Reports 2023). These findings set up the conceptual framework and tools for the search for novel regulators in this process (ongoing work, aim 1.2).
In AIM 2 we are interested in uncovering the mechanisms that drive the transcriptional shutdown upon mitotic entry. We have established innovative quantitative methods to monitor different stages of the transcriptional cycle and revealed that multiple mechanisms govern mitotic transcriptional shutdown. On one hand, we estimated that transcription initiation is inhibited several minutes after mitotic entry. However, most of these transcripts are not transcribed to termination and are sharply evicted from chromatin by premature transcription abortion.
In AIM 3 we have focused on establishing tools to uncover the role of this transcriptional shutdown in the fidelity of nuclear division. We revealed that abnormal retention of nascent transcripts on mitotic fidelity compromises chromosome alignment and sister chromatid resolution. Consequently, cells with impaired shutdown of transcription accumulate several errors during mitosis. We thus identified a novel route to chromosomal instability.
In AIM 2 we are interested in uncovering the mechanisms that drive the transcriptional shutdown upon mitotic entry. We have established innovative quantitative methods to monitor different stages of the transcriptional cycle and revealed that multiple mechanisms govern mitotic transcriptional shutdown. On one hand, we estimated that transcription initiation is inhibited several minutes after mitotic entry. However, most of these transcripts are not transcribed to termination and are sharply evicted from chromatin by premature transcription abortion.
In AIM 3 we have focused on establishing tools to uncover the role of this transcriptional shutdown in the fidelity of nuclear division. We revealed that abnormal retention of nascent transcripts on mitotic fidelity compromises chromosome alignment and sister chromatid resolution. Consequently, cells with impaired shutdown of transcription accumulate several errors during mitosis. We thus identified a novel route to chromosomal instability.
ChromoSilence is centred on two major novel hypotheses: 1) the transcriptional shutdown upon mitotic entry is governed by dedicated mechanisms and not a mere byproduct of the structural changes that occur on mitotic chromatin, as largely assumed. 2) this transient reset is a vital process to ensure genome stability and transcriptional control.
In the reporting period, we made significant advances that validated our central hypotheses. These novel concepts break current dogmas as they establish that the shutdown of transcription is not a mere byproduct of mitotic progression. In parallel, we have established experimental tools to explore this understudied process in far more detail. Until the end of the project, we anticipate to expand these results on three major fronts:
1. Building on the preliminary results from an ongoing screen for novel MTI regulators, we expect to uncover new players in this process (aim 1)
2. We expect to gain further information on how the MTI regulators (Lds and others) are regulated during mitosis to ensure a sharp transcriptional shutdown that is readily reversed upon mitotic exit (aim 2)
3. We will expand the analysis of the consequences of failures in transcriptional shutdown beyond our initial focus on mitotic fidelity. For this, we will explore how failures in this process can impair the maintenance of transcriptional programmes across various developmental systems, including early embryogenesis and in stem cells.
In the reporting period, we made significant advances that validated our central hypotheses. These novel concepts break current dogmas as they establish that the shutdown of transcription is not a mere byproduct of mitotic progression. In parallel, we have established experimental tools to explore this understudied process in far more detail. Until the end of the project, we anticipate to expand these results on three major fronts:
1. Building on the preliminary results from an ongoing screen for novel MTI regulators, we expect to uncover new players in this process (aim 1)
2. We expect to gain further information on how the MTI regulators (Lds and others) are regulated during mitosis to ensure a sharp transcriptional shutdown that is readily reversed upon mitotic exit (aim 2)
3. We will expand the analysis of the consequences of failures in transcriptional shutdown beyond our initial focus on mitotic fidelity. For this, we will explore how failures in this process can impair the maintenance of transcriptional programmes across various developmental systems, including early embryogenesis and in stem cells.