Periodic Reporting for period 2 - GENECARE (GENome and Epigenome integrity Consecutive to DSBs in Active REgion)
Periodo di rendicontazione: 2023-04-01 al 2024-09-30
DNA double-strand breaks (DSBs) are considered to be the most toxic lesions, holding the potential to generate mutations, copy number alterations and translocations. Beyond programmed DSBs induced during meiosis and antibody repertoire generation, DSBs were historically seen as rare, mostly therapy-induced DNA lesions. However, recent work unequivocally revealed that endogenous DSBs occur far more frequently that initially believed, including in physiological conditions, such as neuron stimulation, and mainly fall within transcribed loci, partly due to topoisomerase II activity. Notably, altered repair of these DSBs is not only emerging as a driver of oncogenesis but also of many developmental, neurological and aging-associated diseases.
Our lab has pioneered the discovery of a dedicated pathway that, alike TC-NER for damaged nucleotides, is actually mobilized to repair such DSBs in transcribed loci, coined as Transcription Coupled DSB repair (TC-DSBR). This pathway entails chromatin signaling, nuclear reorganization and the regulation of transcription, but remains poorly characterized overall. Using advanced genomics, proteomics and microscopy, we intend to perform an in-depth characterization of the TC-DSBR protein network, of the function of chromatin and nuclear organization in this process, and to explore its potential as a target for cancer therapy.
By deciphering the mechanisms that ensure repair of DSBs in transcribed loci, this project holds the potential to expand our knowledge on the biogenesis of translocations and cancer onset, to provide new topoisomerase-poison-based therapeutic strategies but also to provide major breakthroughs in our understanding of neurodegenerative diseases.
Our lab has pioneered the discovery of a dedicated pathway that, alike TC-NER for damaged nucleotides, is actually mobilized to repair such DSBs in transcribed loci, coined as Transcription Coupled DSB repair (TC-DSBR). This pathway entails chromatin signaling, nuclear reorganization and the regulation of transcription, but remains poorly characterized overall. Using advanced genomics, proteomics and microscopy, we intend to perform an in-depth characterization of the TC-DSBR protein network, of the function of chromatin and nuclear organization in this process, and to explore its potential as a target for cancer therapy.
By deciphering the mechanisms that ensure repair of DSBs in transcribed loci, this project holds the potential to expand our knowledge on the biogenesis of translocations and cancer onset, to provide new topoisomerase-poison-based therapeutic strategies but also to provide major breakthroughs in our understanding of neurodegenerative diseases.
To decipher the mechanisms of TC-DSBR, we are making use of the DIvA cell system, a potent model to study TC-DSBR, where about a hundred DSBs can be induced at annotated positions, mainly in transcriptionally active loci on the human genome.
One of the main questions addressed in GENECARE is the contribution of chromatin, chromosome architecture and nuclear organization in TC-DSBR.
Our previous work revealed that DSBs can move toward each other within the nuclear space to form DSBs clusters. We now recently reported that upon DSB induction in transcribing loci, a novel chromatin compartment forms, that we called the D-compartment (for DSB-induced compartment), which comprises not only in DSBs but also on a subclass of DNA Damage Responsive genes. We further found that the physical localization of these DDR genes in the D-compartment contributes to activate the DNA Damage response (Arnould, et al, Nature, 2023). Hence, altogether we discovered that DSBs, in mammalian cells, cluster together to form a novel chromatin compartment that is potentiating the response to DNA damage, while increasing the risk of translocations.
Furthermore, during the first period of GENECARE we have also reported that the TC-DSBR is regulated by the circadian clock. The circadian rhythm follows a daily cycle and regulates multiple physiological activities such as sleep cycle or hormone release. It involves cyclic chromatin transitions at a genome wide scale that necessitates the chromatin-modifying CLOCK-BMAL1 and PERIOD (PER) complexes. We found that the PER1/2 proteins, core circadian clock components, are recruited at TC-DSBs and regulates TC-DSBR. We report that TC-DSBs, as other types of persistent DSB in other organisms, are targeted to the nuclear envelope for RAD51 loading and HR repair, and that this step depends on PER1/2 proteins (Le Bozec et al, BioRXiv 2023).
One of the main questions addressed in GENECARE is the contribution of chromatin, chromosome architecture and nuclear organization in TC-DSBR.
Our previous work revealed that DSBs can move toward each other within the nuclear space to form DSBs clusters. We now recently reported that upon DSB induction in transcribing loci, a novel chromatin compartment forms, that we called the D-compartment (for DSB-induced compartment), which comprises not only in DSBs but also on a subclass of DNA Damage Responsive genes. We further found that the physical localization of these DDR genes in the D-compartment contributes to activate the DNA Damage response (Arnould, et al, Nature, 2023). Hence, altogether we discovered that DSBs, in mammalian cells, cluster together to form a novel chromatin compartment that is potentiating the response to DNA damage, while increasing the risk of translocations.
Furthermore, during the first period of GENECARE we have also reported that the TC-DSBR is regulated by the circadian clock. The circadian rhythm follows a daily cycle and regulates multiple physiological activities such as sleep cycle or hormone release. It involves cyclic chromatin transitions at a genome wide scale that necessitates the chromatin-modifying CLOCK-BMAL1 and PERIOD (PER) complexes. We found that the PER1/2 proteins, core circadian clock components, are recruited at TC-DSBs and regulates TC-DSBR. We report that TC-DSBs, as other types of persistent DSB in other organisms, are targeted to the nuclear envelope for RAD51 loading and HR repair, and that this step depends on PER1/2 proteins (Le Bozec et al, BioRXiv 2023).
Altogether our work revealed that TC-DSB repair is tightly regulated by chromosome architecture and nuclear organization. We are currently following up on these studies, to better understand the contribution of the nuclear envelope and chromatin folding in TC-DSBR.
Of importance, Topoisomerase II (TOP2)-derived endogenous DSBs were shown to mainly occur in transcribed loci on the genome, and TOP2 poisons such as etoposide and doxorubicin are used as a first trial to treat a large spectrum of cancer. While powerful to kill cancer cells, these TOP2 poisons can also trigger chromosomal translocations in healthy cells that can result in secondary malignancies (such as leukemia). Thus, delineating the full protein network that handles these types of damage may offer new therapeutic targets, in order to not only potentiate the efficacy but also to protect against the adverse effects of TOP2-poisons based cancer therapies.
Building up on our previous work, we are currently trying to fully characterize the TC-DSBR pathway, to understand its function as a caretaker of genome and epigenome integrity, and to explore its potential as a target for chemotherapy.
Of importance, Topoisomerase II (TOP2)-derived endogenous DSBs were shown to mainly occur in transcribed loci on the genome, and TOP2 poisons such as etoposide and doxorubicin are used as a first trial to treat a large spectrum of cancer. While powerful to kill cancer cells, these TOP2 poisons can also trigger chromosomal translocations in healthy cells that can result in secondary malignancies (such as leukemia). Thus, delineating the full protein network that handles these types of damage may offer new therapeutic targets, in order to not only potentiate the efficacy but also to protect against the adverse effects of TOP2-poisons based cancer therapies.
Building up on our previous work, we are currently trying to fully characterize the TC-DSBR pathway, to understand its function as a caretaker of genome and epigenome integrity, and to explore its potential as a target for chemotherapy.