Final Report Summary - CHROMOREPAIR (Deciphering the role of chromatin remodelling in DNA damage repair)
Maintenance of genome integrity is essential for accurate gene expression and epigenetic inheritance. Equally, a prolonged arrest of transcription due to deoxyribonucleic acid (DNA) damage is detrimental for cells and organisms as it may cause cell death either through apoptosis or by progressively depriving the cell of vital transcripts resulting in genome instability, inborn disorders and syndromes with premature aging and neurodegeneration.
To ensure fidelity of transcription, cells trigger a transcription-dependent genome surveillance pathway, termed transcription-coupled nucleotide excision repair (TC-NER) that ensures rapid removal of chromatin-impeding DNA lesions and prevents persistent stalling of transcription. Defective TC-NER is causatively linked to Cockayne syndrome, a rare severe genetic disorder with multisystem abnormalities that results in patients' death in early adulthood. The molecular mechanisms that couple transcriptional arrest to chromatin alteration and repair has proven quite intractable for many years now. Nevertheless, their elucidation is crucial to understand related disorders and their clinical features.
The aim of CHROMOREPAIR was to study the role of chromatin dynamics at sites of transcriptional arrest in ensuring repair of transcription-blocking lesions and resumption of transcription. During this period a number of key issues in TC-NER and associated chromatin dynamics at sites of damage-arrested transcription have been addressed:
(A) In collaboration with the Vermeulen-Marteijn group in Erasmus, Rotterdam, we identified KIAA1530 protein as the product of UVSSA gene. Mutations on UVSSA gene cause the UVsS, an unresolved NER deficiency disorder. More specifically, cells from UVsS patients have defective TC-NER and are unable to restore DNA damage-inhibited ribonucleic acid (RNA) synthesis. Using a stable isotope labelling with amino acids in cell culture (SILAC)-based proteomic approach we have shown that UVSSA is part of an ubiquitinated protein complex in response to UV irradiation. Employing a chromatin-based assay that allows immunoprecipitation of protein factors residing in close proximity to damage-stalled RNA polymerase II (RNAPII) complexes (ChIP-on-western) we have shown that the UVSSA protein interacts with the elongating RNAPII, localises specifically to UV-induced lesions, resides in chromatin-associated TC-NER complexes and is implicated in stabilising the TC-NER master organising protein ERCC6 (also known as CSB) by delivering the de-ubiquitinating enzyme USP7 to TC-NER complexes. Together, our findings indicate that UVSSA/USP7-mediated stabilisation of ERCC6 represents a critical regulatory mechanism of TC-NER in restoring gene expression (Schwertman et al. 2012, doi:10.1038/ng.2230).
(B) To study chromatin remodelling during DNA repair, we used ChIP-on-western assay in combination with fluorescence microscopy methods. In this particular study and in collaboration with the Vermeulen-Marteijn group, we show that H2A and H2B are evicted and replaced at an accelerated pace in nucleosomes at sites of UV-induced DNA damage, whereas H3.1 and H4 are not. This accelerated exchange of H2A / H2B is dependent on facilitating chromatin transcription (FACT) and in particular its subunit SPT16. This activity of FACT occurred in parallel to and independently of recruitment of TC-NER or GG-NER proteins. However, we found that both subunits of FACT were associated with active TC-NER complexes and RNAPII at sites of damage arrested transcription. Importantly, SPT16 was found to be implicated in the cellular response to UV-C damage by stimulating resumption of damage-inhibited transcription, indicating that transcription-associated DNA repair requires extensive chromatin remodelling. Our data uncover a crucial role for chromatin dynamics at the crossroads of transcription and the DNA damage response (Dinant et al., submitted).
(C) This part of the study was centred on the function of histone modifications and their modifiers in the repair of transcription-blocking damage. Employing ChIP-on-western, knock-down strategies and a number of patient derived cell lines with TC-NER deficiencies we found that nucleosomes flanking damage-arrested RNAPII / repair complexes are extensively modified at the N-terminal tails of histone H3 and H4 .These damage-induced chromatin changes are fully dependent on Cockayne Syndrome A and (CS)B proteins whereas proteins involved in chromatin modulation, such as the non-histone nucleosomal protein HMGN1, p300 and the histone acetyltransferase (HAT) GCN5, are required for the repair of transcription blocking lesions and the recovery of RNA synthesis. These CS-specific chromatin changes are not restricted to sites ultraviolet-photolesions or DNA helix-distortions that undergo TC-NER, but are also induced in response to other obstacles that arrest transcription, such as single strand breaks and trapped protein / DNA complexes.
These data strengthen the hypothesis that histone modifications play key regulatory roles in the resumption of transcription after the repair of DNA damages. We speculate that a regulatory cascade of specific histone modifications in concert with chromatin remodelling factors, presumably upstream of RNAPII arrest, is required to create accessibility and/or effectuate RNAPII backtracking. These early observations will be further investigated and completed in subsequent studies.
Impact
Deficiencies in DNA damage response pathways contribute significantly to a diversity of human diseases ranging from neurodegenerative syndromes to cancer. Despite the relatively large number of studies, we still lack important insights into the aetiology of CS and related disorders. The recent identification of the UVSSA gene (Schwertman et al., 2012) may help in dissecting the mechanistic difference between TC-NER-deficiency in UVSS and the additional pleiotropic effects in CS and thereby assist in our understanding of the molecular basis of the associated syndromes.
Another highly important issue is the regulation of the resumption of transcription after repair, given that improper restart leads to cellular malfunction and apoptosis, and DNA damage-induced inhibition of transcription is an important contributor to aging. Our studies, on chromatin dynamics and modification of histones at sites of damage-induced transcriptional arrest have revealed novel links between chromatin remodelling and a specific transcriptional event; the transcriptional restart upon DNA damage-induced arrest. Furthermore, the FP7-PEOPLE-2009-IEF grant has given the opportunity to MF to acquire a successful training and develop her leading abilities. In summary, during these two years, MF has been largely driving her own research that has led to one joined publication in a top Journal (Nature Genetics), one publication that is just submitted and an invitation as an expert of the field to contribute an article on 'transcription-coupled excision repair' on Cold Spring Harbour's Perspectives series that will be published in Spring 2013. Moreover, MF has been invited as a Speaker to two International Scientific Conferences in the Netherlands and has established long-lasting collaborations with leading labs in the Field. Importantly, the scientific achievements and abilities of MF have led to her election as Group Leader at BSRC 'Alexander Fleming' and to obtain an ERC-StG-2012 grant that will allow her to pursue further a successful scientific career.
To ensure fidelity of transcription, cells trigger a transcription-dependent genome surveillance pathway, termed transcription-coupled nucleotide excision repair (TC-NER) that ensures rapid removal of chromatin-impeding DNA lesions and prevents persistent stalling of transcription. Defective TC-NER is causatively linked to Cockayne syndrome, a rare severe genetic disorder with multisystem abnormalities that results in patients' death in early adulthood. The molecular mechanisms that couple transcriptional arrest to chromatin alteration and repair has proven quite intractable for many years now. Nevertheless, their elucidation is crucial to understand related disorders and their clinical features.
The aim of CHROMOREPAIR was to study the role of chromatin dynamics at sites of transcriptional arrest in ensuring repair of transcription-blocking lesions and resumption of transcription. During this period a number of key issues in TC-NER and associated chromatin dynamics at sites of damage-arrested transcription have been addressed:
(A) In collaboration with the Vermeulen-Marteijn group in Erasmus, Rotterdam, we identified KIAA1530 protein as the product of UVSSA gene. Mutations on UVSSA gene cause the UVsS, an unresolved NER deficiency disorder. More specifically, cells from UVsS patients have defective TC-NER and are unable to restore DNA damage-inhibited ribonucleic acid (RNA) synthesis. Using a stable isotope labelling with amino acids in cell culture (SILAC)-based proteomic approach we have shown that UVSSA is part of an ubiquitinated protein complex in response to UV irradiation. Employing a chromatin-based assay that allows immunoprecipitation of protein factors residing in close proximity to damage-stalled RNA polymerase II (RNAPII) complexes (ChIP-on-western) we have shown that the UVSSA protein interacts with the elongating RNAPII, localises specifically to UV-induced lesions, resides in chromatin-associated TC-NER complexes and is implicated in stabilising the TC-NER master organising protein ERCC6 (also known as CSB) by delivering the de-ubiquitinating enzyme USP7 to TC-NER complexes. Together, our findings indicate that UVSSA/USP7-mediated stabilisation of ERCC6 represents a critical regulatory mechanism of TC-NER in restoring gene expression (Schwertman et al. 2012, doi:10.1038/ng.2230).
(B) To study chromatin remodelling during DNA repair, we used ChIP-on-western assay in combination with fluorescence microscopy methods. In this particular study and in collaboration with the Vermeulen-Marteijn group, we show that H2A and H2B are evicted and replaced at an accelerated pace in nucleosomes at sites of UV-induced DNA damage, whereas H3.1 and H4 are not. This accelerated exchange of H2A / H2B is dependent on facilitating chromatin transcription (FACT) and in particular its subunit SPT16. This activity of FACT occurred in parallel to and independently of recruitment of TC-NER or GG-NER proteins. However, we found that both subunits of FACT were associated with active TC-NER complexes and RNAPII at sites of damage arrested transcription. Importantly, SPT16 was found to be implicated in the cellular response to UV-C damage by stimulating resumption of damage-inhibited transcription, indicating that transcription-associated DNA repair requires extensive chromatin remodelling. Our data uncover a crucial role for chromatin dynamics at the crossroads of transcription and the DNA damage response (Dinant et al., submitted).
(C) This part of the study was centred on the function of histone modifications and their modifiers in the repair of transcription-blocking damage. Employing ChIP-on-western, knock-down strategies and a number of patient derived cell lines with TC-NER deficiencies we found that nucleosomes flanking damage-arrested RNAPII / repair complexes are extensively modified at the N-terminal tails of histone H3 and H4 .These damage-induced chromatin changes are fully dependent on Cockayne Syndrome A and (CS)B proteins whereas proteins involved in chromatin modulation, such as the non-histone nucleosomal protein HMGN1, p300 and the histone acetyltransferase (HAT) GCN5, are required for the repair of transcription blocking lesions and the recovery of RNA synthesis. These CS-specific chromatin changes are not restricted to sites ultraviolet-photolesions or DNA helix-distortions that undergo TC-NER, but are also induced in response to other obstacles that arrest transcription, such as single strand breaks and trapped protein / DNA complexes.
These data strengthen the hypothesis that histone modifications play key regulatory roles in the resumption of transcription after the repair of DNA damages. We speculate that a regulatory cascade of specific histone modifications in concert with chromatin remodelling factors, presumably upstream of RNAPII arrest, is required to create accessibility and/or effectuate RNAPII backtracking. These early observations will be further investigated and completed in subsequent studies.
Impact
Deficiencies in DNA damage response pathways contribute significantly to a diversity of human diseases ranging from neurodegenerative syndromes to cancer. Despite the relatively large number of studies, we still lack important insights into the aetiology of CS and related disorders. The recent identification of the UVSSA gene (Schwertman et al., 2012) may help in dissecting the mechanistic difference between TC-NER-deficiency in UVSS and the additional pleiotropic effects in CS and thereby assist in our understanding of the molecular basis of the associated syndromes.
Another highly important issue is the regulation of the resumption of transcription after repair, given that improper restart leads to cellular malfunction and apoptosis, and DNA damage-induced inhibition of transcription is an important contributor to aging. Our studies, on chromatin dynamics and modification of histones at sites of damage-induced transcriptional arrest have revealed novel links between chromatin remodelling and a specific transcriptional event; the transcriptional restart upon DNA damage-induced arrest. Furthermore, the FP7-PEOPLE-2009-IEF grant has given the opportunity to MF to acquire a successful training and develop her leading abilities. In summary, during these two years, MF has been largely driving her own research that has led to one joined publication in a top Journal (Nature Genetics), one publication that is just submitted and an invitation as an expert of the field to contribute an article on 'transcription-coupled excision repair' on Cold Spring Harbour's Perspectives series that will be published in Spring 2013. Moreover, MF has been invited as a Speaker to two International Scientific Conferences in the Netherlands and has established long-lasting collaborations with leading labs in the Field. Importantly, the scientific achievements and abilities of MF have led to her election as Group Leader at BSRC 'Alexander Fleming' and to obtain an ERC-StG-2012 grant that will allow her to pursue further a successful scientific career.