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1.
Proc Natl Acad Sci U S A ; 116(41): 20411-20417, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31548379

RESUMO

Mutational signatures can reveal properties of underlying mutational processes and are important when assessing signals of selection in cancer. Here, we describe the sequence characteristics of mutations induced by ultraviolet (UV) light, a major mutagen in several human cancers, in terms of extended (longer than trinucleotide) patterns as well as variability of the signature across chromatin states. Promoter regions display a distinct UV signature with reduced TCG > TTG transitions, and genome-wide mapping of UVB-induced DNA photoproducts (pyrimidine dimers) showed that this may be explained by decreased damage formation at hypomethylated promoter CpG sites. Further, an extended signature model encompassing additional information from longer contextual patterns improves modeling of UV mutations, which may enhance discrimination between drivers and passenger events. Our study presents a refined picture of the UV signature and underscores that the characteristics of a single mutational process may vary across the genome.


Assuntos
Variação Genética , Genoma Humano , Melanoma , Raios Ultravioleta , Linhagem Celular Tumoral , Biologia Computacional , Dano ao DNA , Metilação de DNA , Humanos , Mutação , Regiões Promotoras Genéticas , Dímeros de Pirimidina
2.
PLoS Genet ; 14(12): e1007849, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30586386

RESUMO

Sequencing of whole cancer genomes has revealed an abundance of recurrent mutations in gene-regulatory promoter regions, in particular in melanoma where strong mutation hotspots are observed adjacent to ETS-family transcription factor (TF) binding sites. While sometimes interpreted as functional driver events, these mutations are commonly believed to be due to locally inhibited DNA repair. Here, we first show that low-dose UV light induces mutations preferably at a known ETS promoter hotspot in cultured cells even in the absence of global or transcription-coupled nucleotide excision repair (NER). Further, by genome-wide mapping of cyclobutane pyrimidine dimers (CPDs) shortly after UV exposure and thus before DNA repair, we find that ETS-related mutation hotspots exhibit strong increases in CPD formation efficacy in a manner consistent with tumor mutation data at the single-base level. Analysis of a large whole genome cohort illustrates the widespread contribution of this effect to recurrent mutations in melanoma. While inhibited NER underlies a general increase in somatic mutation burden in regulatory elements including ETS sites, our data supports that elevated DNA damage formation at specific genomic bases is at the core of the prominent promoter mutation hotspots seen in skin cancers, thus explaining a key phenomenon in whole-genome cancer analyses.


Assuntos
Melanoma/etiologia , Melanoma/genética , Mutação , Neoplasias Induzidas por Radiação/etiologia , Neoplasias Induzidas por Radiação/genética , Dímeros de Pirimidina/biossíntese , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Raios Ultravioleta/efeitos adversos , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Dano ao DNA , DNA de Neoplasias/genética , Humanos , Melanoma/metabolismo , Neoplasias Induzidas por Radiação/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-ets/metabolismo , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/efeitos da radiação , Neoplasias Cutâneas/metabolismo , Sequenciamento Completo do Genoma
3.
PLoS Genet ; 13(5): e1006773, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28489852

RESUMO

Sequencing of whole tumor genomes holds the promise of revealing functional somatic regulatory mutations, such as those described in the TERT promoter. Recurrent promoter mutations have been identified in many additional genes and appear to be particularly common in melanoma, but convincing functional data such as influence on gene expression has been more elusive. Here, we show that frequently recurring promoter mutations in melanoma occur almost exclusively at cytosines flanked by a distinct sequence signature, TTCCG, with TERT as a notable exception. In active, but not inactive, promoters, mutation frequencies for cytosines at the 5' end of this ETS-like motif were considerably higher than expected based on a UV trinucleotide mutational signature. Additional analyses solidify this pattern as an extended context-specific mutational signature that mediates an exceptional position-specific vulnerability to UV mutagenesis, arguing against positive selection. We further use ultra-sensitive amplicon sequencing to demonstrate that cell cultures exposed to UV light quickly develop subclonal mutations specifically in affected positions. Our findings have implications for the interpretation of somatic mutations in regulatory regions, and underscore the importance of genomic context and extended sequence patterns to accurately describe mutational signatures in cancer.


Assuntos
Melanoma/genética , Mutação , Regiões Promotoras Genéticas , Linhagem Celular Tumoral , Humanos , Taxa de Mutação , Motivos de Nucleotídeos , Telomerase/genética
4.
Nat Commun ; 14(1): 2701, 2023 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-37169761

RESUMO

Decades ago, it was shown that proteins binding to DNA can quantitatively alter the formation of DNA damage by UV light. This established the principle of UV footprinting for non-intrusive study of protein-DNA contacts in living cells, albeit at limited scale and precision. Here, we perform deep base-resolution quantification of the principal UV damage lesion, the cyclobutane pyrimidine dimer (CPD), at select human promoter regions using targeted CPD sequencing. Several transcription factors exhibited distinctive and repeatable damage signatures indicative of site occupancy, involving strong (up to 17-fold) position-specific elevations and reductions in CPD formation frequency relative to naked DNA. Positive damage modulation at some ETS transcription factor binding sites coincided at base level with melanoma somatic mutation hotspots. Our work provides proof of concept for the study of protein-DNA interactions at individual loci using light and sequencing, and reveals widespread and potent modulation of UV damage in regulatory regions.


Assuntos
Proteínas de Ligação a DNA , Raios Ultravioleta , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dano ao DNA , Dímeros de Pirimidina/metabolismo , DNA/metabolismo
5.
Sci Rep ; 12(1): 10322, 2022 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-35725896

RESUMO

Recent research on normal human tissues identified omnipresent clones of cells, driven by somatic mutations known to be responsible for carcinogenesis (e.g., in TP53 or NOTCH1). These new insights are fundamentally changing current tumor evolution models, with broad oncological implications. Most studies are based on surgical remnant tissues, which are not available for many organs and rarely in a pan-organ setting (multiple organs from the same individual). Here, we describe an approach based on clinically annotated post-mortem tissues, derived from whole-body donors that are routinely used for educational purposes at human anatomy units. We validated this post-mortem approach using UV-exposed and unexposed epidermal skin tissues and confirm the presence of positively selected NOTCH1/2-, TP53- and FAT1-driven clones. No selection signals were detected in a set of immune genes or housekeeping genes. Additionally, we provide the first evidence for smoking-induced clonal changes in oral epithelia, likely underlying the origin of head and neck carcinogenesis. In conclusion, the whole-body donor-based approach provides a nearly unlimited healthy tissue resource to study mutational clonality and gain fundamental mutagenic insights in the presumed earliest stages of tumor evolution.


Assuntos
Neoplasias , Carcinogênese/genética , Células Clonais/patologia , Humanos , Mutagênese , Mutação , Neoplasias/genética , Neoplasias/patologia
6.
Nat Rev Cancer ; 21(8): 500-509, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34230647

RESUMO

Tumour formation involves random mutagenic events and positive evolutionary selection acting on a subset of such events, referred to as driver mutations. A decade of careful surveying of tumour DNA using exome-based analyses has revealed a multitude of protein-coding somatic driver mutations, some of which are clinically actionable. Today, a transition towards whole-genome analysis is well under way, technically enabling the discovery of potential driver mutations occurring outside protein-coding sequences. Mutations are abundant in this vast non-coding space, which is more than 50 times larger than the coding exome, but reliable identification of selection signals in non-coding DNA remains a challenge. In this Review, we discuss recent findings in the field, where the emerging landscape is one in which non-coding driver mutations appear to be relatively infrequent. Nevertheless, we highlight several notable discoveries. We consider possible reasons for the relative absence of non-coding driver events, as well as the difficulties associated with detecting signals of positive selection in non-coding DNA.


Assuntos
Mutação , Neoplasias/genética , Regiões não Traduzidas , Animais , Humanos , Sequenciamento do Exoma
7.
Sci Rep ; 10(1): 18176, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097743

RESUMO

One of the ways in which genes can become activated in tumors is by somatic structural genomic rearrangements leading to promoter swapping events, typically in the context of gene fusions that cause a weak promoter to be substituted for a strong promoter. While identifiable by whole genome sequencing, limited availability of this type of data has prohibited comprehensive study of the phenomenon. Here, we leveraged the fact that copy number alterations (CNAs) arise as a result of structural alterations in DNA, and that they may therefore be informative of gene rearrangements, to pinpoint recurrent promoter swapping at a previously intractable scale. CNA data from nearly 9500 human tumors was combined with transcriptomic sequencing data to identify several cases of recurrent activating intrachromosomal promoter substitution events, either involving proper gene fusions or juxtaposition of strong promoters to gene upstream regions. Our computational screen demonstrates that a combination of CNA and expression data can be useful for identifying novel fusion events with potential driver roles in large cancer cohorts.


Assuntos
Aberrações Cromossômicas , Neoplasias/genética , Regiões Promotoras Genéticas , Variações do Número de Cópias de DNA , Bases de Dados Genéticas , Humanos
8.
FEBS J ; 286(21): 4209-4214, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31556220

RESUMO

Recent advances in cancer genomics are revolutionising our understanding of the genetic basis of tumour proliferation and of the impact of the immune system and microbiome in drug response. The 4th Cancer Genomics conference hosted by the European Association for Cancer Research covered breakthroughs in this field such as the search for novel cancer antigens and single-cell mapping of tumour evolution. This report focuses on the novel insights obtained from a variety of research groups under the banner of cancer genomics, as well as some insights of the general discussions held at the conference focussing on setting up a successful laboratory and possible alternatives in the scientific career.


Assuntos
Antígenos de Neoplasias/genética , Evolução Molecular , Genômica , Neoplasias/terapia , Humanos , Neoplasias/genética , Neoplasias/imunologia , Medicina de Precisão , Análise de Célula Única
10.
Mol Biol Cell ; 26(15): 2801-9, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26063733

RESUMO

Correct protein folding is fundamental for maintaining protein homeostasis and avoiding the formation of potentially cytotoxic protein aggregates. Although some proteins appear to fold unaided, actin requires assistance from the oligomeric molecular chaperone CCT. Here we report an additional connection between CCT and actin by identifying one of the CCT subunits, CCTε, as a component of the myocardin-related cotranscription factor-A (MRTF-A)/serum response factor (SRF) pathway. The SRF pathway registers changes in G-actin levels, leading to the transcriptional up-regulation of a large number of genes after actin polymerization. These genes encode numerous actin-binding proteins as well as actin. We show that depletion of the CCTε subunit by siRNA enhances SRF signaling in cultured mammalian cells by an actin assembly-independent mechanism. Overexpression of CCTε in its monomeric form revealed that CCTε binds via its substrate-binding domain to the C-terminal region of MRTF-A and that CCTε is able to alter the nuclear accumulation of MRTF-A after stimulation by serum addition. Given that the levels of monomeric CCTε conversely reflect the levels of CCT oligomer, our results suggest that CCTε provides a connection between the actin-folding capacity of the cell and actin expression.


Assuntos
Actinas/metabolismo , Chaperonina com TCP-1/metabolismo , Fator de Resposta Sérica/metabolismo , Animais , Células 3T3 BALB , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Chaperonina com TCP-1/genética , Humanos , Camundongos , Proteínas dos Microfilamentos/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transativadores/metabolismo
11.
BMC Res Notes ; 6: 429, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-24156781

RESUMO

BACKGROUND: The adenomatous polyposis coli (APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of ß-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion. APC mutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein. RESULTS: In order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable purification of recombinant forms of APC. Here we describe the characterisation of these anti-APC monoclonal antibodies (APC-NT) that specifically recognise endogenous APC both in solution and in fixed cells. Full-length APC(1-2843) and cancer-associated, truncated APC proteins, APC(1-1638) and APC(1-1311) were produced in Sf9 insect cells. CONCLUSIONS: Recombinant APC proteins were purified using a two-step affinity approach using our APC-NT antibodies. The purification of APC proteins provides the basis for detailed structure/function analyses of full-length, cancer-truncated and endogenous forms of the protein.


Assuntos
Proteína da Polipose Adenomatosa do Colo/isolamento & purificação , Anticorpos Monoclonais/biossíntese , Cromatografia de Afinidade/métodos , Proteínas Recombinantes/isolamento & purificação , Proteína da Polipose Adenomatosa do Colo/antagonistas & inibidores , Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos/administração & dosagem , Antígenos/química , Baculoviridae/genética , Cães , Expressão Gênica , Humanos , Células Madin Darby de Rim Canino , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera
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