MECHANISTIC EVALUATION OF PALB2 REGULATION AND A NEW PUTATIVE PROTEIN FUNCTION
Details
- Presentation type: DR - Doctoral Student
- Track: 3. Molecular Biology
- Keywords: DNA damage; PALB2; gene expression;
- 1 INCA
- 2 Brazilian National Cancer Institute, Rio de Janeiro, Brazil.
- 3 Lee Moffitt Cancer Center
- 4 Moffitt Cancer Center
- 5 Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro
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Introduction and objectives: Germline mutations in the PALB2 tumor suppressor gene are associated with breast and pancreatic cancer predisposition. It encodes for a homonymous protein with a pivotal role in genomic integrity maintenance through the homologous recombination repair pathway. PALB2 works as a scaffold protein, connecting BRCA1 and BRCA2 to promote the recruitment of the RAD51 recombinase to DNA damaged sites. Despite the growing interest in PALB2, its molecular function and gene regulation are not fully understood. Our goal is to evaluate putative new functions of PALB2 through the identification of new interaction partners and the characterization of PALB2 promoter locus, defining its regulation and minimal promoter region. Materials and methods: We performed a yeast two hybrid screening assay to identify putative new PALB2 partners. ROCK2 was recognized as a direct interaction partner. Confocal microscopy (CM) approach was applied to investigate the complex. Putative PALB2 promoter region was identified through the Genome Browser platform tracking down in silico H3k27Ac signal (euchromatin marker) upstream PALB2 gene. The selected region was generated through PCR using BJ cells genomic DNA as a template. Different deletion fragments (DF) were also generated to map the minimal promoter region. Promoter activity was assessed through a luciferase reporter expression assay in HEK293FT cells. Results and conclusion: ROCK2 was previously described as a player in centrossome together with BRCA2. CM identified PALB2 in centrosome structure, co-localizing with -tubulin (centrosome marker) during interfase and also in different phases of mitosis. We are current investigating a possible role for PALB2 in centrosome duplication together with ROCK2 and BRCA2. Moreover, we identified a 1001 nucleotide sequence upstream of the PALB2 as the putative gene promoter region (hereafter 1001). The 1001 displayed the ability to activate luciferase transcription (the reporter gene). To map 1001, DF were generated and a 224 nucleotide sequence (hereafter 224) was found to exhibit similar levels of transcription activation observed for 1001. Upon ionizing radiation (IR; 10Gy) treatment, both 1001 and 224 promote increased transcriptional activity when compared to non-IR cells. Interestingly, we also demonstrated high levels of PALB2 mRNA upon DNA damage induced both by IR (10Gy) and PARP inhibition (Olaparib 2,5uM – 10uM). Taking our data together, we demonstrated an new PALB2 subcellular localization and also a new putative interaction partner, shedding light on a novel PALB2 function. We also showed that PALB2 is upregulated upon different types of DNA damage and identified a minimal regulatory region for PALB2 expression.
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Anna Beatriz Ribeiro Elias
Boa tarde, Fábio. Muito obrigada pelas perguntas.
Bem, quanto a sua primeira pergunta, já foi realizada uma validação dos resultados obtidos no ensaio de Y2H, inclusive a possível interação entre PALB2 e ROCK2. Porém essa validação foi realizada de maneira in vitro utilizando fragmentos de ambas as proteínas (porção N-terminal de PALB2 utilizado no ensaio de Y2H e a porção N-terminal de ROCK2 pescada no mesmo ensaio). Nós agora estamos validando essa interação de duas formas: através de um ensaio de Pulldown em um sistema de células de mamífero (HEK293FT) utilizando a cadeia completa de ambas as proteínas e através da co-localização de PALB2 com ROCK2 no centrossomo através da microscopia confocal. Pretendemos ver também se essa interação ocorre de forma ciclo celular dependente, uma vez que nossa hipótese seja de que essa interação ocorra para regular a duplicação dos centrossomos - papel já caracterizado de ROCK2.
Quanto a sua segunda pergunta, nós ainda estamos investigando melhor esse fenótipo, mas nossa teoria até o momento é de que essa redução nos níveis de expressão do gene de PALB2 ocorre devido ao encaminhamento das células submetidas às altas doses de irradiação ou de Olaparibe para um quadro de morte celular e possivelmente uma mudança no quadro de expressão gênica.