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The role of bHLH-LZ transcription factors on epigenetic programming during cell proliferation and differentiation

Final Activity Report Summary - UMSCEP (The role of bHLH-LZ transcription factors on epigenetic programming during cell proliferation and differentiation)

Metastatic melanoma represents a great challenge for cancer therapies because of its increasing frequency and its notorious resistance to chemotherapy. Constitutive pigmentation of the skin and the tanning response in response to ultra-violet (UV) increase protection against UV-induced deoxyribonucleic acid (DNA) damage and thus prevent melanoma formation. A key question is to identify the intracellular and genetic mechanisms that control differentiation in melanocytes leading to pigmentation and proliferation in melanoma. In melanocytes, the expression of several key genes is highly regulated by a cell type-specific bHLH-LZ transcription factor Mitf that controls differentiation, cytoskelatal organisation, survival and proliferation of melanoblasts, melanocyte stem cells and melanoma. The ubiquitously expressed basic-helix-loop-helix leucine zipper upstream stimulating transcription factors (USF-1 and USF-2) also regulate the expression of the same target genes after binding specific consensus elements E-box (CANNTG).

The main goal of this project was to understand which were the mechanisms that controlled the respective and specific actions of these transcription factors, essential for melanocytes and melanoma progression. We focussed our study on the master regulator gene of pigmentation tyrosinase (TYR), whose expression was constitutively and after UV irradiation induced by the USF and Mitf transcription factors. By the use of different cellular models and molecular techniques, such as immunoprecipitation of chromatin and real time polymerase chain reaction (PCR), we described for the first time an accurate regulation of the transcription of TYR after UV, associated with a spatial and temporal regulation of promoter occupancy by the bHLH-LZ. We showed that it was not only Mitf but also USF transcription factors that played a crucial and cooperative function for the regulation of TYR expression. Regulation of this expression was associated with the recruitment of the transcriptional machinery onto the promoter. Since the comprehension of molecular processes was crucial for the understanding of normal cells stability and melanoma formation, we planned to widen these results for other target genes, involved in cytoskelatal organisation, survival and proliferation of melanoblasts, melanocyte stem cells and melanoma.

During the two years of the fellowship, I also addressed an important question regarding how transcription factors interpreted the output from signal transduction pathways to drive distinct programs of gene expression that underpinned development and disease. The ubiquitously expressed USF-1 was a key component of the tanning process. Following UV irradiation, USF-1 was phosphorylated by the p38 stress-activated kinase on threonine 153 and directly up-regulated the expression of several genes, such as proopiomelanocortin (POMC), melanocortin-1 receptor (MC1R), TYR, tyrosine-related protein (TYRP-1) and dopachrome tautomerase (DCT). However, the way in which phosphorylation on T153 might affect the activity of USF-1 remained unclear. We showed here that, in response to DNA-damage, oxidative stress and cellular infection USF-1 was acetylated on K199 in a phospho-T153 dependent fashion. Phospho-acetylated USF-1 was nuclear and interacted with DNA, but displayed altered gene regulatory properties. Phospho-acetylated USF-1 was thus proposed to be associated with loss of transcriptional activation properties towards several target genes implicated in pigmentation process and cell cycle regulation. The identification of this critical stress-dependent USF-1 modification offered new insights into understanding USF-1 gene expression modulation associated with cancer development.

The project set out to address a key question in biology, i.e. how cells established and maintained a programme of gene expression that determined their identity. This was relevant to both genetic diseases of development and cancer where mechanisms that implemented the cell identity or the role of USF were deregulated. Moreover the project would also address the role of USF in gene regulation in general, which was particularly important given that the deregulation of USF was also implicated in a wide number of diseases, such as hyperlipidemia and diabetes. Understanding how USF influenced Mitf activity would therefore be of particular relevance to cancer metastasis in general.