Priming da cromatina: Desde a regulação no desenvolvimento a terapias inovadoras contra o cancro

The main GOAL of this proposal is to characterize how pioneer factors regulate chromatin status during epithelial differentiation to develop strategies to attenuate the responsiveness of epithelial cells to oncogenic signals. This investigation presents a challenge due to the complex nature of human gene networks implicated in cancer evolution. Additionally, manipulating chromatin modulators like pioneer factors often results in developmental defects [7–10]. To address this, novel experimental methodologies are required to elucidate the impact of pioneer factor-mediated chromatin modulation on cellular responses to proliferative signals. We will be to take advantage of the conservation of key elements and the simplicity of the Drosophila eye imaginal disc model (Fig. 2A) to study how the differences in the epigenetic states modulate cellular response to an oncogene. Preliminary data indicates that committed retinal cells respond to abnormal oncogenic Hippo signalling because their chromatin accessibility profile is distinct to undifferentiated progenitor cells resistant to this transformation (Fig. 3). In particular, we found a clear switch of chromatin accessibility on motifs recognized by the pioneer factor grainy head (grh) and a striking correlation between enrichment on these motifs and promotion of cell over-proliferation by the activated form of Yki (Fig. 3B). The HYPOTHESIS that chromatin priming by pioneer factors is important for cell differentiation-dependent oncogenesis will be tested by following specific objectives: Objective 1: Characterize Grh and Yki crosstalk in tissue growth regulation. While multiple factors have been proposed to regulate the cellular output of Hippo signalling at the tissue level, little is known about the cell-intrinsic differences that can dictate its proliferative response. Here we will investigate how Grainy head (Grh) regulates Yorkie (Yki) induced proliferation, focusing on its impact on cellular response to oncogenic stimuli and whether this effect is mediated by direct regulation of chromatin accessibility on enhancers of Yki target genes. Objective 2: Investigate Grh Pioneering Activity regulation during development and in Oncogenic Signalling. Our preliminary results show that despite its ubiquitous expression pattern, Grh activity only peaks in intermediate states of differentiation (Fig. 3A). Characterizing how Grh is regulated is key for the development of the best therapeutical strategy to regulate the cellular response to abnormal Hippo signalling. We will develop new tools using the CRISPR-Cas9 technologies to manipulate and characterize the different Grh isoforms and/or its interactors and assess their role in cellular response to oncogenic signals. Objective 3: Manipulation of Grh Priming for Tumour Resistance. The overarching goal of this project is to learn how to manipulate Grh priming of chromatin to restrict cellular response to strong oncogenic signals. Once determined the optimal strategy to modulate Grh-dependent chromatin accessibility dynamics, we will develop novel tools (RNAis or using Grh DNA binding properties to deliver chromatin repressive factors) to turn cells insensitive to Yki oncogenic signals. These strategies will then be tested for efficiency in human breast cancer cell lines. Achieving these objectives has the potential to lead to innovative approaches for preventing or restricting tumour development.