High throughput mapping of MLKL transcriptional regulatory elements

• Grant number: 19/13813-6
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Effective date (Start): October 25, 2019
• Effective date (End): October 24, 2020
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Ricardo Weinlich
• Grantee: Larissa Cardoso Zanetti
• Supervisor: Richard Sherwood
• Host Institution: Instituto Israelita de Ensino e Pesquisa Albert Einstein (IIEPAE). Sociedade Beneficente Israelita Brasileira Albert Einstein (SBIBAE). São Paulo , SP, Brazil
• Research place: Brigham and Women's Hospital (BWH), United States
• Associated to the scholarship: 17/25009-1 - Gene modulation of RIPK3 and MLKL and its impact in the sensibility to death by necroptosis, BP.DD

Abstract

Necroptosis is a caspase-independent cell death pathway that presents morphological hallmarks of accidental necrosis. It can be activated by different stimuli, which culminate in the activation of MLKL, the effector molecule of this pathway. Necroptosis importance has been demonstrated in many pathophysiological conditions, including bacterial and viral infections, cancer, ischemic insults and autoimmune diseases. In many of these settings, MLKL expression level was shown to vary significantly, impacting on the sensitivity towards cell death. However, there are currently very limited data concerning the transcriptional regulation of MLKL. Our aim is to elucidate the transcriptional regulatory elements of MLKL gene expression, characterizing enhancers, transcription factors and other mechanisms that are involved in its regulation. Using a multiplexed editing regulatory assay, we intend to perform a high throughput CRISPR-Cas9-mediated screening across 10kb of the cis-regulatory genome near MLKL gene and use GFP knock-in expression as an easier read out of MLKL gene activity. Combined with our previous results that had identified a minimal promoter region responsible for basal and interferon gamma activation, this project aims to reveal some novel regulatory elements of MLKL that have not yet been described. This knowledge will contribute to understand how MLKL is regulated and will help laying the basis for exploring the manipulation of MLKL expression in different pathophysiology. (AU)