Large-scale identification of genes that mediate cellular quiescence and differentiation for lactogenesis induced by laminin

Author(s)

Mayara Carolinne Silva Botelho

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2022-09-21

Examining board members

Alexandre Bruni Cardoso; Vanessa Morais Freitas; Pedro Alexandre Favoretto Galante

• Advisor: Alexandre Bruni Cardoso

Abstract

Proliferation is one of the most fundamental attributes of living systems. But unlike unicellular organisms, complex organisms cells stop proliferating, entering a state termed quiescence, even in the presence of nutrients. Factors present in the cell microenvironment, such as the basement membrane (a specialized extracellular matrix (ECM) compartment assembled over a laminin network), trigger and sustain quiescence, contributing to tissue differentiation observed in multicellular organisms. To elucidate the mechanisms underlying acquisition and maintenance of quiescence and cell differentiation, we performed a forward genetic screen in cells exposed to a laminin-111 rich ECM (lrECM) that induce quiescence and differentiation. For this, we generated non-tumoral mammary epithelial cells expressing the fluorescent cell cycle sensor FUCCI, as well as a differentiation fluorescence reporter for lactogenesis (βCas-CFP). Cells were transfected with a CRISPR/Cas9 genome-wide lentiviral pooled library and treated with lrECM and prolactin for 72h, time that most cells enter quiescence and differentiate. The cells were sorted in three distinct populations: mCitrine+ (proliferative), mCherry+ (quiescent) mCherry+/CFP+ (quiescent and differentiated) and sequenced for determining enrichment of CRISPR guideRNAs targeting genes mediating acquisition of quiescence and differentiation. Among the hits we found genes involved in cell cycle, epithelial cell organization and metabolic pathways. Using gene silencing in fluorescence microscopy-based assays, we validated novel mediators of quiescence and differentiation. This work expands our basic understanding of signaling proteins that act during quiescence and differentiation acquisition in physiological contexts, laying the groundwork for translational studies in lactation and for cancer, a disease in which quiescence regulation is lost. (AU)

• FAPESP's process: 17/25437-3 - High-throughput identification of genes that mediate cell quiescence induced by laminin
• Grantee: Mayara Carolinne Silva Botelho
• Support Opportunities: Scholarships in Brazil - Doctorate