Unraveling the role of RNF12 and REX1 in the initiation of human X chromosome inactivation.

• Grant number: 17/23243-7
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Effective date (Start): December 01, 2017
• Effective date (End): November 30, 2018
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Maria Dulcetti Vibranovski
• Grantee: Joana Carvalho Moreira de Mello
• Supervisor: Joost Gribnau
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Research place: Erasmus University Rotterdam (EUR), Netherlands
• Associated to the scholarship: 15/03610-0 - In silico analysis of the X-chromosome epigenetic state in human pre-implantation embryos, BP.PD

Abstract

In female mammals, one X chromosome is transcriptionally inactivated (XCI), leading to dosage compensation between sexes, which is fundamental for embryo viability. We recently analyzed X chromosome allele-specific expression globally during human embryonic development, and identified the XCI process to start during human preimplantation development at the blastocyst stage in a random pattern, different from mouse where XCI begins at 4-cell stage in an imprinted way.Most studies addressing the regulation of XCI involve mouse studies. Using mouse as a model system, the Gribnau laboratory revealed that, in vivo and in vitro, X-encoded RNF12 and autosomally encoded REX1 play a crucial role in XCI initiation, by dose dependent break down of REX1 by RNF12. Mouse cells with an Rnf12 deletion fail to initiate XCI due to stabilization of REX1. However, in compound double knockouts of Rnf12 and Rex1, the XCI phenotype observed in Rnf12-/- cells is rescued. So far, human embryonic stem cell lines could not be used to study XCI, as the inactive X was already established. More recently, improvements in tissue culture conditions resulted in the establishment of naïve human pluripotent stem cells that initiate XCI upon differentiation, opening exciting opportunities for studying the regulation of XCI in a human model system for the first time.We propose to investigate whether, similar to mouse, human RNF12 and REX1 are key regulators of XCI, using naïve female human pluripotent stem cells. The CRISPR/Cas9 technology will be applied to generate different combinations of RNF12, REX1 and XIST mutations to study their respective role in human XCI.