Effect of Aire gene mutations (APS1 Syndrome) induced by CRISPR-Cas9 in protein conformation, transcriptome of mTEC cells and its interaction with thymocytes

• Grant number: 19/23448-3
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: January 01, 2020
• End date: August 31, 2022
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Eduardo Antônio Donadi
• Grantee: Ana Carolina Monteleone Cassiano
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 17/10780-4 - Effect of CRISPR-Cas9-induced mutations in the Aire gene (APS1 syndrome) on protein conformation, mTEC cell transcriptome and their interaction with thymocytes, AP.TEM

Abstract

The Autoimmune regulator (Aire) gene (human chromosome 21q22.3 and murine Mus musculus 10qC1), is a controller of central immune tolerance and of the emergence of autoimmune diseases. The Aire sequence shows ~ 80% man-mouse similarity and this gene regulates the transcription of Peripheral Tissue Antigens (PTAs) genes in medullary Thymic Epithelial Cells (mTECs). This phenomenon was called PGE (promiscuous gene expression). The meaning of the PGE is immunological, that is, the self-representation in the thymus. More than 100 recessive mutations in the human Aire gene have been described, many of which are associated with manifestations of the polyglandular autoimmune syndrome type 1 (APS1) (genotype-phenotype association) (OMIM # 240300). The Aire protein works in conjunction with several "partners" (eg, DNA-PK, Sirt1 ...) in the transcription elongation step by releasing the RNA Pol II anchored to the PTA and other promoter regions. The genes controlled by Aire are referred to as Aire-dependent. Recently, researchers found that the Fezf2 gene controls, in the thymus, the expression of another set of PTAs (Fezf2 -dependent) genes. Our group works with the expression of Aire and PGE in murine mTEC cells and we have already shown that variations in the expression of Aire are associated with changes in PGE, in the posttranscriptional control of PTAs and in the onset of autoimmune type 1 diabetes in NOD mice. In addition, we assigned two "new" functions to the Aire gene: 1) regulation of the expression of microRNAs (miRNAs) in mTECs and 2) control of mTECs-thymocyte adhesion. In addition, we observed some interesting properties of mTECs: 1) when these cells are cultured together with thymocytes (co-culture as a model for mTEC-thymocyte adhesion), there is an increase in the expression of Aire and also of CD80, MHC-II and Fezf2 and 2) the induction of mutation in Aire exon 3 by means of the CRISPR-Cas9 system disrupts the location of the Aire protein in the nucleus of the mTEC cells and also decreases the adhesion of these cells with thymocytes. These findings opened important perspectives to better understand the biology of mTEC cells and to explore points still elusive. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

MONTELEONE-CASSIANO, ANA CAROLINA; DERNOWSEK, JANAINA A.; MASCARENHAS, ROMARIO S.; ASSIS, AMANDA FREIRE; PITOL, DIMITRIUS; SANTOS MOREIRA, NATALIA CHERMONT; SAKAMOTO-HOJO, ELZA TIEMI; MARDEGAN ISSA, JOAO PAULO; DONADI, EDUARDO A.; PASSOS, GERALDO ALEIXO. The absence of the autoimmune regulator gene (AIRE) impairs the three-dimensional structure of medullary thymic epithelial cell spheroids. BMC MOLECULAR AND CELL BIOLOGY, v. 23, n. 1, p. 17-pg., 2022-03-24. (19/23448-3, 17/10780-4)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

CASSIANO, Ana Carolina Monteleone. Mutation in the Aire gene induced by the CRISPR-Cas9 system in an in vitro murine model (thymic medullary epithelial cells): impact on intercellular adhesion and antigen presentation. 2024. Doctoral Thesis - Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) Ribeirão Preto.