Effect of CRISPR-Cas9-induced mutations in the Aire gene (APS1 syndrome) on protein conformation, mTEC cell transcriptome and their interaction with thymocytes

• Grant number: 17/10780-4
• Support Opportunities: Research Projects - Thematic Grants
• Duration: February 01, 2019 - January 31, 2024
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Geraldo Aleixo da Silva Passos Júnior
• Grantee: Geraldo Aleixo da Silva Passos Júnior
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Pesquisadores principais: Eduardo Antônio Donadi
• Associated researchers: Carlos Alberto Moreira Filho ; Daniella Arêas Mendes da Cruz ; Karina Fittipaldi Bombonato Prado ; Nuno Miguel de Oliveira Lages Alves
• Associated scholarship(s): 22/01801-6 - Effect of Crispr-Cas9-induced mutations in the Aire gene on transcriptional, post-transcriptional activity of mTEC cells and their function on thymocyte migration, BP.TT; 21/02081-4 - Effect of aire gene mutations induced by CRISPR-Cas9 on the transcriptional and post-transcriptional activity of mTEC cells and in its function on the thymocyte migration, BP.PD; 21/02459-7 - Effect of aire gene mutations induced in vivo in the thymus by CRISPR-Cas9 on alternative splicing of mRNAS that encode peripheral tissue antigens in mTEC cells, BP.PD; + associated scholarships 20/09664-2 - Transcriptional and post-transcriptional activity of thymocytes adhered to Aire mutant mTEC cells, BP.DD; 20/01879-0 - Induction of mutations in the Aire gene by means of CRISPR-Cas9 system in mTEC 3.10 cells, BP.IC; 19/23448-3 - Effect of Aire gene mutations (APS1 Syndrome) induced by CRISPR-Cas9 in protein conformation, transcriptome of mTEC cells and its interaction with thymocytes, BP.DD - associated scholarships

Abstract

The Autoimmune regulator (Aire) gene located on the human chromosome 21q22.3 or on mouse (Mus musculus) 10qC1, is a controller of the central immune tolerance and consequently the onset of autoimmune diseases. Aire share ~ 80% human-mouse similarity and it regulates the transcription of genes that encode peripheral tissue antigens (PTAs) in medullary thymic epithelial cells (mTECs) (promiscuous gene expression or PGE). The consequence of PGE is immunologic, i.e. the self-representation in the thymus, which is essential to negative selection of autoreactive thymocytes. Over 100 recessive mutations of human Aire gene have been described, which are associated with the manifestations of autoimmune polyglandular syndrome type 1 through genotype-phenotype association (APS1, OMIM # 240300). The Aire protein works with various "partners" (eg. DNA-PK, Sirt1 ...) in transcription elongation stage, releasing the RNA Pol II anchored next to the promoter regions of PTA genes. The genes controlled by Aire are referred to as Aire-dependent. Recently was discovered that the Fezf2 gene controls, in the thymus, the expression of an additional gene set (Fezf2-dependent genes). Our research group works with the expression of Aire and PGE in murine mTEC cells and we evidenced that changes in Aire expression are associated with changes in PGE, post-transcriptional control PTAs and the emergence autoimmune type 1 diabetes in NOD mice. Additionally, we assigned two "new" functions to the Aire gene: 1) regulation of miRNA expression in mTECs and 2) control of mTEC-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 system for mTEC-thymocyte adhesion), there is an increase of Aire expression as well as CD80, MHC-II and Fezf2 and 2) mutation induction within the Aire exon 3 through-CRISPR-Cas9 system disturbs the appearing of the Aire protein in the nucleus of mTECs and reduces the adhesion of these cells with thymocytes. These findings have opened important perspectives to better understand the biology of mTECs and explore points that still are elusive. Therefore, we have formulated the following hypotheses: 1) mTEC-thymocyte adhesion changes the transcriptome (mRNAs and miRNAs) of mTECs and 2) point mutations in the nucleotide sequence of the Aire gene (natural variants), the same mutations found in patients with APS1 (Aire PHD domain on exon 7) change the structural conformation of the Aire protein. Such changes might have a functional consequence in mTEC cells, e.g. changes in the transcriptome and / or mTEC-thymocyte adhesion and / or thymocyte migration towards mTECs. In this project, we make use of RNA-Seq and microarrays and as well as the mTEC-thymocyte adhesion model (murine model) and mTEC à thymocyte transwell migration to test these hypotheses. The technological innovation of the project is the use of CRISPR-Cas9 system for induction of "human" Aire mutations in murine mTEC cells. We will isolate mTEC Aire mutant clones to evaluate the expression of this gene, the modulation of the transcriptome (mRNAs and miRNAs) using RNA-Seq and / or microarrays. With the in silico modeling of the Aire mutant proteins, we have the opportunity to evaluate the effect of such changes in a structure-function basis. The scientific innovation is related to a better understanding on two levels: 1) the transcriptional response of mTECs when these cells adhere to thymocytes and 2) how Aire mutations alter the conformation of its protein and how these changes could be associated, even indirectly, with the mTEC-thymocyte adhesion, which is the key point for negative selection, the central immunological tolerance and preventing autoimmune diseases. (AU)

Scientific publications

(7)

(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)

OLIVEIRA, ERNNA H.; ASSIS, AMANDA F.; SPECK-HERNANDEZ, CESAR A.; DUARTE, MAX JORDAN; PASSOS, GERALDO A.. Aire Gene Influences the Length of the 3 ` UTR of mRNAs in Medullary Thymic Epithelial Cells. FRONTIERS IN IMMUNOLOGY, v. 11, MAY 28 2020. (13/17481-1, 17/10780-4)

COTRIM-SOUSA, LARISSA; FREIRE-ASSIS, AMANDA; PEZZI, NICOLE; TANAKA, PEDRO PARANHOS; OLIVEIRA, ERNNA HERIDA; PASSOS, GERALDO ALEIXO. Adhesion between medullary thymic epithelial cells and thymocytes is regulated by miR-181b-5p and miR-30b. Molecular Immunology, v. 114, p. 600-611, OCT 2019. (13/17481-1, 17/10780-4)

OLIVEIRA, ERNNA H.; ASSIS, AMANDA F.; SPECK-HERNANDEZ, CESAR A.; DUARTE, MAX JORDAN; PASSOS, GERALDO A.. Aire Gene Influences the Length of the 3 ' UTR of mRNAs in Medullary Thymic Epithelial Cells. FRONTIERS IN IMMUNOLOGY, v. 11, p. 12-pg., 2020-05-28. (17/10780-4, 13/17481-1)

ALVES, CINTHIA C.; ARNS, THAIS; OLIVEIRA, MARIA L.; MOREAU, PHILIPPE; ANTUNES, DINLER A.; CASTELLI, ERICK C.; MENDES-JUNIOR, CELSO T.; GIULIATTI, SILVANA; DONADI, EDUARDO A.. Computational and atomistic studies applied to the understanding of the structural and behavioral features of the immune checkpoint HLA-G molecule and gene. HUMAN IMMUNOLOGY, v. 84, n. 8, p. 10-pg., 2023-08-01. (17/10780-4)

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)

SANTOS, JADSON C.; DAMETTO, MARIANGELA; MASSON, ANA PAULA; FACA, VITOR M.; BONACIN, RODRIGO; DONADI, EDUARDO A.; PASSOS, GERALDO ALEIXO. The AIRE G228W mutation disturbs the interaction of AIRE with its partner molecule SIRT1. FRONTIERS IN IMMUNOLOGY, v. 13, p. 10-pg., 2022-09-06. (17/10780-4)

DUARTE, MAX JORDAN; MASCARENHAS, ROMARIO S.; ASSIS, AMANDA FREIRE; TANAKA, PEDRO PARANHOS; SPECK-HERNANDEZ, CESAR A.; PASSOS, GERALDO ALEIXO. Autoimmune regulator act in synergism with thymocyte adhesion in the control of lncRNAs in medullary thymic epithelial cells. Molecular Immunology, v. 140, p. 127-135, DEC 2021. (13/17481-1, 17/10780-4)