Biology subjects

Mascarenhas, R. S.

Publications and source records attributed to Mascarenhas, R. S..

3 recordsLinked to original sources

bioRxiv2023

The ribonucleoprotein-mediated CRISPR-Cas9 system induces recurrent Aire gene mutations in contrast to the nickase expression vector in murine in vitro or in vivo models

Although in vitro mTEC cultures can help determine the role of the autoimmune regulator (Aire) gene, the mechanisms of Aire mutations were identified using Aire mutant mice. Nevertheless, long-term cultures of mTECs from mice are difficult to establish. To overcome this, we used a CRISPR-Cas9 system to edit Aire in a murine mTEC line in vitro and mouse embryos. Two ribonucleoprotein (RNP) complexes were designed to separately target Aire exons 6 and 8. NHEJ-derived indels or HDR-derived mutations were obtained. Recurrent NHEJ-derived mutations were observed among editions, one in exon 6 (del 3554G) and the other in exon 8 (del 5676_5677TG), i.e., the exon 6 mutation was kept in an mTEC clone edited in vitro and in vivo in a mouse, and the exon 8 mutation was kept in mTECs in vitro. None of the mutations obtained with the nickase system were recurrent, indicating the participation of the RNP complex.

molecular biology↗

bioRxiv2021

The absence of the autoimmune regulator gene (AIRE) impairs the three-dimensional structure of medullary thymic epithelial cell spheroids

Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown that AIRE knockout (KO) mice have a small and disorganized thymic medulla; however, whether Aire influences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed an in vitro model system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. The effect of Aire on mTEC-mTEC adhesion was evaluated by comparing AIRE wild type (AIREWT) versus Aire KO (AIRE-/-) mTECs. Considering the 3D spheroid model evaluated, we reported that the absence of AIRE disorganizes the 3D structure of mTEC spheroids, promotes a differential regulation of mTEC classical surface markers, and modulates genes encoding adhesion and other molecules.

immunology↗

bioRxiv2021

Autoimmune regulator act in synergism with thymocyte adhesion in the control of lncRNAs in medullary thymic epithelial cells

The autoimmune regulator (Aire) gene in medullary thymic epithelial cells (mTECs) encodes the AIRE protein, which interacts with its partners within the nucleus. This "Aire complex" induces stalled RNA Pol II on chromatin to proceed with transcription elongation of a large set of messenger RNAs and microRNAs. Considering that RNA Pol II also transcribes long noncoding RNAs (lncRNAs), we hypothesized that Aire might be implicated in the upstream control of this RNA species. To test this, we employed a loss-of-function approach in which Aire knockout mTECs were compared to Aire wild-type mTECs for lncRNA transcriptional profiling both in vitro and in vivo model systems. RNA sequencing enables the differential expression profiling of lncRNAs when these cells adhere in vitro to thymocytes or do not adhere to them as a way to test the effect of cell adhesion. Sets of lncRNAs that are unique and that are shared in vitro and in vivo were identified. Among these, we found the Aire-dependent lncRNAs as for example, Platr28, Ifi30, Morrbid, Malat1, and Xist. This finding represents the first evidence that Aire mediates the transcription of lncRNAs in mTECs. Microarray hybridizations enabled us to observe that temporal thymocyte adhesion modulates the expression levels of such lncRNAs as Morrbid, Xist, and Fbxl12o after 36h of adhesion. This finding shows the existence of a synergistic mechanism involving a link between thymocyte adhesion, Aire, and lncRNAs in mTECs that might be important for immune self-representation.

molecular biology↗