Beyond B cells
Vasco Barreto, Gene Editing Lab, UCIBIO-NOVA
Host: Paula Videira, UCIBIO NOVA
ZOOM link: https://ucibio.pt/l/GuestSeminars
ID da reunião: 865 7786 0516
We study the mechanisms of gene editing in cells, including the natural gene editing of the antigen receptor genes that form the immunoglobulin and TCR genes and the artificial gene editing in Mammalian cells driven by CRISPR/Cas. Currently, we pursue three lines of research.
Because the process of somatic V(D)J rearrangement that each of the immunoglobulin alleles undergoes is error-prone, most B lymphocytes express only one immunoglobulin heavy chain (IgH) allele, a phenomenon known as allelic exclusion. Using murine hematopoietic systems generated from a single hematopoietic stem cell, we concluded that the IgH alleles rearrange independently (1). More recently, we used the same system to approach the epigenetics of monoallelic expression at the genome-wide level. Our in vivo transcriptomics approach revealed the first case of monoallelic expression in B cells that is already present in the original hematopoietic stem cell (2).
Activation-induced Cytidine Deaminase (AID) is the enzyme responsible for the editing of immunoglobulin genes in mature B cells, namely class switch recombination (CSR) and somatic hypermutation (SHM). During CSR, the IgH locus replaces the constant region, and thus the cell switches from the default expression of IgM to one of the IgGs, IgA or IgE. In 2003, we discovered that the C-terminus of AID is specifically required for CSR (but not SHM; 3). Several groups have replicated this finding, but identifying the underlying mechanism is one of the lingering problems in the field. I will present unpublished data suggesting that AID's C-terminus could promote a post-translation modification to displace AID from the deaminated base and allow recombination to proceed.
The parallels between the natural and artificial gene editing processes have attracted us to the CRISPR/Cas-driven editing in Mammalian cells (4). We have started a company that generates knock-ins and knockouts in cell lines. Most importantly, we are working on several experiments to improve the efficiency and enlarge the scope of this technology (e.g., 5).
1. Alves-Pereira CF, de Freitas R, Lopes T, Gardner R, Marta F, Vieira P, Barreto VM. Independent recruitment of Igh alleles in V(D)J recombination. Nat Commun. 2014 Dec 17;5:5623. doi: 10.1038/ncomms6623
2. Kubasova N, Alves-Pereira CF, Gupta S, Vinogradova S, Gimelbrant A, Barreto VM. In Vivo Clonal Analysis Reveals Random Monoallelic Expression in Lymphocytes That Traces Back to Hematopoietic Stem Cells. Front Cell Dev Biol. 2022 Aug 8;10:827774. doi: 10.3389/fcell.2022.827774
3. Barreto V, Reina-San-Martin B, Ramiro AR, McBride KM, Nussenzweig MC. C-terminal deletion of AID uncouples class switch recombination from somatic hypermutation and gene conversion. Mol Cell. 2003 Aug;12(2):501-8. doi: 10.1016/s1097-2765(03)00309-5
4. Vicente MM, Chaves-Ferreira M, Jorge JMP, Proença JT, Barreto VM. The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing. Front Cell Dev Biol. 2021 Sep 17;9:718466. doi: 10.3389/fcell.2021.718466
5. Vicente MM, Mendes A, Cruz M, Vicente JR, Barreto VM. A CyclinB2-Cas9 fusion promotes the homology-directed repair of double-strand breaks. BioRxiv 2019
Vasco M. Barreto (VMB) teaches Genetics at Faculdade de Ciências e Tecnologia da Universidade Nova (assistant professor) and at Escola Superior de Saúde Egas Moniz (invited professor), where he also heads Genetagus, a small biotech company specialized in gene editing. He has independently secured eight external competitive grants and fellowships, and has papers in some of the top journals in the life sciences (e.g., Nature Commun, Mol Cell, JEM), including some that have been highlighted in Nature Immunol, Faculty of 1000, and JEM.
VMB graduated in Biology at the Univ. of Lisbon (1994) and did his doctoral studies at the Inst. Pasteur in Paris, obtaining a Ph.D. in Immunology from the Université Pierre et Marie Curie (2001). He then moved to The Rockefeller University in New York, first as a Postdoc and then as a Research Associate. He returned to Portugal in 2008 to launch his group at the Instituto Gulbenkian de Ciência. He then obtained an Associate Researcher position (IF 2015) and moved to CEDOC (Universidade Nova), where he stayed until 2022.
Since his days as a graduate student, VMB has worked on two hallmarks of B cell molecular biology: the allelic exclusion of antigen receptor genes and the molecular mechanisms of antibody formation. This has allowed him to make key contributions to understanding the regulation of Activation-Induced Deaminase (AID), the editor of immunoglobulin genes, and the process of V(D)J recombination. More recently, he has developed a genome-wide approach to study monoallelic expression and has been exploring the parallels between the natural processes of gene editing in mammals and CRISPR-based gene editing.