High resolution dissection of non-coding determinants of disease

Información del proyecto

B-ALLeles

Identificador del acuerdo de subvención: 101061151

DOI

10.3030/101061151

Proyecto cerrado

Fecha de la firma de la CE 8 Agosto 2022

Fecha de inicio 1 Noviembre 2022

Fecha de finalización 31 Octubre 2024

Financiado con arreglo a

• Marie Skłodowska-Curie Actions (MSCA)

Coste total

Sin datos

Aportación de la UE

€ 183 600,96

Coordinado por

ST. ANNA KINDERKREBSFORSCHUNG GMBH
Austria

Periodic Reporting for period 1 - B-ALLeles (High resolution dissection of non-coding determinants of disease)

Período documentado: 2022-11-01 hasta 2024-10-31

Resumen del contexto y de los objetivos generales del proyecto

While mutations at protein-coding sequences have been extensively characterized, the functional role of disease-associated non-coding variants remains largely elusive. In fact, germline non-coding variants are often associated with increased risk of childhood cancers, especially in the case of B cell acute lymphoblastic leukemia (B-ALL). Furthermore, non-coding variants have been shown to affect transcription factor (TF) binding, therefore altering the activity of gene regulatory elements and the expression of their associated gene. In B-ALL, while some risk-variants are associated with B cell lineage-specific genes, others are associated with genes with uncharacterized function. Thus, in this project, we hypothesize that non-coding variants associated with B-ALL affect the activity of distal regulatory elements, modulating the expression of their target genes and other B-cell lineage-specific genes (objective 1). We further postulate that risk-associated genes with uncharacterized function regulate lineage-specific genes (objective 2), playing a crucial role in B-cell development and B-ALL initiation and progression (objective 3). To test these hypotheses, I have knocked-out risk-variant containing regions (towards objective 1), characterized the molecular mechanism of a risk-associated genes using genomics and proteomics (towards objective 2) and am currently studying the physiological relevance of the associated genes and its interaction partners in B cell development in the mouse (towards objective 3).

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

Focusing on non-coding variants clustering in one genomic locus, we have identified a 7.8kb intronic region containing one lead risk-variant which acts as an enhancer of the risk-associated gene. We further characterized the molecular mechanisms of this gene using a combination of proteomics, genomics and transcriptomics. By performing proteomics, we have identified the interactors of our protein of interest and have further used AlphaFold to model these interactions. Following, we knocked-out our gene of interest and performed transcriptomics and genome-wide occupancy profiling of the protein of interest, its interactors and several active and repressive histone marks in WT and KO cells. This informed us on the targets of our protein and its molecular mechanism. Finally, our protein of interest and its interactors are being targeted in inducible-Cas9 murine fetal livers, which are in turn used for in vivo reconstitution experiments. Based on the molecular studies, we expect the depletion of interacting proteins to phenocopies the depletion of the protein of interest.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

While, in this project, we have investigated the role of non-coding variant clusters by knocking-out large genomic regions, this work has served as a basis for the establishment of dense mutagenesis CRISPR screens of other risk-variant containing regions and the development of specialized analysis tools. This work has recently been published in the form of a pre-print (https://doi.org/10.1101/2024.09.09.612085). Furthermore, important genomics (CUT&Run) and proteomics (AP-MS) protocols have been established in the lab during this project and have been employed by members of the Seruggia group. The use of these methods to answer various other questions has resulted in several new collaborations. Finally, during this project, we benchmarked protocols to study B cell development upon genetic perturbation in inducible Cas9-mice, which will be employed in the study of other hematological malignancies in the lab.