Investigating the role of HEB and its interaction with GFI1 in t-cell acute lymphoblastic leukaemia (T-ALL)

Alazmi, Shimaa (2024). Investigating the role of HEB and its interaction with GFI1 in t-cell acute lymphoblastic leukaemia (T-ALL). University of Birmingham. Ph.D.

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Abstract

An intricate network of transcription factors determines gene expression patterns that define cellular identity. Development of T-cells involves dynamic changes, including the activation and silencing of transcription factors. As part of the transcriptional landscape, TAL1 forms a heterodimer with E-proteins such as E2A, E2-2, and HEB, which bind DNA and activate transcription as part of a complex, including LIM-only protein 2 (LMO2), Lim domain-binding protein 1 (LDB1), and GATA proteins. The overexpression of LMO proteins, as well as an abnormal expression of TAL1, has been identified as hallmarks of T-cell Acute Lymphoblastic Leukaemia (T-ALL). Considering the high relapse rates, drug resistance, and poor prognosis of T-ALL, it is imperative to investigate the molecular mechanisms underlying T-ALL in order to develop effective therapeutic strategies.

E-proteins have important roles in the progression of haematopoiesis, orchestrating thymocyte maturation and developmental progression. HEB, a member of the E-protein protein family, plays a pivotal role in guiding thymocytes toward the double positive stage and beyond. HEB exists in two isoforms: HEB canonical and HEB alternative. A previous study of HEB immunoprecipitation followed by mass spectrometry identified the transcription factor GFI1 as a potential new member of the complex. Recent research has shown that GFI1 has dual functions, acting as a transcriptional activator in conjunction with IKAROS and as a transcriptional repressor by recruiting KDM1A. However, the role of GFI1 in T-ALL remains unexplored.

This study identified the interaction between HEB and GFI1, particularly with the HEB alternative isoform, in four human T-ALL cell lines (ARR, DU528, HSB2, and CCRFCEM), using proteomic analysis and genome-wide studies. The genomic distribution of GFI1 was examined using chromatin immunoprecipitation (ChIP)followed by next generation sequencing (ChIP-seq). Comparisonof these data with previous ChIP-seqdatarevealed that distinct binding complexes exists in ARR and in the other three cell lines.

The role of HEB in T-ALL was further dissected using CRISPR-Cas9 gene editing. Targeting \(\textit{TCF12}\), the gene encoding HEB, led to the loss of the expression of the canonical isoform of HEB. This reduced the expression of its alternative isoform, providing evidence for a regulatory interaction between the two isoforms, as well as reduced the expression of GFI1. There was no significant morphological change or chromosomal instability in the ARR cells following HEB deletion, indicating that \(\textit{TCF12}\) deletion does not cause overt cellular abnormalities. In response to the loss of HEB, LMO2 is observed to possess increased association with the DNA, increasing chromatin accessibility, and potentially compensating for the loss of HEB through enhanced DNA-cofactor interactions. Overall, these findings provide valuable insight into the molecular basis of T-ALL, which could lead to the development of targeted therapeutic interventions.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):