1. E2A – bHLH transcription factor-fusion proteins in Leukemia
Sarah Woznicki

2. E2A Gene TCF3 (Transcription Factor 3)
ITF1 Immunoglobulin Enhancer Binding
Location – chromosome 19
19p13.3

3. E Protein Family E2A protein – part of family of E proteins
Includes HEB and E2-2 proteins
E proteins are part of the basic helix-loop-helix (bHLH) family of DNA-binding transcription factors
Conserved helix-loop-helix (HLH) domain – imparts dimerization capabilities between HLH members
Conserved basic region – amino to HLH; allows HLH dimers to bind to DNA

4. E Protein Family
Mammalian E proteins also share 2 conserved transcriptional activation domains
AD1 domain
Loop-helix (LH) domain

5. E Protein Family Class I HLH proteins
First identified based on their ability (as homo-, heterodimers) to bind with high affinity to DNA sequence – C-A-N-N-T-G – the E box site
E box sites present broadly in the genome

6. E2A Gene Encodes 2 E proteins E12 E47
Arise through differential splicing of the exon encoding for the HLH domain

7. E2A – function in normal cells
Transcription regulation
Heterdimerizes with tissue-specific bHLH proteins
Essential for normal B-cell hematopoiesis

8. E2A – Role in Myogenesis
3 stages of myogenesis- development of skeletal muscle cells
1- determination of precursor myoblasts
2- proliferation (and possible migration) of these precursor muscle cells
3- differentiation into mature muscle
4 genes – signal conversion of cell lines into muscle cells- myoD, myogenin, myf5, mrf4
Members of bHLH family

9. E2A – Role in Myogenesis
E2A protein -when bound to one of these myogenic proteins- increases the myogenic protein’s affinity for DNA binding
The muscle regulatory protein- E2A heterodimer then works directly or with other transcription factors to regulate expression of muscle-specific proteins needed in development

11. E2A – Role in Lymphocyte Development
Common lymphoid progenitor cell can develop into 3 distinct cell types
B lymphocytes
T lymphocytes
NK cells
During lineage commitment / maturation, B and T lymphocyte development defined at different stages by
rearrangement of antigen receptor genes
gain/loss of cell surface and intracellular proteins
responses to growth survival factors
Gene expression at these levels controlled by bHLH transcription factors

12. E2A – Role in Lymphocyte Development
E box – found in promoter and enhancer regions of a wide variety of B and T lineage specific genes – to which E proteins can bind
E2A – works with other transcription factors to maintain expression of target genes that promote lymphocyte maturation appropriately at each stage of development
Experimental support
E2A deficiency – leads to complete lack of B lineage cells
Over-expression of E12 – sufficient to activate transcription of several B lineage- specific genes in non-lymphoid cells

14. Function of E2A at developmental stages
B-cell development
Commitment
IgH gene rearrangement
Proliferation/ survival
IgL gene rearrangement
Isotype switching
T-cell development
Works in similar mechanisms to promote stage progression

15. E2A proteins and Leukemia
Implicated in cellular proliferation and apoptosis
2 influences supported by current models
E2A acts as a tumor suppressor
Promoting B cell differentiation
Inhibiting cell cycle progression
Promoting apoptosis
Suggest that mutations in E2A gene inhibit normal E2A activity and lead to abnormal proliferation/ survival of early B cells – resulting in ALL
Paradoxically, ectopic expression of E2A mutants shown to decrease proliferation/ survival of early B cells

16. E2A gene
E2A locus- target of 2 chromosomal translocations associated with human leukemia
Result in Gene Fusions
t (1; 19) (q23; p13) – E2A transactivating domains join to DNA binding domain of PBX1 homeodomain protein
t (17; 19) (q22; p13) – E2A transactivating domains join to bZIP domain of hepatic leukemic factor (HLF)
These gene fusions alter the action of proteins that normally function in the control of of growth and differentiation during hematopoiesis
Changes in control in this highly regulated process can result in blockades that permit accumulation of immature proliferating cells

17. Gene Fusion Proteins - Translocations

18. E2A- PBX1 fusion gene Present in 25% of pre-B ALL cases
Induces T-cell lymphomas in transgenic mice
Binding of both PBX1 and E2A-PBX1 to consensus PBX1 DNA sequence stimulated by direct interactions between PBX1 and other HOX proteins
HOX proteins appear to direct E2A-PBX1 to DNA sites recognized by HOX: PBX1 complexes- therefore, likely that E2A-PBX1 interferes with hematopoietic differentiation by disrupting gene expression that is normally regulated by HOX proteins (apoptosis)

19. E2A- PBX1
Also works by reducing the number of copies and, therefore expression of E2A gene