Antibody-producing B cells arise from hematopoietic stem cells through a series of developmental steps that involve a hierarchical transcription factor network. The transcription factor E2A plays a central role in early B cell development through synergistic functions with other lineage-restricted transcription factors. Whereas much is known about the direct genetic targets and cooperative functions of E2A and co-regulatory factors, little is known about their actual mechanism of action through interactions with various co-activators that act on the general transcription machinery or on chromatin structure. E2A is also of significance for cancer as a result of chromosomal translocations that fuse the activation domain of E2A to the DNA- binding domain of pre-B-cell leukemia factor 1 (PBX1) or hepatic leukemia factor (HLF), generating fusion proteins (E2A-PBX1 and E2A-HLF) that result in pediatric acute lymphoblastic leukemias (ALL) through dysregulation of oncogenic target genes that are largely unknown. Since E2A and E2A fusion proteins share common activation domains (AD1, AD2 and a newly described AD3), they are likely to utilize some common mechanisms of action on respective target genes. Toward an understanding of these mechanisms, preliminary studies have demonstrated novel physical and functional interactions of AD1/2 with the KIX domain of the histone acetyltransferase/coactivator p300 and of AD3 with the TAFH domain of the TAF4 subunit of initiation factor TFIID. With the general objective of understanding the mechanism of action of E2A and E2A fusion proteins in B cell development and in ALL, respectively, our specific aims are: (i) to employ biochemically defined systems reconstituted with purified factors and either DNA templates or corresponding recombinant chromatin templates to define the mechanism of action of E2A activation domain (AD)-coactivator interactions in gene activation by E2A and E2A fusion proteins~ (ii) to determine high-resolution structures of AD3-TAFH and AD1/2-KIX interactions in order to identify mutations that block these interactions (for genetic analyses) and to design peptidomimetic inhibitors with therapeutic potential~ (iii) To investigate the biological functions of E2A-coactivator interactions in B cell development through differentiation assays with bone marrow cells from E2A-/- mice and through the generation and analysis of knockin E2A (AD1/2/3mutant), TAF4(TAFHmutant) and p300(KIXmutant) mice~ and (iv) to investigate the biological functions of E2A PBX1/HLA AD-coactivator interactions in B cell leukemogenesis (ALL), using various cell transformation and mouse models and by analysis of the effects of inhibitory peptidomimetics on patient-derived leukemic cells.