Differential control of gene expression by specific E protein dimers

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Metazoan developmental processes are regulated by the interplay between transcription factors and signaling molecules. In vertebrates, most transcription factors belong to multigene families with shared protein motifs coupled with unique domains that endow them with the ability to regulate different genes. One of the most important of these is the E protein family, encoded in the vertebrate genome by three gene loci: E2A, HEB, and E2-2. E proteins, which act as obligate dimers, are instrumental in the regulation of genes involved in development, physiological function, cell cycle, and cell death. Remarkably, however, the specific roles of different E protein dimers are virtually unknown. Another critical aspect of E protein function that has been largely overlooked thus far is the existence of different E protein forms. The HEB and E2-2 gene loci each encode a canonical form (HEBCan or E2-2Can) and an alternative form (HEBAlt or E2-2Alt), whereas E2A encodes two canonical forms, E47 and E12. I discovered and first described HEBAlt, and my laboratory has pioneered studies of alternative E protein function. Here, we will address the fundamental questions of how distinct E protein dimers regulate gene expression, and how dimer formation and function are modulated by intracellular signaling. Due to our expertise and the multiple tools that we have developed, we will use pre-T cells as our model system. In Aim 1, we will define the compositions of the dimers that form in the context of pre-T cells by conducting co-immunoprecipitation studies in cells transfected with epitope-tagged versions of HEBAlt, HEBCan, and/or E2A. We will evaluate the impact on dimer formation by distinct signaling pathways that operate in T cell precursors using inhibitors of those pathways. These experiments will reveal the landscape of dimers available to pre-T cells, and how it shifts in response to signaling events that lead to post-translational modifications of E proteins. In Aim 2, we will identify distinct gene targets of different E protein dimers in pre-T cells. To achieve this aim we will use mice that have conditional knockout alleles of HEB, E2A, or both, bred to mice that carry the inducible Cre-ER transgene. Pre-T cells generated in OP9-DL1 co-cultures will be treated with tamoxifen to induce deletion of HEB, E2A, or both, and whole transcriptome analysis will be performed by RNA sequencing. We will evaluate which dimers regulate the expression of these genes using forced dimers, in which distinct E protein partners are linked to each other by flexible tethers. These studies will reveal the genes that are regulated by distinct E protein dimers in the context of pre-T cells. In Aim 3, we will determine the relative affinities of different E proteins for each other and for their antagonists, the Id factors, using FLIM (fluorescence live imaging microscopy)-FRET (fluorescence resonance energy transfer). We will generate expression constructs encoding E proteins or Id proteins fused to fluorescent molecules such as Venus, mTurquoise, and mCherry. Combinations of E protein or Id protein fusion constructs will be transfected into HEB and E2A deficient cells, and the lifetime decrease of the FRET donor Venus will be measured over time by fluorescence microscopy. We will perturb the cells with signaling inhibitors as in Aim 1 and evaluate the impact on dimer formation and subcellular localization. Collectively, these approaches will reveal how HEBAlt, HEBCan, and E2A cooperate or compete to regulate gene programs that control cell fate and function.