Homeodomain (HD) proteins comprise a large family of transcription factors (TFs) that regulate numerous aspects of animal development. For example, members of the Hox-like (HoxL) and Nkx-like (NKL) HD proteins regulate processes ranging from patterning of the anterior-posterior axis (A-P) of the embryo to specifying individual cell fates within different organ systems. Intriguingly, the HoxL and NKL proteins have highly similar HDs that bind largely overlapping AT-rich DNA sequences in vitro. These findings provide a classic TF specificity paradox: How do TFs with highly similar in vitro DNA binding activities achieve sufficient in vivo specificity to ensure the accurate regulation of genetic programs in different cell types? To address this paradox, my lab is focused on defining how HD TFs achieve in vivo specificity by forming cooperative TF complexes on cis- regulatory modules. Our preliminary and published data reveal that members of the HoxL and NKL TFs differ in their ability to form homo- and heterodimer TF complexes on DNA. For instance, we unexpectedly found that the Gsx/Ind TFs, which specify neuronal cell fates in animals from flies to mammals, differentially regulate gene expression when bound to DNA as monomers versus homodimers. In contrast, the Abdominal-A (Abd-A) Hox TF, which specifies distinct cell fates in the Drosophila abdomen, does not bind DNA as a homodimer, but instead cooperatively binds DNA with three other HD proteins: Extradenticle (Exd), Homothorax (Hth), and Engrailed (En). These data support the hypothesis that HD TFs achieve target and regulatory specificity by binding distinct combinations of AT-rich DNA sites as monomers, cooperative homodimers, or cooperative heterodimers. To test this hypothesis, we propose two aims:
In Aim1, we propose to determine how HD monomer versus homodimer binding impacts target gene binding and regulation. To achieve this goal, we will (1) systematically define which HoxL and NKL HDs cooperatively bind DNA as homodimers; (2) assess the regulatory potential of each HD on monomer vs dimer sites in cell culture assays; and (3) define the mechanism and function of Ind homodimer formation on Drosophila neuroblast gene expression using structural biology and transgenic reporter, CUT&RUN, and RNA-seq assays.
In Aim2, we propose to define how the choice of Hox heterodimer partner impacts the DNA binding and regulatory specificity of the Abd-A Hox TF. To achieve this goal, we will (1) define the DNA motifs and molecular domains required for cooperative Abd-A/Hth and Abd-A/En complexes; (2) test the role of Abd-A heterodimerization domains in gene activation and repression assays in the Drosophila embryo; (3) define the in vivo binding motifs and target genes regulated by Abd-A with a focus on identifying heterodimer binding events using CUT&RUN and RNA-seq assays. Since the TFs and biological processes studied are highly conserved between flies and mammals, we are optimistic our studies will uncover gene regulatory mechanisms relevant to human health and development.
/ LAY ABSTRACT The precise control of gene expression by transcription factors is essential for the proper development of specialized cell types within each organ system. Unfortunately, we lack an understanding of how transcription factors are integrated to yield cell-specific gene expression within a complex organism. The goal of this research proposal is to determine how homeodomain transcription factors regulate cell-specific gene expression during development. Our proposed studies utilize powerful genetic, genomics, bioinformatics, and biochemical approaches in the fruit-fly Drosophila melanogaster, and we have already used this approach to uncover novel gene interactions relevant to human development and leukemia. Since both the processes and transcription factors studied are highly conserved from flies to mammals, our findings will reveal fundamental new insights relevant to vertebrate development and human health.
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