Segmentation of the embryonic vertebrate hindbrain into rhombomeres ensures proper positioning of hindbrain derivatives and is essential for normal neural activity. Rhombomere formation is regulated by hox genes whose expression is carefully coordinated to ensure correct timing and spatial distribution of transcripts. hox expression is regulated by a combination of retinoic acid (RA) mediated signaling and by hox genes regulating each other's expression. Our preliminary data reveal a nucleosome-depleted region (NDR) forming at hox promoters in response to RA. We also find that Prep and Pbx cofactors occupy hox promoters well before onset of transcription and that the presence of these cofactors correlates with 'active' histone modifications and recruitment of RNA Polymerase II. We find this constellation of factors to be insufficient to drive transcription - instead, Hox proteins appear required for initiation of transcription. Based on these data we hypothesize that activation of hox expression is achieved by: 1) RA inducing an NDR at hox promoters, 2) Prep and Pbx cofactors binding at the NDR, 3) recruitment of histone-modifying enzymes and RNA Polymerase by Pbx and Prep, 4) binding of Hox proteins to activate transcription. We have developed two aims to test this hypothesis: 1) Define sequential action of cofactors and Hox proteins in initiation of hox gene transcription. In this aim we will establish te order of action and specific roles for each factor required for hox gene expression, 2) Define the role of nucleosome-depleted regions in the activation of hindbrain hox gene expression. In this aim we will determine how NDRs are formed and what role they play in control of hox gene expression. Our work will impact human health by exploring how the embryonic nervous system forms. The importance is underscored by the fact that human hox mutations produce developmental disruptions of the brainstem and cause cognitive defects such as autism. Since our work will improve our understanding of how neural gene expression is initiated in undifferentiated cells in general, our results will also inform studies aimed at driving the differentiation of precursor cells (such as ES and iPS cells) towards a neural fate for basic and clinical applications.
The embryonic hindbrain gives rise to many essential structures - sensory ganglia and branchiomotor neurons of the nervous system, as well as bone, cartilage and muscle of the head. The developing hindbrain is sensitive to disruptions by a variety of factors (e.g. environmental toxins, infectious agents and genetic conditions) that give rise to a range of birth defects - motor control problems such as ataxia, cognitive defects such as autism and craniofacial defects. In addition, the genes studied in this proposal regulate other aspects of neural development (e.g. dorsoventral patterning of the neural tube), and other aspects of embryogenesis (e.g. hematopoiesis). These genes are also proto oncogenes involved in leukemia. The results from our proposed experiments will therefore be applicable to a broad set of biological processes and human disease conditions.
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