REGULATION OF MAPS IN NERVOUS SYSTEM

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MAP1B is a prominent microtubule associated protein whose expression is developmentally regulated, showing the highest levels in growing axons during early postnatal brain development. Our interest in the expression of MAPs in the nervous system has been related to the role of cytoskeletal proteins during neuronal differentiation, but the experiments that we propose will also address fundamental problems concerning the regulatory elements that control the expression of other brain- specific genes. Specific questions addressed in this proposal include: What are the mechanisms that regulate MAP1B expression? What are the regulatory elements that control the cell and stage-specificity during transcription of the MAP1B gene and its response to physiological signals (e.g., NGF, cAMP)? What is the role of MAP1B during axonal growth in neurons? The first set of experiments utilizes nuclear run-on assays to confirm that the control of MAP1B expression is exerted at the transcriptional level. Specific regulatory mechanisms can then be identified from studies of the structure and function of MAP1B promoter. We hypothesize that, as with other brain-specific genes, the basic elements of the MAP1B promoter are defined within a relatively small region upstream from the initiation site of transcription. In preliminary experiments we have already isolated genomic clones that contain part of the MAP1B promoter and determined the initiation site of transcription. Additional distal elements will be identified by nuclease hypersensitivity assays of neuronal cells. Our approach is to follow a set of experiments that are based on in vitro assays of chimeric constructs in which regulatory elements are linked to a reporter CAT gene for functional analysis of MAP1B promoter in both neuronal and non-neuronal cells. These experiments will be followed by physical mapping of specific regulatory regions by footprinting, methylation interference, and gel shift assays. Additional mapping experiments using nuclease hypersensitivity assays will be directed at more distal regulatory elements and help to design promoter constructs that can be studied in vivo with transgenic animals. Finally, the requirement for MAP1B during axonal differentiation will be examined by suppression of its expression in cultured primary neurons using antisense oligonucleotides and by microinjection of MAP1B peptides and antibodies prepared against selected regions of the molecule. These strategies can be extended to study promoters of other MAPs, for which, thus far, no promoters have been described, and to isolate specific transcription factors that control these genes. These studies will also become the basis for in vivo work and development of experimental tools for manipulation of gene expression in transgenic animals. Ultimately, the characterization of these genes and their promoters will provide an understanding of the molecular pathways of neuronal differentiation during development of the nervous system and in response to changes in the environment.