Thyroid hormone deficiency during fetal and early childhood development is a major world-wide public health problem causing mental retardation and irreversible brain damage. The long term goal of this program is to understand the mechanism by which thyroid hormone (T3) regulates mammalian brain development. T3 regulates late brain development by activating several brain genes at a precise time. However, prior to this activation these genes are resistant to thyroid hormone despite the presence of T3 and thyroid hormone receptors. Thus, the loss of repression allows T3 to activate these genes at the appropriate time. To define the mechanism of this novel repressor effect we will study two different brain genes, Purkinje cell protein-2 (Pcp-2) and myelin basic protein (MBP), each expressed in two different cell types, cerebellar Purkinje cells and oligodenrocytes, respectively. We propose three specific aims: (1) to define the sequences within the Pcp-2 gene responsible for the repression of T3-dependent activation; (2) establish the role of the orphan receptor protein COUP-TF in repressing the response of Pcp-2 to T3; (3) determine the mechanism of repression for T3 induction of MBP gene in developing oligodendrocyte cultures.
The first aim will utilize gel shift assays and DNAse I footprinting to localize the repressor sequence within an identified repressor region of the Pcp-2 gene. The identified sequence will be mutated and tested for loss of repressor action in transgenic mice using a Pcp-2 promoter construct shown to duplicate the in vivo developmental response of the Pcp-2 gene. Preliminary results suggest that the orphan nuclear receptor COUP-TF may be involved in the repressor activity. We will test the potential role of COUP- TFI by measuring Pcp-2 in mice deficient in this protein. Primary Purkinje cell cultures will be used to test the potential role of COUP- TFII in repressing the response to T3. We will use anti-sense technology to knockout the protein in culture and transient transfection assays to overexpress the protein.
The third aim will be accomplished by examining the T3 response of other TREs to test if the MBP TRE is involved in repression of the T3 induction of MBP. We will determine if COUP-TF is the repressor protein that binds to the MBP TRE, and if not we will clone the repressor protein. These studies will provide critical information on the role of thyroid hormone in allowing synchronous development of the mammalian brain.
