UCSF DIABETES, ENDOCRINOLOGY & METABOLISM TRAINING PROGRAM FACULTY RESEARCH SUMMARIES

Our laboratory studies the intranuclear organization of the proteins that regulate the genome to be selectively transcribed in different cell types. Our hypothesis is that the intranuclear positions of gene regulatory factors control their access to specific sets of genes. In cell types associated with metabolism (anterior pituitary, fat) or that respond to estradiol (pituitary, breast and uterus), we have found that each factor takes up characteristic positions within the nucleus. Moreover, those positions of these factors are highly plastic and are altered by conditions such as the expression of another gene regulatory factor or the addition of a hormone.

As an example, we found the transcription factor C/EBPa to concentrate in transcriptionally quiescent subregions of both anterior pituitary and adipocyte cell nuclei. Certain C/EBPa mutations resulted in the re-distribution of C/EBPa to more transcriptionally active regions. A pituitary-specific transcription factor Pit-1 distinguishes anterior pituitary cells from adipocytes. We found co-expression of Pit-1 to be required for the transcriptional activation of pituitary-specific promoters by C/EBPa. Pit-1 expression in pituitary progenitor cells was associated with the re-location of C/EBPa from the transcriptionally inactive subcompartment to the transcriptionally active subcompartment where C/EBPa activated pituitary-specific promoters.

Pit-1 mutants, found in human patients with combined pituitary hormone deficiency, did not co-activate C/EBPa transcriptional activity, and no longer re-located C/EBPa from the transcriptionally inactive subcompartment. We also characterized mutations within C/EBPa that, even in the absence of Pit-1 co-expression, re-located C/EBPa to the transcriptionally active subcompartment. These studies proved that C/EBPa was held inactive when localized to the transcriptionally quiescent subregion and that Pit-1 re-location of C/EBPa permitted activity. We also found that the anti-proliferative actions of C/EBPa, common to both pituitary and adipocyte differentiation, was not affected by the intranuclear compartmentalization of C/EBPa. Thus, transcription factor compartmentalization can affect transcriptional capacity without affecting the molecular mechanisms involved in the control of cell replication.

Other factors, including nuclear receptors and co-factors that mediate estrogen, thyroid hormone and retinoid response localized to specific subregions of the nucleus. Each location may permit location-selective activities or interactions for each factor. We pioneered methods for studying the biochemical interactions and conformations of the gene regulatory factors at specific locations within the cell nucleus. This was measured as the transfer of fluorescence energy between fluorophores attached to different gene regulatory factors or to different domains within the factor. For example, we determined that the positions of the DNA binding domains in C/EBPa dimers were ~2? further apart at the transcriptionally inactive regions of the nucleus than at the transcriptionally active regions.

We also use these high-precision, live-cell, FRET techniques to measure the effects of drugs, used in the treatment of breast cancer, prostate cancer and diabetes on the interactions of the relevant transcription factors with themselves or with other factors.

Selected References

Schaufele F, Wang X, Liu X, Day RN 2003 Conformation of CCAAT/enhancer binding protein alpha dimers varies with intranuclear location in living cells. J Biol Chem: 278: 10578-10587.

Schaufele F, Day RN 2005 Imaging molecular interactions in living cells. Mol Endocrinol 19:1675-1686.

Schaufele F, Carbonell X, Guerbadot M, Borngraeber S, Chapman M, Ma AAK, Miner JN, Diamond MI 2005 The structural basis of AR activation: intra and intermolecular amino-carboxy interactions. Proc Natl Acad Sci USA, 102(28): 9802-7.

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