We have investigated the pathophysiologic mechanism of familial/sporadic generalized glucocorticoid resistance syndrome, a prototype of glucocorticoid resistance caused by mutations in the glucocorticoid receptor (GR) gene. We found three new heterozygotic cases with mutations in the GR gene (GR V423A, GR V575G and GR H726R) in collaboration with Dr. Evangelia Charmandari, Univ. of Athens Medical School, Athens, Greece. We characterized molecular defects of the mutant receptors GR V423A and V575G: V423A is located close to the 2nd zinc finger of GR DNA-binding domain. Replacement of valine to alanine at amino acid position 423 reduced hydrophobic environment around this amino acid, which is necessary for GR DNA-binding domain to bind DNA GREs. This resulted in reduction of the mutant receptor for binding to GREs in vivo, and reduced transcriptional activity of this mutant receptor. On the other hand, V575G is located in the 5th helix of the GR ligand-binding domain and its side chain is exposed to the activation function 2 (AF2) surface of the receptor, which physically interacts with the LXXLL motif of the p160 type nuclear receptor coactivators. Replacement of valine to glycine at this amino acid position destroys the AF2 surface, leading to reduction in the transcriptional activity of the mutant receptor by attenuating its binding to p160 type nuclear receptor coactivators. In this fiscal year, we published 2 manuscripts explaining clinical characteristics of the subjects carrying the GR V423A and GR V575G and molecular defects of these mutant receptors. We have also completed the computer-based 3-dimensional structural analysis for the ligand-binding domain of all pathologic GR mutations ever reported and found that there are novel rules for the mutations to damage ligand-binding pocket and AF2 surface. The manuscript explaining these findings is ready for submitting to Journal. We encountered a 7-year old boy with mild resistance to glucocorticoids, androgens and thyroid hormones who carried 1.1 Mb size heterozygotic 16p11.2 microdeletion. We found and published that heterozygotic deletion of the ZNF764 gene by his microdeletion is responsible for his multi-hormone resistance, as this protein acts as a specific coactivator for the glucocorticoid, androgen and thyroid hormone receptors. We have examined molecular mechanisms underlying ZNF764-mediated coactivation of steroid hormone receptors by employing GR as a model system. We found that ZNF764 physically interacts with the ligand-binding domain of GR through its N-terminal half containing a KRAB domain in vitro and in vivo, while C-terminal portion of this molecule containing seven C2H2-type zinc fingers was required for its enhancement of GR transcriptional activity. To reveal molecular action of ZNF764 on GR-induced transcriptional activity genome-wide, we performed the chromatin immunoprecipitation (ChIP) followed by next generation sequence. Preliminary results indicated that binding regions of GR and ZNF764 significantly overlapped with each other. We are now under analysis of obtained results. Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion, and regulate the transcriptional activity of GR in target tissues. The basic helix-loop-helix protein CLOCK, a histone acetyltransferase (HAT), and its heterodimer partner BMAL1 are self-oscillating transcription factors that generate circadian rhythms both in the central nervous system and periphery. We previously reported that CLOCK/BMAL1 repressed GR-induced transcriptional activity by acetylating GR at several lysine residues located in its hinge region and by suppressing binding of GR to promoter GREs. These findings indicate that CLOCK/BMAL1 functions as a reverse phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing to the biologic actions of diurnally fluctuating circulating glucocorticoids. We focused on the longevity-associated and histone deacetylase sirtuin Sirt1 as a candidate of deaceylating the GR acetylated by CLOCK, as this protein is known to interact with the CLOCK/BMAL1 heterodimer and to deaceylate BMAL1. In non-synchronized culture cells, Sirt1 bound GR at latters ligand-binding domain and enhanced its transcriptional activity of both glucocorticoid-responsive reporter constructs and endogenous glucocorticoid-responsive genes. However, this action of Sirt1 on GR was independent to its deacetylase activity, suggesting that Sirt1 is not the deacetylase that deaceylates the GR acetylated by CLOCK. As biological impact of longevity-associated Sirt1 to glucocorticoid hormonal action is worth pursuing, we performed an RNA sequence assay to examine genome-wide effects of Sirt1 on glucocorticoid-responsive transcriptome. We also performed a human study in which we sampled peripheral blood in the morning and evening from normal subjects, and measured mRNA expression of known glucocorticoid-responsive genes and GR acetylation in circulating lymphocytes. We found that GR was acetylated higher in the morning than in the evening, positively correlating with mRNA expression of CLOCK and BMAL1, while circulating glucocorticoid-stimulated mRNA expression of glucocorticoid-responsive genes were repressed by CLOCK/BMAL1 in a gene-specific fashion. These results indicate that the peripheral CLOCK system negatively regulates GR transcriptional activity through acetylation of GR not only in cultured cells but also in humans. In another study where we measured mRNA expression of 190 GR action-regulating and glucocorticoid-responsive genes in subcutaneous fat biopsied from 25 obese subjects, we found that levels of evening cortisol is much more important than those in the morning to regulate mRNA expression of glucocorticoid responsive genes. Ratios of morning/evening serum cortisol levels also have a unique effect. It appears that higher sensitivity of tissues to circulating glucocorticoids in the evening due to reduced GR acetylation by CLOCK underlies stronger impact of evening serum cortisol levels to glucocorticoid-regulated gene expression compared to its morning levels. In connection with our study on circadian rhythms, we found one interesting family with seasonal alteration of circadian rhythmicity in collaboration with Dr. F. Halberg, the Univ. of Minnesota. The proband, 61-year old female, has suffered from an annual cycle of severe fatigue including inability to get out of bed, which lasts 2-3 months in summer and winter. She, however, is free from symptoms with high achievement during the unaffected months between these bad periods. The proposita demonstrated elongation of circadian rhythmicity (24.84 hours) in blood pressure and vigor rates during the affected periods, while she was in normal 24-hour rhythms in the unaffected months. The cycle of 24.84 hours is exactly double of the tidal cycle, which is hypothesized to come about gravity-changes caused by revolution of the moon around the earth. To identify genetic cause(s) of these manifestations, we have completed the whole exome sequence analysis for 3 affected subjects and 5 unaffected members. We found several gene areas associated with high LOD score values, as well as 4 nucleotide replacements specific to affected subjects. One of such replacements is located in the gene known to have impact on circadian rhythms and sleep in drosophila. We also performed SNP array analysis for an additional 3 family members to increase LOD scores of the gene areas identified in the whole exosome sequence. We are now analyzing obtained raw data.
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