Ok-Kyong Park-Sarge

SUMO-1 Modification Regulates the DNA Binding Activity of Heat Shock Transcription Factor 2, a Promyelocytic Leukemia Nuclear Body Associated Transcription Factor

Heat shock transcription factor 2 (HSF2) is a transcription factor that regulates heat shock protein gene expression, but the mechanisms regulating the function of this factor are unclear. Here we report that HSF2 is a substrate for modification by the ubiquitin-related protein SUMO-1 and that HSF2 colocalizes in cells with SUMO-1 in nuclear granules. Staining with anti-promyelocytic leukemia antibodies indicates that these HSF2-containing nuclear granules are PML bodies. Our results identify lysine 82 as the major site of SUMO-1 modification in HSF2, which is located in a “wing” within the DNA-binding domain of this protein. Interestingly, SUMO-1 modification of HSF2 results in conversion of this factor to the active DNA binding form. This is the first demonstration that SUMO-1 modification can directly alter the DNA binding ability of a transcription factor and reveals a new mechanism by which SUMO-1 modification can regulate protein function.

Differential Expression of the Estrogen Receptors α and β in the Rat Corpus Luteum of Pregnancy: Regulation by Prolactin and Placental Lactogens1

Estradiol, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat corpus luteum of pregnancy. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat corpus luteum of pregnancy expresses the estrogen receptor (ER) genes. In this investigation, we have analyzed the expression of the two ER genes (ERα and ERβ) (by RT-PCR and in situ hybridization) in the rat corpus luteum, studied their developmental changes throughout pregnancy, and investigated the regulation of ERα and ERβ messenger RNA (mRNA) expression by PRL and placental lactogens. The RT-PCR studies showed that both ER mRNA species (ERα and ERβ) are coexpressed in the rat corpus luteum during pregnancy. Whereas ERα mRNA increased from early pregnancy, reached a maximum at midpregnancy, and had a remarkable decline before parturition; ERβ mRNA remained constant throughout pregnancy, with a significant decline at parturition. Examinati...

Sumoylation and human disease pathogenesis

Covalent modification by SUMO polypeptides, or sumoylation, is an important regulator of the functional properties of many proteins. Among these are several proteins implicated in human diseases including cancer, Huntingtons, Alzheimers, and Parkinsons diseases, as well as spinocerebellar ataxia 1 and amyotrophic lateral sclerosis. Recent reports reveal two new examples of human disease-associated proteins that are SUMO modified: amyloid precursor protein and lamin A. These findings point to a function for sumoylation in modulating amyloid-beta peptide levels, indicating a potential role in Alzheimers disease, and for decreased lamin A sumoylation as a causative factor in familial dilated cardiomyopathy.

Tissue-dependent expression of heat shock factor 2 isoforms with distinct transcriptional activities.

Heat shock factor 2 (HSF2) functions as a transcriptional regulator of heat shock protein gene expression in mammalian cells undergoing processes of differentiation and development. Our previous studies demonstrated high regulated expression and unusual constitutive DNA-binding activity of the HSF2 protein in mouse testes, suggesting that HSF2 functions to regulate heat shock protein gene expression in spermatogenic cells. The purpose of this study was to test whether HSF2 regulation in testes is associated with alterations in the HSF2 polypeptide expressed in testes relative to other mouse tissues. Our results show that mouse cells express not one but two distinct HSF2 proteins and that the levels of these HSF2 isoforms are regulated in a tissue-dependent manner. The testes express predominantly the 71-kDa HSF2-alpha isoform, while the heart and brain express primarily the 69-kDa HSF2-beta isoform. These isoforms are generated by alternative splicing of HSF2 pre-mRNA, which results in the inclusion of an 18-amino-acid coding sequence in the HSF2-alpha mRNA that is skipped in the HSF2-beta mRNA. HSF2 alternative splicing is also developmentally regulated, as our results reveal a switch in expression from the HSF2-beta mRNA isoform to the HSF2-alpha isoform during testis postnatal developmental. Transfection analysis shows that the HSF2-alpha protein, the predominant isoform expressed in testis cells, is a more potent transcriptional activator than the HSF2-beta isoform. These results reveal a new mechanism for the control of HSF2 function in mammalian cells, in which regulated alternative splicing is used to modulate HSF2 transcriptional activity in a tissue-dependent manner.

Role of Progesterone Receptor Activation in Pituitary Adenylate Cyclase Activating Polypeptide Gene Expression in Rat Ovary

It is well known that the pituitary gonadotropin surge induces progesterone receptor (PR) gene expression in luteinizing granulosa cells and that PR activation is critical for successful ovulation. To further understand the molecular mechanism(s) by which PR plays a role critical for granulosa cell functions, we wanted to identify progesterone-induced genes in granulosa cells. We employed a PCRbased subtraction cloning strategy to screen for genes expressed differentially in granulosa cells that were challenged with forskolin in the presence of progesterone or ZK98299. One such differentially expressed clone was identified as the pituitary adenylate cyclase activating polypeptide (PACAP). To begin to understand the relationship between PR activation and PACAP gene expression in luteinizing granulosa cells, we examined whether PR and PACAP messenger RNA (mRNA) expression is temporally correlated. In cultured granulosa cells, both human CG and forskolin induced PR and PACAP mRNA levels in a dose-dependent manner, as determined by semiquantitative RT-PCR assays. However, the peak expression for PR and PACAP mRNAs was observed at 3 h and 6 h after hormone treatment, respectively. This time difference in cAMP-responsive expression of the PR and PACAP genes is due, at least in part, to the requirement of ongoing protein synthesis for PACAP expression, as demonstrated by the inhibitory effect of cycloheximide on cAMPinduced PACAP, but not PR, mRNA levels. To determine whether PR synthesis is prerequisite for PACAP expression, we examined the effect of ZK98299, a specific PR antagonist, on cAMP-induced PACAP mRNA expression. This compound blocked cAMP-induced PACAP mRNA expression in a dose-dependent manner, indicating that PR activation is required for PACAP gene expression in granulosa cells. We then compared cellular localization and hormonal regulation of ovarian PR and PACAP gene expression in immature rats treated with gonadotropins as well as in adult rats during the preovulatory period by using in situ hybridization and semiquantitative RT-PCR assays. Results show that both PR and PACAP mRNAs are induced in granulosa cells of preovulatory follicles by human CG, but that the PR gene is expressed before the PACAP gene. Taken together, these results demonstrate that PRs mediate the LH-induced PACAP gene expression in rat granulosa cells. (Endocrinology 140: 5185‐5194, 1999)

SUMO and Its Role in Human Diseases

The covalent attachment of small ubiquition-like modifier (SUMO) polypeptides, or sumoylation, is an important regulator of the functional properties of many proteins. Among these are many proteins implicated in human diseases including cancer and Huntingtons, Alzheimers, and Parkinsons diseases, as well as spinocerebellar ataxia 1 and amyotrophic lateral sclerosis. The results of two more recent studies identify two additional human disease-associated proteins that are sumoylated, amyloid precursor protein (APP), and lamin A. APP sumoylation modulates Aβ peptide levels, suggesting a potential role in Alzheimers disease, and decreased lamin A sumoylation due to mutations near its SUMO site has been implicated in causing some forms of familial dilated cardiomyopathy.

Characterization of Estrogen Receptor-β (ERβ) Messenger Ribonucleic Acid and Protein Expression in Rat Granulosa Cells1

We have examined estrogen-responsiveness of ovarian granulosa cells by focusing on estrogen receptor (ER) expression. Estrogen responsiveness was determined by examining the effect of 17β-estradiol (1–10 nm) on luciferase reporter activity in rat granulosa cells transfected with an ERE-luciferase construct. The results demonstrate an estrogen-induced (approximately 3-fold) increase in luciferase reporter activity, indicating that granulosa cells contain functional estrogen response element (ERE)-binding transcriptional activators. Gel mobility shift assays in combination with ER antibodies show that ERβ is the predominant ERE-binding protein in granulosa cells. Western blotting results show that granulosa cells contain ERβ-immunoreactive protein(s) migrating at a size substantially larger than the recombinant protein generated from the originally proposed 485 amino acid open-reading frame. This size discrepancy is not due to granulosa cell expression of ERβ isoforms with insertions within the coding regio...

Detection of proteins sumoylated in vivo and in vitro.

Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is instead involved in regulating multiple protein functional properties including protein-protein interactions and subcellular targeting, to name a few. Protein sumoylation has been particularly well characterized as a regulator of many nuclear processes as well as nuclear structure, making the characterization of this modification vital for understanding nuclear structure and function. Consequently, there has been intense interest in identifying new proteins that are targets of this modification and determining what role it plays in regulating their functions. This chapter presents methodologies for determining whether a particular protein is a substrate of sumoylation, and for identifying the lysine residue(s) where the modification occurs.

Gonadotropins Decrease Estrogen Receptor-β Messenger Ribonucleic Acid Stability in Rat Granulosa Cells1

We have previously shown that the preovulatory LH surge down-regulates estrogen receptor-β (ERβ) messenger RNA (mRNA) levels selectively in the granulosa cells of preovulatory follicles. To gain insight into the underlying mechanisms, we examined whether the LH-induced loss of ERβ mRNA expression in rat granulosa cells is attributable to the hormone-induced changes at the level of transcription and/or mRNA degradation. When the rate of ERβ gene transcription was assessed in cultured granulosa cells, by nuclear run-off assays, we observed only a marginal effect of hCG on ERβ gene transcription. In contrast, when ERβ mRNA levels were estimated in granulosa cells that were cultured in the presence of 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), an RNA synthesis inhibitor, we observed a significant inhibitory effect of human CG (hCG) on ERβ mRNA expression at a magnitude similar to that observed in the absence of DRB. Forskolin (FSK) and 2-O-tetradecanol-phorbol-13-acetate (TPA), pharmacological agent...

Suppression of the Human Parathyroid Hormone Promoter by Vitamin D Involves Displacement of NF-Y Binding to the Vitamin D Response Element

An earlier report in the literature indicated the vitamin D response element (VDRE) in the human parathyroid hormone (hPTH) promoter could be specifically bound by an unidentified transcription factor in addition to the vitamin D receptor (VDR) complex. We confirmed that OK and HeLa cell nuclear extracts formed a specific complex with the hPTH VDRE that was insensitive to competition with other VDRE sequences. However, this factor could be competed for by a consensus NF-Y DNA-binding site, and an anti-NF-Y antibody was able to supershift the bound band. Mutational analysis indicated that the NF-Y-binding site partially overlapped the 3′ portion of the VDRE. Transfection studies using an hPTH promoter construct in Drosophila SL2 cells demonstrated strong synergistic transactivation by NF-Y interactions with both the VDRE site and a previously described distal NF-Y-binding site. Finally, mobility shift studies indicated that the VDR heterodimer competed with NF-Y for binding to the VDRE sequence, and NF-Y-stimulated activity of the hPTH promoter could be suppressed in a hormone-dependent manner when the VDR heterodimer complex was coexpressed in SL2 cells. In summary, these findings establish the presence of a proximal NF-Y-binding site in the hPTH promoter and highlight the potential for synergism between distal and proximal NF-Y DNA elements to strongly enhance transcription. Furthermore, findings suggest that the repressive effects of vitamin D on hPTH gene transcription may involve displacement of NF-Y binding to the proximal site by the VDR heterodimer, which subsequently attenuates synergistic transactivation.