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Kevin D Sarge

Publications and source records attributed to Kevin D Sarge.

10 recordsLinked to original sources
PubMed2005

Gene bookmarking: keeping the pages open.

'Gene bookmarking' is a mechanism of epigenetic memory that functions to transmit through mitosis the pattern of active genes and/or genes that can be activated to daughter cells. It is thought that, at a point before mitosis, genes that exist in an open, transcriptionally competent state are bound by proteins or marked by some kind of modification event. This is thought to facilitate the assembly of transcription complexes on the promoters in early G1, thereby ensuring that daughter cells have the same pattern of gene expression as the cell from which they derived. Little is known, however, about these 'bookmarking factors' and modifications or the mechanisms by which they mediate the transmission of transcriptional competence after mitosis is complete. Recent findings have provided new insights into the mechanisms, regulation and biological importance of gene bookmarking in eukaryotic cell function.

Animals↗
PubMed2005

Transactivation of the parathyroid hormone promoter by specificity proteins and the nuclear factor Y complex.

We previously identified a highly conserved specificity protein 1 (Sp1) DNA element in mammalian PTH promoters that acted as an enhancer of gene transcription and bound Sp1 and Sp3 proteins present in parathyroid gland nuclear extracts. More recently, a nuclear factor (NF)-Y element (NF-Y(prox)) was also described by our group, which was located approximately 30 bp downstream from the Sp1 site in the human PTH (hPTH) promoter and by itself acted as a weak enhancer of gene transcription. We now report that Sp proteins and NF-Y can synergistically enhance transcription of a minimal hPTH promoter construct. Positioning of the Sp1 DNA element appears to be critical for this synergism because deviations of one half of a helical turn caused an approximate 60% decrease in transactivation. Finally, examination of the bovine PTH (bPTH) promoter also revealed Sp1/NF-Y synergism, in conjunction with the identification of an analogous NF-Y binding site similarly positioned downstream from the bPTH Sp1 element. In summary, synergistic transactivation of the hPTH and bPTH promoters is observed by Sp proteins and the NF-Y complex. The conservation of this transactivation in the human and bovine promoters suggests that this may be a principle means of enhancing PTH gene transcription.

Animals↗
PubMed2005

Mechanism of hsp70i gene bookmarking.

In contrast to most genomic DNA in mitotic cells, the promoter regions of some genes, such as the stress-inducible hsp70i gene that codes for a heat shock protein, remain uncompacted, a phenomenon called bookmarking. Here we show that hsp70i bookmarking is mediated by a transcription factor called HSF2, which binds this promoter in mitotic cells, recruits protein phosphatase 2A, and interacts with the CAP-G subunit of the condensin enzyme to promote efficient dephosphorylation and inactivation of condensin complexes in the vicinity, thereby preventing compaction at this site. Blocking HSF2-mediated bookmarking by HSF2 RNA interference decreases hsp70i induction and survival of stressed cells in the G1 phase, which demonstrates the biological importance of gene bookmarking.

Adenosine Triphosphatases↗
PubMed2005

Detection of sumoylated proteins.

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 involved in regulation of protein function, nuclear targeting, and the formation of subcellular structures. Because SUMO is involved in such a plethora of functions and modifies numerous proteins it is important to identify proteins that are sumoylated in order to increase our understanding of how this modification affects protein function and localization. This overview describes techniques utilized for the detection of sumoylated proteins. The techniques covered include immunoprecipitation, an in vitro sumoylation assay, and gel shift mobility assays that have been used to identify SUMO-modified proteins.

Blotting, Western↗
PubMed2004

HSF1 modulation of Hsp70 mRNA polyadenylation via interaction with symplekin.

Induction of heat shock protein (HSP) gene expression by stress is initiated by binding of HSF1 to HSP gene promoters to increase their transcription. The cytoprotective functions of these HSPs are essential for cell survival, and thus it is critical that inducible HSP gene expression be executed rapidly and efficiently. Here we report an interaction between heat shock factor 1 (HSF1) and symplekin, a protein known to form a complex with the polyadenylation factors CstF and CPSF. HSF1-symplekin complexes are detected only after stress treatment, and these two proteins co-localize in punctate nuclear structures in stressed cells. HSF1 also complexes in a stress-induced manner with the 3' processing factor CstF-64. Interfering with HSF1-symplekin interaction by overexpressing a non-DNA-binding mutant HSF1 protein significantly decreases Hsp70 mRNA polyadenylation in stressed cells, supporting the functional role for HSF1 in promoting 3' processing of this transcript. Importantly, this was also found to result in a significant loss of Hsp70 protein induction and increased cell death in response to stress exposure. These results indicate that the HSF1-symplekin interaction functions as a mechanism for recruiting polyadenylation factors to HSP genes to enhance the efficiency/kinetics of production of mature Hsp mRNA transcripts to achieve the critical cellular need for rapid HSP expression after stress. Thus, HSF1 regulates HSP gene expression at not one but two different steps of the expression pathway, functioning both as a transcription factor and a polyadenylation stimulatory factor.

Animals↗
PubMed2004

Identification of Xenopus heat shock transcription factor-2: conserved role of sumoylation in regulating deoxyribonucleic acid-binding activity of heat shock transcription factor-2 proteins.

Heat shock transcription factor (Hsf)-1 and Hsf2 are members of the heat shock factor (HSF) protein family involved in heat shock protein (hsp) gene regulation, a regulation that is critical for the ability of cells to survive exposure to stress conditions. Although the role of Hsf1 in binding and activating transcription of hsp gene promoters in response to cell stress is well established, how Hsf2 enhances stress-induced hsp expression is not understood. To gain an insight into the critical conserved features of the regulation and function of Hsf2, we have identified and characterized the Hsf2 protein from Xenopus laevis. We found that, similar to its human counterpart, Xenopus Hsf2 is sumoylated at lysine 82 and that, as it does in human Hsf2, the modification event of the small ubiquitin-related modifier 1 functions to increase the deoxyribonucleic acid-binding activity of this transcription factor in Xenopus. These results indicate that sumoylation is an evolutionarily conserved modification of Hsf2 proteins, supporting the position of this modification as a critical regulator of Hsf2 function.

Amino Acid Sequence↗
PubMed2003

Interaction between protein phosphatase 2A and members of the importin beta superfamily.

While performing a yeast two-hybrid library screen to uncover novel PP2A-interacting proteins, we discovered a specific interaction between a member of the importin beta/karyopherin beta superfamily, importin 9, and the A subunit of PP2A (PR65). This interaction between importin 9 and the A subunit was confirmed by in vitro pulldown, immunoprecipitation, and microcystin-Sepharose chromatography. We also found that another family member, importin beta, interacted specifically with the A subunit of PP2A. Finally, we showed that treatment of cells with a concentration of okadaic acid known to inhibit PP2A impeded the nuclear localization of an NLS-containing protein. These results provide evidence that these importins can exist in a native complex with endogenous PP2A and that this serine/threonine phosphatase plays a role in regulating the nuclear import of NLS-containing proteins in vivo.

Active Transport, Cell Nucleus↗
PubMed2003

Insights into the regulation of heat shock transcription factor 1 SUMO-1 modification.

The transcriptional regulatory protein HSF1 is the key mediator of induced heat shock protein gene expression in response to elevated temperature and other stresses. Our previous studies identified stress-induced SUMO-1 modification of HSF1 as an important regulator of the DNA-binding activity of this factor. The underlying molecular mechanism by which stress leads to sumoylation of HSF1 was unknown. Prompted by previous studies indicating stress-induced phosphorylation at serine 307 of HSF1, a site very near the sumoylation site at lysine 298, we examined the role of this phosphorylation event in regulating SUMO-1 modification of HSF1. Using a combination of transfection and in vitro phosphorylation/sumoylation experiments, our results indicate that phosphorylation at serine 307 stimulates sumoylation of HSF1. Our results also reveal a role for a conserved leucine zipper sequence in the C-terminal region of HSF1 in inhibiting its SUMO-1 modification. Based on these data, we postulate that phosphorylation at serine 307 could stimulate HSF1 sumoylation by causing a conformation change that relieves the inhibitory effect of the C-terminal leucine zipper.

Amino Acid Sequence↗
PubMed2003

Lowered temperature set point for activation of the cellular stress response in T-lymphocytes.

The induction of heat shock protein gene expression in response to stress is critical for the ability of organisms to cope with and survive exposure to these stresses. However, most studies on HSF1-mediated induction of hsp70 gene expression have utilized immortalized cell lines and temperatures above the physiologically relevant range. For these reasons much less is known about the heat shock response as it occurs in mammalian cells within tissues in the intact organism. To gain insight into this area we determined the temperature thresholds for activation of HSF1 DNA binding in different mouse tissues. We have found that HSF1 DNA binding activity and hsp70 synthesis are induced in spleen cells at significantly lower temperatures relative to cells of other tissues, with a temperature threshold for activation (39 degrees C) that is within the physiological range for fever. Furthermore, we found that the lowered temperature set point for induction of the stress response in spleen is specific to T-lymphocytes residing within this tissue and is not exhibited by B-lymphocytes. This lowered threshold is also observed in T-lymphocytes isolated from lymph nodes, suggesting that it is a general property of T-lymphocytes, and is seen in different mouse strains. Fever is an early event in the immune response to infection, and thus activation of the cellular stress response in T-lymphocytes by fever temperatures could serve as a way to give these cells enough time to express hsps in anticipation of their function in the coming immune response. The induced hsps likely protect these cells from the stressful conditions that can exist during the immune response, for example increasing their protection against stress-induced apoptosis.

Animals↗