Heat shock transcription factor 1 is activated as a consequence of lymphocyte activation and regulates a major proteostasis network in T cells critical for cell division during stress.

Heat shock transcription factor 1 (HSF1) is a major transcriptional regulator of the heat shock response in eukaryotic cells. HSF1 is evoked in response to a variety of cellular stressors, including elevated temperatures, oxidative stress, and other proteotoxic stressors. Previously, we demonstrated that HSF1 is activated in naive T cells at fever range temperatures (39.5°C) and is critical for in vitro T cell proliferation at fever temperatures. In this study, we demonstrated that murine HSF1 became activated to the DNA-binding form and transactivated a large number of genes in lymphoid cells strictly as a consequence of receptor activation in the absence of apparent cellular stress. Microarray analysis comparing HSF1(+/+) and HSF1(-/-) gene expression in T cells activated at 37°C revealed a diverse set of 323 genes significantly regulated by HSF1 in nonstressed T cells. In vivo proliferation studies revealed a significant impairment of HSF1(-/-) T cell expansion under conditions mimicking a robust immune response (staphylococcal enterotoxin B-induced T cell activation). This proliferation defect due to loss of HSF1 is observed even under nonfebrile temperatures. HSF1(-/-) T cells activated at fever temperatures show a dramatic reduction in cyclin E and cyclin A proteins during the cell cycle, although the transcription of these genes was modestly affected. Finally, B cell and hematopoietic stem cell proliferation from HSF1(-/-) mice, but not HSF1(+/+) mice, were also attenuated under stressful conditions, indicating that HSF1 is critical for the cell cycle progression of lymphoid cells activated under stressful conditions.

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, Huntington's, Alzheimer's, and Parkinson's 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 Alzheimer's disease, and for decreased lamin A sumoylation as a causative factor in familial dilated cardiomyopathy.

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.

RNA polymerase II interacts with the Hspa1b promoter in mouse epididymal spermatozoa.

The Hspa1b (Hsp70.1) gene is one of the first genes expressed after fertilization, with expression occurring during the minor zygotic genome activation (ZGA) in the absence of stress. This expression can take place in the male pronucleus as early as the one-cell stage of embryogenesis. The importance of HSPA1B for embryonic viability during times of stress is supported by studies showing that depletion of this protein results in a significant reduction in embryos developing to the blastocyte stage. Recently, we have begun addressing the mechanism responsible for allowing expression of Hspa1b during the minor ZGA and found that heat shock transcription factor (HSF) 1 and 2 bind the Hspa1b promoter during late spermatogenesis. In this report, we have extended those studies using western blots and chromatin immunoprecipitation assays and found that RNA polymerase II (Pol II) is present in epididymal spermatozoa and bound to the Hspa1b promoter. These present results, in addition to our previous results, support a model in which the binding of HSF1, HSF2, SP1, and Pol II to the promoter of Hspa1b would allow the rapid formation of a transcription-competent state during the minor ZGA, thereby allowing Hspa1b expression.

PRC1 associates with the hsp70i promoter and interacts with HSF2 during mitosis.

Mitosis is a series of events leading to division of a cell by the process known as cytokinesis. Protein regulating cytokinesis 1 (PRC1) is a CDK substrate that associates with the mitotic spindle and functions in microtubule bundling. Previous studies revealed that loss of PRC1 is associated with chromosomal mis-segregation and atypical chromosome alignment. HSF2 is a DNA binding protein that we previously showed bookmarks the hsp70i gene during mitosis, an epigenetic mechanism which allows the hsp70i gene to re-establish transcriptional competence early in G1. Another study demonstrated that HSF2-/- mouse embryonic fibroblasts (MEFs) exhibit increased numbers of multinucleated cells vs. wild-type MEFs. This suggests that HSF2 is important for proper cytokinesis, but the mechanism was unknown. Here we report the existence of a direct interaction between HSF2 and PRC1. HSF2 and PRC1 associate during mitosis and co-localize during this phase of the cell cycle. PRC1 does not interact with the related protein HSF1, indicating the specificity of the HSF2-PRC1 interaction. Intriguingly, PRC1 is associated with the hsp70i promoter during mitosis. These results provide a potential mechanistic basis for the defective cytokinesis phenotype exhibited by HSF2-/- cells, as well as suggest a potential role for PRC1 in HSF2-mediated gene bookmarking.

Sumoylation of amyloid precursor protein negatively regulates Abeta aggregate levels.

The proteolytic processing of amyloid precursor protein (APP) to produce Abeta peptides is thought to play an important role in the mechanism of Alzheimer's disease. Here, we show that lysines 587 and 595 of APP, which are immediately adjacent to the site of beta-secretase cleavage, are covalently modified by SUMO proteins in vivo. Sumoylation of these lysine residues is associated with decreased levels of Abeta aggregates. Further, overexpression of the SUMO E2 enzyme ubc9 along with SUMO-1 results in decreased levels of Abeta aggregates in cells transfected with the familial Alzheimer's disease-associated V642F mutant APP, indicating the potential of up-regulating activity of the cellular sumoylation machinery as an approach against Alzheimer's disease. The results also provide the first demonstration that the SUMO E2 enzyme (ubc9) is present within the endoplasmic reticulum, indicating how APP, and perhaps other proteins that enter this compartment, can be sumoylated.

Mel-18 interacts with RanGAP1 and inhibits its sumoylation.

Our previous results showed that the polycomb protein mel-18 binds to a protein called HSF2 and inhibits HSF2 sumoylation, thereby functioning as an anti-SUMO E3 factor. This study also suggested that mel-18 regulates the sumoylation of other cellular proteins, but the identities of these other proteins were unknown. Here we show that mel-18 interacts with the RanGAP1 protein and inhibits its sumoylation, and that these activities do not require the RING domain of mel-18. The results also show that RanGAP1 sumoylation is decreased during mitosis, and that this is associated with increased interaction between RanGAP1 and mel-18 during this stage of the cell cycle. Intriguingly, this regulatory relationship is the opposite of that found for mel-18 and HSF2, in which the interaction between these two proteins decreases during mitosis, resulting in elevated HSF2 sumoylation. The results of this study strengthen the conclusion that mel-18 functions as an anti-SUMO E3 factor, and extend its targets to include regulation of the sumoylation of the important cellular protein RanGAP1.

Phosphorylation of CAP-G is required for its chromosomal DNA localization during mitosis.

Condensin is a 5 subunit complex that plays an important role in the structure of chromosomes during mitosis. It is known that phosphorylation of condensin subunits by cdc2/cyclin B at the beginning of mitosis is important for condensin activity, but the sites of these phosphorylation events have not been identified nor has their role in regulating condensin function. Here we identify two threonine residues in the CAP-G subunit of condensin, threonines 308 and 332, that are targets of cdc2/cyclin B phosphorylation. Mutation of these threonines to alanines results in defects in CAP-G localization with chromosomes during mitosis. These results are the first to identify phosphorylation sites within the condensin complex that regulate condensin localization with chromosomal DNA.

Celastrol inhibits polyglutamine aggregation and toxicity though induction of the heat shock response.

Heat shock proteins (hsps) are protective against the harmful effects of mutant expanded polyglutamine repeat proteins that occur in diseases such as Huntington's, prompting the search for pharmacologic compounds that increase hsp expression in cells as potential treatments for this and related diseases. In this paper, we show that celastrol, a compound recently shown to up-regulate hsp gene expression, significantly decreases killing of cells expressing mutant polyglutamine protein. This effect requires the presence of the transcription factor responsible for mediating inducible hsp gene expression, HSF1, and is correlated with decreased amounts and increased sodium dodecyl sulfate (SDS) solubility of polyglutamine aggregates. These results suggest the potential of celastrol as a therapeutic agent in the treatment of human polyglutamine expansion diseases.

Identification of the PP2A-interacting region of heat shock transcription factor 2.

Previous work in our laboratory demonstrated the existence of an association between heat shock transcription factor 2 (HSF2) and the serine/threonine phosphatase 2A, which is mediated by interaction between HSF2 and the A subunit (also called PR65) of this protein phosphatase. In light of the importance of HSF2-PP2A association for HSF2 cellular function, in this study, we have sought to dissect the sequences within HSF2 that are important for interaction with the A subunit of PP2A. The results of these experiments indicate that the HSF2 region comprising amino acids 343-363 is important for A subunit interaction. This region includes part of the C-terminal leucine zipper motif of HSF2 called heptad repeat C (HR-C). The results of transfection/immunoprecipitation experiments also show that deletion of the 6 amino acids from 343 to 348 from HSF2 (HSF2 (delta343-348)), is sufficient to prevent HSF2 from interacting with PP2A. These data provide insight into a new functional domain of HSF2, the PP2A A subunit-interacting region.

HSF2 binds to the Hsp90, Hsp27, and c-Fos promoters constitutively and modulates their expression.

Although the vast majority of genomic DNA is tightly compacted during mitosis, the promoter regions of a number of genes remain in a less compacted state throughout this stage of the cell cycle. The decreased compaction of these promoter regions, which is referred to as gene bookmarking, is thought to be important for the ability of cells to express these genes during the following interphase. Previously, we reported a role for the DNA-binding protein heat shock factor (HSF2) in bookmarking the stress-inducible 70,000-Da heat shock protein (hsp70) gene. In this report, we have extended those studies and found that during mitosis, HSF2 is bound to the HSE promoter elements of other heat shock genes, including hsp90 and hsp27, as well as the proto-oncogene c-fos. The presence of HSF2 is important for expression of these genes because blocking HSF2 levels by RNA interference techniques leads to decreased levels of these proteins. These results suggest that HSF2 is important for constitutive as well as stress-inducible expression of HSE-containing genes.

Analysis of Protein Sumoylation.

Sumoylation, wherein small ubiquitin-like modifier (SUMO) proteins are covalently attached to specific lysine residues of target proteins, plays an important role in regulating many diverse cellular processes via its control of the functional properties of the modified proteins. Identification of new sumoylated proteins is expected to expand understanding of the role this modification has in cell function. This unit describes two different assays for determining whether a particular protein is sumoylated: the first method employs immunoprecipitation of the protein followed by SUMO immunoblot. The second involves incubating the protein (either an in vitro translation product or a purified recombinant protein) with a reconstituted in vitro sumoylation reaction followed by examination for increased molecular-weight bands in SDS-PAGE as sumoylated forms of the protein. Either of these approaches can also be used to determine the sumoylated lysine residue(s) by comparing modification of the normal protein versus lysine-to-arginine substitutions of potential sumoylation sites, which once determined allows analysis of the effect of sumoylation on the protein's function.

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.

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.

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.

WITHDRAWN: Protein sumoylation and human diseases.

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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 Huntington's, Alzheimer's, and Parkinson's 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 Aß peptide levels, suggesting a potential role in Alzheimer's disease, and decreased lamin A sumoylation due to mutations near its SUMO site has been implicated in causing some forms of familial dilated cardiomyopathy.

PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit of the Cul3-RING ubiquitin ligase.

Cullin-RING ubiquitin ligases promote the polyubiquitination and degradation of many important cellular proteins, which previous studies indicated can be targeted for degradation via interaction with BTB domain-containing subunits of this E3 ligase complex. PEST domains are known to promote the degradation of proteins that contain them. However, the molecular mechanism by which PEST sequences promote degradation of these proteins is not understood. Here we show that the PEST sequences of a short-lived protein called HSF2 interact with Cullin3, a subunit of a Cullin-RING E3 ubiquitin ligase, and that this interaction mediates the Cul3-dependent ubiquitination and degradation of HSF2. These results indicate how, at the molecular level, PEST sequences can promote the proteolysis of proteins that contain them. They also expand understanding of the mechanisms by which substrates can be recruited to Cullin-RING E3 ubiquitin ligases to include interactions between PEST sequences and Cul3.

Identification of a polymorphism in the RING finger of human Bmi-1 that causes its degradation by the ubiquitin-proteasome system.

Bmi-1 is a polycomb protein that plays an important role in tumor cell development and maintaining stem cell populations of many cell lineages. Here we identify a polymorphism in human Bmi-1 that changes a cysteine within its RING domain to tyrosine. This C18Y polymorphism is associated with a significant decrease in Bmi-1 level and its elevated ubiquitination, suggesting that it is being destroyed by the ubiquitin-proteasome system. Consistent with this, treating cells with the proteasome inhibitor MG-132 significantly increases C18Y Bmi-1 levels. This is the first example of a polymorphism in Bmi-1 that reduces levels of this important protein.

Mitotic bookmarking of formerly active genes: keeping epigenetic memories from fading.

In order for cell lineages to be maintained, daughter cells must have the same patterns of gene expression as the cells from which they were divided so that they can have the same phenotypes. However, during mitosis transcription ceases, chromosomal DNA is compacted, and most sequence-specific binding factors dissociate from DNA, making it difficult to understand how the "memory" of gene expression patterns is remembered and propagated to daughter cells. The process of remembering patterns of active gene expression during mitosis for transmission to daughter cells is called gene bookmarking. Here we discuss current knowledge concerning the factors and mechanisms involved in mediating gene bookmarking, including recent results on the mechanism by which the general transcription factor TBP participates in the mitotic bookmarking of formerly active genes.