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1.
Curr Protoc Protein Sci ; 83: 14.8.1-14.8.8, 2016 Feb 02.
Article in English | MEDLINE | ID: mdl-26836406

ABSTRACT

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.


Subject(s)
Electrophoresis, Polyacrylamide Gel , Immunoprecipitation/methods , SUMO-1 Protein/isolation & purification , SUMO-1 Protein/metabolism , Sumoylation/physiology , Animals , Humans , SUMO-1 Protein/chemistry
2.
J Immunol ; 191(8): 4068-79, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-24043900

ABSTRACT

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.


Subject(s)
DNA-Binding Proteins/metabolism , Lymphocyte Activation , Stress, Physiological , T-Lymphocytes/immunology , Transcription Factors/metabolism , Animals , Cell Cycle , Cell Division , Cell Proliferation , Cells, Cultured , Cyclin A/biosynthesis , Cyclin E/biosynthesis , DNA-Binding Proteins/genetics , Enterotoxins/immunology , Fever/immunology , Gene Expression Regulation , Heat Shock Transcription Factors , Heat-Shock Proteins/metabolism , Heat-Shock Response/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Reactive Oxygen Species/metabolism , Transcription Factors/genetics
3.
Biochimie ; 2012 Sep 26.
Article in English | MEDLINE | ID: mdl-23022145

ABSTRACT

This review has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

4.
Int Rev Cell Mol Biol ; 288: 167-83, 2011.
Article in English | MEDLINE | ID: mdl-21482412

ABSTRACT

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.


Subject(s)
Disease , Neoplasms/physiopathology , Neurodegenerative Diseases/physiopathology , Small Ubiquitin-Related Modifier Proteins/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Ataxin-1 , Ataxins , Humans , Huntingtin Protein , Intracellular Signaling Peptides and Proteins/metabolism , Neoplasms/metabolism , Nerve Tissue Proteins/metabolism , Neurodegenerative Diseases/metabolism , Nuclear Proteins/metabolism , Oncogene Proteins/metabolism , Protein Deglycase DJ-1 , Small Ubiquitin-Related Modifier Proteins/genetics , Sumoylation , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , alpha-Synuclein/metabolism , tau Proteins/metabolism
5.
Cell Stress Chaperones ; 15(3): 301-8, 2010 May.
Article in English | MEDLINE | ID: mdl-19768582

ABSTRACT

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.


Subject(s)
Cullin Proteins/metabolism , Heat-Shock Proteins , Protein Subunits/metabolism , Transcription Factors , Ubiquitin-Protein Ligases/metabolism , Animals , Base Sequence , Cullin Proteins/genetics , HeLa Cells , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Protein Subunits/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases/genetics
6.
Methods Mol Biol ; 590: 223-33, 2009.
Article in English | MEDLINE | ID: mdl-19763507

ABSTRACT

In order to fully understand the functions of a DNA-binding protein it is necessary to identify all of its binding sites in chromosomes and assess the role of each site in the overall biological function of the factor. An approach ChIP-on-Chip which combines the chromatin immunoprecipitation technique with chromosomal DNA microarray analysis, has proven to be a powerful means for the chromosome-wide identification of protein binding sites. This approach can also be used to characterize chromosome-wide variations in patterns of post-translational protein modifications, for example histone modifications. This chapter presents methodologies for the ChIP-on-Chip analysis, using as an example the identification of chromosome-wide binding sites for the TATA-binding protein in mitotic cells.


Subject(s)
Chromosomes, Human , Base Sequence , Chromatin Immunoprecipitation , DNA Primers , Fluorescent Dyes , Humans , Protein Binding
7.
Methods Mol Biol ; 590: 265-77, 2009.
Article in English | MEDLINE | ID: mdl-19763510

ABSTRACT

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.


Subject(s)
Small Ubiquitin-Related Modifier Proteins/metabolism , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Humans , Immunoprecipitation , Recombinant Proteins/metabolism
8.
Reproduction ; 137(6): 923-9, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19336471

ABSTRACT

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.


Subject(s)
Epididymis/enzymology , HSP70 Heat-Shock Proteins/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , Spermatozoa/enzymology , Animals , Binding Sites , Blotting, Western , Cell Nucleus/enzymology , Chromatin Immunoprecipitation , Gene Expression Regulation, Developmental , HSP70 Heat-Shock Proteins/genetics , Male , Mice
9.
Trends Biochem Sci ; 34(4): 200-5, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19282183

ABSTRACT

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.


Subject(s)
Disease , SUMO-1 Protein/metabolism , Humans , Protein Binding , SUMO-1 Protein/genetics
10.
Cell Cycle ; 8(6): 818-23, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19221503

ABSTRACT

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.


Subject(s)
Adenosine Triphosphatases/metabolism , DNA-Binding Proteins/metabolism , Epigenesis, Genetic , Gene Expression , Mitosis/genetics , Multiprotein Complexes/metabolism , Promoter Regions, Genetic , Transcription Factors/metabolism , Animals , Base Sequence , Humans
11.
FEBS Lett ; 583(6): 960-4, 2009 Mar 18.
Article in English | MEDLINE | ID: mdl-19233177

ABSTRACT

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.


Subject(s)
Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Polymorphism, Single Nucleotide , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RING Finger Domains/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Amino Acid Sequence , Cells, Cultured , Cysteine/genetics , Cysteine/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Humans , Leupeptins/pharmacology , Nuclear Proteins/chemistry , Polycomb Repressive Complex 1 , Polymorphism, Single Nucleotide/physiology , Protein Denaturation/genetics , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/genetics , Proto-Oncogene Proteins/chemistry , Repressor Proteins/chemistry , Transfection , Tyrosine/genetics , Ubiquitin/metabolism
12.
Methods Mol Biol ; 464: 255-65, 2009.
Article in English | MEDLINE | ID: mdl-18951189

ABSTRACT

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 a variety of different 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 of nuclear structure, making the characterization of this modification vital for understanding nuclear structure and function. Because sumoylation plays an important role in regulating so many important cellular processes, there has been intense interest in identifying new proteins that are targets of this modification and determining what role sumoylation plays in regulating the protein 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.


Subject(s)
Proteins/chemistry , Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/chemistry , Small Ubiquitin-Related Modifier Proteins/metabolism , Animals , Chromatography, Affinity , Humans , Mass Spectrometry , Signal Transduction/physiology
13.
Biochem Biophys Res Commun ; 377(3): 1007-11, 2008 Dec 19.
Article in English | MEDLINE | ID: mdl-18977199

ABSTRACT

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.


Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Chromosomes, Human/metabolism , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Mutation , Adenosine Triphosphatases/genetics , Alanine/genetics , Alanine/metabolism , Amino Acid Sequence , Animals , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/genetics , Chromosomes, Human/genetics , Cyclin B/metabolism , DNA/metabolism , DNA-Binding Proteins/genetics , HeLa Cells , Humans , Mitosis , Molecular Sequence Data , Multiprotein Complexes/genetics , Phosphorylation , Threonine/genetics , Threonine/metabolism , Xenopus
14.
Nat Cell Biol ; 10(11): 1318-23, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18931662

ABSTRACT

To maintain phenotypes of cell lineages, cells must 'remember' which genes were active before mitosis entry and transmit this information to their daughter cells so that expression patterns can be faithfully re-established in G1. This phenomenon is called gene bookmarking. However, during mitosis transcription ceases, most sequence-specific proteins dissociate from DNA and the chromatin is tightly compacted, making it difficult to understand how gene activity 'memory' is maintained through this stage of the cell cycle. A feature of gene bookmarking is that in mitotic cells, the promoters of formerly active genes lack compaction, but how compaction of these regions is inhibited is unknown. Here we show that during mitosis, TATA-binding protein (TBP), which remains bound to DNA during mitosis, recruits PP2A. TBP also interacts with condensin to allow efficient dephosphorylation and inactivation of condensin near these promoters to inhibit their compaction. Further, ChIP-on-chip data show that TBP is bound to many chromosomal sites during mitosis, and is higher in transcribed regions but low in regions containing pseudogenes and genes whose expression is tissue-restricted. These results suggest that TBP is involved not only in gene transcription during interphase but also in preserving the memory of gene activity through mitosis to daughter cells.


Subject(s)
Adenosine Triphosphatases/metabolism , DNA-Binding Proteins/metabolism , Genes , Mitosis , Multiprotein Complexes/metabolism , Protein Phosphatase 2/metabolism , TATA-Box Binding Protein/metabolism , Escherichia coli/genetics , Glutathione Transferase/metabolism , HeLa Cells , Humans , Protein Phosphatase 2/genetics , RNA, Small Interfering/pharmacology , Recombinant Proteins/metabolism , TATA-Box Binding Protein/genetics
15.
Biochem Biophys Res Commun ; 375(2): 252-5, 2008 Oct 17.
Article in English | MEDLINE | ID: mdl-18706886

ABSTRACT

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.


Subject(s)
DNA-Binding Proteins/metabolism , GTPase-Activating Proteins/metabolism , Repressor Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/antagonists & inhibitors , Cell Line , DNA-Binding Proteins/genetics , Humans , Polycomb Repressive Complex 1 , RING Finger Domains/genetics , Repressor Proteins/genetics , Ubiquitin-Conjugating Enzymes/metabolism
16.
Biochem Biophys Res Commun ; 374(4): 673-8, 2008 Oct 03.
Article in English | MEDLINE | ID: mdl-18675254

ABSTRACT

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.


Subject(s)
Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Protein Processing, Post-Translational , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/genetics , Amyloid beta-Protein Precursor/genetics , Endoplasmic Reticulum/enzymology , HeLa Cells , Humans , Lysine/genetics , Lysine/metabolism , Transfection , Ubiquitin-Conjugating Enzymes/genetics
17.
J Cell Biol ; 182(1): 35-9, 2008 Jul 14.
Article in English | MEDLINE | ID: mdl-18606848

ABSTRACT

Lamin A mutations cause many diseases, including cardiomyopathies and Progeria Syndrome. The covalent attachment of small ubiquitin-like modifier (SUMO) polypeptides regulates the function of many proteins. Until now, no examples of human disease-causing mutations that occur within a sumoylation consensus sequence and alter sumoylation were known. We show that lamin A is sumoylated at lysine 201 and that two lamin A mutants associated with familial dilated cardiomyopathy, E203G and E203K, exhibit decreased sumoylation. E203 occupies the conserved +2 position in the sumoylation consensus Psi KXE. Lamin A mutants E203G, E203K, and K201R all exhibit a similar aberrant subcellular localization and are associated with increased cell death. Fibroblasts from an individual with the E203K lamin A mutation also exhibit decreased lamin A sumoylation and increased cell death. These results suggest that SUMO modification is important for normal lamin A function and implicate an involvement for altered sumoylation in the E203G/E203K lamin A cardiomyopathies.


Subject(s)
Cardiomyopathies/metabolism , Lamin Type A/metabolism , Mutant Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Animals , Cell Death , Cell Nucleus/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , HeLa Cells , Humans , Lysine/metabolism , Mice , Protein Transport
18.
Exp Cell Res ; 314(11-12): 2224-30, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18570919

ABSTRACT

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.


Subject(s)
Cell Cycle Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Mitosis/physiology , Promoter Regions, Genetic , Transcription Factors/metabolism , Animals , Cell Cycle Proteins/genetics , HSP70 Heat-Shock Proteins/genetics , HeLa Cells , Heat-Shock Proteins/genetics , Humans , Jurkat Cells , Mice , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Two-Hybrid System Techniques
19.
Biol Reprod ; 79(2): 283-8, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18434628

ABSTRACT

The Hspa1b gene is one of the first genes expressed after fertilization, with expression observed in the male pronucleus as early as the one-cell stage of embryogenesis. This expression can occur in the absence of stress and is initiated during the minor zygotic genome activation. There is a significant reduction in the number of embryos developing to the blastocyte stage when HSPA1B levels are depleted, which supports the importance of this protein for embryonic viability. However, the mechanism responsible for allowing expression of Hspa1b during the minor zygotic genome activation (ZGA) is unknown. In this report, we investigated the role of HSF1 and HSF2 in bookmarking Hspa1b during late spermatogenesis. Western blot results show that both HSF1 and HSF2 are present in epididymal spermatozoa, and immunofluorescence analysis revealed that some of the HSF1 and HSF2 proteins in these cells overlap the 4',6'-diamidino-2-phenylindole-stained DNA region. Results from chromatin immunoprecipitation assays showed that HSF1, HSF2, and SP1 are bound to the Hspa1b promoter in epididymal spermatozoa. Furthermore, we observed an increase in HSF2 binding to the Hspa1b promoter in late spermatids versus early spermatids, suggesting a likely period during spermatogenesis when transcription factor binding could occur. These results support a model in which the binding of HSF1, HSF2, and SP1 to the promoter of Hspa1b would allow the rapid formation of a transcription-competent state during the minor ZGA, thereby allowing Hspa1b expression.


Subject(s)
DNA-Binding Proteins/metabolism , Epididymis/metabolism , HSP70 Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Promoter Regions, Genetic , Spermatozoa/metabolism , Transcription Factors/metabolism , Animals , Cell Nucleus/metabolism , Chromatin Immunoprecipitation , Heat Shock Transcription Factors , Male , Mice , Protein Binding , Sp1 Transcription Factor/metabolism , Spermatogenesis/genetics , Tissue Distribution , Transcription, Genetic/physiology , Zygote/metabolism
20.
J Biol Chem ; 283(12): 7464-9, 2008 Mar 21.
Article in English | MEDLINE | ID: mdl-18211895

ABSTRACT

In a previous study we found that sumoylation of the DNA-binding protein heat shock factor 2 (HSF2) is up-regulated during mitosis, but the mechanism that mediates this regulation was unknown. Here we show that HSF2 interacts with the polycomb protein MEL-18, that this interaction decreases during mitosis, and that overexpression and RNA interference-mediated reduction of MEL-18 result in decreased and increased HSF2 sumoylation, respectively. Other results suggest that MEL-18 may also function to inhibit the sumoylation of other cellular proteins. The results also show that MEL-18 is able to interact with the small ubiquitin-like modifier (SUMO) ubiquitin carrier protein (E2) enzyme UBC9 and that MEL-18 inhibits the ability of UBC9 to transfer the SUMO protein to target proteins. Together, the results in this work suggest a mechanism in which MEL-18 bound to HSF2 inhibits its sumoylation by binding to and inhibiting the activity of UBC9 enzymes in the vicinity of HSF2. These results provide an explanation for how mitotic HSF2 sumoylation is regulated and suggest that MEL-18, in contrast to the sumoylation-stimulating activities of the polycomb protein PC2, actually functions like an anti-SUMO ubiquitin-protein isopeptide ligase (E3), interacting both with HSF2 and the SUMO E2 UBC9 but acting to inhibit UBC9 activity to decrease sumoylation of a target protein, in this case that of HSF2.


Subject(s)
DNA-Binding Proteins/metabolism , Heat-Shock Proteins/metabolism , Mitosis/physiology , Protein Processing, Post-Translational/physiology , Repressor Proteins/metabolism , SUMO-1 Protein/metabolism , Transcription Factors/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , DNA-Binding Proteins/genetics , HeLa Cells , Heat-Shock Proteins/genetics , Humans , Ligases , Polycomb Repressive Complex 1 , Polycomb-Group Proteins , Repressor Proteins/genetics , SUMO-1 Protein/genetics , Transcription Factors/genetics , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
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