Michael L. Goodson

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.

Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay

A stable luciferase reporter gene assay was developed based on the thyroid hormone responsive rat pituitary tumor GH3 cell line that constitutively expresses both thyroid hormone receptor isoforms. Stable transfection of the pGL4CP-SV40-2xtaDR4 construct into the GH3 cells resulted in a highly sensitive cell line (GH3.TRE-Luc), which was further optimized into an assay that allowed the detection of Triiodothyronine (T(3)) and Thyroxine (T(4)) concentrations in the picomolar range after only 24 h of exposure. The greater than 20-fold induction of T(3) relative to the solvent control is illustrative of the high responsiveness of the system. The assay was validated by the quantification of the agonistic effect of the natural hormones (T(3) and T(4)), the acetic acid derivatives of T(3) (triiodothyroaceticacid, or Triac) and T(4) (tetraiodothyroacetic acid, or Tetrac), hydroxy polybrominated diphenylethers (OH-PBDEs), hydroxy polychlorinated biphenyls (OH-PCBs) and the antagonistic action of sodium arsenite (NaAsO(2)). The putative antagonist Amiodarone, Bisphenol A (BPA) and its halogenated derivatives (TCBPA and TBBPA) for which effects reported in the literature are not consistent, showed comparable dose-response curves with a slight agonistic effect (5% of T(3)-max) followed by a slight antagonistic effect. The magnitude and reproducibility of the responses to various compounds confirms this assay as a promising tool for the identification and quantification of specific thyroid hormone receptor disrupting potency of compounds.

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.

Corepressors: custom tailoring and alterations while you wait

A diverse cadre of metazoan transcription factors mediate repression by recruiting protein complexes containing the SMRT (silencing mediator of retinoid and thyroid hormone receptor) or N-CoR (nuclear receptor corepressor) corepressors. SMRT and N-CoR nucleate the assembly of still larger corepressor complexes that perform the specific molecular incantations necessary to confer transcriptional repression. Although SMRT and N-CoR are paralogs and possess similar molecular architectures and mechanistic strategies, they nonetheless exhibit distinct molecular and biological properties. It is now clear that the functions of both SMRT and N-CoR are further diversified through alternative mRNA splicing, yielding a series of corepressor protein variants that participate in distinctive transcription factor partnerships and display distinguishable repression properties. This review will discuss what is known about the structure and actions of SMRT, N-CoR, and their splicing variants, and how alternative splicing may allow the functions of these corepressors to be adapted and tailored to different cells and to different developmental stages.

Nuclear receptor coregulators as a new paradigm for therapeutic targeting.

The complex function and regulation of nuclear receptors cannot be fully understood without a thorough knowledge of the receptor-associated coregulators that either enhance (coactivators) or inhibit (corepressors) transcription. While nuclear receptors themselves have garnered much attention as therapeutic targets, the clinical and etiological relevance of the coregulators to human diseases is increasingly recognized. Aberrant expression or function of coactivators and corepressors has been associated with malignant and metabolic disease development. Many of them are key epigenetic regulators and utilize enzymatic activities to modify chromatin through histone acetylation/deacetylation, histone methylation/demethylation or chromatin remodeling. In this review, we showcase and evaluate coregulators--such as SRCs and ANCCA--with the most promising therapeutic potential based on their physiological roles and involvement in various diseases that are revealed thus far. We also describe the structural features of the coactivator and corepressor functional domains and highlight areas that can be further explored for molecular targeting.

Alternative mRNA splicing of corepressors generates variants that play opposing roles in adipocyte differentiation.

Background: The SMRT and NCoR corepressors play key biological roles in transcriptional repression. Results: Alternative mRNA splicing produces corepressor variants that can exert opposite effects on adipocyte differentiation. Conclusion: Corepressors are diversified by alternative mRNA splicing, allowing one locus to encode multiple proteins with distinct functions. Significance: Changes in alternative splicing may help drive the differentiation and customize the physiology of specific cell types. The SMRT and NCoR corepressors partner with, and help mediate repression by, a wide variety of nuclear receptors and non-receptor transcription factors. Both SMRT and NCoR are expressed by alternative mRNA splicing, resulting in the production of a series of interrelated corepressor variants that differ in their tissue distribution and in their biochemical properties. We report here that different corepressor splice variants can exert opposing transcriptional and biological effects during adipocyte differentiation. Most notably, the NCoRω splice variant inhibits, whereas the NCoRδ splice variant promotes, adipogenesis. Furthermore, the ratio of NCoRω to NCoRδ decreases during adipogenic differentiation. We propose that this alteration in corepressor splicing helps convert the cellular transcriptional program from one that maintains the pre-adipocyte in an undifferentiated state to a new transcriptional context that promotes differentiation and helps establish the proper physiology of the mature adipocyte.

MEL-18 Interacts with HSF2 and the SUMO E2 UBC9 to Inhibit HSF2 Sumoylation

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.

NCoR1 regulates thyroid hormone receptor isoform-dependent adipogenesis

We previously showed that two thyroid hormone receptor (TR) isoforms--TRα1 and TRβ1--differentially regulate thyroid hormone (triiodothyroxine, T(3))-stimulated adipogenesis in vivo. This study aims to understand the role of the nuclear receptor corepressor, NCoR1, in TR isoform-dependent adipogenesis. We found that T(3)-stimulated adipogenesis of 3T3-L1 cells was accompanied by progressive loss of NCoR1 protein levels. In 3T3-L1 cells stably expressing a mutated TRα1, PV (L1-α1PV cells), the T(3)-stimulated adipogenesis was more strongly inhibited than that in 3T3-L1 cells stably expressing an identical mutation in TRβ1 (L1-β1PV cells). The stronger inhibition of adipogenesis in L1-α1PV cells was associated with a higher NCoR1 protein level. These results indicate that the degree of loss of NCoR1 correlates with the extent of adipogenesis. siRNA knockdown of NCoR1 promoted adipogenesis of control 3T3-L1 cells and reversed the inhibited adipogenesis of L1-α1PV and L1-β1PV cells, indicating that NCoR1 plays an essential role in TR isoform-dependent adipogenesis. An ubiquitin ligase, mSiah2, that targets NCoR1 for proteasome degradation was upregulated on day 1 before the onset of progressive loss of NCoR1. NCoR1 was found to associate with mSiah2 and with TR, TRα1PV, or TRβ1PV, but a stronger interaction of NCoR1 with TRα1PV than with TRβ1PV was detected. Furthermore, TRα1PV-NCoR1 complex was more avidly recruited than TRβ1PV-NCoR1 to the promoter of the C/ebpα gene, leading to more inhibition in its expression. These results indicate that differential interaction of NCoR1 with TR isoforms accounted for the TR isoform-dependent regulation of adipogenesis and that aberrant interaction of NCoR1 with TR could underlie the pathogenesis of lipid disorders in hypothyroidism.

Aberrant Corepressor Interactions Implicated in PML-RARα and PLZF-RARα Leukemogenesis Reflect an Altered Recruitment and Release of Specific NCoR and SMRT Splice Variants

Human acute promyelocytic leukemia is causally linked to chromosomal translocations that generate chimeric retinoic acid receptor-α proteins (x-RARα fusions). Wild-type RARα is a transcription factor that binds to the SMRT/NCoR family of corepressors in the absence of hormone but releases from corepressor and binds coactivators in response to retinoic acid. In contrast, the x-RARα fusions are impaired for corepressor release and operate in acute promyelocytic leukemia as dominant-negative inhibitors of wild-type RARα. We report that the two most common x-RARα fusions, PML-RARα and PLZF-RARα, have gained the ability to recognize specific splice variants of SMRT and NCoR that are poorly recognized by RARα. These differences in corepressor specificity between the normal and oncogenic receptors are further magnified in the presence of a retinoid X receptor heteromeric partner. The ability of retinoids to fully release corepressor from PML-RARα differs for the different splice variants, a phenomenon relevant to the requirement for supraphysiological levels of this hormone in differentiation therapy of leukemic cells. We propose that this shift in the specificity of the x-RARα fusions to a novel repertoire of corepressors contributes to the dominant-negative and oncogenic properties of these oncoproteins and helps explain previously paradoxical aspects of their behavior.

An improved high throughput protein-protein interaction assay for nuclear hormone receptors

The Glutathione-S-Transferase (GST) “pulldown” assay has been used extensively to assay protein interactions in vitro. This methodology has been especially useful for investigating the interactions of nuclear hormone receptors with a wide variety of their interacting partners and coregulatory proteins. Unfortunately, the original GST-pulldown technique relies on multiple binding, washing and elution steps performed in individual microfuge tubes, and requires repeated centrifugation, aspiration, and suspension steps. This type of batch processing creates a significant liquid handling bottleneck, limiting the number of sample points that can be incorporated into one experiment and producing inherently less efficient washing and elution than would a flow-through methodology. In this manuscript, we describe the adaptation of this GST-pulldown assay to a 96-well filter plate format. The use of a multi-well filter plate makes it possible to assay more samples in significantly less time using less reagents and more efficient sample processing than does the traditional single tube assay.