HSF1 regulation of ß-catenin in mammary cancer cells through control of HuR/elavL1 expression.

There is now compelling evidence to indicate a place for heat shock factor 1 (HSF1) in mammary carcinogenesis, tumour progression and metastasis. Here we have investigated a role for HSF1 in regulating the expression of the stem cell renewal factor ß-catenin in immortalized human mammary epithelial and carcinoma cells. We found HSF1 to be involved in regulating the translation of ß-catenin, by investigating effects of gain and loss of HSF1 on this protein. Interestingly, although HSF1 is a potent transcription factor, it was not directly involved in regulating levels of ß-catenin mRNA. Instead, our data suggest a complex role in translational regulation. HSF1 was shown to regulate levels of the RNA-binding protein HuR that controlled ß-catenin translation. An extra complexity was added to this scenario when it was shown that the long non-coding RNA molecule lincRNA-p21, known to be involved in ß-catenin mRNA (CTNNB1) translational regulation, was controlled by HSF1 repression. We have shown previously that HSF1 was positively regulated through phosphorylation by mammalian target of rapamycin (mTOR) kinase on a key residue, serine 326, essential for transcriptional activity. In this study, we found that mTOR knockdown not only decreased HSF1-S326 phosphorylation in mammary cells, but also decreased ß-catenin expression through a mechanism requiring HuR. Our data point to a complex role for HSF1 in the regulation of HuR and ß-catenin expression that may be significant in mammary carcinogenesis.

mTOR is essential for the proteotoxic stress response, HSF1 activation and heat shock protein synthesis.

The target of rapamycin (TOR) is a high molecular weight protein kinase that regulates many processes in cells in response to mitogens and variations in nutrient availability. Here we have shown that mTOR in human tissue culture cells plays a key role in responses to proteotoxic stress and that reduction in mTOR levels by RNA interference leads to increase sensitivity to heat shock. This effect was accompanied by a drastic reduction in ability to synthesize heat shock proteins (HSP), including Hsp70, Hsp90 and Hsp110. As HSP transcription is regulated by heat shock transcription factor 1 (HSF1), we examined whether mTOR could directly phosphorylate this factor. Indeed, we determined that mTOR could directly phosphorylate HSF1 on serine 326, a key residue in transcriptional activation. HSF1 was phosphorylated on S326 immediately after heat shock and was triggered by other cell stressors including proteasome inhibitors and sodium arsenite. Null mutation of S326 to alanine led to loss of ability to activate an HSF1-regulated promoter-reporter construct, indicating a direct role for mTOR and S326 in transcriptional regulation of HSP genes during stress. As mTOR is known to exist in at least two intracellular complexes, mTORC1 and mTOR2 we examined which complex might interact with HSF1. Indeed mTORC1 inhibitor rapamycin prevented HSF1-S326 phosphorylation, suggesting that this complex is involved in HSF1 regulation in stress. Our experiments therefore suggest a key role for mTORC1 in transcriptional responses to proteotoxic stress.

The role of heat shock factors in stress-induced transcription.

Heat shock proteins (HSPs) are rapidly induced after stresses, such as heat shock, and accumulate at high concentrations in cells. HSP induction involves a family of heat shock transcription factors that bind the heat shock elements of the HSP genes and mediate transcription in trans. We discuss methods for the study of HSP binding to HSP promoters and the consequent increases in HSP gene expression in vitro and in vivo.

Protein kinase A binds and activates heat shock factor 1.

BACKGROUND: Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1), a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. METHODOLOGY/PRINCIPAL FINDINGS: In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA). HSF1 binds avidly to the catalytic subunit of PKA, (PKAcα) and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320), both in vitro and in vivo. Intracellular PKAcα levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1) and activate hsp70.1 after stress. Reduction in PKAcα levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, null mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. CONCLUSIONS: These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease.

Spatial distribution of histone methylation during MHC class II expression.

We have previously reported that Major Histocompatibility Complex (MHC) class II can be induced by histone deacetylase inhibitors (HDACi) in the absence of class II transactivator (CIITA). Here we characterized the histone modifications associated with the CIITA-dependent (IFN-gamma induced) and -independent (HDACi induced) MHC class II expression. We demonstrate that both IFN-gamma and HDACi induced MHC class II expression exhibited enhanced histone H3, H4 acetylation and H3K4me3 at the MHC class II promoter while H3K9me3 was decreased. In contrast, high levels of H3K36me3 were detected at exons 3 and 5 but not at the promoter or the locus control region (LCR). Interestingly, high levels of H3K79me2 were only detected at the promoter and exon 3 of the B cell lines while the level remained low and unchanged despite active MHC class II expression induced by either IFN-gamma or HDACi treatment. Constitutive expression of the CIITA protein by stable transfection of a CIITA deficient B cell line restored the H3K79me2 to a level comparable to its cell of origin. This data demonstrates that, although regulated by different pathways, both IFN-gamma and HDACi treatments resulted in similar patterns of histone modifications and that HDACi induce both histone methylation and acetylation. In addition, the different spatial distribution of the lysine methylation markers along the gene suggests that these modifications play a distinctive role during different phases of the transcription process.

Histone acetylation regulates the cell type specific CIITA promoters, MHC class II expression and antigen presentation in tumor cells.

The regulation of MHC class II expression by the class II transactivator (CIITA) is complex and differs in various cell types depending on the relative activity of three CIITA promoters. Here we show that, in plasma cell tumors, the deacetylase inhibitor trichostatin A (TSA) elicits PIII-CIITA but does not activate the IFN-gamma-inducible PIV-CIITA promoter. In trophoblast cells, all CIITA promoter types are constitutively silent and not induced by IFN-gamma or TSA treatment. TSA induction of PI-CIITA was restricted to macrophage and dendritic cell lines. In the Colon 26 tumor IFN-gamma induced endogenous PIV-CIITA but not PIII-CIITA while TSA activated class II in the apparent absence of CIITA. Reporter assays in Colon 26 showed that TSA induced PIII-CIITA but not PIV-CIITA. Transfection of a dominant negative CIITA plasmid in Colon 26 inhibited induction of class II by IFN-gamma but not TSA. Thus, the potential for both CIITA-dependent and -independent pathways of MHC induction exists within a single cell. Further evidence of CIITA-independent class II expression elicited by TSA was obtained using knockout mice with defects in CIITA, STAT-1alpha and IRF-1 expression. TSA treatment can also activate class II expression in mutant cell lines with deficiencies in signaling molecules, transcription factors and the BRG-1 cofactor that are required for IFN-gamma-induced CIITA expression. Importantly, after epigenetic activation by the deacetylase inhibitor, MHC class II is transported and displayed on the cell surface of a plasma cell tumor and it is converted to an efficient antigen presenting cell for protein and class II-peptide presentation.