Novel nuclear matrix protein HET binds to and influences activity of the HSP27 promoter in human breast cancer cells

Abstract

Since the small heat shock protein hsp27 enhances both growth and drug resistance in breast cancer cells, and is a bad prognostic factor in certain subsets of breast cancer patients, we have characterized the transcriptional regulation of hsp27, with the long-term goal of targeting its expression clinically. The majority of the promoter activity resides in the most proximal 200 bp. This region contains an imperfect estrogen response element (ERE) that is separated by a 13-bp spacer that contains a TATA box. Gel-shift analysis revealed the binding of a protein (termed HET for Hsp27-ERE-TATA-binding protein) to this region that was neither the estrogen receptor nor TATA-binding protein. We cloned a complete cDNA (2.9 kb) for HET from an MCF-7 cDNA library. To confirm the identity of the HET clone, we expressed a partial HET clone as a glutathione S-transferase fusion protein, and showed binding to the hsp27 promoter fragment in gel-retardation assays. The HET clone is almost identical to a recently published scaffold attachment factor (SAF-B) cloned from a HeLa cell cDNA library. Scaffold attachment factors are a subset of nuclear matrix proteins (NMP) that interact with matrix attachment regions. Analyzing how HET could act as a regulator of hsp27 transcription and as a SAF/NMP, we studied its subnuclear localization and its effect on hsp27 transcription in human breast cancer cells. We were able to show that HET is localized in the nuclear matrix in various breast cancer cell lines. Furthermore, in transient transfection assays using hsp27 promoter-luciferase reporter constructs, HET overexpression resulted in a dose-dependent decrease of hsp27 promoter activity in several cell lines.

Novel role of the RET finger protein in estrogen receptor-mediated transcription in MCF-7 cells

“…We first confirmed that RFP functions as a transcriptional repressor [22]; however, we were unable to detect any synergistic interaction between SAFB1 and RFP in heterologous transient transfections using expression constructs for Gal4 DNA-binding domain (DBD) fusion proteins and a UAS-TK-Luciferase reporter [8] (data not shown). Likewise, we could not shown an RFP effect on SAFB1-mediated ESR1 corepression [29].…”

A 5-Kilobase Pair Promoter Fragment of the Murine Epididymal Retinoic Acid-binding Protein Gene Drives the Tissue-specific, Cell-specific, and Androgen-regulated Expression of a Foreign Gene in the Epididymis of Transgenic Mice

“…However, gene expression was not correlated with transgene copy number, suggesting that, although these promoter fragments contain tissue-specific cis-DNA regulatory elements, they may lack important elements such as matrix attachment regions or locus control regions. Matrix attachment regions are believed to facilitate gene expression and may serve as specific sequence landmarks as they anchor DNA to the nuclear scaffold (32)(33)(34). On the other hand, locus control regions are strong enhancers that bind multiple transcription factors that function in a cooperative manner (for review, see Ref.…”

“…On the other hand, locus control regions are strong enhancers that bind multiple transcription factors that function in a cooperative manner (for review, see Ref. 34). Both of these DNA elements are believed …”

Role of BRCA1 in heat shock response

“…We utilized two HSP27 promoter/luciferase reporter constructs (as described earlier by Oesterreich et al, 1997) to determine if exogenous BRCA1 is capable of activating the HSP27 promoter. In transient transfection assays using promoter constructs containing 15-1090 bp and 15-722 bp upstream of the transcription start site of human HSP27, exogenous wtBRCA1 induced (2.1 to 4.1)-fold increases in HSP27-Luc activity in both DU-145 prostate cancer and T47D breast cancer cells (Po0.001) (see Figure 6a).…”