We are working on regulation of transcription and development by nuclear receptors. In our previous work, we characterized the development role of SKIP (skp-1) on regulation of development in C. elegans. SKIP is a transcription cofactor participating in regulation by NHRs, Notch and TGF-beta and is present in all eukaryotes which genome was sequenced to date. We have studied the developmental role of SKIP on C. elegans. We showed that SKIP is required for C. elegans viablity and development. Expression of CeSKIP assayed by RT-PCR and by GFP fluoresence in transgenic lines starts in embryos and continues to adulthood. Loss of CeSKIP activity by RNA-mediated inhibition (RNAi) results in early embryonic arrest similar to that seen following inhibition of RNA Polymerase II. RNA Polymerase II phosphorylation appears normal early in CeSKIP RNAi treated embryos although the expression of several embryonic GFP reporter genes is severely restricted or absent. SKIP is an indispensable factor for Caenorhabditis elegans development. We have found the SKIP is organized in an operon with another gene that is an orthologue of Survivin, a gene which expression is clearly linked to cancer. Since genes arranged in operons are frequently linked functionally, we have asked if BIR-1 also functions in transcription. bir-1 inhibition resulted in multiple developmental defects that overlapped with CeSKIP loss of function phenotypes: retention of eggs, dumpy, movement defects and lethality. bir-1 RNAi decresed expression of several gfp transgenes and endogenous genes dpy-7 and hlh-1. Immunoblot analysis revealed decreased phosphacetylated histones in bir-1 RNAi treated worms. In a heterologous transfection system, BIR-1 augments thyroid hormone regulated transcription and has an additive effect with SKIP. Thus we show that BIR-1 functions in the regulation of transcription and development. In our third project, we studied the expression of thyroid hormone receptor alpha 1 (TR alpha1) (N1RA1) in 40 cases of infiltrating breast carcinoma. RT-PCR detected expression from the TR alpha locus in all cancers examined and in breast cancer cell lines MCF-7 and MDA-MB-231. Western blot analysis using a monoclonal antibody directed against TR alpha1 revealed expression of multiple N-terminally deleted isoforms in most cases examined and in both cell lines. Full size TR alpha1 was detected as a minor band and was missing in ~10% of cancers. Thyroid hormone receptor regulated promoters are not activated by triodothyronine without co-expression of exogenous TRs in MCF-7 cells. Responsiveness to T3 is not restored by histone deacetylase inhibitor, Trichostatin A. TR alpha1 amplified from MCF-7 cells and cloned in the pCDNA3 expression vector repressed expression from thyroid hormone responsive promoters. Thus, N-terminally truncated isoforms originating from TR alpha gene are commonly expressed in infiltrative breast cancers and are likely to function as inhibitors of thyroid hormone receptor dependent gene expression.
| Kostrouchova, Marta; Housa, Daniel; Kostrouch, Zdenek et al. (2002) SKIP is an indispensable factor for Caenorhabditis elegans development. Proc Natl Acad Sci U S A 99:9254-9 |
| Kostrouchova, M; Krause, M; Kostrouch, Z et al. (2001) Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans. Proc Natl Acad Sci U S A 98:7360-5 |
