Metabolic Effects of Thyroid Hormone

New York University School Of Medicine, New York NY

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Abstract

PROJECT SUMMARY/ABSTRACT Nuclear hormone receptors (NRs) modulate transcription through interaction with co-activators and co- repressors which leads to chromatin modifications involving histone acetylation and methylation. In addition to the DRIP/TRAP "Mediator" and the p160/SRC family of co-activators, other co-activators play important roles in NR function and gene expression. We cloned and characterized NRC, NIF-1, and NRIF3 which modulate the activity of NRs and other transcription factors. NRC (Nuclear Receptor Co-regulator) is a novel 2063 amino acid nuclear protein with two LxxLL motifs. LxxLL-1 interacts with all NRs while LxxLL-2 interacts with ER and LXR. NIF-1 (NRC Interacting Factor-1), identified as an NRC-interactor in a yeast two-hybrid screen, is a novel 1,342 amino acid nuclear protein. Although NIF-1 does not directly interact with liganded NRs, it enhances transcriptional activation by NRs, presumably through association with NRC. We have identified the components of a novel NIF-1 complex(es) by mass spectrometry. Based on chromatographic analysis we have identified high and low molecular weight (MW) NIF-1 complexes. The high MW (1-~2 MDa) complex contains the novel factors EMSY, DBC-1 and Ki-67. The lower MW NIF-1 complex contains WDR5, RbBP5 and ASH2L. Knockdown of either DBC-1, EMSY, or NIF-1 leads to a marked reduction in stimulation of Sox9, an RAR responsive gene in MCF-7 cells suggesting that the high MW NIF-1 complex plays a role in transcriptional activation by NRs. The NIF-1 complex in vitro methylates histone H3 but at residues other than H3K4. This was unexpected because of the presence of WDR5, RbBP5 and ASH2L which are involved in H3K4 methylation. NRIF3 (Nuclear Receptor Interacting Factor 3) is a 177 amino acid nuclear protein that interacts with thyroid hormone receptors (TRs) and retinoid x receptors (RXRs) and contains both activation and repression domains. Surprisingly, in a wide variety of breast cancer cell lines (but not other cell types), expression of NRIF3 rapidly leads to caspase-2 mediated apoptosis. This effect is mediated by a short region of NRIF3 (amino acids 20-50) containing Ser28 which we refer to as Death Domain-1 (DD1). Mutation of Ser28 to Ala28 eliminates DD1-mediated apoptosis in breast cancer cells. We identified a factor by yeast two-hybrid screen, DIF-1 (DD1 Interacting Factor-1), that mediates apoptosis by NRIF3 in breast cancer cells. DIF-1 is a repressor which acts through the Class III histone and protein deacetylase, SirT1. We have identified two other proteins by mass spectrometry that form a repressor complex with DIF-1. In this application we propose to identify the role of components of the NIF-1 complex on NR activity, histone modification, and transcriptional regulation. Lastly, we will further characterize the biological role of DIF-1 in breast cancer cells, understand how the proteins in the repressor complex interact and function, how repression is modulated by NRIF3/DD1, and determine whether DIF-1 represses one or more pro-apoptotic genes that are expressed when NRIF3/DD1 reverses repression by DIF-1. These studies should provide a more comprehensive picture of how these factors modulate NR function and other important biological processes in cells.