Nicotinamide Uncouples Hormone-Dependent Chromatin Remodeling from Transcription Complex Assembly

Abstract

Sirtuins, homologs of the yeast SIR2 family, are protein deacetylases that require nicotinamide adenosine dinucleotide as cofactor. To determine whether the sirtuin family of deacetylases is involved in progesterone receptor (PR)-mediated transcription, the effect of sirtuin inhibitor, nicotinamide (NAM), was monitored in T47D breast cancer cells. NAM suppressed hormone-dependent activation of PR-regulated genes in a dosedependent manner. Surprisingly, NAM-mediated inhibition of PR-mediated transcription occurs independently of SIRT1 and PARP1. Chromatin immunoprecipitation experiments did not show that PR binding nor that of the coactivators CBP and SRC3 was compromised. Consistent with the recruitment of the BRG1 chromatin remodeling complex, promoter chromatin remodeling still occurs despite NAM inhibition of PR transactivation. Rather, we show that this inhibition of transcription is due to dramatic loss of recruitment of the basal transcriptional machinery to the promoter. These results show that NAM uncouples promoter chromatin remodeling from transcription preinitiation complex assembly and suggest the existence of vital NAM-regulated steps required for promoter chromatin remodeling and basal transcription complex communication.Steroid hormone receptors such as the progesterone receptor (PR) and the glucocorticoid receptor (GR) are part of a large nuclear receptor (NR) family of eukaryotic transcription factors (41, 59). NRs play essential roles in numerous biological processes such as growth and development, reproduction, homeostasis, and metabolism by eliciting a transcriptional output from target genes in response to their cognate ligands which include steroids, retinoids, thyroid hormone, and vitamin D 3 , among others (12,41,59). Studies of NR action have not only provided insight into their physiological roles but have been vital in the overall understanding of the mechanism of transcription by transcription activators (34,42,49,55).The process of ligand-dependent transcription initiation by steroid hormone receptors (SHRs) such as PR and GR involve ligand binding, followed by receptor binding to the hormone responsive elements at the promoter DNA of target genes as dimers. The promoter-bound SHR leads to recruitment of a large number of coactivators that work in sequence and/or in combination to ultimately facilitate the recruitment of RNA polymerase II (RNAP II) and the transcription machinery to elicit a transcriptional response (34,49,59). These coactivators include the p160/SRC family of proteins (SRC1, -2, and -

Mutational Analysis of Progesterone Receptor Functional Domains in Stable Cell Lines Delineates Sets of Genes Regulated by Different Mechanisms

“…2C). Additionally, SRC-3, recently described to mediate PR activation of MMTV in breast cancer cells (27), was also recruited similarly in WT-and AF2-expressing cells. We also tested recruitment of total and activated (phospho-Ser5) RNA polymerase II in response to progestin.…”

Sirtuin 1 (SIRT1) and steroid hormone receptor activity in cancer

“…Steroid hormone receptors, such as the estrogen (ER) and androgen receptors (AR), as well as the progesterone (PR), glucocorticoid (GR), and mineralocorticoid receptors (MR) are part of a large nuclear receptor family of eukaryotic transcription factors (Aoyagi and Archer 2008; Mangelsdorf, et al 1995; Tsai and Omalley 1994). Steroid hormone receptors play essential roles in numerous biological processes, such as homeostasis, metabolism, cell growth and development (Chawla, et al 2001; Mangelsdorf et al 1995; Tsai and Omalley 1994).…”

Identification of a Novel LXXLL Motif in α-Actinin 4-spliced Isoform That Is Critical for Its Interaction with Estrogen Receptor α and Co-activators

“…The functional importance of the p160 family lies in the fact that mice lacking p160 genes are defective in nuclear receptor-mediated developmental processes (8 -10). Several studies have indicated that hormone-induced interactions between NRs and co-activators is mediated through a single or multiple copies of conserved motif LXXLL (where L is leucine, and X can be any amino acid) (11), termed the nuclear receptor interaction box or nuclear receptor signature motif (12)(13)(14)(15)(16)(17). NR boxes and flanking residues are grouped into four classes depending on the amino acid residues present at the Ϫ1 and Ϫ2 positions upstream of the LXXLL motif.…”

“…NR boxes and flanking residues are grouped into four classes depending on the amino acid residues present at the Ϫ1 and Ϫ2 positions upstream of the LXXLL motif. The first three classes were identified by a phage display approach, and the fourth class was identified following analysis of naturally occurring motifs among co-activators (11)(12)(13)(14)(15)(16)(17)(18).…”