Regulation of poly(ADP-ribose) polymerase-1-dependent gene expression through promoter-directed recruitment of a nuclear NAD+ synthase.

NMNAT-1 and PARP-1, two key enzymes in the NAD(+) metabolic pathway, localize to the nucleus where integration of their enzymatic activities has the potential to control a variety of nuclear processes. Using a variety of biochemical, molecular, cell-based, and genomic assays, we show that NMNAT-1 and PARP-1 physically and functionally interact at target gene promoters in MCF-7 cells. Specifically, we show that PARP-1 recruits NMNAT-1 to promoters where it produces NAD(+) to support PARP-1 catalytic activity, but also enhances the enzymatic activity of PARP-1 independently of NAD(+) production. Furthermore, using two-photon excitation microscopy, we show that NMNAT-1 catalyzes the production of NAD(+) in a nuclear pool that may be distinct from other cellular compartments. In expression microarray experiments, depletion of NMNAT-1 or PARP-1 alters the expression of about 200 protein-coding genes each, with about 10% overlap between the two gene sets. NMNAT-1 enzymatic activity is required for PARP-1-dependent poly(ADP-ribosyl)ation at the promoters of commonly regulated target genes, as well as the expression of those target genes. Collectively, our studies link the enzymatic activities of NMNAT-1 and PARP-1 to the regulation of a set of common target genes through functional interactions at target gene promoters.

A role for BAF57 in cell cycle-dependent transcriptional regulation by the SWI/SNF chromatin remodeling complex.

The SWI/SNF complex is an ATP-dependent chromatin remodeling complex that plays pivotal roles in gene regulation and cell cycle control. In the present study, we explored the molecular functions of the BAF57 subunit of SWI/SNF in cell cycle control via transcriptional regulation of cell cycle-related genes. We affinity purified SWI/SNF from HeLa cells stably expressing FLAG-tagged BAF47/Ini1 with or without stable short hairpin RNA-mediated knockdown of BAF57. The subunit composition of the holo-SWI/SNF and BAF57-depleted SWI/SNF complexes from these cells was determined using a quantitative SILAC (stable isotope labeling by amino acids in cell culture)-based proteomic approach. Depletion of BAF57 resulted in a significant codepletion of BAF180 from the SWI/SNF complex without decreasing total cellular BAF180 levels. In biochemical assays of SWI/SNF activity, the holo-SWI/SNF and BAF57/BAF180-depleted SWI/SNF complexes exhibited similar activities. However, in cell proliferation assays using HeLa cells, knockdown of BAF57 resulted in an accumulation of cells in the G(2)-M phase, inhibition of colony formation, and impaired growth in soft agar. Knockdown of BAF57 also caused transcriptional misregulation of various cell cycle-related genes, especially genes involved in late G(2). Collectively, our results have identified a new role for BAF57 within the SWI/SNF complex that is required for (a) maintaining the proper subunit composition of the complex and (b) cell cycle progression through the transcriptional regulation of a subset of cell cycle-related genes.

The Zn3 domain of human poly(ADP-ribose) polymerase-1 (PARP-1) functions in both DNA-dependent poly(ADP-ribose) synthesis activity and chromatin compaction.

PARP-1 is involved in multiple cellular processes, including transcription, DNA repair, and apoptosis. PARP-1 attaches ADP-ribose units to target proteins, including itself as a post-translational modification that can change the biochemical properties of target proteins and mediate recruitment of proteins to sites of poly(ADP-ribose) synthesis. Independent of its catalytic activity, PARP-1 binds to chromatin and promotes compaction affecting RNA polymerase II transcription. PARP-1 has a modular structure composed of six independent domains. Two homologous zinc fingers, Zn1 and Zn2, form the DNA-binding module. Zn1-Zn2 binding to DNA breaks triggers catalytic activity. Recently, we have identified a third zinc binding domain in PARP-1, the Zn3 domain, which is essential for DNA-dependent PARP-1 activity. The crystal structure of the Zn3 domain revealed a novel zinc-ribbon fold and a homodimeric Zn3 structure that formed in the crystal lattice. Structure-guided mutagenesis was used here to investigate the roles of these two features of the Zn3 domain. Our results indicate that the zinc-ribbon fold of the Zn3 domain mediates an interdomain contact crucial to assembly of the DNA-activated conformation of PARP-1. In contrast, residues located at the Zn3 dimer interface are not required for DNA-dependent activation but rather make important contributions to the chromatin compaction activity of PARP-1. Thus, the Zn3 domain has dual roles in regulating the functions of PARP-1.

Biochemical analyses of nuclear receptor-dependent transcription with chromatin templates.

Chromatin, the physiological template for transcription, plays important roles in gene regulation by nuclear receptors (NRs). It can (1) restrict the binding of NRs or the transcriptional machinery to their genomic targets, (2) serve as a target of regulatory posttranslational modifications by NR coregulator proteins with histone-directed enzymatic activities, and (3) function as a binding scaffold for a variety of transcription-related proteins. The advent of in vitro or "cell-free" systems that accurately recapitulate ligand-dependent transcription by NRs with chromatin templates has allowed detailed analyses of these processes. Biochemical studies have advanced our understanding of the mechanisms of gene regulation, including the role of ligands, coregulators, and nucleosome remodeling. In addition, they have provided new insights about the dynamics of NR-mediated transcription. This chapter reviews the current methodologies for assembling, transcribing, and analyzing chromatin in vitro, as well as the new information that has been gained from these studies.

The DNA binding and catalytic domains of poly(ADP-ribose) polymerase 1 cooperate in the regulation of chromatin structure and transcription.

We explored the mechanisms of chromatin compaction and transcriptional regulation by poly(ADP-ribose) polymerase 1 (PARP-1), a nucleosome-binding protein with an NAD(+)-dependent enzymatic activity. By using atomic force microscopy and a complementary set of biochemical assays with reconstituted chromatin, we showed that PARP-1 promotes the localized compaction of chromatin into supranucleosomal structures in a manner independent of the amino-terminal tails of core histones. In addition, we defined the domains of PARP-1 required for nucleosome binding, chromatin compaction, and transcriptional repression. Our results indicate that the DNA binding domain (DBD) of PARP-1 is necessary and sufficient for binding to nucleosomes, yet the DBD alone is unable to promote chromatin compaction and only partially represses RNA polymerase II-dependent transcription in an in vitro assay with chromatin templates (approximately 50% of the repression observed with wild-type PARP-1). Furthermore, our results show that the catalytic domain of PARP-1, which does not bind nucleosomes on its own, cooperates with the DBD to promote chromatin compaction and efficient transcriptional repression in a manner independent of its enzymatic activity. Collectively, our results have revealed a novel function for the catalytic domain in chromatin compaction. In addition, they show that the DBD and catalytic domain cooperate to regulate chromatin structure and chromatin-dependent transcription, providing mechanistic insights into how these domains contribute to the chromatin-dependent functions of PARP-1.

Purification of a human SRCAP complex that remodels chromatin by incorporating the histone variant H2A.Z into nucleosomes.

The Snf-2-related CREB-binding protein activator protein (SRCAP) serves as a coactivator for a number of transcription factors known to interact with CBP. Swr1, the closest Saccharomyces cerevisiae ortholog of SRCAP, is a component of the chromatin remodeling complex SWR-C, which catalyzes exchange of the histone variant H2A.Z into nucleosomes. In this report, we use a combination of conventional chromatography and anti-SRCAP immunoaffinity chromatography to purify a native human SRCAP complex with a polypeptide composition similar to that of SWR-C, and we show for the first time that this SRCAP-containing complex supports ATP-dependent exchange of histone dimers containing H2B and H2A.Z into mononucleosomes reconstituted with recombinant H2A, H2B, H3, and H4. Our findings, together with previous evidence implicating H2A.Z in transcriptional regulation, suggest that SRCAP's coactivator function may depend on its ability to promote incorporation of H2A.Z into chromatin.