Clickable NAD analogues for labeling substrate proteins of poly(ADP-ribose) polymerases.

Poly(ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple adenine diphosphate ribose (ADP-ribose) units from nicotinamide adenine dinucleotide (NAD) to substrate proteins. There are 17 PARPs in humans. Several PARPs, such as PARP-1 and Tankyrase-1, are known to play important roles in DNA repair, transcription, mitosis, and telomere length maintenance. To better understand the functions of PARPs at a molecular level, it is necessary to know what substrate proteins PARPs modify. Here we report clickable NAD analogues that can be used to label PARP substrate proteins. The clickable NAD analogues have a terminal alkyne group which allows the conjugation of fluorescent or affinity tags to the substrate proteins. Using this method, PARP-1 and tankyrase-1 substrate proteins were labeled by a fluorescent tag and visualized on SDS-PAGE gel. Using a biotin affinity tag, we were able to isolate and identify a total of 79 proteins as potential PARP-1 substrates. These include known PARP-1 substrate proteins, including histones and heterogeneous nuclear ribonucleoproteins. About 40% of the proteins were also identified in recent proteomic studies as potential PARP-1 substrates. Among the identified potential substrates, we further demonstrated that tubulin and three mitochondrial proteins, TRAP1 (TNF receptor-associated protein 1), citrate synthase, and GDH (glutamate dehydrogenase 1), are substrates of PARP-1 in vitro. These results demonstrate that the clickable NAD analogue is useful for labeling, in-gel detection, isolation, and identification of the substrate proteins of PARPs and will help to understand the biological functions of PARPs.

The histone variant macroH2A1 marks repressed autosomal chromatin, but protects a subset of its target genes from silencing.

MacroH2A1 is a histone variant that is enriched on the inactive X chromosome (Xi) in mammals and is postulated to play an important, but unknown, role in the repression of gene expression. Here we show that, although macroH2A1 marks repressed autosomal chromatin, it positively regulates transcription when located in the transcribed regions of a subset of its target genes. We used chromatin immunoprecipitation (ChIP) coupled with tiling microarrays (ChIP-chip) to determine the genomic localization of macroH2A1 in IMR90 human primary lung fibroblasts and MCF-7 breast cancer cells. The patterns of macroH2A1 deposition are largely similar across the autosomes of both cell lines. Our studies revealed a genomic localization pattern unique among histone variants; namely, the occupation by macroH2A1 of large chromatin domains (>500 kb in some cases) that contain repressive chromatin marks (e.g., histone H3 Lys 27 trimethylation). The boundaries of macroH2A1-containing domains tend to occur in promoter-proximal regions. Not all promoters, however, serve as macroH2A1 boundaries; many macroH2A1-containing chromatin domains invade the transcribed regions of genes whose products play key roles in development and cell-cell signaling. Surprisingly, the expression of a subset of these genes is positively regulated by macroH2A1. MacroH2A1 also plays a role in augmenting signal-regulated transcription, specifically for genes responsive to serum starvation. Collectively, our results document an unexpected role for macroH2A1 in the escape from heterochromatin-associated silencing and the enhancement of autosomal gene transcription.

PARP inhibitors and the treatment of breast cancer: beyond BRCA1/2?

Poly(ADP-ribose) polymerase (PARP) inhibitors have been explored as therapeutic agents for the treatment of hereditary breast and ovarian cancers harboring mutations in BRCA1 or BRCA2. In a new study, Inbar-Rozensal and colleagues show that phenanthridine-derived PARP inhibitors promote cell cycle arrest and cell death in breast cancer cells lacking BRCA1 and BRCA2 mutations and prevent the growth of tumors from xenografts of these cells in immunocompromised mice. These results suggest a potential broader utility of PARP-1 inhibitors in the treatment of breast cancer, although further mechanistic studies are needed.

Global analysis of transcriptional regulation by poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase in MCF-7 human breast cancer cells.

Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs to stably knock down PARP-1 or PARG in MCF-7 cells followed by expression microarray analyses. Correlation analyses showed that the majority of genes affected by the knockdown of one factor were similarly affected by the knockdown of the other factor. The most robustly regulated common genes were enriched for stress-response and metabolic functions. In chromatin immunoprecipitation assays, PARP-1 and PARG localized to the promoters of positively and negatively regulated target genes. The levels of chromatin-bound PARG at a given promoter generally correlated with the levels of PARP-1 across the subset of promoters tested. For about half of the genes tested, the binding of PARP-1 at the promoter was dependent on the binding of PARG. Experiments using stable re-expression of short hairpin RNA-resistant catalytic mutants showed that PARP-1 and PARG enzymatic activities are required for some, but not all, target genes. Collectively, our results indicate that PARP-1 and PARG, nuclear enzymes with opposing enzymatic activities, localize to target promoters and act in a similar, rather than antagonistic, manner to regulate gene expression.

Enzymes in the NAD+ salvage pathway regulate SIRT1 activity at target gene promoters.

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 1 (NMNAT-1) constitute a nuclear NAD(+) salvage pathway which regulates the functions of NAD(+)-dependent enzymes such as the protein deacetylase SIRT1. One of the major functions of SIRT1 is to regulate target gene transcription through modification of chromatin-associated proteins. However, little is known about the molecular mechanisms by which NAD(+) biosynthetic enzymes regulate SIRT1 activity to control gene transcription in the nucleus. In this study we show that stable short hairpin RNA-mediated knockdown of NAMPT or NMNAT-1 in MCF-7 breast cancer cells reduces total cellular NAD(+) levels and alters global patterns of gene expression. Furthermore, we show that SIRT1 plays a key role in mediating the gene regulatory effects of NAMPT and NMNAT-1. Specifically, we found that SIRT1 binds to the promoters of genes commonly regulated by NAMPT, NMNAT-1, and SIRT1 and that SIRT1 histone deacetylase activity is regulated by NAMPT and NMNAT-1 at these promoters. Most significantly, NMNAT-1 interacts with, and is recruited to target gene promoters by SIRT1. Collectively, our results reveal a mechanism for the direct control of SIRT1 deacetylase activity at a set of target gene promoters by NMNAT-1. This mechanism, in collaboration with NAMPT-dependent regulation of nuclear NAD(+) production, establishes an important pathway for transcription regulation by NAD(+).

Reciprocal binding of PARP-1 and histone H1 at promoters specifies transcriptional outcomes.

Nucleosome-binding proteins act to modulate the promoter chromatin architecture and transcription of target genes. We used genomic and gene-specific approaches to show that two such factors, histone H1 and poly(ADP-ribose) polymerase-1 (PARP-1), exhibit a reciprocal pattern of chromatin binding at many RNA polymerase II-transcribed promoters. PARP-1 was enriched and H1 was depleted at these promoters. This pattern of binding was associated with actively transcribed genes. Furthermore, we showed that PARP-1 acts to exclude H1 from a subset of PARP-1-stimulated promoters, suggesting a functional interplay between PARP-1 and H1 at the level of nucleosome binding. Thus, although H1 and PARP-1 have similar nucleosome-binding properties and effects on chromatin structure in vitro, they have distinct roles in determining gene expression outcomes in vivo.

Regulation of chromatin structure and chromatin-dependent transcription by poly(ADP-ribose) polymerase-1: possible targets for drug-based therapies.

Poly(ADP-ribose) Polymerase-1 (PARP-1) is the prototypical and most abundantly expressed member of a family of PARPs that catalyze the polymerization of ADP-ribose (ADPR) units from donor NAD' molecules on target proteins. PARP-1 plays roles in a variety of genomic processes, including the regulation of chromatin structure and transcription in response to specific cellular signals. PARP-1 also plays important roles in many stress-induced disease states. In this chapter, we review the molecular and cellular aspects of PARP-1's chromatin-modulating activities, as well as the impact that these chromatin-modulating activities have on the regulation of gene expression. In addition, we highlight the potential therapeutic use of drugs that target PARP-1's enzymatic activity for the treatment of human diseases