The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of rapamycin proteins), ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational arrest. We measured the immediate transcriptional response of yeast grown in rich media and treated with rapamycin to investigate the direct effects of Tor proteins on nutrient-sensitive signaling pathways. The results suggest that Tor proteins directly modulate the glucose activation and nitrogen discrimination pathways and the pathways that respond to the diauxic shift (including glycolysis and the citric acid cycle). Tor proteins do not directly modulate the general amino acid control, nitrogen starvation, or sporulation (in diploid cells) pathways. Poor nitrogen quality activates the nitrogen discrimination pathway, which is controlled by the complex of the transcriptional repressor Ure2p and activator Gln3p. Inhibiting Tor proteins with rapamycin increases the electrophoretic mobility of Ure2p. The work presented here illustrates the coordinated use of genome-based and biochemical approaches to delineate a cellular pathway modulated by the protein target of a small molecule.

The dynamic assembly and remodelling of eukaryotic chromosomes facilitate fundamental cellular processes such as DNA replication and gene transcription. The repeating unit of eukaryotic chromosomes is the nucleosome core, consisting of DNA wound about a defined octamer of histone proteins. Two enzymatic processes that regulate transcription by targeting elements of the nucleosome include ATP-dependent nucleosome remodelling and reversible histone acetylation. The histone deacetylases, however, are unable to deacetylate oligonucleosomal histones in vitro. The protein complexes that mediate ATP-dependent nucleosome remodelling and histone acetylation/deacetylation in the regulation of transcription were considered to be different, although it has recently been suggested that these activities might be coupled. We report here the identification and functional characterization of a novel ATP-dependent nucleosome remodelling activity that is part of an endogenous human histone deacetylase complex. This activity is derived from the CHD3 and CHD4 proteins which contain helicase/ATPase domains found in SWI2-related chromatin remodelling factors, and facilitates the deacetylation of oligonucleosomal histones in vitro. We refer to this complex as the nucleosome remodelling and deacetylating (NRD) complex. Our results establish a physical and functional link between the distinct chromatin-modifying activities of histone deacetylases and nucleosome remodelling proteins.

Here we describe the components of a histone deacetylase (HDAC) complex that we term the CoREST-HDAC complex. CoREST-HDAC is composed of polypeptides distinct from previously characterized HDAC1͞2-containing complexes such as the mSin3 and nucleosome remodeling and deacetylating (NRD, also named NURD, NuRD) complex. Interestingly, we do not observe RbAp46 and RbAp48 in this complex, although these proteins have been observed in all previously identified complexes and are thought to be part of an HDAC1͞2 core. We identify the transcriptional corepressor CoREST and a protein with homology to polyamine oxidases as components of CoREST-HDAC. The HDAC1͞2-interacting region of CoREST is mapped to a 179-aa region containing a SANT domain, a domain found in other HDAC1͞2-interacting proteins such as NCoR, MTA1, and MTA2. Furthermore, we demonstrate that the corepressor function of CoREST depends on this region. Although CoREST initially was cloned as a corepressor to REST (RE1 silencing transcription factor͞neural restrictive silencing factor), we find no evidence for the existence of the eight-zinc finger REST transcription factor as an interacting partner in this complex; however, we do find evidence for association of the putative oncogene ZNF 217 that contains eight zinc fingers. M ammalian human histone deacetylases (HDACs) have been observed to exist in large, multisubunit protein complexes (1). HDAC1 and HDAC2, in particular, have been well characterized and previously found predominantly in either an mSin3-containing complex or in a complex containing the ATP-dependent chromatin remodeling protein CHD4 (also known as Mi-2) and MTA2 (2-5). The former complex is referred to as the mSin3 complex and the latter, as the nucleosome remodeling and deacetylating (NRD) complex. The mSin3 complex is recruited by DNA-binding transcription factors such as the unliganded nuclear hormone receptors and the Mad͞Max heterodimer, whereas the NRD complex has been shown to be recruited by the transcription factors Ikaros and hunchback (6, 7). The emerging model of mSin3 function is one in which the complex is recruited as a repressor under one set of conditions and released and exchanged for histone acetyltransferase coactivators under a different set of conditions. In contrast, less is currently understood about the regulation and function of the NRD complex at the transcriptional level.In the course of purifying HDAC1͞2-associated proteins, we obtained peptide sequence corresponding to a hypothetical protein of unknown function (KIAA0071). The partially translated product possesses homology to MTA1 and MTA2 in the region of the SANT domain, and we hypothesized that the full-length protein derived from KIAA0071 might be a component of an uncharacterized HDAC1͞2-containing complex. Antibodies were generated against KIAA0071 and used to purify associated polypeptides. At the same time, an effort was undertaken to clone the full-length cDNA of KIAA0071. While this work was in progress, Andres et al. (8) reported the cloning of full-...