The functions of the SAGA and SWI/SNF complexes are interrelated and can form stable "epigenetic marks" on promoters in vivo. Here we show that stable promoter occupancy by SWI/SNF and SAGA in the absence of transcription activators requires the bromodomains of the Swi2/Snf2 and Gcn5 subunits, respectively, and nucleosome acetylation. This acetylation can be brought about by either the SAGA or NuA4 HAT complexes. The bromodomain in the Spt7 subunit of SAGA is dispensable for this activity but will anchor SAGA if it is swapped into Gcn5, indicating that specificity of bromodomain function is determined in part by the subunit it occupies. Thus, bromodomains within the catalytic subunits of SAGA and SWI/SNF anchor these complexes to acetylated promoter nucleosomes.

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the plateletspecific integrin ␣ IIb ␤ 3 , all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, ␣-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.It is now well established that the mitogen-activated protein (MAP) kinase cascade is a key regulator of mammalian cell proliferation (44). This pathway includes the MAP kinases extracellular-signal-regulated kinase 1 (ERK1) and ERK2, which are phosphorylated and activated by MAP kinase kinase 1 (MKK1) and MKK2 (44). ERKs are able to phosphorylate and activate the MAP kinase-activated protein (MAPKAP) kinases, including pp90 rsk (47), MAPKAP kinase 2 (46), and 3pK (45). MKK1 and MKK2 can be phosphorylated and activated by any of three protein kinases, Raf-1, MEK kinase, and Mos, and thus represent convergence points for diverse signalling pathways triggered upon cell surface receptor activation. Downstream targets of the MAP kinase cascade include several transcription factors which may be regulated by direct phosphorylation by the ERKs or MAPKAP kinases (9,13,20,32,50,56). Thus, the mitogen-activated MAP kinase cascade is a key mechanism for the control of transcription by extracellular signals.Several observations underscore the essential role for the MAP kinase pathway in cell transformation and cell cycle regulation. Signalling components further upstream, including receptor tyrosine kinases, Src, Ras, Raf-1, and the G

The regulation of histone deacetylases (HDACs) by phosphorylation was examined by elevating intracellular phosphorylation in cultured cells with the protein phosphatase inhibitor okadaic acid. After fractionation of extracts from treated versus untreated cells, HDAC 1 and 2 eluted in several peaks of deacetylase activity, assayed using mixed acetylated histones or acetylated histone H4 peptide. Stimulation of cells with okadaic acid led to hyperphosphorylation of HDAC 1 and 2 as well as changes in column elution of both enzymes. Hyperphosphorylated HDAC2 was also observed in cells synchronized with nocodazole or taxol, demonstrating regulation of HDAC phosphorylation during mitosis. Phosphorylated HDAC1 and 2 showed a gel mobility retardation that correlated with a small but significant increase in activity, both of which were reversed upon phosphatase treatment in vitro. However, the most pronounced effect of HDAC phosphorylation was to disrupt protein complex formation between HDAC1 and 2 as well as complex formation between HDAC1 and corepressors mSin3A and YY1. In contrast, interactions between HDAC1/2 and RbAp46/48 were unaffected by okadaic acid. These results establish a novel link between HDAC phosphorylation and the control of protein-protein interactions and suggest a mechanism for relief of deacetylase-catalyzed transcriptional repression by phosphorylation-dependent signaling.Acetylation of nucleosomal histones by histone acetyltransferases generally stimulates transcription, whereas deacetylation of nucleosomes by histone deacetylases (HDACs) 1 is correlated with transcriptional repression. Thus, histone acetylases and deacetylases are potential targets for regulation of chromatin acetylation at targeted promoters by signal transduction pathways. Our previous studies showed that global acetylation of nucleosomal histones changes in response to enhanced phosphorylation induced by inhibition of intracellular phosphatases (1). Therefore, we examined the potential control of mammalian HDACs 1 and 2 by phosphorylation.Two classes of HDAC in mammalian cells are catalogued based on their homology to yeast catalytic subunits. Class I includes HDACs 1, 2, and 3, enzymes homologous to yeast RPD3 (2-5). Class II includes HDACs 4, 5, 6, and 7, most similar to the yeast histone deacetylase A repressor (6 -9). Of these forms, HDAC1 and 2 are expressed ubiquitously (10). Neither of these enzymes is active when expressed in bacteria, but both are active when expressed in insect cells, suggesting that post-translational modifications or other eukaryotic factors are needed for enzymatic activity (11, 12).HDAC1 and 2 isolated from tissue culture extracts are able to deacetylate free and nucleosome-bound histones as well as histone peptides, but they are unable to deacetylate SV40 minichromosomes, suggesting that other factors must direct enzyme access to acetylated histones in higher ordered chromatin structures (3, 12, 13). In agreement, both are found as catalytic subunits of multiprotein complexes involved in tra...