Transcriptional silencing by many transcription factors is mediated by the nuclear receptor corepressor (N-CoR). The mechanism by which N-CoR represses basal transcription involves the direct or indirect recruitment of histone deacetylases (HDACs). We have isolated two multiprotein N-CoR complexes, designated N-CoR-1 and N-CoR-2, which possess histone deacetylase activity that is mediated by distinct HDACs. Based on Western blotting using antibodies against known subunits, the only HDAC found in the N-CoR-1 complex was HDAC3. In contrast, N-CoR-2 contained predominantly HDAC1 and HDAC2 as well as several other subunits that are found in the Sin3A⅐HDAC complex. Using mass spectrometry and Western blotting, we have identified several novel components of the N-CoR-1 complex including the SWI/SNF-related proteins BRG1, BAF 170, BAF 155, BAF 47/INI1, and the corepressor KAP-1 that is involved in silencing heterochromatin. Indirect immunofluorescence has revealed that both KAP-1 and N-CoR colocalize throughout the nucleus. These results suggest that N-CoR is found in distinct multiprotein complexes, which are involved in multiple pathways of transcriptional repression.Cellular proliferation and differentiation is critically dependent on the ability of specific DNA-binding transcription factors to activate or repress the transcription of target genes in a coordinated fashion. This is accomplished through transcription factor-mediated recruitment of coactivators and corepressors, which can regulate transcription through the modification of the local chromatin environment and by specifically interacting with components of the core transcriptional machinery (1, 2). The nuclear hormone receptor superfamily provides a unique model to study the mechanism of transcriptional activation and repression and the role of reversible chromatin modification in the control of gene expression. In the absence of ligand, nuclear hormone receptors such as the thyroid hormone (TR) 1 and retinoic acid receptors (RAR) function as potent repressors by interacting with specific corepressor proteins (3, 4). Ligand binding induces a conformational change in these receptors that results in corepressor release and the recruitment of coactivator proteins (2, 5). The nuclear receptor corepressor (N-CoR), and its related family member silencing mediator for retinoid and thyroid hormone receptor (SMRT), were initially identified by yeast two hybrid screening using unliganded TR or RAR as bait, respectively (3, 4). Several lines of evidence suggest that both N-CoR and SMRT mediate the repressive effects of nuclear hormone receptors. First, both N-CoR and SMRT contain two nuclear receptor interaction domains (IDs) in the carboxyl terminus. Molecular characterization of the IDs reveals the presence of a signature CoR box motif that is necessary and sufficient for receptor interaction and ligand-induced release of N-CoR or SMRT (6 -8). More recently, a strong correlation between repression mediated by the TR and recruitment of N-CoR or SMRT have also been ...

The transcriptional coactivator p/CIP is a member of a family of nuclear receptor coactivator/steroid receptor coactivator (NCoA/SRC) proteins that mediate the transcriptional activities of nuclear hormone receptors. We have found that p/CIP is predominantly cytoplasmic in a large proportion of cells in various tissues of the developing mouse and in a number of established cell lines. In mouse embryonic fibroblasts, serum deprivation results in the redistribution of p/CIP to the cytoplasmic compartment and stimulation with growth factors or tumor-promoting phorbol esters promotes p/CIP shuttling into the nucleus. Cytoplasmic accumulation of p/CIP is also cell cycle dependent, occurring predominantly during the S and late M phases. Leptomycin B (LMB) treatment results in a marked nuclear accumulation, suggesting that p/CIP undergoes dynamic nuclear export as well as import. We have identified a strong nuclear import signal in the N terminus of p/CIP and two leucine-rich motifs in the C terminus that resemble CRM-1-dependent nuclear export sequences. When fused to green fluorescent protein, the nuclear export sequence region is cytoplasmic and is retained in the nucleus in an LMB-dependent manner. Disruption of the leucine-rich motifs prevents cytoplasmic accumulation. Furthermore, we demonstrate that cytoplasmic p/CIP associates with tubulin and that an intact microtubule network is required for intracellular shuttling of p/CIP. Immunoaffinity purification of p/CIP from nuclear and cytosolic extracts revealed that only nuclear p/CIP complexes possess histone acetyltransferase activity. Collectively, these results suggest that cellular compartmentalization of NCoA/SRC proteins could potentially regulate nuclear hormone receptor-mediated events as well as integrating signals in response to different environmental cues.Nuclear receptors (NRs) are a superfamily of structurally related proteins that function as ligand-regulated transcription factors. Members of this family include receptors for estrogen, glucocorticoids, nonsteroidal ligands such as thyroid hormone, and retinoic acid, as well as receptors that bind by-products of lipid metabolism such as fatty acids and prostaglandins. These receptors control a complex array of genes involved in many biological functions including cell proliferation and differentiation, metabolism and growth, morphogenesis, programmed cell death, and homeostasis. In the absence of hormone, some NRs such as the thyroid hormone receptor and retinoic acid receptor function as transcriptional repressors by interacting with corepressor proteins. Hormone binding results in a conformational change in the receptor that results in the release of corepressor proteins and in the recruitment of coactivator proteins (17).The nuclear receptor coactivator/steroid receptor coactivator (NCoA/SRC) proteins were among the first coactivators to be identified. This family includes steroid receptor coactivator 1 (SRC-1) (51) also designated nuclear receptor coactivator 1 (NCoA-1) (28); GRIP1 (25), also kno...

Two models of biogenesis have been proposed for the development of platelet granules. One model postulates that granule contents develop in the maturing megakaryocyte via a direct biosynthetic pathway from the golgi, whereas the second model is thought to involve the multivesicular body for sorting and targeting granular constituents via an endosomal pathway. While these models may appropriately apply to the mechanism of the development of platelet alpha granules, dense granule biosynthesis is not readily explained. In animal studies and in human cultured megakaryocyte experiments, molecular markers (CD63 and serotonin) have been reported to appear concomitant with alpha granule formation, yet the characteristic density of the delta granule is not present until later in the maturation of the megakaryocyte. This suggests that dense granule vesicles must be “loaded” for a fully functioning platelet. Our evaluations of platelets obtained from umbilical cord blood would suggest that circulating platelets may become fully functional after birth via a mechanism that loads specific vesicles. The neonate is known to have a transient platelet dysfunction at the time of birth which has been explained in the literature as being due to 1) a diminished response to physiologic agonists, 2) defective platelet dense granule secretion, and/or 3) ineffective mobilization of intracellular calcium in platelets. Studies to define the transient platelet dysfunction in the neonate have been limited. We and others have found that the adenine content of platelets obtained from umbilical cord blood is significantly less than seen in adults. We have found that platelets obtained from umbilical cord blood have an average of 0.79 ± 0.08 DG/PL (n=62), which is significantly decreased compared to the adult population and that the ATP:ADP ratio in these platelets is 1.95 (n=61), consistent with established adult normal range ratio values (1.4–1.95). The actual measured adenine content was found to be decreased in the neonatal platelets (ATP = 2.76 ± 0.53 mmol/1011 PL; ADP=0.96±0.19 mmol/1011 PL). The normal adenine nucleotide DG contents have been reported to be 3.5–7 mmol/1011 PL for ATP and 1.9–3.7 mmol/1011 PL for ADP in adults. Using the uranaffin reaction technique to classify the four types (I–IV) of dense granules present in neonatal platelets, we have found a significant difference in the percentage of each type than is found in adult controls. We have employed an image analysis technique (Blood, 90: 140a, 1997) to calculate the mean volume of dense granules in neonatal platelets. We report that not only do neonatal platelets have a deficiency of dense granules, but that these granules are also extremely small in size having an average volume of 6.45 X 105 nm3 (n =36) (normal adult = 8–12 X 106 nm3). These results would suggest that the neonatal platelet has an incomplete incorporation of constituents (i.e., calcium, ADP) into the dense granules at the time of birth and that an unknown mechanism exists that induces dense vesicle “loading” at some time after birth. These observations may prove useful in pursuit of understanding the mechanism of platelet dense granule biosynthesis.