Biomedical subjects

T P Yao

Publications and source records attributed to T P Yao.

17 recordsLinked to original sources

PubMed2001

The modular nature of histone deacetylase HDAC4 confers phosphorylation-dependent intracellular trafficking.

In C2C12 myoblasts, endogenous histone deacetylase HDAC4 shuttles between cytoplasmic and nuclear compartments, supporting the hypothesis that its subcellular localization is dynamically regulated. However, upon differentiation, this dynamic equilibrium is disturbed and we find that HDAC4 accumulates in the nuclei of myotubes, suggesting a positive role of nuclear HDAC4 in muscle differentiation. Consistent with the notion of regulation of HDAC4 intracellular trafficking, we reveal that HDAC4 contains a modular structure consisting of a C-terminal autonomous nuclear export domain, which, in conjunction with an internal regulatory domain responsive to calcium/calmodulin-dependent protein kinase IV (CaMKIV), determines its subcellular localization. CaMKIV phosphorylates HDAC4 in vitro and promotes its nuclear-cytoplasmic shuttling in vivo. However, although 14-3-3 binding of HDAC4 has been proposed to be important for its cytoplasmic retention, we find this interaction to be independent of CaMKIV. Rather, the HDAC4.14-3-3 complex exists in the nucleus and is required to confer CaMKIV responsiveness. Our results suggest that the subcellular localization of HDAC4 is regulated by sequential phosphorylation events. The first event is catalyzed by a yet to be identified protein kinase that promotes 14-3-3 binding, and the second event, involving protein kinases such as CaMKIV, leads to efficient nuclear export of the HDAC4.14-3-3 complex.

14-3-3 Proteins↗

PubMed2001

Role of T-bet in commitment of TH1 cells before IL-12-dependent selection.

How cytokines control differentiation of helper T (TH) cells is controversial. We show that T-bet, without apparent assistance from interleukin 12 (IL-12)/STAT4, specifies TH1 effector fate by targeting chromatin remodeling to individual interferon-gamma (IFN-gamma) alleles and by inducing IL-12 receptor beta2 expression. Subsequently, it appears that IL-12/STAT4 serves two essential functions in the development of TH1 cells: as growth signal, inducing survival and cell division; and as trans-activator, prolonging IFN-gamma synthesis through a genetic interaction with the coactivator, CREB-binding protein. These results suggest that a cytokine does not simply induce TH fate choice but instead may act as an essential secondary stimulus that mediates selective survival of a lineage.

Alleles↗

PubMed2001

p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2.

The tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post-translational modification. Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress-inducing agents including hypoxia, anti-metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase-deficient mutant, efficiently induces specific p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBP-mediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19(ARF), indicating that regulation of acetylation is a central target of the p53-MDM2-p19(ARF) feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP-mediated acetylation may be a universal and critical modification for p53 function.

Acetylation↗

PubMed2000

A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor alpha gene expression.

The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor alpha (TNF-alpha) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF-alpha gene induction by TCR activation is inhibited, whereas virus induction of the TNF-alpha gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-alpha gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

Animals↗

PubMed2000

Gene dose-dependent control of hematopoiesis and hematologic tumor suppression by CBP.

Mice with monoallelic inactivation of the CBP gene develop highly penetrant, multilineage defects in hematopoietic differentiation and, with advancing age, an increased incidence of hematologic malignancies. The latter are characterized, at least in some cases, by loss of heterozygosity (LOH) at the CBP locus. No such pathology was observed in wild-type or p300 heterozygous null mice of the same age and genetic background. Thus, a full complement of CBP, but not p300, is required for normal hematopoietic differentiation. These results also provide the first experimental evidence for the hypothesis that CBP has tumor-suppressing activity.

Animals↗

PubMed1999

SMRTER, a Drosophila nuclear receptor coregulator, reveals that EcR-mediated repression is critical for development.

The Drosophila ecdysone receptor (EcR)/ultraspiracle (USP) heterodimer is a key regulator in molting and metamorphoric processes, activating and repressing transcription in a sequence-specific manner. Here, we report the isolation of an EcR-interacting protein, SMRTER, which is structurally divergent but functionally similar to the vertebrate nuclear corepressors SMRT and N-CoR. SMRTER mediates repression by interacting with Sin3A, a repressor known to form a complex with the histone deacetylase Rpd3/HDAC. Importantly, we identify an EcR mutant allele that fails to bind SMRTER and is characterized by developmental defects and lethality. Together, these results reveal a novel nuclear receptor cofactor that exhibits evolutionary conservation in the mechanism to achieve repression and demonstrate the essential role of repression in hormone signaling.

Amino Acid Sequence↗

PubMed1998

Lineage-specific signaling in melanocytes. C-kit stimulation recruits p300/CBP to microphthalmia.

During melanocyte development, the cytokine Steel factor activates its receptor c-Kit, initiating a signal transduction cascade, which is vital for lineage determination via unknown downstream nuclear targets. c-Kit has recently been found to trigger mitogen-activated protein kinase-mediated phosphorylation of Microphthalmia (Mi), a lineage-restricted transcription factor, which, like Steel factor and c-Kit, is essential for melanocyte development. This cascade results in increased Mi-dependent transcriptional reporter activity. Here we examine the mechanism by which Mi is activated by this pathway. Phosphorylation does not significantly alter Mi's nuclear localization, DNA binding, or dimerization. However, the transcriptional coactivator p300/CBP selectively associates with mitogen-activated protein kinase-phosphorylated Mi, even under conditions in which non-MAPK phospho-Mi is more abundant. Moreover, p300/CBP coactivates Mi transcriptional activity in a manner dependent upon this phosphorylation. Mi thus joins CREB as a transcription factor whose signal-responsive phosphorylation regulates coactivator recruitment, in this case modulating lineage development in melanocytes.

Animals↗

PubMed1998

Distinct roles of the co-activators p300 and CBP in retinoic-acid-induced F9-cell differentiation.

The related proteins p300 and CBP (cAMP-response-element-binding protein (CREB)-binding protein)) are transcriptional co-activators that act with other factors to regulate gene expression and play roles in many cell-differentiation and signal transduction pathways. Both proteins have intrinsic histone-acetyltransferase activity and may act directly on chromatin, of which histone is a component, to facilitate transcription. They are also involved in growth control pathways, as shown by their interaction with the tumour suppressor p53 and the viral oncogenes E1A and SV40 T antigen. Here we report functional differences of p300 and CBP in vivo. We examined their roles during retinoic-acid-induced differentiation, cell-cycle exit and programmed cell death (apoptosis) of embryonal carcinoma F9 cells, using hammerhead ribozymes capable of cleaving either p300 or CBP messenger RNAs. F9 cells expressing a p300-specific ribozyme became resistant to retinoic-acid-induced differentiation, whereas cells expressing a CBP-specific ribozyme were unaffected. Similarly, retinoic-acid-induced transcriptional upregulation of the cell-cycle inhibitor p21Cip1 required normal levels of p300, but not CBP, whereas the reverse was true for p27Kip1. In contrast, both ribozymes blocked retinoic-acid-induced apoptosis, indicating that both co-activators are required for this process. Thus, despite their similarities, p300 and CBP have distinct functions during retinoic-acid-induced differentiation of F9 cells.

Apoptosis↗

PubMed1998

Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300.

The transcriptional coactivator and integrator p300 and its closely related family member CBP mediate multiple, signal-dependent transcriptional events. We have generated mice lacking a functional p300 gene. Animals nullizygous for p300 died between days 9 and 11.5 of gestation, exhibiting defects in neurulation, cell proliferation, and heart development. Cells derived from p300-deficient embryos displayed specific transcriptional defects and proliferated poorly. Surprisingly, p300 heterozygotes also manifested considerable embryonic lethality. Moreover, double heterozygosity for p300 and cbp was invariably associated with embryonic death. Thus, mouse development is exquisitely sensitive to the overall gene dosage of p300 and cbp. Our results provide genetic evidence that a coactivator endowed with histone acetyltransferase activity is essential for mammalian cell proliferation and development.

Animals↗

PubMed1996

The nuclear hormone receptor coactivator SRC-1 is a specific target of p300.

p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.

Amino Acid Sequence↗

PubMed1996

Interaction and functional collaboration of p300/CBP and bHLH proteins in muscle and B-cell differentiation.

Differentiation of skeletal muscle cells and B lymphocytes is regulated by basic helix-loop-helix (bHLH) proteins. Both differentiation programs are inhibited by the adenovirus E1A oncoprotein. Analysis of E1A mutants has implicated two of its cellular-binding proteins, p300 and CBP, in controlling certain aspects of differentiation. We find that p300 can cooperate with tissue-specific bHLH proteins in activating target genes and requires only the bHLH domain of such proteins to stimulate E box-directed transcription. Importantly, the ability of bHLH proteins to activate transcription correlates with the presence of p300/CBP in E box-dependent DNA-binding complexes, because both phenomena require at least two adjacent E-box motifs. Microinjection of p300/CBP antibodies into myoblasts blocks terminal differentiation, cell fusion, and transcriptional activity of myogenic bHLH proteins. These results suggest that the function of p300/CBP is essential for the execution of key aspects of cellular differentiation.

3T3 Cells↗

PubMed1996

Ecdysone-inducible gene expression in mammalian cells and transgenic mice.

During metamorphosis of Drosophila melanogaster, a cascade of morphological changes is triggered by the steroid hormone 20-OH ecdysone via the ecdysone receptor, a member of the nuclear receptor superfamily. In this report, we have transferred insect hormone responsiveness to mammalian cells by the stable expression of a modified ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified ecdysone receptor can activate an integrated ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy.

Animals↗

PubMed1995

Identification and characterization of a Drosophila nuclear receptor with the ability to inhibit the ecdysone response.

In a search for retinoid X receptor-like molecules in Drosophila, we have identified an additional member of the nuclear receptor superfamily, XR78E/F. In the DNA-binding domain, XR78E/F is closely related to the mammalian receptor TR2, as well as to the nuclear receptors Coup-TF and Seven-up. We demonstrate that XR78E/F binds as a homodimer to direct repeats of the sequence AGGTCA. In transient transfection assays, XR78E/F represses ecdysone signaling in a DNA-binding-dependent fashion. XR78E/F has its highest expression in third-instar larvae and prepupae. These experiments suggest that XR78E/F may play a regulatory role in the transcriptional cascade triggered by the hormone ecdysone in Drosophila.

Amino Acid Sequence↗

PubMed1995

Seven-up inhibits ultraspiracle-based signaling pathways in vitro and in vivo.

Seven-up (Svp), the Drosophila homolog of the chicken ovalbumin upstream transcription factor (COUP-TF); Ultraspiracle (Usp), the Drosophila homolog of the retinoid X receptor; and the ecdysone receptor are all members of the nuclear/steroid receptor superfamily. COUP-TF negatively regulates hormonal signaling involving retinoid X receptor in tissue culture systems. Here we demonstrate that Svp, like COUP-TF, can modulate Ultraspiracle-based hormonal signaling both in vitro and in vivo. Transfection assays in CV-1 cells demonstrate that Seven-up can inhibit ecdysone-dependent transactivation by the ecdysone receptor complex, a heterodimeric complex of Usp and ecdysone receptor. This repression depends on the dose of Svp and occurs with two different Drosophila ecdysone response elements. Ectopic expression of Svp in vivo induces lethality during early metamorphosis, the time of maximal ecdysone responsiveness. Concomitant overexpression of Usp rescues the larvae from the lethal effects of Svp. DNA binding studies show that Svp can bind to various direct repeats of the sequence AGGTCA but cannot bind to one of the ecdysone response elements used in the transient transfection assays. Our results suggest that Svp-mediated repression can occur by both DNA binding competition and protein-protein interactions.

Animals↗

PubMed1993

Functional ecdysone receptor is the product of EcR and Ultraspiracle genes.

Although the biological activity of the insect moulting hormone ecdysone, is manifested through a hormonally regulated transcriptional cascade associated with chromosomal puffing, a direct association of the receptor with the puff has yet to be established. The cloned ecdysone receptor (EcR) is by itself incapable of high-affinity DNA binding or transcriptional activation. Rather, these activities are dependent on heterodimer formation with Ultraspiracle (USP) the insect homologue of vertebrate retinoid X receptor. Here we report that native EcR and USP are co-localized on ecdysone-responsive loci of polytene chromosomes. Moreover, we show that natural ecdysones selectively promote physical association between EcR and USP, and conversely, that high-affinity hormone binding requires both EcR and USP. Replacement of USP with retinoid X receptor produces heterodimers with distinct pharmacological and functional properties. These results redefine the ecdysone receptor as a dynamic complex whose activity may be altered by combinatorial interactions among subunits and ligand.

Animals↗

PubMed1992

Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation.

The vertebrate retinoid X receptor (RXR) has been implicated in the regulation of multiple hormonal signaling pathways through the formation of heteromeric receptor complexes that bind DNA with high affinity. We now demonstrate that ultraspiracle (usp), a Drosophila RXR homolog, can substitute for RXR in stimulating the DNA binding of receptors for retinoic acid, T3, vitamin D, and peroxisome proliferator activators. These observations led to the search and ultimate identification of the ecdysone receptor (EcR) as a Drosophila partner of usp. Together, usp and EcR bind DNA in a highly cooperative fashion. Cotransfection of both EcR and usp expression vectors is required to render cultured mammalian cells ecdysone responsive. These results implicate usp as an integral component of the functional EcR. By demonstrating that receptor heterodimer formation precedes the divergence of vertebrate and invertebrate lineages, these data underscore a central role for RXR and its homolog usp in the evolution and control of the nuclear receptor-based endocrine system.

Animals↗

PubMed1991

Alternative translation initiation site usage results in two structurally distinct forms of Pit-1.

Pit-1 is a pituitary-specific transcription factor that plays a critical role in the normal development of the anterior pituitary gland. Previous analyses have shown that this protein exists in the rat pituitary gland and in rat pituitary-derived cell lines as two forms of relative molecular mass 33 and 31 kDa. This aspect of Pit-1 expression has been conserved throughout the evolution from rodents to humans. Here, we determine the origin of these structurally distinct forms of Pit-1 protein and find that these arise as a consequence of the alternative usage of translation initiation sites present in Pit-1 mRNA.

Amino Acid Sequence↗