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Biomedical subjects

Richard H Goodman

Publications and source records attributed to Richard H Goodman.

18 recordsLinked to original sources
PubMed2007

Metabolic regulation of SIRT1 transcription via a HIC1:CtBP corepressor complex.

The Sir2 histone deacetylases are important for gene regulation, metabolism, and longevity. A unique feature of these enzymes is their utilization of NAD(+) as a cosubstrate, which has led to the suggestion that Sir2 activity reflects the cellular energy state. We show that SIRT1, a mammalian Sir2 homologue, is also controlled at the transcriptional level through a mechanism that is specific for this isoform. Treatment with the glycolytic blocker 2-deoxyglucose (2-DG) decreases association of the redox sensor CtBP with HIC1, an inhibitor of SIRT1 transcription. We propose that the reduction in transcriptional repression mediated by HIC1, due to the decrease of CtBP binding, increases SIRT1 expression. This mechanism allows the specific regulation of SIRT1 in response to nutrient deprivation.

Alcohol Oxidoreductases↗
PubMed2006

An antisense transcript induced by Wnt/beta-catenin signaling decreases E2F4.

Wnt signaling induces the nuclear accumulation of beta-catenin and transcription of specific target genes via the DNA-binding proteins TCF/Lef. Although all known beta-catenin target genes encode proteins, genome-wide RNA profiling studies indicate that many transcripts do not have this capability. Transcription factor-binding sites associated with these noncoding transcripts can be identified using unbiased techniques such as serial analysis of chromatin occupancy (SACO). We used this method to identify a beta-catenin-regulated antisense RNA expressed in HCT116 colorectal carcinoma cells, a cellular model of activated beta-catenin signaling. Genomic signature tags designating putative beta-catenin-binding sites mapped to the 3'-untranslated region (3'-UTR) of the E2F4 gene. We showed that both beta-catenin and TCF4 bind to the E2F4 3'-UTR site in vivo, inducing expression of an E2F4 antisense transcript. LiCl, which mimics Wnt signaling, also induced expression of the E2F4 antisense transcript and decreased E2F4 protein levels. This effect was blocked by a cDNA expressing the E2F4 3'-UTR sense strand. The antisense-mediated decrease in E2F4 protein was reflected by reduced E2F4 association with specific target genes, including CCNA2, CDC2, PCNA, and Rad54. We propose that Wnt/beta-catenin signaling may contribute to colorectal carcinogenesis by reducing the level of the E2F4 cell cycle repressor via an antisense mechanism.

Antisense Elements (Genetics)↗
PubMed2006

Redox sensor CtBP mediates hypoxia-induced tumor cell migration.

The rapid growth and poor vascularization of solid tumors expose cancer cells to hypoxia, which promotes the metastatic phenotype by reducing intercellular adhesion and increasing cell motility and invasiveness. In this study, we found that hypoxia increased free NADH levels in cancer cells, promoting CtBP recruitment to the E-cadherin promoter. This effect was blocked by pyruvate, which prevents the NADH increase. Furthermore, hypoxia repressed E-cadherin gene expression and increased tumor cell migration, effects that were blocked by CtBP knockdown. We propose that CtBP senses levels of free NADH to control expression of cell adhesion genes, thereby promoting tumor cell migration under hypoxic stress.

Alcohol Oxidoreductases↗
PubMed2005

A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis.

MicroRNAs (miRNAs) regulate cellular fate by controlling the stability or translation of mRNA transcripts. Although the spatial and temporal patterning of miRNA expression is tightly controlled, little is known about signals that induce their expression nor mechanisms of their transcriptional regulation. Furthermore, few miRNA targets have been validated experimentally. The miRNA, miR132, was identified through a genome-wide screen as a target of the transcription factor, cAMP-response element binding protein (CREB). miR132 is enriched in neurons and, like many neuronal CREB targets, is highly induced by neurotrophins. Expression of miR132 in cortical neurons induced neurite outgrowth. Conversely, inhibition of miR132 function attenuated neuronal outgrowth. We provide evidence that miR132 regulates neuronal morphogenesis by decreasing levels of the GTPase-activating protein, p250GAP. These data reveal that a CREB-regulated miRNA regulates neuronal morphogenesis by responding to extrinsic trophic cues.

Animals↗
PubMed2005

Homeodomain-interacting protein kinase-2 mediates CtBP phosphorylation and degradation in UV-triggered apoptosis.

Homeodomain-interacting protein kinase-2 (HIPK2) is a serine/threonine kinase involved in transcriptional regulation and apoptosis. The transcriptional corepressor CtBP (carboxyl-terminal binding protein) also plays a fundamental role in these processes. Our previous studies indicate that HIPK2 participates in a pathway of UV-triggered CtBP clearance that results in cell death. HIPK2 phosphorylates CtBP at Ser-422 in vitro. We developed a Ser-422 phospho-specific antibody to demonstrate that CtBP is phosphorylated on this residue in response to UV irradiation. HIPK2 knock-down blocked the UV-induced Ser-422 phosphorylation and degradation. The proteasomal inhibitor MG-132 treatment increased levels of ubiquitinated CtBP, which was induced by UV. Interference with HIPK2 function via the kinase-dead mutant decreased CtBP ubiquitination. Furthermore, a phosphopeptide spanning Ser-422 blocked UV-triggered CtBP degradation, confirming that Ser-422 phosphorylation marks CtBP for clearance. Consequently, interference with HIPK2 action in H1299 cells rescued UV-triggered apoptosis.

Alcohol Oxidoreductases↗
PubMed2005

Role reversal: the regulation of neuronal gene expression by microRNAs.

In a similar fashion to transcription factors, non-coding RNAs can be essential regulators of gene expression. The largest class of non-coding RNAs is the microRNAs. These approximately 22 nt double-stranded RNA molecules can repress translation or target mRNA degradation. There has been a surge of research in the past year stimulated by the recent availability of specialized techniques, both in vitro and in silico, for predicting and characterizing microRNAs. The accumulating evidence suggests that microRNAs are ubiquitous regulators of gene expression during development. The combined actions of microRNAs and transcription factors are able to tune the expression of proteins on a global level in a manner that cannot be achieved by transcription factors alone.

Animals↗
PubMed2004

Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions.

The CREB transcription factor regulates differentiation, survival, and synaptic plasticity. The complement of CREB targets responsible for these responses has not been identified, however. We developed a novel approach to identify CREB targets, termed serial analysis of chromatin occupancy (SACO), by combining chromatin immunoprecipitation (ChIP) with a modification of SAGE. Using a SACO library derived from rat PC12 cells, we identified approximately 41,000 genomic signature tags (GSTs) that mapped to unique genomic loci. CREB binding was confirmed for all loci supported by multiple GSTs. Of the 6302 loci identified by multiple GSTs, 40% were within 2 kb of the transcriptional start of an annotated gene, 49% were within 1 kb of a CpG island, and 72% were within 1 kb of a putative cAMP-response element (CRE). A large fraction of the SACO loci delineated bidirectional promoters and novel antisense transcripts. This study represents the most comprehensive definition of transcription factor binding sites in a metazoan species.

Animals↗
PubMed2004

Cell-type-specific binding of the transcription factor CREB to the cAMP-response element.

The cAMP-response element-binding protein (CREB) transcription factor was initially identified as a mediator of cAMP-induced gene expression. CREB binds to a target sequence termed the cAMP-response element (CRE) found in many cellular and viral gene promoters. One of the best-characterized CREs resides in the promoter of the gene encoding the neuropeptide somatostatin, and this element has served as a model for studies of CREB function. Phosphorylation of CREB by protein kinase A allows recruitment of the coactivator CREB-binding protein (CBP). A central tenet of the CREB-CBP model is that CREB binds constitutively to the CRE and that regulation occurs through the phosphorylation-dependent recruitment of CBP. In this report, we use chromatin immunoprecipitation assays to show that CREB does not interact in vivo with the somatostatin CRE, or similar elements in several other genes, in PC12 cells, a standard model for studies of CREB function. Rather, CREB binding in vivo is regulated in a cell-specific manner, a finding that was confirmed by using in vivo genomic footprinting assays. The CREs in other genes were also found to interact differentially with CREB in PC12 cells, hepatoma cells, and cortical neurons. We conclude that the family of CREB target genes differs from one cell type to another and that the ability of CREB to bind to a particular CRE represents an important component of gene regulation.

Animals↗
PubMed2003

Homeodomain interacting protein kinase 2 promotes apoptosis by downregulating the transcriptional corepressor CtBP.

Genetic knockout of the transcriptional corepressor CtBP in mouse embryo fibroblasts upregulates several genes involved in apoptosis. We predicted, therefore, that a propensity toward apoptosis might be regulated through changes in cellular CtBP. To identify pathways involved in this regulation, we screened a mouse embryo cDNA library with an E1A-CtBP complex and identified the homeodomain interacting protein kinase 2 (HIPK2), which had previously been linked to UV-directed apoptosis through its ability to phosphorylate p53. Expression of HIPK2 or exposure to UV irradiation reduced CtBP levels via a proteosome-mediated pathway. The UV effect was prevented by coexpression of kinase-inactive HIPK2 or reduction in HIPK2 levels via siRNA. Mutation of the residue phosphorylated by HIPK2 prevented UV- and HIPK2-directed CtBP clearance. Finally, reduction in CtBP levels, either by genetic knockout or siRNA, promoted apoptosis in p53-deficient cells. These findings provide a pathway for UV-induced apoptosis in cells lacking p53.

Alcohol Oxidoreductases↗
PubMed2003

Deacetylase activity is required for cAMP activation of a subset of CREB target genes.

Many hormones activate transcription by raising the level of cAMP within cells. In one well studied pathway, cAMP induces protein kinase A to phosphorylate the transcription factor CREB, which binds to a consensus sequence, the cAMP-regulated enhancer, found in many target genes. A generally accepted model suggests that phosphorylated CREB recruits the histone acetyltransferase CBP to activate transcription. In contrast, histone deacetylases have been linked to the cessation of CREB-dependent transcription. Here we tested this model in the regulation of endogenous CREB target genes. We used a constitutively active CREB mutant and microarray analysis to identify target genes in PC12 cells. We then tested the role of histone deacetylase activity in cAMP activation of four of these genes (c-FOS, ICER, NOR-1, and NUR77) by treating cells with the histone deacetylase inhibitor trichostatin A. Consistent with the generally accepted model, trichostatin A enhanced activation of c-FOS and NUR77 by cAMP. Surprisingly, trichostatin A blocked activation of ICER and NOR-1. The block of ICER and NOR-1 activation persisted in the presence of cycloheximide, indicating that the trichostatin A effect did not depend on new protein synthesis. This unexpected role of histone deacetylases in transcriptional activation of certain endogenous CREB target genes was not apparent in transfected reporter genes. Chromatin immunoprecipitation analysis indicated that the differential roles of histone deacetylases in activating or repressing CREB target genes was manifested at the level of preinitiation complex recruitment. These data indicate that histone deacetylases differentially regulate CREB target genes by contributing to either activation or cessation of transcription.

Acetyltransferases↗
PubMed2003

Differential binding of NAD+ and NADH allows the transcriptional corepressor carboxyl-terminal binding protein to serve as a metabolic sensor.

Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor originally identified through its ability to interact with adenovirus E1A. The finding that CtBP-E1A interactions were regulated by the nicotinamide adeninine dinucleotides NAD+ and NADH raised the possibility that CtBP could serve as a nuclear redox sensor. This model requires differential binding affinities of NAD+ and NADH, which has been controversial. The structure of CtBP determined by x-ray diffraction revealed a tryptophan residue adjacent to the proposed nicotinamide adenine dinucleotide binding site. We find that this tryptophan residue shows strong fluorescence resonance energy transfer to bound NADH. In this report, we take advantage of these findings to measure the dissociation constants for CtBP with NADH and NAD+. The affinity of NADH was determined by using fluorescence resonance energy transfer. The binding of NADH to protein is associated with an enhanced intensity of NADH fluorescence and a blue shift in its maximum. NAD+ affinity was estimated by measuring the loss of the fluorescence blue shift as NADH dissociates on addition of NAD+. Our studies show a >100-fold higher affinity of NADH than NAD+, consistent with the proposed function of CtBP as a nuclear redox sensor. Moreover, the concentrations of NADH and NAD+ required for half-maximal binding are approximately the same as their concentrations in the nuclear compartment. These findings support the possibility that changes in nuclear nicotinamide adenine dinucleotides could regulate the functions of CtBP in cell differentiation, development, or transformation.

Alcohol Oxidoreductases↗
PubMed2003

C-terminal-binding protein corepresses epithelial and proapoptotic gene expression programs.

The genesis of carcinoma cells often involves epithelial-to-mesenchymal transitions and the acquisition of apoptosis resistance, but it is unclear whether these alterations are controlled coordinately or independently. Our previously reported effects of adenovirus E1a in human tumor cells raised the possibility that the E1a-interacting corepressor protein C-terminal-binding protein (CtBP) might selectively repress epithelial cell adhesion and proapoptotic genes. Here, we report that CtBP-knockout cells were hypersensitive to apoptosis. Correspondingly, microarray analysis of CtBP-knockout vs. CtBP-rescued mouse embryo fibroblasts revealed that many epithelial-specific and proapoptotic genes were indeed regulated by CtBP. Neither the apoptosis nor the repression activities of CtBP required histidine-315, suggesting that the proposed dehydrogenase activity is not essential for CtBP function. The results presented herein establish two functional roles of CtBP: to corepress epithelial genes, thus permitting epithelial-to-mesenchymal transitions, and to modulate the cellular threshold for apoptotic responses.

Alcohol Oxidoreductases↗
PubMed2002

Phosphorylation of CBP mediates transcriptional activation by neural activity and CaM kinase IV.

Activity-regulated transcription has been implicated in adaptive plasticity in the CNS. In many instances, this plasticity depends upon the transcription factor CREB. Precisely how neuronal activity regulates CREB remains unclear. To address this issue, we examined the phosphorylation state of components of the CREB transcriptional pathway. We show that NMDA activates transcription of CREB-responsive genes in hippocampal neurons, with ERK responsible for persistent CREB phosphorylation and CaM kinase IV (CaMKIV) responsible for phosphorylating the CREB coactivator, CBP. Ser301 of CBP was identified as a major target of CaMKIV phosphorylation in vitro and in vivo. CaM kinase inhibitors attenuated phosphorylation at Ser301 and blocked CBP-dependent transcription. Additionally, mutation of Ser301 impaired NMDA- and CaMKIV-stimulated transcription. These findings demonstrate that activity-induced CaMKIV signaling contributes to CREB/CBP-dependent transcription by phosphorylating CBP at Ser301.

Amino Acid Sequence↗
PubMed2002

The interaction between the coactivator dCBP and Modulo, a chromatin-associated factor, affects segmentation and melanotic tumor formation in Drosophila.

The development of Drosophila requires the function of the CREB-binding protein, dCBP. In flies, dCBP serves as a coactivator for the transcription factors Cubitus interruptus, Dorsal, and Mad, and as a cosuppressor of Drosophila T cell factor. Current models propose that CBP, through its intrinsic and associated histone acetyltransferase activities, affects transient chromatin changes that allow the preinitiation complex to access the promoter. In this report, we provide evidence that dCBP may regulate the formation of chromatin states through interactions with the modulo (mod) gene product, a protein that is thought to be involved in chromatin packaging. We demonstrate that dCBP and Modulo bind in vitro and in vivo, that mutations in mod enhance the embryonic phenotype of a dCBP mutation, and that dCBP mutations enhance the melanotic tumor phenotype characteristic of mod homozygous mutants. These results imply that, in addition to its histone acetyltransferase activity, dCBP may affect higher-order chromatin structure.

Acetyltransferases↗
PubMed2002

Regulation of corepressor function by nuclear NADH.

The corepressor CtBP (carboxyl-terminal binding protein) is involved in transcriptional pathways important for development, cell cycle regulation, and transformation. We demonstrate that CtBP binding to cellular and viral transcriptional repressors is regulated by the nicotinamide adenine dinucleotides NAD+ and NADH, with NADH being two to three orders of magnitude more effective. Levels of free nuclear nicotinamide adenine dinucleotides, determined using two-photon microscopy, correspond to the levels required for half-maximal CtBP binding and are considerably lower than those previously reported. Agents capable of increasing NADH levels stimulate CtBP binding to its partners in vivo and potentiate CtBP-mediated repression. We propose that this ability to detect changes in nuclear NAD+/NADH ratio allows CtBP to serve as a redox sensor for transcription.

Adenovirus E1A Proteins↗
PubMed2002

The acetyltransferase activity of CBP is required for wingless activation and H4 acetylation in Drosophila melanogaster.

CBP is a critical coactivator of transcription, but little is understood about the importance of its intrinsic acetyltransferase (AT) activity in gene activation in vivo. We show that the intrinsic AT function of CBP in Drosophila melanogaster (dCBP) is necessary to maintain a dCBP overexpression phenotype in the eye, for the in vivo activation of a specific target gene, wingless, and for the global acetylation of histone H4. These findings indicate that a point mutation which alters the intrinsic AT activity of CBP (only one of many CBP functions) has profound effects on CBP-induced gene activation in a physiologically intact transcription system. Furthermore, the effects of CBP AT activity are not limited to a few specific promoters, but rather CBT AT activity may play a role in regulating global histone acetylation throughout the developing organism.

Acetyl Coenzyme A↗
PubMed2002

Calmodulin-dependent protein kinase IV mediated antagonism of BMP signaling regulates lineage and survival of hematopoietic progenitors.

In the current study, we show that bone morphogenetic proteins (BMPs) play a role in hematopoiesis that is independent of their function in specifying ventral mesodermal fate. When BMP activity is upregulated or inhibited in Xenopus embryos hematopoietic precursors are specified properly but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of BMP activity induces erythroid precursors to undergo apoptotic cell death, whereas constitutive activation of BMPs causes an increase in commitment of hematopoietic progenitors to myeloid differentiation and a concomitant decrease in erythrocytes that is not due to enhanced apoptosis. These blood defects are observed even when BMP activity is misregulated solely in non-hematopoietic (ectodermal) cells, demonstrating that BMPs generate extrinsic signals that regulate hematopoiesis independent of mesodermal patterning. Further analysis revealed that endogenous calmodulin-dependent protein kinase IV (CaM KIV) is required to negatively modulate hematopoietic functions of BMPs downstream of receptor activation. Our data are consistent with a model in which CaM KIV inhibits BMP signals by activating a substrate, possibly cAMP-response element-binding protein (CREB), that recruits limiting amounts of CREB binding protein (CBP) away from transcriptional complexes functioning downstream of BMPs.

Animals↗