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Biomedical subjects

Neil D Perkins

Publications and source records attributed to Neil D Perkins.

At least 19 recordsLinked to original sources
PubMed2005

SIRT1 deacetylation and repression of p300 involves lysine residues 1020/1024 within the cell cycle regulatory domain 1.

The SIR2 family of nicotinamide adenosine dinucleotide (NAD)-dependent deacetylases modulates diverse biological functions in different species, including longevity, apoptosis, cell cycle exit, and cellular differentiation. SIRT1, the closest mammalian ortholog of the yeast SIR2 (silent information regulator 2) gene, represses several transcription factors, including p53, NFkappaB and forkhead proteins. The p300 protein serves as a rate-limiting transcriptional cointegrator of diverse transcription factors either to activate or to repress transcription through modular subdomains. Herein, SIRT1 physically interacted with and repressed p300 transactivation, requiring the NAD-dependent deacetylase activity of SIRT1. SIRT1 repression involved the CRD1 transcriptional repression domain of p300. Two residues within the CRD1 domain (Lys-1020 and Lys-1024) were required for SIRT1 repression and served as substrates for SIRT1 deacetylation. These residues also serve as acceptor lysines for modification by the ubiquitin-like SUMO protein. The SUMO-specific protease SSP3 relieved SIRT1 repression of p300. SSP3 antagonism of SIRT1 required the SUMO-deconjugating function of SSP3. Thus, p300 serves as a deacetylase substrate for SIRT1 through a conserved SUMO consensus motif. Because p300 is a limiting transcriptional cofactor, deacetylation and repression of p300 by SIRT1 may serve an important integration point during metabolism and cellular differentiation.

Amino Acid Motifs↗
PubMed2004

The FHA domain protein SNIP1 is a regulator of the cell cycle and cyclin D1 expression.

Smad nuclear interacting protein 1 (SNIP1) is an evolutionarily conserved protein containing a forkhead-associated (FHA) domain that regulates gene expression through interactions with multiple transcriptional regulators. Here, we have used short interfering RNAs (siRNAs) to knockdown SNIP1 expression in human cell lines. Surprisingly, we found that reduction in SNIP1 levels resulted in significantly reduced cell proliferation and accumulation of cells in the G1 phase of the cell cycle. Consistent with this result, we observed that cyclin D1 protein and mRNA levels were reduced. Moreover, SNIP1 depletion results in inhibition of cyclin D1 promoter activity in a manner dependent upon a previously characterized binding site for the AP-1 transcription factor family. SNIP1 itself is induced upon serum stimulation immediately prior to cyclin D1 expression. These effects were independent of the tumour suppressors p53 and retinoblastoma (Rb), but were consistent with an interaction with BRG1, a component of the ATP-dependent chromatin remodelling complex, Swi/Snf. These results define both a new function for SNIP1 and identify a previously unrecognized regulator of the cell cycle and cyclin D1 expression.

Base Sequence↗
PubMed2004

The p68 and p72 DEAD box RNA helicases interact with HDAC1 and repress transcription in a promoter-specific manner.

BACKGROUND: p68 (Ddx5) and p72 (Ddx17) are highly related members of the DEAD box family and are established RNA helicases. They have been implicated in growth regulation and have been shown to be involved in both pre-mRNA and pre-rRNA processing. More recently, however, these proteins have been reported to act as transcriptional co-activators for estrogen-receptor alpha (ER alpha). Furthermore these proteins were shown to interact with co-activators p300/CBP and the RNA polymerase II holoenzyme. Taken together these reports suggest a role for p68 and p72 in transcriptional activation. RESULTS: In this report we show that p68 and p72 can, in some contexts, act as transcriptional repressors. Targeting of p68 or p72 to constitutive promoters leads to repression of transcription; this repression is promoter-specific. Moreover both p68 and p72 associate with histone deacetylase 1 (HDAC1), a well-established transcriptional repression protein. CONCLUSIONS: It is therefore clear that p68 and p72 are important transcriptional regulators, functioning as co-activators and/or co-repressors depending on the context of the promoter and the transcriptional complex in which they exist.

Adenocarcinoma↗
PubMed2004

Induction of transcription by p21Waf1/Cip1/Sdi1: role of NFkappaB and effect of non-steroidal anti-inflammatory drugs.

p21(Waf1/Cip1/Sdi1) is the primary mediator of cell cycle arrest in response to different forms of stress and in the programs of senescence and differentiation. p21 interacts with many regulatory proteins and has broad effects on cellular gene expression. p21 was previously shown to stimulate NFkappaB transcriptional activity through its effect on the p300/CBP transcription cofactor family. p21 expression in human cells increases mRNA levels of different genes, some of which have been implicated in carcinogenesis and age-related diseases. Here we report that p21 expression stimulates promoters of six p21-responsive human genes and the cytomegalovirus promoter, as well as an artificial promoter containing NFkappaB response elements. The IkappaBalpha super-repressor blocked the effect of p21 on all but one of the promoters, and the response to p21 was abrogated by the mutagenesis of an NFkappaB element. p21 inducibility of all the tested promoters and of the endogenous p21-responsive genes was strongly inhibited by adenoviral E1A protein and its deletion mutants that bind p300/CBP but not p21 or Rb. Sulindac and some other non-steroidal anti-inflammatory drugs that inhibit NFkappaB decrease the effects of p21 on the responsive promoters and endogenous genes. These findings suggest the feasibility of developing agents that will counteract p21-mediated induction of disease-associated genes.

Adenovirus E1A Proteins↗
PubMed2004

Reprogramming RelA.

The diversity of activators and targets of the NF-kappaB transcription factor family demands that there be regulatory mechanisms in place to control the specificity with which genes under their control are induced. In part this can be achieved through selective induction of different NF-kappaB subunits and through co-operative interactions with heterologous DNA-binding proteins and co-activators. Recent work from our laboratory indicates another critical mechanism regulating NF-kappaB. We find that the RelA(p65) NF-kappaB subunit does not always function as an inducer of gene expression, but under certain circumstances can be programmed to actively repress these same target genes. This repressor form of NF-kappaB appears to be induced by distinct, atypical pathways of activation and also through the action of some tumor suppressors. The identification of these pathways not only allows a reinterpretation of NF-kappaB function in normal cells and during tumorigenesis but could also have implications for both traditional and NF-kappaB based cancer therapy.

Animals↗
PubMed2004

Active repression of antiapoptotic gene expression by RelA(p65) NF-kappa B.

With the emerging role of NF-kappa B in cancer it is important that its responses to stimuli relevant to tumor progression and therapy are understood. Here, we demonstrate that NF-kappa B induced by cytotoxic stimuli, such as ultraviolet light (UV-C) and the chemotherapeutic drugs daunorubicin/doxorubicin, is functionally distinct to that seen with the inflammatory cytokine TNF and is an active repressor of antiapoptotic gene expression. Surprisingly, these effects are mediated by the RelA(p65) NF-kappa B subunit. Furthermore, UV-C and daunorubicin inhibit TNF-induced NF-kappa B transactivation, indicating that this is a dominant effect. Consistent with this, mechanistic studies reveal that UV-C and daunorubicin induce the association of RelA with histone deacetylases. RelA can therefore be both an activator and repressor of its target genes, dependent upon the manner in which it is induced. This has important implications for the role of NF-kappa B in tumorigenesis and the use of NF-kappa B inhibitors in cancer therapy.

Animals↗
PubMed2004

NF-kappaB: tumor promoter or suppressor?

A role for the NF-kappaB family of transcription factors as tumor promoters is firmly established. However, other data suggest that NF-kappaB can also inhibit tumor growth. Moreover, NF-kappaB activity is modulated by tumor suppressors, such as p53 and ARF, whereby NF-kappaB subunits repress, rather than activate, the expression of tumor-promoting genes. This suggests a dual function of NF-kappaB during tumor progression - in the early stages, NF-kappaB inhibits tumor growth but, as further mutations lead to a loss of tumor suppressor expression, the oncogenic functions of NF-kappaB become unleashed, allowing it to actively contribute to tumorigenesis. Here, I discuss this hypothesis, its implications for NF-kappaB function, and how this might influence the use of NF-kappaB-based anticancer therapies.

Animals↗
PubMed2003

Oncogenes, tumor suppressors and p52 NF-kappaB.

A role for the p52 NF-kappaB subunit in tumorigenesis has been steadily emerging since its discovery as a gene associated with chromosomal translocations in B- and T-cell lymphomas. Now Eliopoulos and co-workers have extended these studies to examine the effect of the Epstein-Barr virus (EBV)-encoded latent infection membrane protein 1 (LMP1) on p52. They find that LMP1 stimulates the processing of p100 to p52 NF-kappaB. Moreover, nuclear p52 is also associated with LMP1 expression in tumor tissue biopsies. They also demonstrate that the pathway leading to p100/p52 processing is distinct from that engaged by LMP1 to activate other NF-kappaB subunits through IkappaBalpha degradation. A clearer picture is now developing of the important role that p52 NF-kappaB plays during normal cell growth and how subverting its function can contribute to oncogenesis.

Gene Expression Regulation, Neoplastic↗
PubMed2003

The p53-inhibitor pifithrin-alpha inhibits firefly luciferase activity in vivo and in vitro.

BACKGROUND: Pifithrin-alpha is a small molecule inhibitor of p53 transcriptional activity. It has been proposed that the use of pifithrin-alpha in conjunction with chemotherapeutic and radiation therapies for cancer will reduce the side effects of these treatments in normal tissue that still contains wild type p53. In addition, pifithrin-alpha provides a useful tool in the laboratory to investigate the function of p53 in model systems. RESULTS: While investigating the effects of pifithrin-alpha on the transcriptional activity of NF-kappaB, we observed a strong inhibition of reporter plasmids containing the firefly luciferase gene. Firefly luciferase is one of the most commonly used enzymes in reporter gene assays. In contrast, no inhibition of reporter plasmids containing Renilla luciferase or chloramphenicol acetyltransferase was observed. The inhibition of firefly luciferase activity by pifithrin-alpha was observed both in vivo and in vitro. Pifithrin-alpha did not inhibit firefly luciferase protein expression, but rather suppressed light production/emission, since addition of exogenous pifithrin-alpha to active extracts inhibited this activity. Furthermore, pifithrin-alpha also inhibited recombinant firefly luciferase protein activity. CONCLUSIONS: Among its other biological activities, pifithrin-alpha is an inhibitor of firefly luciferase activity. Caution must therefore be taken when using this compound, which has been characterised as an inhibitor of p53 transcriptional activity, to investigate effects on gene expression using transiently transfected reporter plasmids. Furthermore, these results demonstrate that when using novel compounds, the choice of vectors used in the experimental procedures might be of great importance for the correct conclusions to be made.

Animals↗
PubMed2003

P300 transcriptional repression is mediated by SUMO modification.

p300 and CREB binding protein can both activate and repress transcription. Here, we locate the CRD1 transcriptional repression domain between residues 1017 and 1029 of p300. This region contains two copies of the sequence psiKxE that are modified by the ubiquitin-like protein SUMO-1. Mutations that reduce SUMO modification increase p300-mediated transcriptional activity and expression of a SUMO-specific protease or catalytically inactive Ubc9 relieved repression, demonstrating that p300 repression was mediated by SUMO conjugation. SUMO-modified CRD1 domain bound HDAC6 in vitro, and p300 repression was relieved by histone deacetylase inhibition and siRNA-mediated ablation of HDAC6 expression. These results reveal a mechanism controlling p300 function and suggest that SUMO-dependent repression is mediated by recruitment of HDAC6.

Amino Acid Sequence↗
PubMed2003

p53- and Mdm2-independent repression of NF-kappa B transactivation by the ARF tumor suppressor.

One mechanism by which a cell affords protection from the transforming effects of oncogenes is via the action of the tumor suppressor, ARF, which activates p53 by inactivating Mdm2. Many oncogenes have also been shown to activate the transcription factor NF-kappa B, which can contribute toward the malignant phenotype in many ways, including an ability to antagonize p53. Here we find that ARF inhibits NF-kappa B function and its antiapoptotic activity independent of Mdm2 and p53. ARF represses the transcriptional activation domain of the NF-kappa B family member RelA by inducing its association with the histone deacetylase, HDAC1. Further, we show that the response of NF-kappa B to the oncogene Bcr-Abl is determined by the ARF status of the cell. These results reveal an important function of ARF that can regulate the NF-kappa B response to oncogene activation.

Cell Transformation, Neoplastic↗
PubMed2003

p53 represses cyclin D1 transcription through down regulation of Bcl-3 and inducing increased association of the p52 NF-kappaB subunit with histone deacetylase 1.

The p53 and NF-kappaB transcription factor families are important, multifunctional regulators of the cellular response to stress. Here we have investigated the regulatory mechanisms controlling p53-dependent cell cycle arrest and cross talk with NF-kappaB. Upon induction of p53 in H1299 or U-2 OS cells, we observed specific repression of cyclin D1 promoter activity, correlating with a decrease in cyclin D1 protein and mRNA levels. This repression was dependent on the proximal NF-kappaB binding site of the cyclin D1 promoter, which has been shown to bind the p52 NF-kappaB subunit. p53 inhibited the expression of Bcl-3 protein, a member of the IkappaB family that functions as a transcriptional coactivator for p52 NF-kappaB and also reduced p52/Bcl-3 complex levels. Concomitant with this, p53 induced a significant increase in the association of p52 and histone deacetylase 1 (HDAC1). Importantly, p53-mediated suppression of the cyclin D1 promoter was reversed by coexpression of Bcl-3 and inhibition of p52 or deacetylase activity. p53 therefore induces a transcriptional switch in which p52/Bcl-3 activator complexes are replaced by p52/HDAC1 repressor complexes, resulting in active repression of cyclin D1 transcription. These results reveal a unique mechanism by which p53 regulates NF-kappaB function and cell cycle progression.

B-Cell Lymphoma 3 Protein↗
PubMed2003

Regulation of RelA (p65) function by the large subunit of replication factor C.

The RelA (p65) subunit of NF-kappaB is an important regulator of inflammation, proliferation, and apoptosis. We have discovered that the large subunit, p140, of replication factor C (RFC) can function as a regulator of RelA. RFC is a clamp loader, facilitating the addition and removal of proliferating-cell nuclear antigen from DNA during replication and repair but can also interact directly with the retinoblastoma tumor suppressor protein and the transcription factor C/EBPalpha. We find that RFC (p140) interacts with RelA both in vitro and in vivo and stimulates RelA transactivation. In contrast, coexpression of fragments of RFC (p140) that mediate the interaction with RelA results in transcriptional inhibition. The significance of this regulation was confirmed by using short interfering RNA oligonucleotides targeted to RFC (p140). Down regulation of endogenous RFC (p140) inhibits expression from a chromosomally integrated reporter plasmid induced by endogenous, TNF-alpha-activated NF-kappaB. Dominant negative fragments of RFC (p140) also cooperate with overexpressed RelA to induce cell death. Interestingly, RFC (p140) also interacts with the tumor suppressor p53. Taken together, these observations suggest that, in addition to its previously described function in DNA replication and repair, RFC (p140) has an important role as a regulator of transcription and NF-kappaB activity.

Amino Acid Motifs↗
PubMed2002

DNA-dependent acetylation of p53 by the transcription coactivator p300.

Reconstitution of the stages in the assembly of the p300.p53 transcription complex has identified a novel type of DNA-dependent regulation of p300-catalyzed acetylation. Phosphorylation at the CHK2 site (Ser(20)) in the N-terminal activation domain of p53 stabilized p300 binding. The phosphopeptide binding activity of p300 was mapped in vitro to two domains: the C-terminal IBiD domain and the N-terminal IHD domain (IBiD homology domain). The IHD or IBiD minidomains can bind to the p53 activation domain in vivo as determined using the mammalian two-hybrid VP16-GAL4 luciferase reporter assay. The IHD and IBiD minidomains of p300 also functioned as dominant negative inhibitors of p53-dependent transcription in vivo. Upon examining the affects of p300 binding on substrate acetylation, we found that the p53 consensus site DNA promotes a striking increase in p53 acetylation in vitro. Co-transfection into cells of the p53 gene and plasmid DNA containing the consensus DNA binding site of p53 activated DNA-dependent acetylation of p53 in vivo. The phosphopeptide binding activity of p300 is critical for DNA-dependent acetylation, as p53 acetylation was inhibited by phospho-Ser(20) peptides. Consensus site DNA-dependent acetylation of p53 stabilized the p300.p53 protein complex, whereas basal acetylation of p53 by p300 in the presence of nonspecific DNA resulted in p300 dissociation. These data identify at least three distinct stages in the assembly of a p300.p53 complex: 1) p300 docking to the activation domain of p53 via the IBiD and/or IHD domains; 2) DNA-dependent acetylation of p53; and 3) stabilization of the p300.p53(AC) complex after acetylation. The ability of DNA to act as an allosteric ligand to activate substrate acetylation identifies a conformational constraint that can be placed on the p300-acetylation reaction that is likely to be an amplification signal and influence protein-protein contacts at a promoter.

Acetylation↗
PubMed2002

A novel form of the RelA nuclear factor kappaB subunit is induced by and forms a complex with the proto-oncogene c-Myc.

Members of both Myc and nuclear factor kappaB (NF-kappaB) families of transcription factors are found overexpressed or inappropriately activated in many forms of human cancer. Furthermore, NF-kappaB can induce c-Myc gene expression, suggesting that the activities of these factors are functionally linked. We have discovered that both c-Myc and v-Myc can induce a previously undescribed, truncated form of the RelA(p65) NF-kappaB subunit, RelA(p37). RelA(p37) encodes the N-terminal DNA binding and dimerization domain of RelA(p65) and would be expected to function as a trans-dominant negative inhibitor of NF-kappaB. Surprisingly, we found that RelA(p37) no longer binds to kappaB elements. This result is explained, however, by the observation that RelA(p37), but not RelA(p65), forms a high-molecular-mass complex with c-Myc. These results demonstrate a previously unknown functional and physical interaction between RelA and c-Myc with many significant implications for our understanding of the role that both proteins play in the molecular events underlying tumourigenesis.

Amino Acid Sequence↗
PubMed2002

The large subunit of replication factor C interacts with the histone deacetylase, HDAC1.

Replication factor C (RFC) is a pentameric complex of five distinct subunits that functions as a clamp loader, facilitating the loading of proliferating cell nuclear antigen (PCNA) onto DNA during replication and repair. More recently the large subunit of RFC, RFC (p140), has been found to interact with the retinoblastoma (Rb) tumor suppressor and the CCAAT/enhancer-binding protein alpha (C/EBP alpha) transcription factor. We now report that RFC (p140) associates with histone deacetylase activity and interacts with histone deacetylase 1 (HDAC1). This complex is functional and when targeted to promoters as a Gal4 fusion, RFC (p140) is a strong, deacetylase-dependent repressor of transcription. Further analysis revealed that RFC (p140) contains two distinct transcriptional repression domains. Moreover, both of these domains interact separately with HDAC1.

Base Sequence↗
PubMed2002

The ETS domain transcription factor Elk-1 contains a novel class of repression domain.

The ETS domain transcription factor Elk-1 serves as an integration point for different mitogen-activated protein (MAP) kinase pathways. Phosphorylation of Elk-1 by MAP kinases triggers its activation. However, while the activation process is well understood, its downregulation-inactivation is less well characterized. The ETS DNA-binding domain plays a role in the downregulation of Elk-dependent promoter activity following mitogenic activation by recruiting the mSin3A-HDAC complex. Here we have identified a novel evolutionarily conserved repression domain in Elk-1, termed the R motif, which serves to reduce the basal transcriptional activity of Elk-1 and dampen its response to mitogenic signals. This domain is highly potent and portable and can repress transcription in trans. The R motif is related to the CRD1 repression domain in p300 and can functionally replace this domain and confer p21(waf1/cip1) inducibility on p300. However, the R motif acts in a context-dependent manner and is not p21(waf1/cip1) responsive in Elk-1. Thus, the Elk-1 R motif and the p300 CRD1 motif represent a new class of repression domains that are regulated in a context-dependent manner.

Amino Acid Motifs↗