Biomedical subjects

E Seto

Publications and source records attributed to E Seto.

At least 19 recordsLinked to original sources

PubMed2007

Differential regulation of human YY1 and caspase 7 promoters by prohibitin through E2F1 and p53 binding sites.

Prohibitin is a 30 kDa growth suppressive protein that has pleiotropic functions in the cell. Although prohibitin has been demonstrated to have potent transcriptional regulatory functions, it has also been proposed to facilitate protein folding in the mitochondria and promote cell migration in association with Raf-1. Our previous studies have shown that prohibitin physically interacts with the marked-box domain of E2F family members and represses their transcriptional activity; in contrast, prohibitin could bind to and enhance the transcriptional activity of p53. Here, we show that promoters of human YY1 (Yin and Yang 1) as well as caspase 7 genes are modulated by prohibitin. YY1 promoter activity was reduced upon overexpression of prohibitin, while it was enhanced when prohibitin was depleted by small interfering RNA techniques. The repressive effects of prohibitin on the YY1 promoter were mediated through E2F binding sites, as seen by mutational analysis and chromatin immunoprecipitation assays. Further, depletion of E2F1 prevented prohibitin from repressing the YY1 promoter. In contrast with YY1, prohibitin overexpression led to enhanced levels of caspase 7, whereas depletion of prohibitin reduced it. Interestingly, the caspase 7 promoter was found to have p53-binding sites and prohibitin activated this promoter through p53. These studies show that prohibitin can have diverse effects on the expression of different genes and the activity of various cellular promoters is affected by prohibitin. Further, it appears very likely that prohibitin carries out many of its cellular functions by affecting the transcription of different genes.

Caspase 7↗

PubMed2001

Yin-Yang 1 activates interleukin-4 gene expression in T cells.

Interleukin-4 (IL-4) is a multifunctional cytokine that plays an important role in immune and inflammatory responses. Expression of the IL-4 gene is tightly controlled at the level of gene transcription by both positive and negative regulatory elements in the IL-4 promoter. Several constitutive nuclear factors have been identified that can interact with IL-4 promoter elements in DNA binding assays. Here we report that the zinc-finger protein YY-1 (Yin-Yang 1) can bind to multiple elements within the human IL-4 promoter. Cotransfection of Jurkat T cells with different IL-4 promoter/reporter constructs together with expression vectors encoding antisense, wild-type, or zinc finger-deleted mutant YY-1 suggested that YY-1 enhanced IL-4 promoter activity in a DNA-binding domain-dependent manner. Site-directed mutagenesis revealed that a proximal YY-1-binding site, termed Y0 ((-59)TCATTTT(-53)), was essential for YY-1-driven IL-4 promoter activity. In addition, cotransfected YY-1 enhanced both IL-4 promoter activity and endogenous IL-4 gene expression in nontransformed peripheral blood T cells. Thus, YY-1 positively regulates IL-4 gene expression in lymphocytes.

Calcimycin↗

PubMed2001

The FK506-binding protein 25 functionally associates with histone deacetylases and with transcription factor YY1.

FK506-binding proteins (FKBPs) are cellular receptors for immunosuppressants that belong to a subgroup of proteins, known as immunophilins, with peptidylprolyl cis-trans isomerase (PPIase) activity. Sequence comparison suggested that the HD2-type histone deacetylases and the FKBP-type PPIases may have evolved from a common ancestor enzyme. Here we show that FKBP25 physically associates with the histone deacetylases HDAC1 and HDAC2 and with the HDAC-binding transcriptional regulator YY1. An FKBP25 immunoprecipitated complex contains deacetylase activity, and this activity is associated with the N-terminus of FKBP25, distinct from the FK506/rapamycin-binding domain. Furthermore, FKBP25 can alter the DNA-binding activity of YY1. Together, our data firmly establish a relationship between histone deacetylases and the FKBP enzymes and provide a novel and critical function for the FKBPs.

Base Sequence↗

PubMed2001

Histone deacetylases in replicative senescence: evidence for a senescence-specific form of HDAC-2.

To analyze mechanisms of senescence-associated gene expression, we have investigated histone deacetylases (HDACs) in human fibroblasts undergoing replicative senescence. We found that the overall acetylation pattern of histones does not vary detectably with replicative senescence. By Northern blot and Western blot, we found a significant decrease in the abundance of HDAC-1 in senescent cells. Biochemical analysis of deacetylase activities in extracts from old and young cells revealed a striking difference. While by anion exchange chromatography we found a single peak of activity in extracts from young cells, which coincided with the elution of both HDAC-1 and HDAC-2, in senescent cells a second peak of activity was found. This second peak of activity is associated with HDAC-2 but does not contain HDAC-1. These results suggest that HDAC-2 is present in at least two distinct forms, one of which is specific for senescent cells. Further biochemical characterization of the enzyme activity revealed that addition of nicotinamide adenine dinucleotide (NAD) did not detectably influence the activity of any fraction, suggesting that NAD is not an essential co-factor for the analyzed HDACs from diploid human fibroblasts.

Blotting, Western↗

PubMed2001

Quantifying head motion associated with motor tasks used in fMRI.

In functional magnetic resonance imaging (fMRI) studies, long experiment times and small intensity changes associated with brain activation frequently lead to image artifacts due to head motion. Methods to minimize and correct for head motion by restraint, fast imaging, and retrospective image registration are typically combined but do not completely solve the problem, particularly for specific patient populations. As an initial step toward optimizing future designs of head restraints and improving motion correction techniques, the head motion characteristics of groups of stroke subjects, age-matched controls, and young adults were investigated with the aid of an MR simulator and a highly accurate position tracking system. Position measurements were recorded during motor tasks involving either the hand or the foot. Head motion was strongly dependent on the subject group and less upon the task conditions based on ANOVA calculations (P < 0.05). The stroke subjects exhibited approximately twice the head motion compared to that of age-matched controls, and the latter's head motion was about twice that of young adults. Moreover, the range of head motion in stroke subjects over all tasks was approximately 2 +/- 1 mm, with the motion occurring predominantly as translation in the superior-inferior direction and pitch rotation (nodding). These results lead to several recommendations on the design of fMRI motor experiments and suggest that improved motion correction strategies are required to examine such patient populations comprehensively.

Adult↗

PubMed2001

Rationale for combination ketoconazole/ vitamin D treatment of prostate cancer.

The high rate of progression of prostate cancer after androgen deprivation therapy mandates that new strategies be developed. Adjuvant therapy combined with androgen deprivation may slow or prevent progression. Ketoconazole plus calcitriol therapy is an example of 1 such a combination with a mechanistic basis for synergistic activity. Ketoconazole is commonly used as a second-line androgen deprivation therapy. This imidazole derivative is an inhibitor of P-450 enzymes, including those involved in steroidogenesis. Other P-450 enzymes that are inhibited by ketoconazole include 1alpha-hydroxylase and 24-hydroxylase, which metabolize vitamin D. Growth inhibition of prostate cancer cells by vitamin D depends on levels of the active metabolite, 1,25-dihydroxyvitamin D(3) (calcitriol). The enzyme 24-hydroxylase converts calcitriol to less active products. The inhibition of 24-hydroxylase by ketoconazole maintains the magnitude and duration of response to calcitriol. Combined ketoconazole/calcitriol therapy might therefore potentiate the antitumor activity of calcitriol. Because androgen-independent prostate cancer cells often remain responsive to growth inhibition by calcitriol, it is also possible that calcitriol would slow or prevent development of androgen-independent cancer growth. Another consideration is that ketoconazole blocks 1alpha-hydroxylase activity, which is the key enzyme that creates calcitriol in the body. Therefore, patients receiving ketoconazole therapy are likely to be deficient in vitamin D. The detrimental consequences of vitamin D deficiency in these patients would also be alleviated by the addition of calcitriol to the therapeutic regimen.

Androgen Antagonists↗

PubMed2001

Regulation of transcription factor YY1 by acetylation and deacetylation.

YY1 is a sequence-specific DNA-binding transcription factor that has many important biological roles. It activates or represses many genes during cell growth and differentiation and is also required for the normal development of mammalian embryos. Previous studies have established that YY1 interacts with histone acetyltransferases p300 and CREB-binding protein (CBP) and histone deacetylase 1 (HDAC1), HDAC2, and HDAC3. Here, we present evidence that the activity of YY1 is regulated through acetylation by p300 and PCAF and through deacetylation by HDACs. YY1 was acetylated in two regions: both p300 and PCAF acetylated the central glycine-lysine-rich domain of residues 170 to 200, and PCAF also acetylated YY1 at the C-terminal DNA-binding zinc finger domain. Acetylation of the central region was required for the full transcriptional repressor activity of YY1 and targeted YY1 for active deacetylation by HDACs. However, the C-terminal region of YY1 could not be deacetylated. Rather, the acetylated C-terminal region interacted with HDACs, which resulted in stable HDAC activity associated with the YY1 protein. Finally, acetylation of the C-terminal zinc finger domain decreased the DNA-binding activity of YY1. Our findings suggest that in the natural context, YY1 activity is regulated through intricate mechanisms involving negative feedback loops, histone deacetylation, and recognition of the cognate DNA sequence affected by acetylation and deacetylation of the YY1 protein.

Acetylation↗

PubMed2001

Histone deacetylase activity represses gamma interferon-inducible HLA-DR gene expression following the establishment of a DNase I-hypersensitive chromatin conformation.

Expression of the retinoblastoma tumor suppressor protein (Rb) is required for gamma interferon (IFN-gamma)-inducible major histocompatibility complex class II gene expression and transcriptionally productive HLA-DRA promoter occupancy in several human tumor cell lines. Treatment of these Rb-defective tumor cell lines with histone deacetylase (HDAC) inhibitors rescued IFN-gamma-inducible HLA-DRA and -DRB mRNA and cell surface protein expression, demonstrating repression of these genes by endogenous cellular HDAC activity. Additionally, Rb-defective, transcriptionally incompetent tumor cells retained the HLA-DRA promoter DNase I-hypersensitive site. Thus, HDAC-mediated repression of the HLA-DRA promoter occurs following the establishment of an apparent nucleosome-free promoter region and before transcriptionally productive occupancy of the promoter by the required transactivators. Repression of HLA-DRA promoter activation by HDAC activity likely involves a YY1 binding element located in the first exon of the HLA-DRA gene. Chromatin immunoprecipitation experiments localized YY1 to the HLA-DRA gene in Rb-defective tumor cells. Additionally, mutation of the YY1 binding site prevented repression of the promoter by HDAC1 and partially prevented activation of the promoter by trichostatin A. Mutation of the octamer element also significantly reduced the ability of HDAC1 to confer repression of inducible HLA-DRA promoter activation. Treatment of Rb-defective tumor cells with HDAC inhibitors greatly reduced the DNA binding activity of Oct-1, a repressor of inducible HLA-DRA promoter activation. These findings represent the first evidence that HDAC activity can repress IFN-gamma-inducible HLA class II gene expression and also demonstrate that HDAC activity can contribute to promoter repression following the establishment of a DNase I-hypersensitive chromatin conformation.

Butyrates↗

PubMed2001

The growth suppressor PML represses transcription by functionally and physically interacting with histone deacetylases.

The growth suppressor promyelocytic leukemia protein (PML) is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL). PML plays a key role in multiple pathways of apoptosis and regulates cell cycle progression. The present study demonstrates that PML represses transcription by functionally and physically interacting with histone deacetylase (HDAC). Transcriptional repression mediated by PML can be inhibited by trichostatin A, a specific inhibitor of HDAC. PML coimmunoprecipitates a significant level of HDAC activity in several cell lines. PML is associated with HDAC in vivo and directly interacts with HDAC in vitro. The fusion protein PML-RARalpha encoded by the t(15;17) breakpoint interacts with HDAC poorly. PML interacts with all three isoforms of HDAC through specific domains, and its expression deacetylates histone H3 in vivo. Together, the results of our study show that PML modulates histone deacetylation and that loss of this function in APL alters chromatin remodeling and gene expression. This event may contribute to the development of leukemia.

Animals↗

PubMed2001

RBP1 recruits the mSIN3-histone deacetylase complex to the pocket of retinoblastoma tumor suppressor family proteins found in limited discrete regions of the nucleus at growth arrest.

Retinoblastoma (RB) tumor suppressor family pocket proteins induce cell cycle arrest by repressing transcription of E2F-regulated genes through both histone deacetylase (HDAC)-dependent and -independent mechanisms. In this study we have identified a stable complex that accounts for the recruitment of both repression activities to the pocket. One component of this complex is RBP1, a known pocket-binding protein that exhibits both HDAC-dependent and -independent repression functions. RB family proteins were shown to associate via the pocket with previously identified mSIN3-SAP30-HDAC complexes containing exclusively class I HDACs. Such enzymes do not interact directly with RB family proteins but rather utilize RBP1 to target the pocket. This mechanism was shown to account for the majority of RB-associated HDAC activity. We also show that in quiescent normal human cells this entire RBP1-mSIN3-SAP30-HDAC complex colocalizes with both RB family members and E2F4 in a limited number of discrete regions of the nucleus that in other studies have been shown to represent the initial origins of DNA replication following growth stimulation. These results suggest that RB family members, at least in part, drive exit from the cell cycle by recruitment of this HDAC complex via RBP1 to repress transcription from E2F-dependent promoters and possibly to alter chromatin structure at DNA origins.

Binding Sites↗

PubMed2001

The orphan nuclear receptor TR2 interacts directly with both class I and class II histone deacetylases.

A combination of in vivo and in vitro assays was employed to describe the ligand-independent interaction of the orphan nuclear receptor TR2 and histone deacetylase proteins. The repressive effect of TR2 on transcription of a luciferase reporter driven by a promoter containing a direct repeat-5 (DR5) derived from the human RARbeta gene was suppressed by the addition of the histone deacetylase inhibitor trichostatin A. Immunoprecipitation with FLAG-epitope (MDYKDDDDK)-tagged histone deacetylase proteins was used to demonstrate that TR2 and histone deacetylases 3 or 4 are present in the same immunoprecipitated complex. Deacetylase activity was demonstrated for these coimmunoprecipitates, further confirming the in vivo interaction of TR2 and histone deacetylases. Immunoprecipitation with anti-TR2 antibody was used to demonstrate interaction of TR2 with endogenously expressed histone deacetylases 3 and 4 in COS-1 cells. Dissection of TR2 domains showed that the DNA binding domain of the receptor was responsible for interaction with both histone deacetylases 3 and 4 in glutathione-S-transferase pull-down assays, while the ligand binding domain did not interact. The pull-down data were confirmed with far Western blots that also showed a direct interaction between labeled histone deacetylase proteins and TR2. It is suggested that repression mediated by unliganded TR2 is mediated, in part, by a direct interaction of this receptor with histone deacetylase proteins.

Animals↗

PubMed2000

Receptor-interacting protein 140 directly recruits histone deacetylases for gene silencing.

Receptor-interacting protein 140 (RIP140) encodes a histone deacetylase (HDAC) inhibitor-sensitive repressive activity. Direct interaction of RIP140 with HDAC1 and HDAC3 occurs in vitro and in vivo as demonstrated in co-immunoprecipitation and glutathione S-transferase pull-down experiments. The HDAC-interacting domain of RIP140 is mapped to its N-terminal domain, between amino acids 78 and 303 based upon glutathione S-transferase pull-down experiments. In chromatin immunoprecipitation assays, it is demonstrated that histone deacetylation occurs at the chromatin region of the Gal4 binding sites as a result of Gal4 DNA binding domain-tethered RIP expression. The immunocomplexes of RIP140 from cells transfected with RIP140 and HDAC are able to deacetylate histone proteins in vitro. This study presents the first evidence for RIP140 as a negative coregulator for nuclear receptor actions by directly recruiting histone deacetylases and categorizes RIP140 as a novel negative coregulator that is able to directly interact with HDACs.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Inhibition of mitogenesis in Balb/c-3T3 cells by Trichostatin A. Multiple alterations in the induction and activation of cyclin-cyclin-dependent kinase complexes.

Trichostatin A (TSA), a global repressor of histone deacetylase activity, inhibits the proliferation of a number of cell types. However, the identification of the mechanisms underlying TSA-mediated growth arrests has remained elusive. In order to resolve in more detail the cellular process modulated during the growth inhibition induced by TSA, we studied the effect of the drug on G(0)/G(1) traverse in mitogen-stimulated quiescent Balb/c-3T3 cells. Cyclin D1 and retinoblastoma proteins were induced following the mitogenic stimulation of both control and TSA-treated cells, and cyclin D1 formed complexes with CDK4 under both conditions. However, cyclin D1-associated kinase was not increased in growth-arrested cells. The lack of cyclin D-associated kinase was paralleled by an accumulation of RB in a hypophosphorylated form, as would be expected. In contrast, p130 became partially phosphorylated, accompanied by a marked increase in p130-dependent E2F DNA binding activity and a partial release of free E2F-4. Despite the presence of E2F complexes not bound to pocket proteins, late G(1) E2F-dependent gene expression was not observed. The lack of cyclin D1-associated kinase in TSA-treated cultures was potentially due to high levels of the cyclin-dependent inhibitor p27(kip1). However, the modulation of p27(kip1) levels by the deacetylase inhibitor cannot be responsible for the induction of the cell cycle arrest, since the growth of murine embryo fibroblasts deficient in both p27(kip1) and p21(cip1) was also inhibited by TSA. These data support a model in which TSA inhibits very early cell cycle traverse, which, in turn, leads to a decrease in cyclin D1-associated kinase activation and a repression of late cell cycle-dependent events. Alterations in early G(0)/G(1) gene expression accompany the TSA-mediated growth arrest.

3T3 Cells↗

PubMed2000

Combinatorial roles of the nuclear receptor corepressor in transcription and development.

Transcriptional repression plays crucial roles in diverse aspects of metazoan development, implying critical regulatory roles for corepressors such as N-CoR and SMRT. Altered patterns of transcription in tissues and cells derived from N-CoR gene-deleted mice and the resulting block at specific points in CNS, erythrocyte, and thymocyte development indicated that N-CoR was a required component of short-term active repression by nuclear receptors and MAD and of a subset of long-term repression events mediated by REST/NRSF. Unexpectedly, N-CoR and a specific deacetylase were also required for transcriptional activation of one class of retinoic acid response element. Together, these findings suggest that specific combinations of corepressors and histone deacetylases mediate the gene-specific actions of DNA-bound repressors in development of multiple organ systems.

Animals↗

PubMed2000

Histone deacetylases specifically down-regulate p53-dependent gene activation.

p53, the most commonly mutated gene in cancer cells, directs cell cycle arrest or induces programmed cell death (apoptosis) in response to stress. It has been demonstrated that p53 activity is up-regulated in part by posttranslational acetylation. In agreement with these observations, here we show that mammalian histone deacetylase (HDAC)-1, -2, and -3 are all capable of down-regulating p53 function. Down-regulation of p53 activity by HDACs is HDAC dosage-dependent, requires the deacetylase activity of HDACs, and depends on the region of p53 that is acetylated by p300/CREB-binding protein (CBP). These results suggest that interactions of p53 and HDACs likely result in p53 deacetylation, thereby reducing its transcriptional activity. In support of this idea, GST pull-down and immunoprecipitation assays show that p53 interacts with HDAC1 both in vitro and in vivo. Furthermore, a pre-acetylated p53 peptide was significantly deacetylated by immunoprecipitated wild type HDAC1 but not deacetylase mutant. Also, co-expression of HDAC1 greatly reduced the in vivo acetylation level of p53. Finally, we report that the activation potential of p53 on the BAX promoter, a natural p53-responsive system, is reduced in the presence of HDACs. Taken together, our findings indicate that deacetylation of p53 by histone deacetylases is likely to be part of the mechanisms that control the physiological activity of p53.

Acetylation↗

PubMed2000

The histone deacetylase-3 complex contains nuclear receptor corepressors.

Acetylation and deacetylation of nucleosomal histones have profound effects on gene transcription in all eukaryotes. In humans, three highly homologous class I and four class II histone deacetylase (HDAC) enzymes have been identified to date. The class I deacetylases HDAC1 and HDAC2 are components of multisubunit complexes, one of which could associate with the nuclear hormone receptor corepressor, N-CoR. N-CoR also interacts with class II deacetylases HDAC4, HDAC5, and HDAC7. In comparison with HDAC1 and HDAC2, HDAC3 remains relatively uncharacterized, and very few proteins have been shown to interact with HDAC3. Using an affinity purification approach, we isolated an enzymatically active HDAC3 complex that contained members of the nuclear receptor corepressor family. Deletion analysis of N-CoR revealed that HDAC3 binds multiple N-CoR regions in vitro and that all of these regions are required for maximal binding in vivo. The N-CoR domains that interact with HDAC3 are distinct from those that bind other HDACs. Transient overexpression of HDAC3 and microinjection of Abs against HDAC3 showed that a component of transcriptional repression mediated by N-CoR depends on HDAC3. Interestingly, data suggest that interaction with a region of N-CoR augments the deacetylase activity of HDAC3. These results provide a possible molecular mechanism for HDAC3 regulation and argue that N-CoR is a platform in which distinct domains can interact with most of the known HDACs.

Acetylation↗

PubMed2000

A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia.

AML1 is one of the most frequently translocated genes in human leukemia. Here we demonstrate that acute myeloid leukemia-1 (AML-1) (Runx-1) represses transcription from a native promoter, p21(Waf1/Cip1). Unexpectedly, this repression did not require interactions with the Groucho co-repressor. To define the mechanism of repression, we asked whether other co-repressors could interact with AML-1. We demonstrate that AML-1 interacts with the mSin3 co-repressors. Moreover, endogenous AML-1 associated with endogenous mSin3A in mammalian cells. A deletion mutant of AML-1 that did not interact with mSin3A failed to repress transcription. The AML-1/mSin3 association suggests a mechanism of repression for the chromosomal translocation fusion proteins that disrupt AML-1.

Acute Disease↗

PubMed2000

Histone deacetylases, transcriptional control, and cancer.

A key event in the regulation of eukaryotic gene expression is the posttranslational modification of nucleosomal histones, which converts regions of chromosomes into transcriptionally active or inactive chromatin. The most well studied posttranslational modification of histones is the acetylation of epsilon-amino groups on conserved lysine residues in the histones' amino-terminal tail domains. Significant advances have been made in the past few years toward the identification of histone acetyltransferases and histone deacetylases. Currently, there are over a dozen cloned histone acetyltransferases and at least eight cloned human histone deacetylases. Interestingly, many histone deacetylases can function as transcriptional corepressors and, often, they are present in multi-subunit complexes. More intriguing, at least some histone deacetylases are associated with chromatin-remodeling machines. In addition, several studies have pointed to the possible involvement of histone deacetylases in human cancer. The availability of the cloned histone deacetylase genes has provided swift progress in the understanding of the mechanisms of deacetylases, their role in transcription, and their possible role in health and disease.

Animals↗