Identification of a panel of MYC and Tip60 co-regulated genes functioning primarily in cell cycle and DNA replication.

We recently reported that adenovirus E1A enhances MYC association with the NuA4/Tip60 histone acetyltransferase (HAT) complex to activate a panel of genes enriched for DNA replication and cell cycle. Genes from this panel are highly expressed in examined cancer cell lines when compared to normal fibroblasts. To further understand gene regulation in cancer by MYC and the NuA4 complex, we performed RNA-seq analysis of MD-MB231 breast cancer cells following knockdown of MYC or Tip60 - the HAT enzyme of the NuA4 complex. We identify here a panel of 424 genes, referred to as MYC-Tip60 co-regulated panel (MTcoR), that are dependent on both MYC and Tip60 for expression and likely co-regulated by MYC and the NuA4 complex. The MTcoR panel is most significantly enriched in genes involved in cell cycle and/or DNA replication. In contrast, genes repressed by shMYC but not by shTip60 (224 genes) have a low significance of enrichment in identifiable biological processes other than cell cycle and DNA replication. Genes repressed by shTip60 but not by shMYC (102 genes) have no significant identifiable gene enrichment. We propose that MYC cooperates with the NuA4 complex to activate the MTcoR panel of genes to promote DNA replication and cell cycle.

Synchronous occurrence of primary right ovarian endometrioid adenocarcinoma and primary left ovarian clear cell adenocarcinoma: A case report.

RATIONALE: Ovarian malignancy is associated with one of the highest rates of death among gynecological reproductive system malignancies. While progress has been made in surgical and postoperative adjuvant treatment approaches, the early atypical clinical manifestations, quick progression, and lack of the effective early screening means imply that the prognosis remains poor. Bilateral ovarian cancers are common, but different types of primary bilateral ovarian carcinomas are extremely rare. PATIENT CONCERNS: According to clinical pathologic, immunohistochemistry, and medical imaging data, a 51-year-old Chinese woman with abdominal pain was diagnosed as having right ovarian well-differentiated endometrioid adenocarcinoma with mucinous adenocarcinoma and left ovarian clear cell adenocarcinoma. DIAGNOSES: Immunohistochemistry confirmed the diagnosis of primary bilateral ovarian cancers. INTERVENTIONS: She received multimodal treatment including surgery and chemotherapy. OUTCOMES: The patient's recovery was uneventful, and she responded well to the chemotherapy. LESSONS: We speculate that the different types of primary bilateral ovarian carcinomas presented in this case may be due to different malignant transformations of the endometriotic lesions. Therefore, clinicians should pay special attention to the possible malignant transformation of endometriosis.

[Effects of Thalidomide on the Ratio of Th17 to Treg Cells in Peripheral Blood and Expression of IL-17 and IL-35 in Patients with Multiple Myeloma].

OBJECTIVE: To explore the effects of thalidomide on the ratio of Th17 to Treg cells in peripheral blood and expression of IL-17 and IL-35 in patients with multiple myeloma(MM), so as to provide reference for the clinical treatment of patients with MM. METHODS: A total of 82 MM patients treated with thalidomide from January 2014 to December 2016 were enrolled in MM group, 30 healthy subjects were selected as control (control group). The ratio of T cell subsets and Treg cells accounted for CD4+T cell were detected by flow cytometer. The levels of IL-17 and IL-35 in serum were measured by ELISA, and the differences of various indexes were compared between 2 groups. RESULTS: Compared with the control group, the ratio of Th17 cells in peripheral blood and serum levels of IL-17 of MM patients were significantly increased, the ratio of Treg cells and the level of IL-35 were significantly decreased and the ratio of Th17/Treg cells was significantly increased in the patients with multiple myeloma before treatment (P<0.05). The ratio of Th17 cells in peripheral blood and serum levels of IL-17 in patients with multiple myeloma after treatment with thalidomide were significantly lower than those before treatment, and the ratio of Treg cells and levels of IL-35 were significantly higher than those before treatment, and the ratio of Th17 / Treg cells was higher than that before treatment (P<0.05). The indexes in ineffective treatment were not significantly changed (P> 0.05). CONCLUSION: The unbanlace of Th17/Treg cell ratio and abnormality of IL-17, IL-35 levels play an important role in the progression of multiple myeloma. The anti-MM mechanism of thalidomide may relate with the regulation of Th17 / Treg cell ratio and expression levels of IL-17 and IL-35.

Adenovirus E1A TRRAP-targeting domain-mediated enhancement of MYC association with the NuA4 complex activates a panel of MYC target genes enriched for gene expression and ribosome biogenesis.

Cellular transformation by adenovirus E1A requires targeting TRRAP, a scaffold protein which helps assemble histone acetyltransferase complexes, including the NuA4 complex. We recently reported that E1A and E1A 1-80 (N-terminal 80 aa) promote association of the proto-oncogene product MYC with the NuA4 complex. The E1A N-terminal TRRAP-targeting (ET) domain is required for E1A 1-80 to interact with the NuA4 complex. We demonstrate that an ET-MYC fusion associates with the NuA4 complex more efficiently than does MYC alone. Because MYC regulates genes for multiple cellular pathways, we performed global RNA-sequence analysis of cells expressing MYC or ET-MYC, and identified a panel of genes (262) preferentially activated by ET-MYC and significantly enriched in genes involved in gene expression and ribosome biogenesis, suggesting that E1A enhances MYC association with the NuA4 complex to activate a set of MYC target genes likely involved in cellular proliferation and cellular transformation by E1A and by MYC.

[Clinical Value of Prophylactic Salpingectomy in Hysterectomy due to Uterine Benign Lesions].

OBJECTIVES: To explore the clinical value of resection of bilateral fallopian tubes in patients with benign uterine diseases who received (laparoscopic) hysterectomy or subhysterectomy through the postoperative pathologic analysis of resected fallopian tubes. METHODS: A retrospective analysis was conducted to review the histopathological examination results in 1 272 women who underwent (laparoscopic) total hysterectomy or subtotal hysterectomy and the removal of bilateral fallopian tube simultaneously due to uterine leiomyoma, adenomyosis and other benign lesions from December 2010 to December 2015. RESULTS: Of the 1 272 patients, laparoscopic resection was underwent in 1 005 patients (79.01%) and laparotomy in 267 patients (20.99%). In the attachment area, 334 patients (26.26%) had tenderness signs, and 401 patients (31.53%) had signs of thickening. Total 2 498 fallopian tubes were removed. There were 1 654 tubal with no obvious abnormal appearance (66.21%), 636 tubal with lumen part of the uplift (25.46%), 128 fallopian tube with congestion and swelling (5.12%), 80 fallopian tube atrophy adhesions (3.20%). Pathological. RESULTS: showed 2 386 (95.52%) fallopian tubes with chronic fallopian tube inflammation, 988 (39.55%) of fallopian tube cyst, 80 (3.20%) of normal fallopian tube, 78 (3.12%) of tubal effusion, 48 (1.92% ) of tubal hyperplasia, 4 (0.26%) of tubal benign tumor, 8 (0.32%) of tubal mucosa atypical hyperplasia change and 2(0.08%) of tubal cancer. In the 10 cases of fallopian tube cancer and atypical hyperplasia, 8 had obvious changes of chronic inflammation in the contralateral fallopian tube, including 7 cases of atypical hyperplasia and 1 case of fallopian tube cancer. CONCLUSION: Prophylactic salpingectomy can prevent the occurrence of tubal inflammation and removal cancer incentives.

Enhanced MYC association with the NuA4 histone acetyltransferase complex mediated by the adenovirus E1A N-terminal domain activates a subset of MYC target genes highly expressed in cancer cells.

The proto-oncogene MYC is a transcription factor over-expressed in many cancers and required for cell survival. Its function is regulated by histone acetyltransferase (HAT) complexes, such as the GCN5 complex and the NuA4/Tip60 complex. However, the roles of the HAT complexes during MYC function in cancer have not been well characterized. We recently showed that adenovirus E1A and its N-terminal 80 aa region, E1A 1-80, interact with the NuA4 complex, through the E1A TRRAP-targeting (ET) domain, and enhance MYC association with the NuA4 complex. We show here that the ET domain mainly targets the MYC-NuA4 complex. By global gene expression analysis using E1A 1-80 and deletion mutants, we have identified a panel of genes activated by targeting the MYC-NuA4 complex and notably enriched for genes involved in ribosome biogenesis and gene expression. A second panel of genes is activated by E1A 1-80 targeting of both the MYC-NuA4 complex and p300, and is enriched for genes involved in DNA replication and cell cycle processes. Both panels of genes are highly expressed in cancer cells. Since the ET domain is essential for E1A-mediated cellular transformation, our results suggest that MYC and the NuA4 complex function cooperatively in cell transformation and cancer.

Ad E1A 243R oncoprotein promotes association of proto-oncogene product MYC with the NuA4/Tip60 complex via the E1A N-terminal repression domain.

The adenovirus E1A 243R oncoprotein targets TRRAP, a scaffold protein that assembles histone acetyltransferase (HAT) complexes, such as the NuA4/Tip60 complex which mediates transcriptional activity of the proto-oncogene MYC and helps determine the cancer cell phenotype. How E1A transforms cells through TRRAP remains obscure. We performed proteomic analysis with the N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) and showed that E1A 1-80 interacts with TRRAP, p400, and three other members of the NuA4 complex - DMAP1, RUVBL1 and RUVBL2 - not previously shown to associate with E1A 243R. E1A 1-80 interacts with these NuA4 components and MYC through the E1A TRRAP-targeting domain. E1A 243R association with the NuA4 complex was demonstrated by co-immunoprecipitation and analysis with DMAP1, Tip60, and MYC. Significantly, E1A 243R promotes association of MYC/MAX with the NuA4/Tip60 complex, implicating the importance of the MYC/NuA4 pathway in cellular transformation by both MYC and E1A.

The adenoviral E1A N-terminal domain represses MYC transcription in human cancer cells by targeting both p300 and TRRAP and inhibiting MYC promoter acetylation of H3K18 and H4K16.

Human cancers frequently arise from increased expression of proto-oncogenes, such as MYC and HER2. Understanding the cellular pathways regulating the transcription and expression of proto-oncogenes is important for targeted therapies for cancer treatment. Adenoviral (Ad) E1A 243R (243 aa residues) is a viral oncoprotein that interacts with key regulators of gene transcription and cell proliferation. We have shown previously that the 80 amino acid N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) can target the histone acetyltransferase (HAT) p300 and repress HER2 in the HER2-overexpressing human breast cancer cell line SKBR3. Expression of E1A 1-80 induces death of SKBR3 and other cancer cell lines. In this study, we performed total cell RNA sequence analysis and identified MYC as the regulatory gene for cellular proliferation most strongly repressed by E1A 1-80. By RT-quantitative PCR analysis we show that repression of MYC in SKBR3 cells occurs early after expression of E1A 1-80, suggesting that MYC may be an early responder of E1A 1-80-mediated transcriptional repression. Of interest, while E1A 1-80 repression of MYC occurs in all eight human cancer cell lines examined, repression of HER2 is cell-type dependent. We demonstrate by ChIP analysis that MYC transcriptional repression by E1A 1-80 is associated with inhibition of acetylation of H3K18 and H4K16 on the MYC promoter, as well as inhibition of RNA Pol II binding to the MYC promoter. Deletion mutant analysis of E1A 1-80 suggests that both p300/CBP and TRRAP are involved in E1A 1-80 repression of MYC transcription. Further, E1A 1-80 interaction with p300/CBP and TRRAP is correlated with inhibition of H3K18 and H4K16 acetylation on the MYC promoter, respectively. Our results indicate that E1A 1-80 may target two important pathways for histone modification to repress transcription in human cancer cells.

The Cellular Protein Complex Associated with a Transforming Region of E1A Contains c-MYC.

UNLABELLED: The cell-transforming activity of human adenovirus 5 (hAd5) E1A is mediated by the N-terminal half of E1A, which interacts with three different major cellular protein complexes, p300/CBP, TRRAP/p400, and pRb family members. Among these protein interactions, the interaction of pRb family proteins with conserved region 2 (CR2) of E1A is known to promote cell proliferation by deregulating the activities of E2F family transcription factors. The functional consequences of interaction with the other two protein complexes in regulating the transforming activity of E1A are not well defined. Here, we report that the E1A N-terminal region also interacted with the cellular proto-oncoprotein c-MYC and the homolog of enhancer of yellow 2 (ENY2). Our results suggested that these proteins interacted with an essential E1A transforming domain spanning amino acid residues 26 to 35 which also interacted with TRRAP and p400. Small interfering RNA (siRNA)-mediated depletion of TRRAP reduced c-MYC interaction with E1A, while p400 depletion did not. In contrast, depletion of TRRAP enhanced ENY2 interaction with E1A, suggesting that ENY2 and TRRAP may interact with E1A in a competitive manner. The same E1A region additionally interacted with the constituents of a deubiquitinase complex consisting of USP22, ATXN7, and ATXN7L3 via TRRAP. Acute short hairpin RNA (shRNA)-mediated depletion of c-MYC reduced the E1A transforming activity, while depletion of ENY2 and MAX did not. These results suggested that the association of c-MYC with E1A may, at least partially, play a role in the E1A transformation activity, independently of MAX. IMPORTANCE: The transforming region of adenovirus E1A consists of three short modules which complex with different cellular protein complexes. The mechanism by which one of the transforming modules, CR2, promotes cell proliferation, through inactivating the activities of the pRb family proteins, is better understood than the activities of the other domains. Our analysis of the E1A proteome revealed the presence of the proto-oncoprotein c-MYC and of ENY2. We mapped these interactions to a critical transforming module of E1A that was previously known to interact with the scaffolding molecule TRRAP and the E1A-binding protein p400. We showed that c-MYC interacted with E1A through TRRAP, while ENY2 interacted with it independently. The data reported here indicated that depletion of c-MYC in normal human cells reduced the transforming activity of E1A. Our result raises a novel paradigm in oncogenic transformation by a DNA viral oncogene, the E1A gene, that may exploit the activity of a cellular oncogene, the c-MYC gene, in addition to inactivation of the tumor suppressors, such as pRb.

The adenovirus E1A oncoprotein N-terminal transcriptional repression domain enhances p300 autoacetylation and inhibits histone H3 Lys18 acetylation.

Expression of the adenovirus E1A N-terminal transcription repression domain alone (E1A 1-80) represses transcription from specific promoters such as HER2 [1] and from reconstituted chromatin [2]. Significantly, E1A 1-80 can induce the death of human breast cancer cells over-expressing the HER2 oncogene [1] as well as other cancer cells. Here, we report that E1A 1-80 alone is sufficient to inhibit H3K18 acetylation in vivo and p300-mediated H3K18 acetylation in reconstituted chromatin. Of interest, hypoacetylation of H3K18 has been correlated with the survival of tumor cells and the poor prognosis of many cancers [3, 4]. E1A 1-80 enhances p300 autoacetylation and concurrently inhibits H3K18 acetylation in chromatin in a dose-dependent manner. Pre-acetylation of p300 by incubation with acetyl-CoA alone reduces p300's ability to acetylate H3K18 in chromatin. Additional acetylation of p300 in the presence of E1A 1-80 produces stronger inhibition of H3K18 acetylation. These findings indicate that autoacetylation of p300 greatly reduces its ability to acetylate H3K18. The results reported here combined with our previous findings suggest that E1A can repress transcription by multiple strategies, including altering the chromatin modifying activity of p300 and dissociating TFIID from the TATA box thus disrupting formation of the transcription pre-initiation complex [5, 6].

CtBP2 proteome: Role of CtBP in E2F7-mediated repression and cell proliferation.

C-terminal binding protein (CtBP) family transcriptional corepressors include CtBP1 and CtBP2. While CtBP1 and CtBP2 share significant amino acid sequence homology, CtBP2 possesses a unique N-terminal domain that is modified by acetylation and contributes to exclusive nuclear localization. Although CtBP1 and CtBP2 are functionally redundant for certain activities during vertebrate development, they also perform unique functions. Previous studies have identified several CtBP1-interacting proteins that included other transcriptional corepressors, DNA-binding repressors and histone modifying enzymatic components such as the histone deacetylases and the histone demethylase LSD-1. Here, we carried out an unbiased proteomic analysis of CtBP2-associated proteins and discovered the association of several components of the CtBP1 proteome as well as novel interactions. The CtBP2 proteome contained components of the NuRD complex and the E2F family member E2F7. E2F7 interacted with the hydrophobic cleft region of CtBP1 and CtBP2 through a prototypical CtBP binding motif, PIDLS. E2F7 repressed E2F1 transcription, inhibited cell proliferation in a CtBP-dependent fashion. Our study identified CtBP as a corepressor of E2F7 and as a regulator of DNA damage response.

[The relationship between lactate clearance rate and delayed encephalopathy after acute carbon monoxide poisoning].

OBJECTIVE: To study the relationship between lactate clearance rate (LCR) and prognosis after acute carbon monoxide poisoning in patients with delayed encephalopathy (DEACMP). METHODS: Data from 354 patients with acute severe carbon monoxide poisoning (ASCOP) were retrospectively analyzed. The patients were divided into hyperlactacidemia group (arterial lactic acid > 2 mmol/L, n=263) and low lactic acidosis group (arterial lactate ≤2 mmol/L, n=91) according to the blood lactic acid level at admission. Arterial blood (1 mL) was collected from all patients before and 6, 24, 72 hours after treatment at ambient air, and arterial blood lactic acid was determined, and LCR was calculated. The initial level of blood lactic acid and LCR at 6, 24, 72 hours were compared between two groups. At the same time, the patients with hyperlactacidemia were divided into high LCR group (LCR more than 10%, n=101) and low LCR group (LCR less than or equal to 10%, n=162) according to 6-hour LCR, and the incidence of DEACMP was compared between two groups. The relationship between LCR and the incidence of DEACMP was analyzed with Spearman linear correlation analysis. The risk factors associated with DEACMP were analyzed with logistic regression analysis. RESULTS: The initial level of blood lactic acid (2.73±0.57 mmol/L vs. 1.69±0.20 mmol/L, t=5.327, P=0.001) and LCR at 6, 24, 72 hours [6 hours: (9.0±2.4)% vs. (1.2±0.6)%, t=9.468, P=0.001; 24 hours: (8.6±3.7)% vs. (1.2±0.4)%, t=4.889, P=0.001; 72 hours: (14.0±3.9)% vs. (1.7±1.0)%, t=5.211, P=0.001] in hyperlactacidemia group were significantly higher than those in low lactic acidosis group. The initial level of blood lactic acid in high LCR group was significantly lower than that in low LCR group (2.41±0.23 mmol/L vs. 2.92±0.63 mmol/L, t=2.429, P=0.023), and LCR at 6 hours and 24 hours were significantly higher than those in low LCR group [6 hours: (11.0±1.2)% vs. (8.0±2.1)%, t=4.487, P=0.001; 24 hours: (12.2±3.0)% vs. (6.3±1.8)%, t=6.264, P=0.001]. But there was no difference in 72-hour LCR between high LCR group and low LCR group [(14.1±3.6)% vs. (13.9±4.1)%, t=0.182, P=0.857]. The incidence of DEACMP in high LCR group was significantly lower than that in low LCR group [15.8% (16/101) vs. 61.1% (99/162), χ(2)=51.814, P=0.001]. The blood LCR at early period (6, 24, 72 hours) in ASCOP patients with hyperlactacidemia was negatively correlated with the incidence of DEACMP (r1=-0.493, P1=0.011; r2=-0.408, P2=0.038; r3=-0.428, P3=0.029). Logistic regression analysis showed that LRC at 6 hours and 24 hours [odds ratio (OR) was 2.701, 1.070, P value was 0.035, 0.001], long-time coma (OR=1.537, P=0.068), contact carbon monoxide (CO) long time (OR=2.686, P=0.014), age (OR=1.464, P=0.017), acute carbon monoxide complications (OR=1.363, P=0.072) patients with ASCOP had an increased risk of DEACMP. CONCLUSIONS: LCR is helpful for the assess of DEACMP patients severity, for the treatment guide and for prognosis judgement.

Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection.

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP-E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP-E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells.

Functional similarity between E6 proteins of cutaneous human papillomaviruses and the adenovirus E1A tumor-restraining module.

The adenovirus E1A C-terminal region restrains oncogenic transformation through interaction with three distinct cellular protein complexes that include the DYRK1A/1B/HAN11 complex. The E6 proteins of beta-human papillomaviruses (beta-HPVs) also interact with the DYRK1/HAN11 complex. A variant of HPV5 E6 frequently found in epidermodysplasia verruciformis skin lesions interacted less efficiently with DYRK1A/HAN11. The E6 variant and E7 of HPV5 efficiently coimmortalized primary epithelial cells, suggesting that naturally arising variants may contribute potential oncogenic activities of beta-HPV E6 proteins.

Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

Interaction of ZEB and histone deacetylase with the PLDLS-binding cleft region of monomeric C-terminal binding protein 2.

BACKGROUND: Proteins of the C-terminal binding protein (CtBP) family, CtBP1 and CtBP2 are closely related transcriptional regulators that are coded by two different gene loci in the vertebrate genomes. They perform redundant and unique functions during animal development. CtBP proteins mediate their transcriptional function through interaction with various DNA-binding repressors that contain PLDLS-like motifs and chromatin modifying enzymes, such as class I histone deacetylases (HDAC) that do not contain such motifs. The N-terminal region of CtBP1/2 forms a hydrophobic cleft and is involved in interaction with both PLDLS-containing factors and non-PLDLS factors. CtBP proteins function as dimers to mediate transcriptional repression and dimerization is modulated by specific binding to NAD/NADH. RESULTS: In this study, we have investigated the role of dimerization of CtBP2 in recruitment of PLDLS-motif cofactors and non-PLDLS cofactors. Our results indicate that mutations in CtBP2 that interfere with dimerization abolish CtBP2 interaction with most cellular factors, except the PLDLS-motif factor zinc-finger E-box binding homeobox (ZEB) and the non-PLDLS factor HDAC2. Unlike most PLDLS-containing CtBP-binding proteins, ZEB contains three PLDLS-like motifs and all three contribute to the interaction with the CtBP2 monomer. Despite the ability to interact with ZEB and HDAC, the CtBP2 monomer fails to mediate ZEB-dependent transcriptional repression. The lack of repression activity of the CtBP2 monomer is correlated with the competition between ZEB and HDAC for interaction with the CtBP2 monomer. CONCLUSION: These results suggest a competition between the canonical PLDLS-motif factors such as E1A and non-PLDLS factor HDAC for interaction with CtBP. They also indicate that the affinity for the CtBP monomer may be determined by the number as well as amino acid sequence compositions of the PLDLS-like motifs. Our results are consistent with a model that the CtBP2 dimer may interact with a PLDLS-containing repressor through one monomer and recruit HDAC and other chromatin modifying enzymes through the second monomer in the CtBP2 dimer.

Role of the PLDLS-binding cleft region of CtBP1 in recruitment of core and auxiliary components of the corepressor complex.

C-terminal binding protein (CtBP) family proteins CtBP1 and CtBP2 are highly homologous transcriptional corepressors and are recruited by a large number of transcription factors to mediate sequence-specific transcriptional repression. In addition to DNA-binding repressors, the nuclear protein complex of CtBP1 consists of enzymatic constituents such as histone deacetylases (HDAC1/2), histone methyl transferases (HMTases; G9a and GLP), and the lysine-specific demethylase (LSD1). Additionally, CtBPs also recruit the components of the sumoylation machinery. The CtBPs contain two different unique structural elements, a hydrophobic cleft, with which factors that contain motifs related to the E1A PLDLS motif bind, and a surface groove that binds with factors containing motifs related to the sequence RRTGXPPXL (RRT motif). By structure-based functional dissection of CtBP1, we show that the PLDLS-binding cleft region functions as the primary recruitment center for DNA-binding factors and for the core and auxiliary enzymatic constituents of the CtBP1 corepressor complex. We identify HDAC1/2, CoREST/LSD1, and Ubc9 (E2) as the core constituents of the CtBP1 complex, and these components interact with the PLDLS cleft region through non-PLDLS interactions. Among the CtBP core constituents, HDACs contribute predominantly to the repression activity of CtBP1. The auxiliary components include an HMTase complex (G9a/Wiz/CDYL) and two SUMO E3 ligases, HPC2 and PIAS1. The interaction of auxiliary components with CtBP1 is excluded by PLDLS (E1A)-mediated interactions. Although monomeric CtBP1 is proficient in the recruiting of both core and auxiliary components, NAD(H)-dependent dimerization is required for transcriptional repression. We also provide evidence that CtBP1 functions as a platform for sumoylation of cofactors.

Evidence for involvement of BH3-only proapoptotic members in adenovirus-induced apoptosis.

Mammalian cells infected with human adenoviruses (Ads) undergo an apoptotic response as a result of expression of the viral E1A proteins, and this process is suppressed by the viral E1B-19K protein. The intermediary steps in the Ad-induced apoptosis pathway are not fully resolved. The apical step in the canonical mammalian apoptosis pathway involves functional activation of one or more of the BH3-only BCL-2 family proapoptotic proteins. Previous reports have suggested that Ad-induced apoptosis may be initiated at checkpoints downstream of the BH3-only proteins. Here, we undertook genetic and biochemical studies to determine the roles of BH3-only proteins in Ad-induced apoptosis. We examined the activities of the cellular antiapoptosis protein BCL-xL and its mutants expressed from the E1B region of the Ad5 genome. Our results showed efficient suppression of Ad-induced apoptosis by a BCL-xL mutant (mt1) deficient in interaction with multidomain proapoptotic proteins BAX and BAK but proficient in interaction with BH3-only proteins, suggesting a role for BH3-only proteins in the initiation of Ad-induced apoptosis. Further, the antiapoptotic activity of BCL-xL mt1 in Ad-infected cells was observed in spite of BAK activation as a consequence of MCL-1 degradation. Analysis of the mRNA levels of various BH3-only members by reverse transcription-PCR revealed prominent activation of the Bik gene. Further, the BIK protein was also modified into an apoptotically enhanced phosphorylated form during the viral infection. In addition to BIK, enhanced level of BIM was observed in Ad-infected cells. Between the two major E1A proteins coded by the 12S and 13S mRNAs, the 13S product appeared to contribute to the activation of these BH3-only members and apoptosis during viral infection. Depletion of BIK by the use of small interfering RNA reduced the level of Ad-induced apoptosis. Our results are consistent with a model that activation of the BH3-only members may initiate Ad-induced apoptosis.

Changes in C-terminal binding protein 2 (CtBP2) corepressor complex induced by E1A and modulation of E1A transcriptional activity by CtBP2.

The N-terminal region of adenovirus E1A interacts with histone acetyl transferases (HATs) such as p300, P/CAF, and GCN5. The C-terminal region interacts with the transcriptional corepressors CtBP1 and CtBP2. The functional significance of co-recruitment of HATs and CtBPs by E1A is not well understood. In this study, we have shown that E1A enhanced acetylation of CtBP2 by recruitment of p300 to the CtBP2 complex. Additionally, E1A also displaced the histone methyltransferase G9a and the E-box repressor ZEB from the CtBP2 complex through the C-terminal CtBP-binding domain. A transcriptional activation function encoded by the E1A N-terminal region was efficiently inhibited by CtBP2 but not by a mutant with an N-terminal deletion or by a mutant deficient in interaction with E1A. Two isoforms of CtBP1 (CtBP1-L and CtBP1-S) poorly inhibited transcriptional activity of the E1A N-terminal region. Thus, the N-terminal domain of CtBP2 may contribute a unique transcriptional regulatory activity of CtBP2. Our results provide new insights by which CtBP might modulate the biochemical activities of E1A.

Acetylation by p300 regulates nuclear localization and function of the transcriptional corepressor CtBP2.

CtBP family members, CtBP1 and CtBP2, are unique transcriptional regulators that adapt a metabolic enzyme fold, and their activities are regulated by NAD(H)-binding. CtBP1 is both cytoplasmic and nuclear, and its subcellular localization is regulated by sumoylation, phosphorylation, and binding to a PDZ protein. In contrast, we showed that CtBP2 is exclusively nuclear. CtBP1 and CtBP2 are highly similar, but differ at the N-terminal 20 amino acid region. Substitution of the N-terminal domain of CtBP1 with the corresponding CtBP2 domain confers a dominant nuclear localization pattern to CtBP1. The N-terminal domain of CtBP2 contains three Lys residues. Our results show that these Lys residues are acetylated by the nuclear acetylase p300. Although all three Lys residues of CtBP2 (Lys-6, Lys-8, and Lys-10) appear to be acetylated, acetylation of Lys-10 is critical for nuclear localization. CtBP2 with a single amino acid substitution at Lys-10 (K10R) is predominantly localized in the cytoplasm. The cytoplasmic localization of the K10R mutant is correlated with enhanced nuclear export that is inhibited by leptomycin B. Furthermore, lack of acetylation at Lys-10 renders CtBP2 to be more efficient in repression of the E-cadherin promoter. Our studies have revealed the important roles of acetylation in regulating subcellular localization and transcriptional activity of CtBP2.