Robert Chiu

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

The related proteins p300 and CBP (cAMP-response-element-binding protein (CREB)-binding protein)) are transcriptional co-activators that act with other factors to regulate gene expression and play roles in many cell-differentiation and signal transduction pathways. Both proteins have intrinsic histone-acetyltransferase activity, and may act directly on chromatin, of which histone is a component, to facilitate transcription. They are also involved in growth control pathways, as shown by their interaction with the tumour suppressor p53 (refs 13–15) and the viral oncogenes E1A (refs 1, 2, 16) and SV40 T antigen. Here we report functional differences of p300 and CBP in vivo. We examined their roles during retinoic-acid-induced differentiation, cell-cycle exit and programmed cell death (apoptosis) of embryonal carcinoma F9 cells, using hammerhead ribozymes capable of cleaving either p300 or CBP messenger RNAs. F9 cells expressing a p300-specific ribozyme became resistant to retinoic-acid-induced differentiation, whereas cells expressing a CBP-specific ribozyme were unaffected. Similarly, retinoic-acid-induced transcriptional upregulation of the cell-cycle inhibitor p21Cip1 required normal levels of p300, but not CBP, whereas the reverse was true for p27Kip1. In contrast, both ribozymes blocked retinoic-acid-induced apoptosis, indicating that both co-activators are required for this process. Thus, despite their similarities, p300 and CBP have distinct functions during retinoic-acid-induced differentiation of F9 cells.

The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

Cloning and sequence analysis of candidate human natural killer-enhancing factor genes.

A cytosol factor from human red blood cells enhances natural killer (NK) activity. This factor, termed NK-enhancing factor (NKEF), is a protein of 44000 Mrconsisting of two subunits of equal size linked by disulfide bonds. NKEF is expressed in the NK-sensitive erythroleukemic cell line K562. Using an antibody specific for NKEF as a probe for immunoblot screening, we isolated several clones from a λgt11 cDNA library of K562. Additional subcloning and sequencing revealed that the candidate NKEF cDNAs fell into one of two categories of closely related but non-identical genes, referred to as NKEF A and B. They are 88% identical in amino acid sequence and 71% identical in nucleotide sequence. Southern blot analysis suggests that there are two to three NKEF family members in the genome. Analysis of predicted amino acid sequences indicates that both NKEF A and B are cytosol proteins with several phosphorylation sites each, but that they have no glycosylation sites. They are significantly homologous to several other proteins from a wide variety of organisms ranging from prokaryotes to mammals, especially with regard to several well-conserved motifs within the amino acid sequences. The biological functions of these proteins in other species are mostly unknown, but some of them were reported to be induced by oxidative stress. Therefore, as well as for immunoregulation of NK activity. NKEF may be important for cells in coping with oxidative insults.

Phosphorylation of the adenovirus E1A-associated 300 kDa protein in response to retinoic acid and E1A during the differentiation of F9 cells.

Transcription of the c‐jun gene is up‐regulated by either retinoic acid (RA) or adenovirus E1A during the differentiation of F9 cells. We show here that RA and E1A induce phosphorylation of the E1A‐associated 300 kDa protein (p300) during the differentiation of F9 cells. The region of E1A that is required for interaction with cellular protein p300 overlaps with the region of E1A required for E1A to induce expression of the c‐jun gene. Treatment of F9 cells with RA or infection of the cells by adenovirus led to a decrease in the electrophoretic mobility of p300. Phosphatase treatment of p300 from RA‐treated or adenovirus‐infected F9 cells reversed the changes in migration of p300, indicating that RA‐ and E1A‐mediated changes in the mobility of p300 were due to phosphorylation. We also found factors, designated DRF1 and DRF2, that bound specifically to a sequence element that is necessary and sufficient for RA‐ and E1A‐mediated up‐regulation of the c‐jun gene. The mobility of DRF complexes was changed by E1A or RA and the complexes were supershifted by addition of a polyclonal p300 antiserum. Moreover, overexpression of p300 resulted in an increase in the level of DRF1 complex. p300 fused to the DNA binding domain of the E2 protein of papilloma virus stimulated E2‐dependent reporter activity in response to RA or E1A in F9 cells. Our results suggest that p300 is part of the DRF complexes, that it is differentially phosphorylated in undifferentiated versus differentiated cells and that it is likely involved in regulating transcription of the c‐jun gene during F9 cell differentiation.

JDP2, a Repressor of AP-1, Recruits a Histone Deacetylase 3 Complex To Inhibit the Retinoic Acid-Induced Differentiation of F9 Cells

ABSTRACT Up-regulation of the c-jun gene is a critical event in the retinoic acid (RA)-mediated differentiation of embryonal carcinoma F9 cells. Activating transcription factor 2 (ATF-2) and p300 cooperate in the activation of transcription of the c-jun gene during the differentiation of F9 cells. We show here that the overexpression of Jun dimerization protein 2 (JDP2), a repressor of AP-1, inhibits the transactivation of the c-jun gene by ATF-2 and p300 by recruitment of the histone deacetylase 3 (HDAC3) complex, thereby repressing the RA-induced transcription of the c-jun gene and inhibiting the RA-mediated differentiation of F9 cells. Moreover, chromatin immunoprecipitation assays showed that the JDP2/HDAC3 complex, which binds to the differentiation response element within the c-jun promoter in undifferentiated F9 cells, was replaced by the p300 complex in response to RA, with an accompanying change in the histone acetylation status of the chromatin, the initiation of transcription of the c-jun gene, and the subsequent differentiation of F9 cells. These results suggest that JDP2 may be a key factor that controls the commitment of F9 cells to differentiation and shed new light on the mechanism by which an AP-1 repressor functions.

Anti-neoplastic effects of gallic acid, a major component of Toona sinensis leaf extract, on oral squamous carcinoma cells.

Extract of Toona sinensis (TS) has been reported to have various effects on cultured cell lines, including anti-proliferative activity in cancer cells. We have studied the effects of TS on various human oral squamous carcinoma cell lines (HOSCC), including UM1, UM2, SCC-4, and SCC-9. These cell lines were treated with TS leaf extract and screened for viability, apoptosis, necrosis, and apoptotic gene expression. Normal human oral keratinocytes (NHOK) served as a control for cytotoxic assays. Viability of TS-treated HOSCC was reduced, whereas that of NHOK was not affected. FACScan analysis revealed that the leaf extract induced apoptosis or a combination of apoptosis and necrosis, depending on cell type. Microarray and semi-quantitative RT-PCR analysis for apoptotic-related gene expression revealed that 3,4,5-trihydroxybenzoic acid (gallic acid, one of the major bioactive compounds purified from TS extract) up-regulated pro-apoptotic genes such TNF-α, TP53BP2, and GADD45A, and down-regulated the anti-apoptotic genes Survivin and cIAP1, resulting in cell death. This study suggests that gallic acid, the major bioactive compound present, is responsible for the anti-neoplastic effect of Toona sinensis leaf extract.

Regulation of histone acetylation and nucleosome assembly by transcription factor JDP2.

Jun dimerization protein-2 (JDP2) is a component of the AP-1 transcription factor that represses transactivation mediated by the Jun family of proteins. Here, we examine the functional mechanisms of JDP2 and show that it can inhibit p300-mediated acetylation of core histones in vitro and in vivo. Inhibition of histone acetylation requires the N-terminal 35 residues and the DNA-binding region of JDP2. In addition, we demonstrate that JDP2 has histone-chaperone activity in vitro. These results suggest that the sequence-specific DNA-binding protein JDP2 may control transcription via direct regulation of the modification of histones and the assembly of chromatin.

Transcriptional regulation of the c-jun gene by retinoic acid and E1A during differentiation of F9 cells.

Differentiation of mouse F9 embryonal carcinoma (EC) cells can be induced by exposure to retinoic acid (RA) or by expression of adenovirus E1A. The transcription of the c‐jun gene is stimulated by either RA or E1A. We report here that both RA and E1A strongly induce the expression of chloramphenicol acetyltransferase (CAT) from c‐jun promoter/CAT reporter construct (c‐jun/CAT), which is stably integrated into F9 cells, in a manner that is independent of both copy number and integration locus. The induction of c‐jun/CAT expression is observed in undifferentiated F9 cells, but not in differentiated F9 cells, adenovirus‐infected F9 cells or HeLa cells. Deletion analysis of the promoter region of the c‐jun gene indicates that the sequence elements required for the RA‐ and E1A‐mediated induction are identical and they have been defined as a region of 145 bp between −190 and −46 of the 5′ flanking region of c‐jun. This RA and E1A response element (RERE) contains five variants of the motif CGCGGTGACGNT. The upstream two motifs are adjacent and extend in opposite directions, creating an imperfect palindrome. The downstream four motifs are located at 35 or 36 bp intervals in the same orientation. Substitution and insertion analysis indicates that these motifs and their regular intervals are important for the activity of the RERE.

Recruitment of the Retinoblastoma Protein to c-Jun Enhances Transcription Activity Mediated through the AP-1 Binding Site

The retinoblastoma susceptibility gene product (RB) is a transcriptional modulator. One of the targets for this modulator effect is the AP-1 binding site within the c-junand collagenase promoters. The physical interactions between RB and c-Jun were demonstrated by co-immunoprecipitation of these two proteins using anti-c-Jun or anti-RB antisera, glutathioneS-transferase affinity matrix binding assays in vitro, and electrophoretic mobility shift assays. The C-terminal site of the leucine zipper of c-Jun mediated the interaction with RB. Although the B-pocket domain of RB alone bound to c-Jun, a second c-Jun binding site in the RB was also suggested. Mammalian two-hybrid-based assay provided corroborative evidence that transactivation of gene expression by RB required the C-terminal region of c-Jun. We conclude that RB enhances transcription activity mediated through the AP-1 binding site. Adenovirus E1A or human papillomavirus E7 inhibits RB-mediated transcription activity. These data reveal that the interactions between these two distinct classes of oncoproteins RB and c-Jun may be involved in controlling cell growth and differentiation mediated by transcriptional regulation.

In vitro activation of the HIV-1 enhancer in extracts from cells treated with a phorbol ester tumor promoter.

The transition from persistent to lytic infection by the human immunodeficiency virus, HIV, is marked by a burst of viral replication and gene expression that occurs when infected cells are stimulated by physiological inducers or tumor promoters like 12‐O‐tetradecanoyl phorbol acetate (TPA). We report here that the HIV enhancer is activated specifically by TPA in several non‐lymphoid cell types, and that this transcriptional regulation can be reproduced in a cell‐free system. In vitro transcription experiments revealed a 6‐fold activation of the HIV promoter in nuclear extracts prepared from TPA‐induced HeLa tk‐ cells, whereas a control (human alpha‐globin) promoter was transcribed with equal efficiency in either induced or uninduced cell extracts. A corresponding increase in the activity of a cellular DNA‐binding protein that interacts with the HIV enhancer was detected in TPA‐treated cells with DNase I footprint experiments. This increase occurred in the absence of de novo protein synthesis, suggesting a post‐transcriptional activation mechanism. Analysis of HIV deletion mutants suggests that the enhancer is the target for the TPA effect both in vitro and in vivo. The cell‐free system described here should facilitate studies on the mechanism of phorbol ester induction of gene‐specific transcription factors.