Food Factors in Health Promotion and Disease Prevention - American

Chapter 15

Role of Histone Acetyltransferase and Deacetylase in the Retinoic Acid-Induced Differentiation of F9 Cells 1,2

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Chunyuan Jin , Hongie Li , Masami Horikoshi , Kailai Sun , and Kazunari K . Yokoyama Downloaded by GEORGETOWN UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: June 19, 2003 | doi: 10.1021/bk-2003-0851.ch015

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Gene Engineering Division, RIKEN (The Institute of Physical and Chemical Research), Bioresource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan Department of Medical Genetics, China Medical University, Shenyang 110001, Peoples Republic of China The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku 113-0032, Japan 2

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Up-regulation of the c-jun gene is a critical event in the retinoic acid (RA)-mediated differentiation of embryonal carcinoma F9 cells. Activation transcription factor-2 (ATF-2), adenovirus E 1 A associated p300 protein (p300) and Jun dimerization protein (JDP2) cooperate in the regulation of transcription of the c-jun gene. ATF-2 was found to be a novel histone acetyltransferase (HAT) and it acetylated specifically both histone H2B and histone H4. Motif A , of the H A T domain, was responsible for stimulation of the transcription of c-jun gene, in conjunction with H A T of p300. B y contrast, JDP2 served as a repressor of AP-1 and inhibited the transactivation of the c-jun gene by p300/ATF-2, by recruitment of histone deacetylase complex (HDAC3), thereby repressing the RA-induced transcription of the c-jun gene and then inhibiting the RA-mediated differentiation of F9 cells. These results suggest that HDAC3/JDP2 and p300/ATF-2 complex play a critical role in controlling the differentiation of F9 cells, in response to R A .

© 2003 American Chemical Society Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Downloaded by GEORGETOWN UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: June 19, 2003 | doi: 10.1021/bk-2003-0851.ch015

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Murine F9 cells, a line of embryonal carcinoma (EC) cells derived from a teratocarcinoma (1), can be induced to differentiate to distinct types of endoderm-like cells by exposure to retinoic acid (RA) (2). The RA-mediated differentiation is associated with dramatic changes in gene expression, including a rapid increase in the rate of transcription of the c-jun gene (3,4). Moreover, constitutive expression of the c-jun gene results in differentiation of various lines of E C cells, such as F9 and P19 cells (4-6), suggesting that the induction of the transcription of c-jun by R A plays an important role in such differentiation. It has been demonstrated that both R A and adenovirus E 1 A can stimulate the activity of the c-jun promoter and increase the level of endogenous c-jun m R N A (4,7-11). The c-jun gene encodes a major component of the AP-1 transcription factor that is expressed in many organs during murine development as well as in the adult mouse (12). Murine embryos with null mutations in the c-jun gene die at midgestation, a phenomenon that suggests an essential function for the product of this gene in murine development (13,14). A sequence element in the c-jun promoter, designated D R E (differentiation response element), is necessary and sufficient for the RA-induced expression of the c-jun gene (11). This element binds the differentiation regulatory factor (DRF) complex, of which one component is the adenovirus ElA-associated protein p300 (16), which has histone acetyltransferase activity (HAT) (17-19), and another component is activating transcription factor-2 (ATF-2), which is a DNA-binding subunit of the D R F complex (20,21). Activation transcription factor-2 (ATF-2; also known as [cAMP-response element (CRE) BP-1] is a member of the ATF/CRE-binding (CREB) family of transcription factors and has a basic region leucine zipper motif (bZip domain) (22,23). This motif is necessary for the formation of heterodimers with other members of the A T F family, as well as with members of the Jun/Fos family of factors. The motif is also required for the binding of homodimers of A T F - 2 to D R E . ATF-2 and p300 interact with each other in the D R F complex and cooperate in the control of transcription in response to differentiation-inducing signals, such as R A or E l A .

ATF-2 has an intrinsic H A T activity Various coactivators of transcription, such as GCN/5 (24), p300/CBP (18,25), P C A F (19), A C T R (25), SRC-1 (26), TAFII250 (27) and p/CIP (28), have recently been identified as histone acetyltransferases (HATs), an observation that suggests that the ability to catalyze acetylation might be linked to the ability to serve as a transcriptional activator and that such an activity might be involved in the regulation of expression of many genes, as well as in the remodeling of nucleosomes (29). We have previously demonstrated that ATF-2 interacts with the region that includes the active site of the H A T domain of the coactivator p300 to control transcription of the c-jun gene upon treatment of F9

Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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cells with retinoic acid (RA) or adenovirus E 1 A (30). Therefore, we postulated that A T F - 2 might affect the intrinsic H A T activity of p300 (18,25). To examine this possibility, the H A T activity of p300 in vitro in the presence and in the absence of ATF-2 was monitored. Both a recombinant glutathione Stransferase-p300 (GST-p300) fusion protein that contained a H A T domain [amino acids (a.a.) 1195-1810] and GST-ATF-2 were generated m Escherichia coli and their relative H A T activities determined. As reported previously (17,18), the H A T domain of p300 was responsible for the H A T activity of p300 in vitro, while GST itself had no H A T activity. The H A T activity of p300 was enhanced after incubation with ATF-2. To our surprise, significant H A T activity was still observed when ATF-2 was incubated with p300 that lacked the H A T domain and that had no detectable H A T activity per se, suggesting that the residual H A T activity might have been due to ATF-2. Possible enhancement of H A T activity by ATF-2 that might be detectable in vivo was then examined. The H A T activity of immunoprecipitates obtained with p300-specific or ATF-2-specific antibodies was assayed. Incubation of proteins in extracts of Hela cells with antibodies against p300 or against ATF-2 immunoprecipitated the respective factors that specifically acetylated histones was carried out. The immunoprecipitates obtained with antibodies against c-Jun or p21 had no H A T activity in vitro. Therefore, it appeared that ATF-2 might have H A T activity. Assuming that nucleosomes would be the targets for H A T activity in vivo, we examined the purified ATF-2 protein for its ability to acetylate free histones and histones in mononucleosomes. A T F - 2 preferentially acetylated human histones H2B and H4, both as free histones and as histones bound within mononucleosomes (Fig. 1A) but it did not acetylate histones H3 and H 2 A (Figs. l A a n d IB). This finding was further confirmed in experiments that involved in vitro phosphorylation by J N K and "in gel H A T assay" using phosphorylated ATF-2 proteins. The phosphorylation of ATF-2 appears to play a key role in stimulating the H A T activity of ATF-2 and in promoting DRE-dependent transcription (31). Moreover, the H A T domain of A T F - 2 exhibited significant homology to motif A of HATs in the P C A F family and the conserved amino acids at 296, 297 and 299 in motif A were critical for the intrinsic H A T activity of ATF-2 [Fig. 1C]. Our findings provide evidence of a novel phenomenon with respect to the functions of sequence-specific transcription factors. It appears that sequence-specific transcription factors target specific promoters and then recruit various H A T s to these specific promoters to enhance transcription. Moreover, it was demonstrated here that a sequence-specific factor, A T F - 2 , itself has intrinsic H A T activity; ATF-2 might also recruit other H A T s that are required for transcriptional activation. Nucleosomal histone acetylation is believed to be a critical step in the modulation of chromatin structures associated with transcriptional activation (32,33). Here, it is demonstrated that R A induces dramatic hyperacetylation at endogenous target genes through the H A T activity



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168 of ATF-2. To assess directly whether RA-dependent induction of gene expression was correlated with changes in states of histone acetylation, levels of histone acetylation at target genes of ATF-2 was analyzed using a chromatin immunoprecipitation (ChIP) assay (34). Thus, an in vivo assay of chromatin acetylation demonstrated that R A enhanced the hyperacetylation of H4 by p300/ATF-2 at the ATF-2 target D R E in the c-jun promoter. The p300- and CBP-specific ribozyme expressing F9 clones, on the other hand, were not associated with such enhancement of acetylation of all four histones species in the p300 specific-immunoprecipitates. Thus, the possibility that the acetylation of H 2 B and H4 is caused by other H A T proteins other than ATF-2 and p300/CBP cannot be ruled out. However, these results suggest that treatment of F9 cells with R A enhances the histone acetyltranferase activity of A T F - 2 . This H A T activity promotes the acetylation of chromatin of ATF-2's target genes, such as c-jun, particularly at the chromatin in the D R E region of the c-jun promoter, in vivo. Role of histone acetyltransferase activity of ATF-2 The specificity of the histone acetylation catalyzed by ATF-2 is intriguing. Individual heterodimers are quite stable during the assembly of a stable nucleosome core (35). Thus, the H2B:H4 interface is a likely site for initial disruption of histone-histone interactions upon unfolding of the nucleosome in vitro and in vivo. It is possible that ATF-2, a specific DNA-binding factor, might play a key role in the control of the stability of the H2B:H4 interaction by acetylation and/or by direct binding to a specific site on D N A . However, the possibility that nonhistone proteins, such as p53 (36), E K L F (37), G A T A - 1 (38), TFIIE (27) and TFIIF (27), might be targets of the H A T activity of ATF-2 cannot be ruled out. Acetylation of the regulatory domains, of these factors by A T F - 2 might lead to a dramatic increase in DNA-binding activity and stimulate transcription, as in the case of p53 (36). The possibility exists that ATF-2 has two distinct H A T activities, namely, the intrinsic activity of ATF-2 itself and the H A T activity associated with the p300 coactivator. ATF-2 and p300 might have overlapping actions in terms of substrate specificity, with each protein being required for optimal acetylation for the activation of transcription. It is now important to determine the way in which ATF-2 and p300 function cooperatively and regulate each others activities. In conclusion, as shown here, ATF-2 has intrinsic H A T activity and that the H A T activity of A T F - 2 and subsequent DRE-mediated transcription of c-jun gene are modulated by phosphorylation. JDP2 is an inhibitory subunit of the DRF In a yeast-two hybrid screening with ATF-2 as the bait to identify additional proteins in D R F complex, JDP2, a repressor of AP-1, was isolated as a candidate protein of other members of D R F (39). Further studies confirmed that JDP2 is


169 an inhibitory DNA-binding subunit of D R F on the basis of the following observations. First, JDP2 bound directly to the D R E as homodimers or as heterodimers with ATF-2; second, in the electrophoresis migration shift assay (EMSA), bands that corresponded to D R F were shifted still further upon addition of antibodies against JDP2 (Fig. 2A); third, JDP2 interacted directly with ATF-2 in vivo and in vitro , as well as in a yeast two hybrid system (39); and, finally, overexpresson of JDP2 repressed DRE-mediated transactivation by ATF-2 and p300.


Repression of the RA-mediated transcription of the c-jun gene by JDP2 Since D R E is both necessary and sufficient for the RA-induced expression of the c-jun gene (11), repression by JDP2 of the DRE-dependent transcription induced by ATF-2 and p300 implies a role for JDP2 in regulation of the R A mediated transcription of the c-jun gene. In a C A T assay using F9 cells, which had been stably transfected with the -730/+874 c-jun-CAT reporter, both transiently and stably expressed JDP2 repressed the C A T activity that was mediated by the c-jun promoter in response to R A . Furthermore, when the cells, which had been stably transfected with JDP2 expressing vector, were treated with R A for different times and then monitored the level of the transcription of the c-jun gene by Northern blotting analysis, overexpression of JDP2 delayed and decreased the extent of RA-induced transcription of the c-jun gene, as compared to that in cells transfected with the empty vector alone (Fig. 2B). These results suggested that JDP2 inhibits the RA-induced expression of the cjun gene mediated through D R E within c-jun promoter. Block of the RA-induced differentiation of F9 cells by JDP2 Inhibition of transcription of the c-jun gene by JDP2 led us to investigate the role of JDP2 in the RA-induced differentiation of F9 cells. A s expected, overexpression of JDP2 repressed RA-mediated differentiation, with a delay in and a decrease in the extent of morphological changes associated with differentiation (Fig. 3A), as well as repression of the transcription of R A inducible genes and genes for markers of differentiation, such as collagen 4 a l , laminin B l and Hoxa-1. The inhibitory effect of JDP2 on differentiation might involve regulation of the transcription of c-jun, in view of the key role of c-jun in differentiation and of the activity of JDP2 as a negative regulator of the expression of this gene. It is also possible that JDP2 inhibits differentiation, not only via suppression of transcription of c-jun but also, by direct repression of expression of other RA-inducible genes and/or genes for markers of differentiation.


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Food Factors in Health Promotion and Disease Prevention - American

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