Cell-Type-Specific Binding of the Transcription Factor CREB to the Camp-Response Element

Cell-type-specific binding of the transcription factor CREB to the cAMP-response element

Hyunjoo Cha-Molstad*, David M. Keller*, Gregory S. Yochum, Soren Impey, and Richard H. Goodman†

Vollum Institute, Oregon Health and Sciences University, Portland, OR 97239

Contributed by Richard H. Goodman, July 30, 2004 The cAMP-response element-binding protein (CREB) transcription genes and recruitment of the CBP coactivator. The first step, factor was initially identified as a mediator of cAMP-induced gene CREB binding, is believed to be constitutive, and the second expression. CREB binds to a target sequence termed the cAMP- step, gene activation, is thought to occur through the regulated response element (CRE) found in many cellular and viral gene recruitment of CBP (5). The idea that CREB binds constitutively promoters. One of the best-characterized CREs resides in the stems primarily from in vitro binding assays by using purified promoter of the gene encoding the neuropeptide somatostatin, DNA. Gel-mobility shift assays, for example, show that phos- and this element has served as a model for studies of CREB phorylation does not affect the interaction of CREB with naked function. Phosphorylation of CREB by protein kinase A allows DNA (17), although this point has been controversial (18). More recruitment of the coactivator CREB-binding protein (CBP). A cen- sensitive fluorescence anisotropy binding assays show that tral tenet of the CREB–CBP model is that CREB binds constitutively CREB binds to the consensus CRE at high affinity (Ϸ2 nM) and to the CRE and that regulation occurs through the phosphoryla- that variant CREs bind only slightly weaker (19). In these tion-dependent recruitment of CBP. In this report, we use chroma- fluorescence studies, phosphorylation also did not affect CREB tin immunoprecipitation assays to show that CREB does not inter- binding (19). In reporter assays, CREB seems to activate most act in vivo with the somatostatin CRE, or similar elements in several CRE-containing promoters in a similar manner (4), supporting other genes, in PC12 cells, a standard model for studies of CREB the idea that CREB binding to its targets in various gene function. Rather, CREB binding in vivo is regulated in a cell-specific promoters is not regulated and that the CREs in most genes are manner, a finding that was confirmed by using in vivo genomic occupied by CREB under basal conditions. footprinting assays. The CREs in other genes were also found to Nonetheless, other studies have suggested mechanisms that interact differentially with CREB in PC12 cells, hepatoma cells, and could conceivably regulate CREB binding in the context of cortical neurons. We conclude that the family of CREB target genes specific CREs (5). Potential regulatory factors include magne- differs from one cell type to another and that the ability of CREB sium, which is required for CREB interaction with consensus but to bind to a particular CRE represents an important component of not variant CREs (20), and associated proteins, such as the Tax gene regulation. protein of the human T cell leukemia virus (21–23), which cooperates with CREB to allow binding to the Tax-responsive he transcription factor cAMP-response element-binding element in the human T cell leukemia virus-1 long-terminal Tprotein (CREB) was discovered through its ability to bind to repeat. Additionally, the existence of a CpG at the center of the the cAMP-response element (CRE) (TGACGTCA) in the CRE makes this element ideally suited for regulation by DNA promoter of the neuropeptide gene, somatostatin (1–3). The methylation. Indeed, studies have shown that methylation of the regulation of somatostatin gene expression by the cAMP-protein central CpG prevents CREB binding in vitro and in vivo (24–26). kinase A pathway, in concert with the presence of a consensus X-ray crystallographic analyses suggest that methylation of the protein kinase A phosphorylation site within the CREB activa- central CpG sterically hinders interaction with arginine 301 in tion domain (4), led to the suggestion that CREB mediates the the CREB DNA-binding domain (27) and reduces the flexibility cAMP-induced activation of somatostatin gene expression. A required for optimal CREB interaction (28). similar involvement of CREB has been proposed for a multitude Probably most relevant, however, are studies examining of other cellular and viral genes (5, 6). In fact, recent studies have whether CREB binding is regulated in vivo. In experiments predicted that there may be as many as 10,000 CREB-binding performed over a decade ago, Schutz and coworkers (29) found sites in the mammalian genome (7). In part because of its that protein kinase A signaling regulated occupancy of a variant simplicity, the cAMP-CREB model is regarded as one of the CRE sequence in the tyrosine aminotransferase promoter. best-characterized transcriptional signaling pathways. CREB is These studies used an in vivo genomic footprinting assay, how- phosphorylated at the same critical serine residue, Ser-133, by a ever, which by itself cannot identify the particular factor involved variety of kinases in addition to protein kinase A (6). Phosphor- in binding. At least in vitro, CRE sequences can interact with ylation at Ser-133 is essential for the recruitment of the tran- many transcription factors in addition to CREB, so it is possible scriptional coactivator, CREB-binding protein (CBP) (8, 9), and that the regulated binding involves a different protein. Wolfl et this association, the first well characterized phosphoserine- al. (30) showed that CREB binding to the corticotropin- dependent protein–protein interaction, has been described in releasing hormone promoter was similarly regulated by cAMP atomic detail (10). CBP and its paralogue, p300, are believed to signaling. This study was also performed before the advent of activate gene expression by interacting with components of the chromatin immunoprecipitation (ChIP) assays, but clearly general transcriptional machinery (11–13) and by acetylating showed regulated CREB occupancy at a variant CRE site. Most chromatin (14–16). Thus, the selection of which genes are recently, Strauss and coworkers (31) used ChIP assays to deter- activated is determined by CREB, and activation, per se,is

determined, for the most part, by CBP. Because of its ability to Abbreviations: CRE, cAMP-response element; CREB, CRE-binding protein; CBP, CREB- serve as a target for multiple kinase pathways, CREB has been binding protein; ChIP, chromatin immunoprecipitation; BDNF, brain-derived neurotrophic implicated in a large number of biological processes, including factor; PEPCK, phosphoenolpyruvate carboxykinase; ICER, inducible cAMP early repressor; long-term neuronal plasticity, cell survival, circadian rhythms, SstR2, type II somatostatin receptor. adaptation to drugs, and hormonal regulation of metabolism (6). *H.C.-M. and D.M.K. contributed equally to this work. The CREB-CBP pathway can be broken down into two †To whom correspondence should be addressed. E-mail: [email protected]. distinct steps: binding of CREB to the CRE sequences in target © 2004 by The National Academy of Sciences of the USA

13572–13577 ͉ PNAS ͉ September 14, 2004 ͉ vol. 101 ͉ no. 37 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0405587101 Downloaded by guest on September 30, 2021 mine that CREB binding to the steroidogenic acute regulatory sulfate treatment was followed by washing and harvesting in 1ϫ protein promoter was increased by cAMP signaling in adrenal PBS. Genomic DNA was isolated, and DNA from the untreated cells. cells was subjected to 0.5% dimethyl sulfate treatment in vitro for In this study, we used both ChIP and in vivo genomic foot- 4 min at room temperature and stopped by addition of 375 mM printing assays to explore whether CREB occupies the CRE sodium acetate (pH 7.0) and 250 mM 2-mercaptoethanol. Cleav- sequences of target promoters in different cell types under basal age of the modified guanosines was carried out by treatment with and stimulated conditions. Our studies suggest that in contrast to 1 M piperidine for 30 min at 90°C. Cleaved DNA (2.5 ␮g) was the currently accepted model, CREB binding varies in a cell used in primer extension reactions at 48°C by using the program type-specific manner. Thus, the family of CREB targets differs SEQUENASE VERSION 2.0 (United States Biochemical) with 40 nM from one cell type to another. Interestingly, the best-studied of the following gene-specific primers designed against the CREB target, the somatostatin CRE, does not interact with bottom strand of the rat sequence: somatostatin (5Ј- CREB in PC12 cells, where it has been so extensively studied. GTCAGTCTAGACGCAGGT-3Ј); c-fos (5Ј-GGCAAAG- CTCGGCGAG-3Ј); and phosphoenolpyruvate carboxykinase Materials and Methods (PEPCK; 5Ј-CCGGATTTCCCCTGTT-3Ј). The primer exten- Cell Culture. PC12 cells were grown in DMEM with 10% neonatal sion products were ligated with a linker (top strand, 5Ј- calf serum and 5% FBS plus 100 units͞ml penicillin and 100 GCGGTGACCCGGGAGATCTGAATTC-3Ј; and bottom units͞ml streptomycin (pen͞strep). H4IIE rat hepatoma cells strand, 5Ј-GAATTCAGATC-3Ј); which served as a fixed end for (American Type Culture Collection) were grown in DMEM ligation-mediated PCR. The top-strand primer and a gene- (American Type Culture Collection) supplemented with 10% specific primer (each 100 nM) were used in 20 cycles of PCR FBS (HyClone) and pen͞strep (Invitrogen). Cortical neurons amplification with TaqDNA polymerase plus Q buffer (Qiagen). were cultured from postnatal day 1–2 rats and plated at a density The amplification primers were somatostatin (5Ј-AGGTCCG- of 4 ϫ 105 cells per well onto 10-cm plates. Cells were maintained CAGGCAGCAGAG-3Ј); c-fos (5Ј-AGGGGTCCAGGGGTA- in Neurobasal A medium (Invitrogen) supplemented with 1% GACAC-3Ј); and PEPCK (5Ј-CTGTTGGCCAAGGGTGTGT- B27 (Invitrogen) and 0.5% FBS, 35 mM glucose, 1 mM L- TCC-3Ј). A final three rounds of primer extension was carried glutamine, and pen͞strep, 5–7 days before experimentation. out by using ␥33P-ATP end-labeled primers [(5 pmol of 1.4 PC12 and H4IIE cells were cultured under serum-free condi- ␮Ci͞pmol (1 Ci ϭ 37 GBq) specific activity, with partial removal tions overnight before treatment with 10 ␮M forskolin (Sigma) of free nucleotides by precipitation with ethanol with addition of for 15 min. 5 M ammonium sulfate]. The end-labeled primers were somatostatin (5Ј-GACGACTCCAGTAGCGTCTCCTTCAG-3Ј); ChIP. Cells were fixed for 20 min with 1% formaldehyde in PBS c-fos (5Ј-ACTGGTGGGAGCTGCAGAGCAG-3Ј); and at room temperature. Cells were harvested with 100 mM PEPCK (5Ј-GAAGGCCAACCGTGCTTGGTAGCTAG-3Ј). Tris⅐HCl, pH 9.4͞10 mM DTT and pelleted by centrifugation for Labeled ligation-mediated PCR products were phenol- 5 min at 2,000 ϫ g. The pellets were washed with 1 ml of ice-cold chloroform-extracted, ethanol-precipitated, and resuspended in PBS, resuspended in 0.6 ml of cell lysis buffer (0.1% SDS͞0.5% formamide gel-loading buffer. Samples were electrophoresed on Triton X-100͞20 mM Tris⅐HCl, pH 8.1͞150 mM NaCl͞1 ϫ a 7% acrylamide-urea gel and the footprints were visualized by protease inhibitor mixture; Roche Molecular Biochemicals), exposure to a PhosphorImager screen (Molecular Dynamics). sonicated, and then centrifuged for 10 min at 13,000 rpm with an Eppendorf microcentrifuge at 4°C. Supernatants were trans- RT-PCR. PC12 cells (1–5 ϫ 104), H4IIE, and cortical neurons were ferred to fresh tubes and precleared with protein A-Sepharose treated as described, and total RNA was isolated by using TRIzol [80 ␮l of 50% slurry in 10 mM Tris⅐HCl, pH 8.1͞1 mM EDTA (Invitrogen) or an RNeasy kit (Qiagen) according to manufac- (TE)͞0.5% BSA͞19.2 ␮g of glycogen] for1hat4°C. Immuno- turer’s instructions. RNA (from 50 ng to 3 ␮g) was reverse- precipitation was performed overnight at 4°C with 10 ␮gof transcribed with Moloney murine leukemia virus reverse tran- anti-CREB antibody (32), 3 ␮g of anti-dimethyl methyl K4 scriptase (Invitrogen) and 50–250 ng of random primers histone H3 (abcam, Cambridge, MA), or anti-␤-Gal (5 Prime 3 (Invitrogen). cDNA was amplified with platinum Taq (Invitro- 3 Prime). Immune complexes were captured with 80 ␮lof50% gen) for 28 cycles (c-fos), 29 cycles [brain-derived neurotrophic protein A-agarose slurry for1hat4°C. The beads were collected factor (BDNF)], or 33 cycles for all other genes. PCR products by centrifugation at 7,600 rpm for 1 min and washed as follows: were resolved in 2% agarose gels. Primers will be provided on two times in lysis buffer for 10 min, two times in washing buffer request. (0.1% SDS͞0.5% Triton X-100͞2 mM EDTA, pH 8.0͞20 mM Tris⅐HCl, pH 8.1͞150 mM NaCl) for 10 min, one time in LiCl Quantitative PCR. Primers surrounding CRE sites were designed buffer (0.25 M LiCl͞1% Nonidet P-40͞1% deoxycholate͞1mM by using MIT’s PRIMER3 software (http:͞͞frodo.wi.mit.edu͞cgi- EDTA, pH 8.0͞10 mM Tris⅐HCl, pH 8.1), and two times with TE. bin͞primer3͞primer3࿝www.cgi) with default parameters, except CREB-DNA complexes were eluted from the beads, first by rodent and simple repeat library was on, product size was 80–200 15-min incubation with 100 ␮l of elution buffer (1% SDS͞0.1 M bp, primer size was 18–27 bases, Tm was 66°–70°C, maximum Ј NaHCO3, pH 8.0) at room temperature. Beads were collected by self-complementarity was 4, and maximum 3 complementarity centrifugation, and a second elution was performed by 10-min was 1. Primer sequences are available on request. PCRs (10 ␮l) ␮ ␮ ϫ incubation with 50 l of elution buffer. After the addition of 10 contained 1 lof10 PCR buffer (Invitrogen), 2.5 mM MgCl2, ␮l of 5 M NaCl, eluates were pooled and heated at 65°C 200 ␮M dNTP (Roche), 0.125–0.25 ␮M primer (Integrated overnight to reverse the formaldehyde cross-linking. DNA frag- DNA Technologies, Coralville, IA), 1ϫ SYBR green I (Invitro- ments were purified by using a Qiagen (Valencia, CA) kit and gen), and 1 unit of Platinum Taq (Invitrogen). PCR was per- quantified by real-time PCR. formed on an Opticon OP346 (MJ Research, Cambridge, MA) for one cycle at 95°C for 35 s, 30–50 cycles at 94°C for 15 s, and In Vivo Genomic Footprinting. Genomic footprints followed the 68–70°C for 40 s. Data were expressed as either nanograms of ligation-mediated PCR technique of Mueller and Wold (33) and gel-purified (Qiagen) amplicon or fold-change over IgG. Riggs et al. (34) with some modifications. Briefly, PC12 and Ϸ ϫ 7 Results H4IIE cells ( 8 10 ) were either not treated or treated with GENETICS 0.2% dimethyl sulfate at room temperature for 5 min to alkylate ChIP assays were used to examine the association of CREB with the guanine bases by the Maxam–Gilbert method (35). Dimethyl endogenous promoters in PC12 cells, a frequently used model

Cha-Molstad et al. PNAS ͉ September 14, 2004 ͉ vol. 101 ͉ no. 37 ͉ 13573 Downloaded by guest on September 30, 2021 binding at c-fos and ICER promoters was observed with cells treated with forskolin, an activator of adenylyl cyclase. The epitope recognized by the anti-CREB antibody could conceivably be masked in specific contexts and thereby cause an artifactually negative ChIP result. No binding of CREB to the somatostatin promoter was observed when we used another anti-CREB antiserum, however (data not shown). Because activation of the somatostatin CRE in PC12 cells has served as a model for CREB function, we sought to examine CREB occupancy by using an independent method, in vivo genomic footprinting. This assay cannot determine the identity of factors interacting with a DNA element, but it can assess whether a binding site is occupied. In vivo genomic footprinting assays were performed to examine the c-fos and somatostatin promoters (Fig. 1B). Consistent with the ChIP studies, the c-fos CRE showed a clear and reproducible footprint, represented by three bands of diminished intensity and one hypersensitive site. A weak but reproducible footprint was also detected immediately 3Ј to the c-fos TATA sequence as a band of diminished intensity. In contrast, no footprints were detected over the CRE or TATA sequences of the somatostatin gene. This experiment confirms the results of the ChIP assays. To determine whether the promoter-specific occupancy of CREB in PC12 cells was a general phenomenon or unique to somatostatin, we performed a similar analysis for other promoters known to interact with CREB in vitro and to be induced by cAMP in transient transfection assays (Fig. 2A). We found that CREB binding was undetectable at a variety of other CRE- containing promoters in PC12 cells, including those in the BDNF, gonadotropin-releasing hormone receptor, PEPCK, or insulin. CREB binding appeared to correlate with the potential for gene expression (Fig. 2B), as transcripts for BDNF, gonadotropin-releasing hormone receptor, PEPCK, and insulin are Fig. 1. Differential binding of CREB to CRE-containing promoters. (A) PC12 undetectable in PC12 cells, whereas c-fos is expressed at high cells were treated without (gray bar) or with (white bar) 10 ␮M forskolin for levels, at least after induction by forskolin. 15 min. ChIP assays were performed by using 10 ␮g of anti-CREB or anti-␤-Gal We next performed ChIP assays on a variety of additional (IgG control, black bar) antibodies, and the levels of immunoprecipitated genes, comparing the results in PC12 cells, H4IIE hepatoma c-fos, ICER, and somatostatin promoter regions were measured by real-time cells, and primary cortical neurons. Because of the difficulty PCR. The data represent 15 individual ChIPs from four experiments. Error is comparing absolute ChIP signals in separate immunoprecipita- SEM. (B) In vivo genomic footprinting was performed on PC12 cells as de- tions, we used real-time PCR to compare the fold-increase in scribed in Materials and Methods. In vitro denotes dimethyl sulfate-cleaved signal obtained by using anti-CREB antiserum versus nonspe- naked genomic DNA. In vivo denotes cleaved DNA from cells that were treated cific IgG. As shown in Fig. 3, some CREs bind CREB in all three with dimethyl sulfate in vivo. Arrows denote footprinted or hypersensitive bands, including several over the CRE and immediately 3Ј of the TATA box in cell types [Vamp-1 and type II somatostatin receptor (SstR2) the c-fos promoter. No footprints were detected over the somatostatin (Fig. 3 A and B), whereas others (the potassium channel, Kv 3.1, promoter. and synatotagmin V) associate with CREB only in PC12 cells and cortical neurons (Fig. 3 C and D). CREB binding to the somatostatin and BDNF CREs was evident only in cortical for studies of CREB action. To determine whether CREB neurons (Fig. 3 E and F). In contrast, the PEPCK CRE interacts constitutively with all CREs in their native context, we interacted with CREB in H4IIE cells, but not in PC12 cells or tested elements in three well studied promoters, the consensus cortical neurons (Fig. 3G). CREB occupancy of the CRE CREs in the somatostatin (3) and inducible cAMP early repres- correlates tightly with gene expression (or the potential for gene sor (ICER) (36) genes and the variant CRE in the c-fos promoter expression). To confirm our hypothesis that CREB binds to (37). PC12 cells, derived from a rat pheochromocytoma, express CREs in a cell type-specific manner, we performed additional in c-fos and ICER but not somatostatin. Of note, although c-fos vivo genomic footprinting assays, this time examining the gene expression is induced strongly by various stimuli, a signif- PEPCK promoter. A clear footprint over the CRE can be icant proportion of the induction is due to the release of a block observed in H4IIE cells, but not in PC12 cells (Fig. 4). We of transcriptional elongation (38, 39). Thus, although considered conclude from these experiments that CREB binds to its target to be an immediate early gene, c-fos expresses a 5Ј-truncated CREs in a cell-type-specific manner, which is not consistent with transcript in the absence of stimulation. The GAPDH gene was the idea that the CRE is occupied constitutively. used as a negative control because its promoter does not bind To test whether CREB binding to DNA correlated with CREB and it is not induced transcriptionally by cAMP. As nucleosomal modifications that reflect the activation state of depicted in Fig. 1A, a high level of CREB binding is detected promoters, we examined the methylation status of histone H3 at over the c-fos and ICER CREs but not over that in somatostatin. Lys-4. This modification is believed to inhibit the binding of the By using in vitro fluorescence anisotropy measurements, we have repressive Nurd complex and seems to be a marker for chromatin shown that CREB interacts better with the somatostatin CRE capable of transcriptional activity (40). Fig. 5 shows that three than it does with the CRE in the c-fos promoter (20). Thus, the genes capable of being activated in PC12 cells, SstR2, c-fos, and ability of CREB to interact with the CRE in vitro does not ICER, bind CREB and are heavily methylated at Lys-4. The high predict its binding in vivo. No significant stimulation of CREB level of Lys-4 methylation is evident even in the absence of

13574 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0405587101 Cha-Molstad et al. Downloaded by guest on September 30, 2021 Fig. 2. CREB does not interact with the promoters of transcriptionally silent genes. (A) PC12 cells were treated without (gray bar) or with (white bar) 10 ␮M forskolin for 15 min. Black bar denotes IgG signal. ChIP assays were performed to measure the level of CREB binding on BDNF, gonadotropin-releasing hormone receptor, PEPCK, and insulin promoters by employing real-time PCR. The data represent 10 individual ChIPs from four experiments. Error is SEM. (B) PC12 cells were treated without (lanes, 2, 5, 8, 11, and 14) or with (lanes, 3, 6, 9, 12, and 15) 10 ␮M forskolin for 1 h. mRNAs for CREB target genes indicated were detected by reverse transcription and PCR. The molecular weight marker (MW) is a 1-kb DNA ladder, and lanes 1, 4, 7, 10, and 13 are no reverse transcriptase controls.

stimulation for c-fos and ICER, which either reflects their potential for transcriptional activation or the basal transcriptional activity of these immediate early genes. Genes that are inactive, including somatostatin, PEPCK, and insulin, do not bind CREB and are not associated with methylated Lys-4. Discussion In this study, we demonstrate that CREB binding is highly Fig. 3. CRE occupancy by CREB is cell type-speciﬁc. ChIPs were performed by tissue-specific. Binding is apparent at genes that are either using PC12 and H4IIE cells, and primary cortical neurons (CN). The level of transcriptionally active or potentially active but not at promoters immunoprecipitated promoter regions was measured by real-time PCR. The that are not expressed. Therefore, the repertoire of genes that black bar indicates IgG control, and the gray bar indicates CREB. Lanes 1, 3, and are potentially regulated by CREB (the CREB regulon) is 5 are no reverse transcriptase controls. Lanes 2, 4, and 6 are reverse transcrip- distinct in different cell types. The presence of the epigenetic tase-PCR products from PC12 cells, H4IIE cells, and cortical neurons, respec- tively. For study of BDNF expression (F, lane 6), cortical neurons were stimu- marker, histone H3 dimethyl-Lys-4, distinguishes active versus lated with Kϩ for 1 h. Data are presented as fold changes over IgG (average of inactive genes and correlates with CREB binding. This finding 12 individual ChIPs from at least two different experiments). Error is SD. suggests that CREB binding is regulated through an epigenetic mechanism (or that binding of CREB determines an active chromatin conformation). finding that CREB does not bind to all CREs was unexpected

Occupancy of many genetic regulatory elements is determined and is entirely contrary to the generally accepted model of GENETICS by the tissue-specific expression of particular transcription fac- CREB function. Most importantly, our results suggest that the tors. CREB expression seems to be ubiquitous, however. The presence of a CRE sequence in a gene promoter does not

Cha-Molstad et al. PNAS ͉ September 14, 2004 ͉ vol. 101 ͉ no. 37 ͉ 13575 Downloaded by guest on September 30, 2021 Fig. 5. Correlation of histone H3 Lys-4 methylation and CREB binding. ChIP was performed by using an antibody against dimethylated histone H3 Lys-4 in PC12 cells. The level of methylation at the indicated promoters was measured by real-time PCR. Black bars represent the IgG control. Histone methylation was detected in SstR2, c-fos, and ICER promoters but not in the Sst, PEPCK, or insulin promoters. Data from two experiments are shown.

proposed to bind to a CRE-like element in the tyrosine aminotransferase gene in hepatoma cells, which express tyrosine aminotransferase, but not in fibrosarcoma cells, where the gene is inactive (42). These studies were performed before the advent of ChIP assays, however, so it is not certain that they actually monitor CREB binding, as opposed to some other transcription factor. Our results clearly show that CREB does not bind to genes that are inactive in PC12 cells, H4IIE cells, and primary Fig. 4. The PEPCK CRE is occupied in H4IIE but not in PC12 cells. In vivo cortical neurons. Therefore, CRE occupancy by CREB is not genomic footprinting was performed as described in Fig. 1. Footprints or constitutive. hypersensitive sites were visualized only in the H4IIE sample. Locations of the Because CREB was believed to bind its target genes consti- CRE and NF-1 sites are indicated. tutively, the mechanisms underlying its tissue-specific binding have received little attention. It is possible, in fact, that CREB could control the tissue-specific expression of some genes, rather necessarily predict that CREB participates in its regulation. than the converse. For example, BDNF is highly expressed in Thus, our findings may require the reevaluation of many genes hippocampal neurons but not in some other neuronal subtypes, proposed to be regulated through the CREB pathway, especially in response to activity (43–45). Because CREB activation occurs those characterized by using reporter genes and transient trans- in both the expressing and nonexpressing neurons, how CREB fection assays. Indeed, the origin of the idea that CREB binds drives BDNF expression in distinct neuronal populations has constitutively to CRE sequences is somewhat obscure. Support been in question. Tao et al. (46) reported that in response to for this model include the findings that CREB is highly ex- membrane depolarization, both CREB and a transcription factor pressed, that it has a high affinity for the CRE, and that CREB termed CaRF are required for BDNF exon III expression in rat binding to DNA in vitro does not seem to be regulated. The embryonic cortical neurons. Interestingly, these authors pointed dogma that CREB binds constitutively to all CRE sequences is out that BDNF expression does not occur in PC12 cells, even not entirely vestigial, however. Euskirchen et al. (7) recently used though these cells contain both CREB and CaRF. It is possible a human chromosome 22 microarray in ChIP-on-ChIP assays to that the failure of CREB to occupy the BDNF exon III CRE examine CREB binding to the CREs of expressed and nonex- accounts for the lack of BDNF expression. pressed promoters in human choriocarcinoma placental cells. What controls whether CREB binds to a specific CRE? The Because CREB seemed to interact with several targets that are binding of transcription factors to their recognition sites in DNA generally classified as ‘‘neuronal’’ rather than ‘‘placental,’’ these can be prevented through multiple mechanisms, including steric workers concluded that CREB binding was constitutive. There occlusion, sequestration by association with other proteins, or are several possible explanations for our discordant interpreta- competition by other transcription factors. The most likely tion. First, it is well known that many neuronal genes are mechanism for the CREB block is steric occlusion. Our genomic expressed in nonneuronal tissues; for example, BDNF and its footprinting data support this idea because of the complete receptor, trkB, are expressed in immune cells (41). Second, genes absence of footprints over the CREs in nonexpressing cells. are often aberrantly regulated in transformed cell lines, which Steric occlusion in this case is likely the result of chromatin, as underscores the need to combine ChIP data with more direct opposed to other competing transcription factors, because nu- measures of transcription. The difference could also relate to the cleosomes are invisible to dimethyl sulfate treatment. Thus, we quality of the antibodies used or other aspects of the ChIP assays. believe that chromatin structure precludes CREB from binding The idea that CREB binds to CRE sequences in a tissue-specific to its target sites in certain situations. This conclusion is con- manner is not unprecedented, however. For example, CREB was sistent with the findings of Ji et al. (47), who used in vivo genomic

13576 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0405587101 Cha-Molstad et al. Downloaded by guest on September 30, 2021 footprinting to show that occupancy of the bcl-2 CRE occurs be detected by RT-PCR, and no transcription was detected from only after its translocation to a transcriptionally active Ig heavy- genes lacking dimethylated Lys-4. These findings suggest that chain locus. epigenetic differences in chromatin from one cell type to another In conclusion, the correlation of gene expression, histone H3 determine the population of genes capable of binding CREB Lys-4 methylation, and CRE occupancy supports the idea that and, in turn, the pattern of gene responses to the wide variety of CREB binding is determined by the methylation status of pathways that activate CREB function. Although the mechanism histones at specific promoters. We found no detectable histone that attenuates binding of CREB to transcriptionally silent target H3 Lys-4 methylation signal at the somatostatin, PEPCK, or genes is not known, this process likely participates in specifying insulin promoters in PC12 cells, and high levels of dimethylated the transcriptional programs that underlie development and Lys-4 at active genes, such as SstR2, c-fos, and ICER. Of interest, differentiation. Future work will be required to determine the c-fos promoter binds TFIIB and RNA polymerase II under whether epigenetic modifications regulate occupancy by CREB basal conditions (48), but the ICER promoter does not and is or vice versa. only minimally active. This result indicates, as has been suggested by others, that Lys-4 methylation correlates with the potential for We thank Jeremy Boss (Emory University School of Medicine, Atlanta) activation, rather than by the absolute levels of gene expression for providing anti-CREB antisera and Gail Mandel for helpful com- (49). However, in all cases where the promoter contained the ments. This work was supported by grants from the National Institutes dimethyl-Lys-4 epigenetic modification, gene expression could of Health.

1. Gonzalez, G. A., Yamamoto, K. K., Fischer, W. H., Karr, D., Menzel, P., Biggs, 25. Martinowich, K., Hattori, D., Wu, H., Fouse, S., He, F., Hu, Y., Fan, G. & Sun, W., III, Vale, W. W. & Montminy, M. R. (1989) Nature 337, 749–752. Y. E. (2003) Science 302, 890–893. 2. Montminy, M. R. & Bilezikjian, L. M. (1987) Nature 328, 175–178. 26. Mancini, D. N., Singh, S. M., Archer, T. K. & Rodenhiser, D. I. (1999) 3. Montminy, M. R., Sevarino, K. A., Wagner, J. A., Mandel, G. & Goodman, Oncogene 18, 4108–4119. R. H. (1986) Proc. Natl. Acad. Sci. USA 83, 6682–6686. 27. Schumacher, M. A., Goodman, R. H. & Brennan, R. G. (2000) J. Biol. Chem. 4. Gonzalez, G. A. & Montminy, M. R. (1989) Cell 59, 675–680. 275, 35242–35247. 5. Mayr, B. & Montminy, M. (2001) Nat. Rev. Mol. Cell. Biol. 2, 599–609. 28. Konig, P. & Richmond, T. J. (1993) J. Mol. Biol. 233, 139–154. 6. Lonze, B. E. & Ginty, D. D. (2002) Neuron 35, 605–623. 29. Weih, F., Stewart, A. F., Boshart, M., Nitsch, D. & Schutz, G. (1990) Genes Dev. 7. Euskirchen, G., Royce, T. E., Bertone, P., Martone, R., Rinn, J. L., Nelson, 4, 1437–1449. F. K., Sayward, F., Luscombe, N. M., Miller, P., Gerstein, M., et al. (2004) Mol. 30. Wolfl, S., Martinez, C. & Majzoub, J. A. (1999) Mol. Endocrinol. 13, 659–669. Cell. Biol. 24, 3804–3814. 31. Hiroi, H., Christenson, L. K., Chang, L., Sammel, M. D., Berger, S. L. & Strauss, 8. Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy, M. R. & J. F., III (2004) Mol. Endocrinol. 18, 791–806. Goodman, R. H. (1993) Nature 365, 855–859. 32. Moreno, C. S., Beresford, G. W., Louis-Plence, P., Morris, A. C. & Boss, J. M. 9. Kwok, R. P., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., (1999) Immunity 10, 143–151. Brennan, R. G., Roberts, S. G., Green, M. R. & Goodman, R. H. (1994) Nature 33. Mueller, P. R. & Wold, B. (1989) Science 246, 780–786. 370, 223–226. 34. Riggs, A. D., Singer-Sam, J. & Pfeifer, G. P. (1998) in Chromatin: A Practical 10. Radhakrishnan, I., Perez-Alvarado, G. C., Parker, D., Dyson, H. J., Montminy, Approach, ed. Gould, H. (Oxford Univ. Press, Oxford), pp. 79–109. M. R. & Wright, P. E. (1997) Cell 91, 741–752. 35. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499–560. 11. Felzien, L. K., Farrell, S., Betts, J. C., Mosavin, R. & Nabel, G. J. (1999) Mol. 36. Molina, C. A., Foulkes, N. S., Lalli, E. & Sassone-Corsi, P. (1993) Cell 75, Cell. Biol. 19, 4241–4246. 875–886. 12. Nakajima, T., Uchida, C., Anderson, S. F., Lee, C. G., Hurwitz, J., Parvin, J. D. 37. Sheng, M., Dougan, S. T., McFadden, G. & Greenberg, M. E. (1988) Mol. Cell. & Montminy, M. (1997) Cell 90, 1107–1112. Biol. 8, 2787–2796. 13. Swope, D. L., Mueller, C. L. & Chrivia, J. C. (1996) J. Biol. Chem. 271, 38. Mechti, N., Piechaczyk, M., Blanchard, J. M., Jeanteur, P. & Lebleu, B. (1991) 28138–28145. Mol. Cell. Biol. 11, 2832–2841. 14. Bannister, A. J. & Kouzarides, T. (1996) Nature 384, 641–643. 39. Pinaud, S. & Mirkovitch, J. (1998) J. Mol. Biol. 280, 785–798. 15. Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., 40. Zegerman, P., Canas, B., Pappin, D. & Kouzarides, T. (2002) J. Biol. Chem. 277, Privalsky, M. L., Nakatani, Y. & Evans, R. M. (1997) Cell 90, 569–580. 11621–11624. 16. Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H. & Nakatani, Y. 41. Besser, M. & Wank, R. (1999) J. Immunol. 162, 6303–6306. (1996) Cell 87, 953–959. 42. Weih, F., Nitsch, D., Reik, A., Schutz, G. & Becker, P. B. (1991) EMBO J. 10, 17. Hagiwara, M., Brindle, P., Harootunian, A., Armstrong, R., Rivier, J., Vale, W., 2559–2567. Tsien, R. & Montminy, M. R. (1993) Mol. Cell. Biol. 13, 4852–4859. 43. Patterson, S. L., Grover, L. M., Schwartzkroin, P. A. & Bothwell, M. (1992) 18. Nichols, M., Weih, F., Schmid, W., DeVack, C., Kowenz-Leutz, E., Luckow, B., Neuron 9, 1081–1088. Boshart, M. & Schutz, G. (1992) EMBO J. 11, 3337–3346. 44. Tao, X., Finkbeiner, S., Arnold, D. B., Shaywitz, A. J. & Greenberg, M. E. 19. Richards, J. P., Bachinger, H. P., Goodman, R. H. & Brennan, R. G. (1996) (1998) Neuron 20, 709–726. J. Biol. Chem. 271, 13716–13723. 45. Shieh, P. B., Hu, S. C., Bobb, K., Timmusk, T. & Ghosh, A. (1998) Neuron 20, 20. Craig, J. C., Schumacher, M. A., Mansoor, S. E., Farrens, D. L., Brennan, R. G. 727–740. & Goodman, R. H. (2001) J. Biol. Chem. 276, 11719–11728. 46. Tao, X., West, A. E., Chen, W. G., Corfas, G. & Greenberg, M. E. (2002) 21. Kwok, R. P., Laurance, M. E., Lundblad, J. R., Goldman, P. S., Shih, H., Neuron 33, 383–395. Connor, L. M., Marriott, S. J. & Goodman, R. H. (1996) Nature 380, 47. Ji, L., Mochon, E., Arcinas, M. & Boxer, L. M. (1996) J. Biol. Chem. 271, 642–646. 22687–22691. 22. Cox, J. M., Sloan, L. S. & Schepartz, A. (1995) Chem. Biol. 2, 819–826. 48. Fass, D. M., Butler, J. E. & Goodman, R. H. (2003) J. Biol. Chem. 278, 23. Yin, M. J., Paulssen, E. J., Seeler, J. S. & Gaynor, R. B. (1995) J. Virol. 69, 43014–43019. 3420–3432. 49. Schneider, R., Bannister, A. J., Myers, F. A., Thorne, A. W., Crane-Robinson, 24. Iguchi-Ariga, S. M. & Schaffner, W. (1989) Genes Dev. 3, 612–619. C. & Kouzarides, T. (2004) Nat. Cell Biol. 6, 73–77. GENETICS

Cha-Molstad et al. PNAS ͉ September 14, 2004 ͉ vol. 101 ͉ no. 37 ͉ 13577 Downloaded by guest on September 30, 2021