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- Materials and methods
GTP cyclohydrolase I (GTPCH) gene expression was investigated in the human monoamine-containing neuroblastoma cell line SK-N-BE(2)M17. Northern blot analysis revealed a single GTPCH mRNA transcript that was confirmed by RNase protection assay to encode for Type 1 GTPCH; no alternatively spliced forms of GTPCH mRNA were detected with this assay. Incubation with 8Br-cAMP, but not nerve growth factor or leukemia inhibitory factor, produced a rapid increase in GTPCH mRNA and protein levels; protein levels remained elevated during the entire treatment period while mRNA content declined rapidly between 10 and 24 h. Treatment with 8Br-cAMP did not significantly modify the stability of GTPCH mRNA but did increase GTPCH transcription as determined by transient transfection assays of a luciferase reporter construct containing 1171 bp of human GTPCH 5′-flanking sequence. Cis-acting elements required for maximal basal and cAMP-dependent transcription were localized by deletion analysis to the 146 bp proximal promoter. DNase I footprint analysis of the proximal promoter using SK-N-BE(2)M17 nuclear extracts identified two protein binding domains: one an upstream Sp1-like site and the other a combined CRE-Sp1-CCAAT-box element. EMSA and supershift assays demonstrated that the combined CRE-Sp1-CCAAT-box element recruits ATF-2 and NF-Y but not Sp1–4 or Egr-1–3. NF-Y binding was confirmed using pure recombinant human NF-Y protein. Transcription of the human GTPCH gene in human SK-N-BE(2)M17 cells is thus enhanced by cAMP acting through regulatory elements located in the proximal promoter and may involve the transcription factors NF-Y and ATF-2.
GTPCH gene expression within rat MA cell types is highly dynamic and regulated by numerous signal transduction pathways, including those that utilize the second messenger cAMP (Zhu et al. 1994; Anastasiadis et al. 1998). Cyclic-AMP-dependent enhancement of GTPCH transcription is observed in only a subset of rat cell types the normally express GTPCH (Kapatos et al. 1981). Located within the proximal promoter of the rat GTPCH gene is a non-canonical cAMP response element (CRE) and an adjacent perfect CCAAT-box that are both required for maximal and cell type-specific cAMP-dependent transcription (Kapatos et al. 2000). This combined CRE and CCAAT-box element acts as an enhancer and can confer sensitivity to cAMP on a heterologous minimal promoter. Moreover, in vitro assays have revealed that this CRE recruits the transcription factors CCAAT enhancer-binding protein β (C/EBPβ) and activating transcription factor 4 (ATF-4), whereas the CCAAT-box is bound by nuclear factor Y (NF-Y). The rat and human GTPCH proximal promoters exhibit almost 80% sequence homology (Kapatos et al. 2000) and the location and spacing of this CRE and CCAAT-box cassette are conserved in the human sequence. This suggests that in the appropriate cellular milieu the human GTPCH gene might also be enhanced by cAMP although, to our knowledge, this has never been demonstrated.
Despite their high level of homology, however, there are also differences between the rat and human promoter sequences that may impart variation in the control of this gene. These include a lack of homology in the 3′ end of the non-canonical CRE, the presence in the human promoter of a GC-rich putative Sp1-like site overlapping the 3′ end of the CRE and an E-box in the rat promoter that is not found in the human sequence (Kapatos et al. 2000). Like the rat and mouse genes (Shimoji et al. 1999; Kapatos et al. 2000) the 5′-flanking region of the human GTPCH gene has recently been shown to support transcription in transient transfection assays of gene reporter constructs (Witter et al. 1996). Unlike the rat gene, deletion analysis of the human promoter did not provide evidence for inhibitory elements located upstream of the proximal promoter. This same study also showed that transcription driven by the human GTPCH promoter was not enhanced in response to a stimulus that is known to induce expression of the endogenous GTPCH gene. There is thus reason to believe that what may appear to be minor variations between human and rat GTPCH promoter sequences may actually be responsible for significant differences in promoter function. Moreover, unlike in the rat, the product of the human GTPCH gene is alternatively spliced to generate three (Togari et al. 1992; Ichinose et al. 1995) or more (Golderer et al. 2001) transcripts with different coding sequences which introduces an additional level of complexity to the study of human GTPCH gene expression.
The major goals of the present study were therefore to use the human MA (Biedler et al. 1978) and BH4 (Albrecht et al. 1978) containing neuroblastoma cell line SK-N-BE(2)M17 to: (1) characterize human GTPCH mRNA expression within a neuronal cell type with regard to alternative splicing; (2) discover a stimulus capable of increasing human GTPCH gene expression at the transcriptional level; and (3) characterize the human GTPCH 5′ flanking sequence with respect to cis-acting elements and trans-acting factors involved in basal and enhanced transcription.
- Top of page
- Materials and methods
Five main conclusions can be reached from the present work. First, only Type 1 GTPCH mRNA can be detected by nuclease protection assay in the human neuroblastoma cell line SK-N-BE(2)M17. Second, the second messenger cAMP increases levels of BH4 in these cells by rapidly stimulating GTPCH gene transcription and increasing GTPCH mRNA and protein content. Third, cis-acting elements required for maximal basal and cAMP-dependent transcription are found within the 146 bp proximal promoter. Fourth, two protein binding domains are located within the proximal promoter, one associated with an upstream Sp1-like site and the other encompassing the entire combined CRE-Sp1-CCAAT-box element. Fifth, the CRE-Sp1-CCAAT-box binding domain recruits the transcription factors NF-Y and ATF-2 in vitro.
The major form of GTPCH mRNA found in human tissues and cell lines (Gutlich et al. 1992; Nomura et al. 1993) is reported to be approximately 2.9–3.6 kb long and encodes for the Type 1 transcript. Only Type 1 GTPCH mRNA was detectable by nuclease protection assay in SK-N-BE(2)M17 cells under conditions of basal and cAMP-enhanced transcription and this transcript was approximately 3.6 kb in size. This is a significant observation because unlike the rat GTPCH gene, which generates two forms of GTPCH mRNA (Gutlich et al. 1992; Hirayama et al. 1993) with identical coding regions (S. I. Lentz and G. Kapatos, unpublished data), the human GTPCH gene is reported to encode for three forms of mRNA (Togari et al. 1992) that differ in their coding regions and are produced by alternative splicing of exon 6 (Ichinose et al. 1995). Moreover, Golderer et al. (2001) have recently reported additional alternatively spliced forms of human GTPCH. Neither Type 2 nor 3 transcripts have ever been observed by northern blot but are predicted to contain at least 1000 fewer bases than Type 1. While we could not detect Type 2 and 3 mRNA by nuclease protection assay, these transcripts can be amplified by RT-PCR using RNA isolated from human liver RNA (Togari et al. 1992) and the human neuroblastoma cell line SK-N-SH (Golderer et al. 2001) and are reported to make up as much as 20% of the GTPCH mRNA found in some human tissues (Hirano et al. 1997). Although Type 2 and 3 and less abundant alternatively spliced forms of GTPCH mRNA may be present in some human cells it should be kept in mind that the proteins translated from these transcripts have never been observed in vivo. Indeed, the physiological significance of Type 2 and 3 GTPCH remains enigmatic because, when expressed in recombinant form in Escherichia coli, neither protein is reported to have enzyme activity (Gutlich et al. 1992).
Incubation of SK-N-BE(2)M17 cells with 8Br-cAMP resulted in a rapid and coordinated increase in GTPCH mRNA and protein levels that was detectable as early as 1 h after treatment. The rapidity of these responses indicates that new protein synthesis is not required for the induction of GTPCH transcription by cAMP. Following 10 h of 8Br-cAMP treatment, however, GTPCH mRNA levels declined while protein levels remained stable. A similar rapid increase and equally rapid decline in GTPCH mRNA in response to continuous stimulation by cAMP has previously been shown to occur within cultured mesencephalic and diencephalic dopamine neurons (Zhu et al. 1994). These data suggest a cellular process that functions to limit the magnitude of the increase in GTPCH protein and thus BH4 synthesis after induction of GTPCH gene transcription by cAMP. At the nucleic acid level this would presumably involve either inhibition of GTPCH transcription or a destabilization of existing GTPCH mRNA molecules. A cAMP-dependent increase of this magnitude in GTPCH mRNA turnover is not supported by the data presented here or by studies showing that cAMP can actually prolong the half-life of GTPCH mRNA (Pluss et al. 1996). On the other hand, the decline in GTPCH mRNA that occurs between 10 and 24 h of continuous 8Br-cAMP treatment is similar to that observed here following inhibition of transcription with actinomycin D. This implies that at some time beyond 10 h of treatment GTPCH transcription is no longer stimulated and/or may actually be inhibited by cAMP. The loss of a transcriptional response to continuous cAMP stimulation is referred to as attenuation and most often results from a cAMP-dependent activation of protein phosphatase 1 and the dephosphorylation of CREB and/or some other phosphorylated transcription factor (Montminy 1997). Alternatively, a switch from an increase to a decrease in transcription in response to the same stimulus has been shown to result from a change in the mix of available trans-acting factors. For example, the Gadd153 (a.k.a. Chop) gene expressed in PC12 cells exhibits a biphasic response to continued presentation of the inducing stimulus that involves the displacement on the Gadd153 promoter of the activator ATF-4 by the newly synthesized repressor protein ATF-3 (Fawcett et al. 1999). With respect to GTPCH gene transcription and cAMP either mechanism may be operating in SK-N-BE(2)M17 cells.
This is not to say the post-transcriptional regulation of GTPCH mRNA stability does not take place. Indeed, the 3′ UTR of human (Nomura et al. 1993) and rat (Hatakeyama et al. 1992) GTPCH mRNA both contain cis-acting elements that are often involved in determining mRNA stability. Furthermore, while cAMP is reported to increase GTPCH mRNA stability (Pluss et al. 1996) glucocorticoids appear to destabilize this transcript (Simmons et al. 1996). Even under basal conditions the stability of GTPCH mRNA differs across cell types, with the estimated half-life reported here for GTPCH mRNA in SK-N-BE(2)M17 cells being two- to seven-fold greater than that observed previously for either rat cardiac endothelial cells (4.8 h) (Simmons et al. 1996) or rat renal mesangial cells (1.5 h) (Pluss et al. 1996). Post-transcriptional control of GTPCH mRNA stability may thus be an important process regulated in a cell type-specific manner.
The results of the present study show that in transient transfection assays the human GTPCH promoter performs very much like that of the rat. This analysis differs from that reported previously by Witter et al. (1996), possibly because in that study a non-neuronal cell type that does not constitutively express GTPCH was used to examine promoter function. While the human and rat proximal promoters demonstrate similarity of form and function there are still a number of interesting differences that should be noted here.
First, in vitro footprinting of the human proximal promoter identified an upstream Sp1-like site and the entire combined CRE-Sp1-CCAAT-box element as the only two protein-binding domains. This is in contrast to the rat promoter, where an identical analysis using PC12 nuclear extracts identified three protein-binding domains including an upstream Sp1-like site, the CCAAT-box and an E-box but not the CRE (Kapatos et al. 2000). In the human sequence the rat E-box is disrupted by a single base change (CAGCTT for human versus CAGCTG for the rat) that presumably eliminates binding of what we believe to be a ubiquitous factor. Located 17 bp upstream from the rat cap site (Kapatos et al. 2000) and imbedded within the E-box is the transcription start site for the human gene (Witter et al. 1996). As protein binding to the E-box and binding of TFIID to the transcription start site are presumably mutually exclusive this evolutionary change in the E-box sequence may have been responsible for the upstream shift in the human start site. The ability of nuclear extracts to footprint the human but not the rat CRE may also be related to a single base change (TGACGCGA for the human vs. TGACGCAA for the rat) and thus the proteins recruited by this DNA element. Alternatively, the proteins available for binding to this site may differ in nuclear extracts prepared from PC12 and SKN-BE(2)M17 cells.
Second, the footprint of the human upstream Sp1-like element was found to span 10 bp and corresponds to the most 5′ Sp1-like site located 20 bp upstream from the CRE. In contrast, the footprint of the Sp1-like element in the rat promoter spans over 20 bp and corresponds to the more 3′ Sp1-like site located within 5 bp of the CRE. The upstream Sp1-like element in the rat GTPCH proximal promoter appears to inhibit transcription (Kapatos et al. 2000).
Third, the human CRE-Sp1-CCAAT-box domain spans almost 50 bp and located within it are three DNase I hypersensitive sites. This differs from the rat promoter, where the CCAAT-box footprint covers only 20 bp and no evidence for protein-dependent DNA deformation was apparent. The CRE-Sp1-CCAAT-box element also binds the transcription factors NF-Y and ATF-2 while the rat CRE-CCAAT-box cassette binds NF-Y, ATF-4 and C/EBPβ. Although these differences could simply be due to the mix of proteins available for binding there is also no doubt that the human CRE-Sp1-CCAAT-box element attracts a protein complex that is larger and able to distort DNA in a way that proteins recruited to the rat CRE-CCAAT-box cassette cannot.
Finally, while the spacing between the CRE and CCAAT-box is identical in the human and rat promoters, the intervening sequence in the human element is GC-rich (GAGGCGGGGC for human versus AAGAGGCTCG for rat). While we predicted that this overlapping Sp1-like site might have a role in human transcription, no members of the Sp1 or Egr-1 families of transcription factors known to bind GC-rich sequences were found associated with it. These negative results do not necessarily mean that the putative element is unoccupied and the hypersensitive C located in the center of this site may indicate otherwise.
The studies reported here identify NF-Y and ATF-2 as proteins recruited to the CRE-Sp1-CCAAT-box cassette. Based upon our previous study of the rat GTPCH promoter it is highly likely that this combined element is involved in the response of the human GTPCH promoter to cAMP and that ATF-2 is recruited to the CRE, while NF-Y is bound by the CCAAT-box. ATF-2 is an activator protein, a member of the family of bZip transcription factors and is able to heterodimerize with c-Jun and thus to bind to non-canonical CRE response elements such as that found in the GTPCH promoter (Hai and Curran 1991). However, c-Jun was not a part of the ATF-2 binding complex reported here. ATF-2 is phosphorylated by MAPK, p38 and JNK on two threonine residues located within the transactivation domain but does not appear to be a substrate for PKA (Kageyama et al. 1991; Gupta et al. 1995). NF-Y is a ubiquitous heterotrimeric transcription factor composed of A, B and C subunits all of which must be present before binding DNA (Chodosh et al. 1988; Li et al. 1992; Sinha et al. 1995). NF-Y is not reported to be a substrate for any protein kinase. While it is likely that ATF-2 is recruited to the human GTPCH CRE, a recent report has shown that NF-Y and ATF-2 can form a stable complex in the absence of a CRE and thus DNA binding by ATF-2 (Fritz and Kaina 2001). Perhaps this direct interaction between ATF-2 and NF-Y also forms the basis for the ability of ATF-2 bound to a CRE to facilitate the binding of NF-Y to an adjacent CCAAT-box (Srebrow et al. 1993). ATF-2 has recently been shown to have intrinsic histone acetyltransferase activity that is controlled by phosphorylation (Kawasaki et al. 2000). Similarly, NF-Y is able to mobilize histone acetyltransferase activity by interacting directly with CBP and P300/CBP-associated factor (Bevilacqua et al. 1997; Currie 1997) as well as now with ATF-2 (Fritz and Kaina 2001). These observations suggest that the combination of NF-Y and ATF-2 binding may activate transcription of the endogenous human GTPCH promoter by direct effects on chromatin structure.
The human and rat GTPCH promoters have much in common with the promoters of the fibronectin (Muro et al. 1992), hexokinase II (Osawa et al. 1996) and arylalkylamine N-acetyltransferase (Baler et al. 1997) genes in that each responds to cAMP with an increase in transcription that is dependent upon a CCAAT-box and a CRE separated by approximately one or two turns of the DNA helix and that bind NF-Y along with the bZip family members CREB, ATF-1, ATF-2, ATF-4 or C/EBPβ. The combination of a CRE and an adjacent CCAAT-box is therefore a highly conserved and evolutionarily successful cis-acting element for the cAMP-dependent enhancement of gene transcription and presumably is capable of stimulating transcription in a manner that a CRE or a CCAAT-box acting alone cannot.