Regulation of the cholinergic gene locus

J Physiology (Paris) (1998) 92, 149-152 © Elsevier, Paris

Regulation of the cholinergic gene locus Hiromitsu Tanaka, Masahito Shimojo, Donghai Wu, Louis B. Hersh Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0084, USA

Astract - - DNase I hypersensitive site mapping of the human cholinergic gene locus has been used to detect cholinergic specific potential regulatory sites. Analysis of mutant PC12 cell lines provides evidence that protein kinase A II is required and coordinately regulates basal expression of both the ChAT and VAChT genes. (©Elsevier, Paris)

R~sum~ - - R~gulation du locus g~n~tique cholinergique Nous avons utilis6 la cartographie des sites hypersensibles h la DNAse I darts le locus g6n6tique cholinergique humain pour identifier des sites r6gulateurs potentiels. L'analyse de lign6es mutantes de cellules PC12 indique que la prot6ine kinase A II est impliqu6e dans la r6gulation coordonn6e de l'expression des g~nes ChAT et VAChT. (©Elsevier, Paris) choline acetyltransferase / vesicular acetylcholine transporter / transcriptional regulation / coordinate regulation / protein kinase A

1, I n t r o d u c t i o n

2. M a t e r i a l s and m e t h o d s

The cholinergic gene locus is comprised of the genes for the biosynthetic enzyme choline acetyltransferase (CHAT) and the gene for the vesicular a c e t y l c h o l i n e t r a n s p o r t e r (VAChT). The V A C h T gene, whose open reading frame is within a single exon, lies within the intron between the first and second exons of the ChAT gene. This unusual gene arrangement is conserved across such diverse species as the n e m a t o d e , Drosophila, and m a m m a l s , and suggests potential coordinate regulation of the gene. The mechanisms responsible for the transcriptional regulation o f the cholinergic gene locus have been studied by both transient cell transfection analysis as well as through transgenic mice models. Based on studies f r o m a n u m b e r o f l a b o r a t o r i e s a complex regulatory pattern at this locus is emerging. Recent transgenic mouse models [10, 13], each utilizing different non-overlapping regions o f the locus, were reported to provide cell specific expression of the locus. This suggests the existence of multiple cell specific regulatory elements in the gene. We report here two approaches to study regulation of the cholinergic gene locus. In one we analyzed the chromatin structure of the human cholinergic gene locus by DNase I hypersensitive mapping to identify potential regulatory sites in the gene. In the other we have used mutant PC12 cell lines to demonstrate c o o r d i n a t e regulation o f the ChAT and V A C h T g e n e s by a p r o t e i n k i n a s e A d e p e n d e n t mechanism.

2.1. Cell lines The cell lines used in this study include CHPI34, a cholinergic neuroblastoma cell line, HeLa, MCF-7, a human breast adenocarcinoma cell line, HeLa, PC12, and two PCI2 mutant cell lines. PC12/A123.7 expresses a mutant regulatory PKA subunit [2, 5] and contains reduced levels of PKA I and PKA II. PC 12/A 126-1 B2 was generated by nitrosoguanidine mutagenesis and lacks PKA II activity, but contains normal levels of PKA I [ 15].

2.2. DNase I hypersensitivity mapping Cell nuclei were isolated from detergent lysed cells by centrifugation and resuspended in digestion buffer (10 mM TrisHCI, pH 7.6, 5 mM MgCI2, 0.5 mM CaClz, 25 mM KC1, 0.5 mM DTT and 0.35 M sucrose). Nuclei were digested with I, 5, or 10 units of DNase I for 10 min at 25 °C. Following termination the sample was digested with proteinase K followed by phenol, phenol/chloroform, and chloroform extraction, and precipitation with ethanol. The DNA pellet was treated with RNase, followed by digestion with proteinase K, and extraction and precipitation as noted above. This DNA was digested with a suitable restriction enzyme and electrophoresed on a 0.8% agarose gel. The DNA was transferred to a Hybond N (+) membrane and hybridized with a probe radiolabeled by the random priming method [14]. The washed membrane was exposed to Kodak XAR-5 film.

2.3. Determination of choline ace~ltransferase activity' Choline acetyltransferase was assayed by a modification of the method of Fonnum [4] as described by Hersh [6].

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2.4. Competitive PCR Total cellular RNA was isolated from cultured cells by the method of Chomczynski and Sacchi [1]. Single-stranded cDNAs were synthesized using Molony murine leukemia virus (M-MLV) reverse transcriptase using specific primers for VAChT or CHAT. Single-stranded cDNA (0.1 lag) was amplified by PCR in a programmable thermal cycler that included variable amounts of an appropriate internal standard and a labeled nucleotide. Following amplification the PCR product was electrophoresed on a 5% polyacrylamide gel and autoradiographed. Signal intensities were quantified directly from the bands with a StormtmPhosphorlmager and Image Quant.

2.5. Other materials RNase inhibitor, dibutyryl cAME and deoxynucleotides were purchased from Boehringer Mannheim (Indianapolis, IN, USA). DNase I was from Promega (Madison, WI, USA). H-89 and H-9 dihydrochloride were obtained from Calbiochem (La Jolla, CA, USA). M-MLV reverse transcriptase and Taq DNA polymerase were purchased from Promega (Madison, WI). 8- (4-chlorophenylthio) adenosine-3',5'-cyclic monophosphate (8-CPT-cAMP), 8-piperidinoadenosine -3',5'-cyclic monophosphate (8-PIP-cAMP), 8- (4-aminohexyl) aminoadenosine-3',5'-cyclic monophosphate (8-AHAcAMP), and N6-benzoyladenosine-3',5'-cyclic monophosphate (N6-BNZ-cAMP) were purchased from BioLog Life Science Institute (Bremen, Germany). All other reagents were from Sigma Chem. Co. (St. Louis, MO, USA).

3. Results

DNase I hypersensitive site mapping was employed as a method to locate specific elements involved in the regulation of the human cholinergic gene locus. CHP134 cells were used as a cholinergic cell line and HeLa cells and MCF-7 cells as noncholinergic cell lines. A variety of probes were emp l o y e d to d e t e c t the D N a s e I d e p e n d e n t fragmentation pattern of nuclear DNA digested with restriction enzymes. This analysis led to the identification of eight DNase I hypersensitive sites in the cholinergic cell line (figure 1). In contrast the two non-cholinergic cell lines showed two DNase I hypersensitive sites (figure 1). Analysis of the two DNase I hypersensitive sites seen in the inactive cholinergic gone from the noncholinergic cell lines indicated that site 1' corresponds to the neuronal restrictive silencer element, NRSE, previously identified in the gene [11, 3]. The other site, site 6, corresponded to an enhancer element identified in this laboratory [9]. Of the eight DNase I hypersensitive sites found in active chromatin from CHP 134 cells six of these appear to correspond to core promoter regions, site 2, R promoter, site 3, VAChT promoter, site 4, VI VAChT

promoter, site 7, N promoter, site 8, M promoter. In addition site 1 is a cholinergic specific site which resides in the vicinity of the NRSE, site 5 appears to be an enhancer element, while site 6 is the previously identified enhancer element noted above. These latter sites may be key regulators in the cell specific expression of this locus. In a different approach to study regulation of the cholinergic gene locus we utilized two protein kinase A (PKA) mutant cell lines, A I 2 6 - 1 B 2 which is deficient in PKA II and A123.7 which has decreased levels of PKA I and PKA II. A PCR based assay was used to demonstrate that both ChAT and VAChT m R N A levels in the mutant cell lines were significantly decreased (figure 2). Treatment of the parental P C I 2 cell line, but not the P K A mutants, with dbcAMP increased ChAT m R N A levels 3.6-fold and VAChT m R N A levels 4.2-fold. Treatment of the parental P C I 2 cell line with two selective PKA inhibitors H-9 or H-89 reduced both ChAT and VAChT m R N A levels to ~ 1 / 3 of that in the untreated cells (figure 2). The reduction of VAChT mRNA again paralleled that of ChAT mRNA. The dbcAMP induced increase in ChAT m R N A was paralleled by a 5-fold increase in ChAT activity, and H89 blocked this increase. H89 and H9 alone decreased ChAT activity to 1/4 and 1/5 the control level, respectively. The above results suggest that PKA II, but not PKA I, may regulate the cholinergic gene locus. We confirmed this by demonstrating 2.5 fold increase increases in ChAT activity produced by a combination of 8-CPT-cAMP and N6-BNZ-cAMP. which selectively activates P K A II, with no effect of a c o m b i n a t i o n of 8 - A H A - c A M P and 8 - P I P - c A M P which selectively activates PKA I.

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Figure 1. DNase I hypersensitive sites of the cholinergic gene locus in CHP134, HeLa and MCF-7 cells. The R, N, M, and first coding exons are illustrated as well as the VAChT gene open reading frame. Arrows indicate the DNase 1 hypersensitive sites.

Xth International Symposium on Cholinergic Mechanisms

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monstrate that both ChAT and VAChT gene transcription are regulated by protein kinase A, and that this regulation is coordinate. The finding that ChAT and VAChT m R N A levels are reduced in a cell line which contains wild type levels of PKA I, but is devoid of PKA II activity, and the ability of PKA II agonists, but not PKA I agonists, to increase ChAT activity shows that regulation of the cholinergic gene is through a PKA II signaling pathway. Transient transfection analysis indicates that protein kinase A can act through a site on the h u m a n ChAT gene upstream of the VAChT promoter. This is clearly a different site than the CRE-like element shown by Misawa et al. [12] to be localized between the M exon and the first coding exon. The mechanism by which PKA II regulates the cholinergic gene locus is currently under investigation.

4. Discussion Acknowledgments For many genes, it has been reported that the elements which correspond to DNase I hypersensitive sites play an important role for transcriptional regulation. We observed a number of cholinergic specific DNase l hypersensitive sites in regions of the gene which do not exhibit cell specific reporter gene activity [7]. It is possible that these sites are cholinergic cell specific as a result of the chromatin structure of the cholinergic gene locus. An interesting finding is the presence of a DNase I hypersensitivity site, site 1, which is located more than 1 kb upstream of the R exon. This is the region of the cholinergic gene locus shown to contain a cholinergic specific repressor in the rat gene [8]. The presence of s i t e - l ' in non-cholinergic cells, which corresponds to the NRSE, is understandable since this element can repress expression of the cholinergic gene in non-neuronal cells [11]. However, the presence of site 6, which corresponds to an enhancer element, is unexpected. We suggest that this site might act in conjunction with the NRSE to silence this locus in non-cholinergic cells. Studies with protein kinase A deficient P C I 2 cells establish the requirement for this kinase for basal expression of both the ChAT and VAChT genes. Furthermore this regulation appears to be coordinate with these two genes, In the parental P C I 2 cell line, dbcAMP induced both ChAT and VAChT mRNAs in a parallel fashion. Similarly a parallel reduction in ChAT and VAChT mRNA levels was produced by the PKA inhibitors H-89 and H-9. In the protein kinase A d e f i c i e n t cell lines c o n s i d e r a b l y lower ChAT and VAChT m R N A levels were observed compared to the P C I 2 parental line. These findings de-

We thank Dr. John Wagner, Cornell University, for providing the PC I2 cell lines used in this study. This work was supported by grants from the NIH, NIA AG05893 and AG05144.

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