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. 1997 Aug 5;94(16):8842-7.
doi: 10.1073/pnas.94.16.8842.

A calcium responsive element that regulates expression of two calcium binding proteins in Purkinje cells

D B Arnold  1 , N Heintz
Affiliations

A calcium responsive element that regulates expression of two calcium binding proteins in Purkinje cells

D B Arnold et al. Proc Natl Acad Sci U S A. .

Abstract

Calbindin D28 encodes a calcium binding protein that is expressed in the cerebellum exclusively in Purkinje cells. We have used biolistic transfection of organotypic slices of P12 cerebellum to identify a 40-bp element from the calbindin promoter that is necessary and sufficient for Purkinje cell specific expression in this transient in situ assay. This element (PCE1) is also present in the calmodulin II promoter, which regulates expression of a second Purkinje cell Ca2+ binding protein. Expression of high levels of exogenous calbindin or calretinin decreased transcription mediated by PCE1 in Purkinje cells 2.5- to 3-fold, whereas the presence of 1 microM ionomycin in the extracellular medium increased expression. These results demonstrate that PCE1 is a component of a cell-specific and Ca2+-sensitive transcriptional regulatory mechanism that may play a key role in setting the Ca2+ buffering capacity of Purkinje cells.

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Figures

Figure 1
Figure 1
P12 cerebellar slices cotransfected with calbindin and CMV promoter constructs. (A) P12 parasagittal slices of murine cerebellum were cotransfected with CMVAP and CMV-nLacZ, incubated for 48 hr, and then fixed and stained with fluorescence-conjugated antibodies. Alkaline phosphatase (red) is located throughout the entire cell, whereas β-galactosidase (yellow) is located only in the nucleus. Purkinje cells are marked with an arrow. (Bar, 100 μm.) (B) Cerebellar slices were cotransfected with CMVAP and a calbindin promoter construct extending from −1,010 to +105 (with respect to the transcription start site) driving nLacZ. Note that while many different cell types express alkaline phosphatase, only Purkinje cells (denoted by arrows) also express β-galactosidase. (Bar, 100 μm.) (C) Two Purkinje cells that illustrate the discrete nature of nLacZ expression in cotransfected cells. One Purkinje cell expresses β-galactosidase in addition to alkaline phosphatase (as indicated by the yellow nucleus) whereas the other expresses only alkaline phosphatase. (Bar, 50 μm.)
Figure 2
Figure 2
Identification of PCE1 by transfection of truncated calbindin promoter constructs into P12 cerebellar slices. (A) Calbindin promoter constructs extending from −1,010, −155, −85, and −45 to +105 and driving expression of nLacZ each were cotransfected along with CMVAP into P12 cerebellar slices. After incubation for 48 hr they were fixed and stained for presence of β-galactosidase and alkaline phosphatase using either immunocytochemistry or chromogenic enzymatic reactions. The percentage of transfected cells expressing β-galactosidase refers to the ratio of the number of cells expressing β-galactosidase to the number expressing alkaline phosphatase both for Purkinje and non-Purkinje cells. n represents the number of transfected cells in each case. Mutations in the −1,010 promoter were introduced into PCE1 in two of 10-bp repeats to prepare the construct named mutated PCE1. The sequence of the mutated region and the results of these assays also are included. (B) One construct consisting of the minimal adeno major late promoter (pML), which extends from −40 to +66 of the adenovirus major late promoter, driving nLacZ, and another consisting of pML with PCE1 (see A for sequence) added at the 5′ end (PCE1/pML) and also driving nLacZ, each were cotransfected along with CMVAP into cerebellar slices. (C) Slices were cotransfected with PCE1/pML and CMVAP and then immunostained as in Fig. 1. Purkinje cells (marked by arrows) express both β-galactosidase and alkaline phosphatase, whereas non-Purkinje cells express only alkaline phosphatase.
Figure 3
Figure 3
Two constructs, the first consisting of the calmodulin II promoter construct extending from −278 to +54, and the second consisting of the same construct, except that 20 of the 40 nucleotides comprising its PCE1 sequence (PCE1*) were altered (as shown in the sequence labeled mutated PCE1*), were cotransfected along with CMVAP into cerebellar slices. In addition, a construct consisting of pML with PCE1* added to its 5′ end (PCE1*/pML) was also cotransfected with CMVAP into cerebellar slices. The three 10 nucleotide repeats are underlined in the sequence labeled PCE1*.
Figure 4
Figure 4
Comparison of PCE1 in the calbindin and calmodulin II promoters of different species. Comparison of the promoter sequence immediately 5′ of the TATA box for mouse calbindin D28k (11), human calbindin D27k (21), mouse calmodulin II (20), dog calmodulin II (20), and human calmodulin II (J. D. Karkera and F. Friedberg, unpublished results). Repeated motifs are surrounded by boxes. Comparison of individual motifs comprising PCE1 from the different promoters shown in part was used to derive the consensus sequence.
Figure 5
Figure 5
The effect of modulating the level of intracellular free Ca2+ on expression mediated by PCE1. (A) Increasing the concentration of intracellular free Ca2+ causes an increase in the activity of PCE1. Cerebellar slices were cotransfected with CMVAP and PCE1/pML driving nLacZ with and without 1.0 μM ionomycin in the medium. The resulting percentages of transfected cells expressing β-galactosidase were calculated and graphed as in Fig. 2A. (B) Decreasing the concentration of intracellular free Ca2+ by cotransfection with calcium buffers causes a decrease in the activity of PCE1, both in the context of the native promoter and when combined with pML. Cerebellar slices were cotransfected with CMVAP, either PCE1/pML driving nLacZ or the 85-bp calbindin promoter driving nLacZ, and one of either the GW1 promoter alone, GW1 driving calbindin, or GW1 driving calretinin. The resulting percentages of transfected cells expressing β-galactosidase were calculated and graphed as in Fig. 2.
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References

    1. Darnell J E, Kerr I M, Stark G R. Science. 1994;264:1415–1421. - PubMed
    1. Treisman R. EMBO J. 1995;14:4905–4913. - PMC - PubMed
    1. Miale I, Sidman R. Exp Neurol. 1961;4:277–296. - PubMed
    1. Enderlin S, Norman A W, Celio M R. Anat Embryol. 1987;177:15–28. - PubMed
    1. Garcia Segura L M, Baetens D, Roth J, Norman A W, Orci L. Brain Res. 1984;296:75–86. - PubMed

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