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Functional characterization of the 5' flanking region of the BACE gene: identification of a 91 bp fragment involved in basal level of BACE promoter expression¹

YUAN-WEN GE^*, BRYAN MALONEY^*, KUMAR SAMBAMURTI and DEBOMOY K. LAHIRI^*,,2

^* Departments of Psychiatry and of
Medical and Molecular Genetics, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA; and
Medical University of South Carolina, Charleston, South Carolina, USA

² Correspondence: 791 N. Union Drive, Indianapolis, IN 46206, USA. Email: dlahiri{at}iupui.edu

SPECIFIC AIMS

The aims of this study are 1) to functionally characterize regulatory domains within the BACE gene (i.e., to understand regulation of BACE expression); 2) to examine regulatory domains within the BACE gene; 3) to determine expression levels of BACE regulatory regions in different cell types; 4) to investigate whether the BACE promoter region binds to cell-type specific proteins; and 5) to examine how the BACE gene is regulated by different transcription factors.

PRINCIPAL FINDINGS

1. BACE1 5'-flanking region contains positive and negative regulatory elements
To map regulatory functions of the BACE1 5'-flanking region including the promoter (BACEP), we subcloned different fragments of the sequence into the pBLCAT3 vector to produce sixteen BACEP-CAT reporter clones. These were transfected into PC12 neuronal cells and adjusted CAT protein expression analyzed. Statistical analysis of adjusted expression revealed both positive and negative regulatory elements throughout the sequence (Fig. 1 A).

View larger version (33K): [in this window] [in a new window]   Figure 1. Transfection of BACE-deletion constructs in PC12, C6, and SK-N-SH cell lines. A) Sixteen BACEP-CAT expression constructs were transfected into PC12 cells in parallel with ß-GAL control vector. CAT activity was measured by ELISA, normalized against ß-galactosidase (ß-GAL) activity, and then adjusted for relative clone length. These results were then divided by the normalized CAT activity signal for the pBLCAT3 vector. Letters indicate whether two deletions differ significantly at P < 0.05. Clones sharing the same category letter are not significantly different. Numbers separated by a slash indicate specific bases in the BACEP sequence. Numbers in square brackets indicate some bases are from vector DNA. B) and C) C6, PC12, and SK-N-SH cells were transfected with pBLCAT3 (vector), pBACE1 and pBACE3 plasmids, and CAT assay was conducted. Plain letters indicate REGWQ categories and underlined letters indicate Waller-Duncan categories. Data that share categories are not significantly different. Results are presented according to plasmid (B), with statistical analysis over all samples and according to cell type (C), with statistics restricted to within cell type.

The full promoter appears to consist of a far upstream positive regulatory element flanked on either side by a negative regulatory element. This is separated from the basic promoter and 5'-UTR by a neutral region of roughly 700 bp in length. The region from –3765 to –2975 (transcription start site, TSS, as +1) appears to have a negative regulatory function, as its deletion significantly enhanced CAT expression. The –2975 to –2062 region exerts a distinct and strong positive influence on expression. This region is insufficient to drive CAT expression on its own. The –2062 to –1056 region is a negative element, as its deletion significantly enhanced expression. Deletion of the –1056 to –327 region yields no significant effect on CAT expression, so this region is likely to not be important in basal BACE1 gene expression. This result does not preclude it from having an inducible function.

2. Determination of the minimum promoter element for basal level of BACE expression
The strongest near-transcription start expression was for a fragment of from –327 to +364. Removing a 141 bp (+224/+364) fragment significantly decreased expression. This 141 bp fragment was able to drive CAT expression at levels significantly higher than background, although promoter activity of –327 to +224 was more than twice that of the 141 bp fragment, suggesting that –327 to +224 contains the core BACE1 promoter while +224 to +364 acts more in an enhancement capacity. 91 bp (+224/+314) and 50 bp (+315/+365) drove basal expression at 4 to 5-fold greater levels than the vector. However, only the former was significant with Dunnett and Waller-Duncan test. The 91 bp fragment most likely constitutes the minimal promoter element for BACE expression. This region and the 141 bp region also specifically bind nuclear proteins as demonstrated by subsequent gel shift assays.

3. Determination of directionality of BACE promoter
Direction of expression was checked by testing activity of 4 different promoter regions in both orientations with respect to TSS. These reverse-oriented constructs, BACEP1R, BACEP3R BACEP10R, and BACEP11R did not show any significant level of activity from the vector (Fig. 1A ). This rules out bidirectional promoter activity within the region studied.

4. Cell-type specific expression of BACE promoter
To determine how specific BACEP activity was to cell type, we transfected glial (C6), neuronal (PC12), and neuroblastoma (SK-N-SH) cells with two BACE promoter regions: pBACEP1 and pBACEP2. Data was analyzed via two-way ANOVA with SAS, followed by REGWQ and Waller-Duncan means separation (Fig. 1B, C ). Variation in expression was very highly significant (P<0.0001) according to both cell type and clone used. Difference in expression (pBACEP1 higher than pBACEP3) mirrors our previous assay (Fig. 1A ). Significant (P<0.0001) interaction was revealed between both cell type and specific clone, suggesting that sub-elements of the BACE1 promoter might themselves have tissue specificity.

5. Binding of 141 bp region with nuclear proteins (AP2 and SP1)
The 141 bp fragment (+224/+364) showed specific protein-DNA interaction in electrophoretic mobility (gel shift) shift assay (EMSA) in nuclear extracts from two independent human neuroblastoma (Kelly and SK-N-BE) cell lines. This interaction did not appear in HeLa extracts to any great extent. In SK-N-BE nuclear extracts, interaction was strongly blocked by excess cold 141 bp fragment and by AP2 binding oligomer. It was less strongly blocked by excess SP1 binding oligomer, but AP1 oligomer does not seem to inhibit DNA–protein interaction (Fig. 2 A). These results confirm predicted binding sites for AP2 and SP1 in this region.

View larger version (97K): [in this window] [in a new window]   Figure 2. Competitive EMSA of 141 bp fragment of BACE 5'-UTR and comparison with certain TF binding sites. A) A BamHI/SalI fragment corresponding to 141 bp of the BACE 5'-UTR was used in the gel shift assay with several different cell nuclear extracts, subject to different oligomer competition. Lane 1 is unbound probe. Lanes 2, 3, and 4 used PC12, U373, and Kelly cell nuclear extracts. Lanes 5–9 used SK-N-BE nuclear extracts; lanes 10–12 used HeLa nuclear extracts. Lane 6 was competed against 300x 141 bp fragment, lane 7 against SP1-binding oligomer, lanes 8 and 12 against AP1-binding oligomer, and lane 9 against AP2-binding oligomer. B) HeLa cell nuclear extracts were used. TPA-induced nuclear extracts were used in lanes 5–7 and 12–14. Lanes 1–7 were probed with AP1 oligomer, lanes 8–14 with AP2 oligomer. Lanes 2, 6, and 9 were competed against 300x AP1 oligomer. Lanes 3, 10, and 13 were competed against 300X AP2 oligomer. Lanes 4, 7, 11, and 14 were competed against 300X 91 bp oligomer. Specific DNA–protein complexes are shown by solid arrows and free probe by broken arrow at bottom of gel.

6. Binding of 91 bp region with nuclear proteins (AP2)
EMSA was carried out with AP1 and AP2 binding oligomers in HeLa nuclear extract showed strong interaction. Competition against the 91 bp fragment revealed that while the AP1 oligomer and the 91 bp fragment would not compete against each other for DNA–protein interaction, the 91 bp fragment interfered with the AP2 oligomer’s binding capacity (Fig. 2B ). An AP2 sequence is predicted to occur in the 91 bp fragment. A supershift assay was carried out with an antibody against the AP2 protein, and there was an effect on the DNA–protein band when labeled with the AP2 fragment. Repetition of this assay substituting the 91 bp fragment oligomer revealed that binding would be reduced in presence of anti-AP2. AP2 or an analog may be operating on this segment of BACE1 promoter/5'-UTR.

CONCLUSIONS AND SIGNIFICANCE

Processing of APP to toxic Aß requires participation of BACE, however structure and function of its gene are poorly understood. We report functional characterization of the human BACE promoter and show that a short regulatory region of 141 bp around TSS containing a likely AP2 site mediates neuron-specific BACE transcription. Transcription of BACE is further modulated by additional regulatory elements that reside over 2000 bp from TSS. Transcription factors (TF) that bind these regions need to be carefully examined in human brain to determine whether any of these elements are affected in aging brain or in AD patients. Gel shift data revealed strong DNA–protein interaction with a small 5'-UTR fragment (+224/+364) in nuclear extracts from human neuroblastoma cells but not from HeLa extracts to any great extent, suggesting a mechanism to investigate for cell-specificity. To specify the binding site for AP2 within 141 bp region, we performed competitive and gel supershift assay with a smaller 91 bp fragment. Our results suggest that AP1 binding is not found in the 91 bp fragment. However, both the AP2-binding oligomer and the 91 bp fragment successfully interfered with each other’s binding capacity. An AP2 sequence is predicted to occur in the 91 bp fragment. Therefore, an AP2 analog may actually be operating on this segment of BACE1 promoter/5'-UTR. This information has led us to propose a specific model for the BACE1 proximal promoter region (Fig. 3 ) While this manuscript was in preparation, reports describing rat and human BACE promoters have been published. These analyses looked at shorter regions of respective BACE promoter regions and only detected proximal regulators. Our study compares the human promoter with these reports, identifies novel regulatory mechanisms for BACE, and identifies regulatory elements far upstream of TSS. These studies will provide the basis for understanding changes in regulation of BACE in the brain as a function of aging as well as genetic and environmental insults. This aspect is likely to be easily missed by studying BACE protein as it is very stable, and transient changes in expression will be overlooked unless the protein is pulse-labeled after appropriate treatment.

View larger version (38K): [in this window] [in a new window]   Figure 3. Models of interaction of BACE proximal promoter region with nuclear proteins. A) Schematic of BACE 5'-flanking region showing six regulatory domains with proximal promoter region highlighted. Selected putative TFs and an alternate TSS are shown. B) The 141 bp fragment (+224/+364) consisting of a 91 bp (white) and a 50 bp element (vertical striped) binds a TF (most likely AP2), driving minimal expression. C) The 550 bp fragment (–327/+223 speckled) binds putative TFs, before and after the TSS or one factor that crosses the TSS, driving greater expression than seen in the 141 bp fragment. D) Combining the 550 bp and 141 bp fragments results in still greater expression, presumably via interaction of transcription elements binding to both. Higher activity is shown by increasing width of arrows. E) If the TSS is further upstream, the factors will most likely all bind within the 5'-UTR, making the BACE minimal promoter element and its 5'-UTR effectively identical.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1379fje;

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