Fetal Alz-50 clone 1, a novel zinc finger protein, binds a specific DNA sequence and acts as a transcriptional regulator

Abstract

Fetal Alz-50 clone 1 (FAC1) is a novel, developmentally regulated gene that exhibits changes in protein expression and subcellular localization during neuronal development and neurodegeneration. To understand the functional implications of altered subcellular localization, we have established a normal cellular function of FAC1. The FAC1 amino acid sequence contains regional homology to transcriptional regulators. Using the polymerase chain reaction-assisted binding site selection assay, we have identified a DNA sequence recognized by recombinant FAC1. Mutation of any 2 adjacent base pairs in the identified binding site dramatically reduced the binding preference of FAC1, demonstrating that the binding is specific for the identified site. Nuclear extracts from neural and non-neural cell lines contained a DNA-binding activity with similar specificity and nucleotide requirements as the recombinant FAC1 protein. This DNA-binding activity can be attributed to FAC1 since it is dependent upon the presence of FAC1 and behaves identically on a nondenaturing polyacrylamide gel as transiently transfected FAC1. In NIH3T3 cells, luciferase reporter plasmids containing the identified binding site (CACAACAC) were repressed by cotransfected FAC1 whether the binding site was proximal or distal to the transcription initiation site. This study indicates that FAC1 is a DNA-binding protein that functions as a transcription factor when localized to the nucleus.

Fig. 1. Alignment of representative DNA sequences obtained in the PCR-assisted binding site selection assay showing the identified consensus sequence

The identified consensus site is listed at the top with the alignment of representative sequences listed underneath. The percentage of the 24 isolated clones containing a specific nucleotide is listed above the indicated consensus base. A core sequence of AACA is present in the majority of identified sequences.

Fig. 2. Recombinant FAC1 specifically recognizes the identified FBE

A, representative EMSA using ³²P-labeled FBE oligonucleotides as probes and incubated with the FBE probe alone (lane 1), affinity-purified GST-FAC1-(1–398) (lane 2), GST-FAC1-(1–398) and unlabeled FBE competitor (30 ng excess) (lane 3), GST-FAC1-(1–398) and unlabeled FBE competitor (100 ng excess) (lane 4), GST-FAC1-(1–398) and unlabeled unrelated (Unrel.) competitor (100 ng excess) (lane 5), and GST-FAC1-(1–398) and unlabeled unrelated competitor (300 ng excess) (lane 6). FBE WT, wild-type FBE. B, mutation of the FBE results in decreased binding affinity. Shown is a representative EMSA using the FBE probe (lane 1) and affinity-purified GST-FAC1-(1–398) (lane 2), which compete for binding with three mutant FBE sites (mutants 1–3 (MT1–MT3)) (Table I) at 30, 100, and 300 ng of unlabeled oligonucleotide added (lanes 3–11). C, quantitation of mutant FAC1 competitors. The amount of unlabeled competitor is shown on the x axis and is plotted against the amount of DNA-protein complex at that concentration of competitor. The y axis values are presented as a percentage of DNA-FAC1 complex when no competitor is added. The values shown are an average of three experiments quantified by NIH Image Version 1.5.8 software. Wild-type competitor (open squares), mutant 1 (open diamonds), mutant 2 (open circles), mutant 3 (open triangles), and unrelated competitor (hatched squares) are shown.

Fig. 3. Endogenous FAC1 specifically recognizes the identified FBE

A, immunoblot for FAC1 in cytosolic (cyto) and nuclear (nuc) extracts from the indicated cell lines. B, EMSA with 30 μg of MSN-2 (lane 2), PC12 (lane 3), and NIH3T3 (lane 4) nuclear extracts, each with 3 μg of salmon sperm DNA. Lane 1 is probe alone. C, representative EMSA using the FBE as a probe (lane 1) and 12 μg of PC12 nuclear extracts plus 1 μg of salmon sperm DNA (lanes 2 and 12). The indicated unlabeled competitors were added at 30, 100, and 300 ng. MT1–MT3, mutants 1–3. D, representative EMSA using the FBE as a probe with nuclear extracts from MSN-2 cells (left panel, lane 1) and COS cells (right panel, lane 1). Wild-type competitors (WT) were added at 30 and 100 ng (lanes 2 and 3 in each panel). The autoradiographs shown in C and D were exposed for different lengths of time so as not to exceed the linear range of the film and demonstrate that the binding specificity is the same in all the cell lines. They are not meant to reflect the relative quantities of FAC1 present in each of the cell lines, and each experiment was performed at least three times.

Fig. 4. DNA-protein complexes contain a protein with the same properties as FAC1

A, EMSA with 30 μg of nuclear extracts (nuc) from MSN-2 cells (lane 1), NIH3T3 cells (lane 2), and NIH3T3 cells transfected with a FAC1 expression vector (lane 3). B, EMSA with 30 μg of nuclear extracts from PT67 cells (lane 2) compared with PT67 cells transfected with a FAC1 expression vector (lanes 3–5). 100 ng each of unlabeled wild-type (WT) and unrelated (Unrel.) competitors was added.

Fig. 5. FAC1 represses transcription through the FBE

A, shown are the SV40 reporter constructs used in the cotransfection experiments. bp, base pairs. B, NIH3T3 cells were transfected with 4 μg of reporter plasmid: SV40 control (SV40), 3×FBE in the promoter (FBE-P), 3×FBE in the enhancer (FBE-E), 3×MTFBE in the promoter (MT-P), and 3×MTFBE in the enhancer (MT-E). Each reporter was cotransfected with 0.3 μg of FAC1 expression vector or control expression vector. The results shown are each reporter construct cotransfected with FAC1 and represent an average of six separate experiments. The results are presented as a percent ratio of reporter without FAC1. S.D. is indicated by the error bars.

Fig. 6. Several endogenous promoters contain FAC1-binding elements

Shown are DNA sequences containing potential FAC1-binding sites from regulatory regions of the APP gene, presenilin-1 (PS1), the dopamine receptor (D2-R), and Cu²⁺, Zn²⁺-superoxide dis-D₂ mutase-1 (SOD1). The potential FAC1-binding site is underlined, and the nucleotide positions are indicated.