Fetal Alz-50 Clone 1, a Novel Zinc Finger Protein, Binds a Specific DNA Sequence and Acts as a Transcriptional Regulator

J L Rhodes,J M Dragich,K L Jordan-Sciutto,Robert Bowser

doi:10.1074/jbc.274.49.35262

J L Rhodes, J M Dragich + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.49.35262

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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.

Highlights

The fetal Alz-50 clone 1 (FAC1)1 protein is a novel member of the plant homeodomain/leukemia-associated protein (PHD/ LAP) zinc finger family [1,2,3]
The first 398 amino acids of FAC1 were chosen for use in this assay because the zinc finger, basic region, and acidic region are present in this protein fragment
A FAC1-(1–398) affinity column was made by producing a fusion between FAC1-(1–398) and glutathione S-transferase (GST), which binds glutathione conjugated to Sepharose beads

Summary

EXPERIMENTAL PROCEDURES

Degenerate Double-stranded Oligonucleotides for the PCR-assisted Binding Site Selection Assay—To produce a degenerate doublestranded nucleotide, a template was synthesized containing 15 degen-. GST-FAC1 protein was purified by the specific interaction between GST and glutathione immobilized on Sepharose beads (Amersham Pharmacia Biotech) At this point, the protein was used as a FAC1 affinity column (for the PCR-assisted binding site selection assay); or the purified protein was eluted from the Sepharose column by addition of 10 mM glutathione in EMSA buffer (20 mM HEPES (pH 7.9), 20% glycerol, 200 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) followed by dialysis in EMSA buffer to remove the glutathione and was used for EMSA.

TABLE I Oligonucleotides used for EMSA and cloning

RESULTS

DISCUSSION