(Received for publication, January 3, 1996)
From the
A screen designed to identify proteins that specifically bind to
retinoic acid response elements resulted in the identification of a rat
cDNA encoding a novel protein containing six Cys-Cys, His-Cys zinc
fingers. This gene is expressed in a restricted fashion exhibiting
distinct temporal and spatial patterns in the developing nervous
system, primarily brain, spinal cord, sensory ganglia, retina, and
nasal epithelia, as well as in the pituitary, and is referred to as
neural zinc finger factor 1 (NZF-1). NZF-1 binds specifically to a cis-regulatory element of the -retinoic acid receptor
(RAR
) gene, as well as to other related DNA elements, including
two in the upstream enhancer region of the mouse Pit-1 gene.
In heterologous cells, NZF-1 activates transcription from promoters
containing specific binding sequences and can synergize with other
factors, such as Pit-1, to regulate gene expression. These results
suggest that NZF-1 may exert regulatory roles in the developing and
mature nervous system and in the pituitary gland. Identification of a
second mouse gene highly homologous to NZF-1, encoded by a distinct
genomic locus, reveals a dispersed gene family encoding proteins
containing Cys-Cys, His-Cys motifs.
Precise temporal and spatial patterns of development are
controlled by sequential activation of a hierarchy of regulatory genes,
which encode transcription factors containing multiple classes of DNA
binding motifs. Zinc coordinated fingers are one of the most common DNA
binding motifs among eukaryotic transcription factors and are
classified based on amino acid sequence of the zinc fingers. The
Cys-Cys, His-His class, which is typified by the Xenopus transcription factor IIIA(1) , contains the largest number
of members. These proteins contain two or more fingers in a tandem
repeat. In contrast, steroid receptors, such as the glucocorticoid
receptor, contain only two zinc coordinated structures with four
(C) and five (C
) conserved cysteines. The third
class of zinc fingers, which also binds to single-stranded nucleic
acids, has a consensus sequence of
Cys-X
-Cys-X
-His-X
-Cys.
Such factors are found in transposable element copia, plants, and
mammalian cells as well as in retroviruses. Other metal-coordinating
proteins have different structures such as C
in the yeast
GAL4 protein and a cysteine-rich structure in the E1A oncoprotein (2) .
In accordance with their structural diversity, zinc finger proteins play a variety of important roles in cell growth, differentiation, and development. Transcription factor IIIA and the ubiquitous transcription factor SP1 are broadly involved in the regulation of transcription, whereas the Drosophila zinc finger proteins Krüppel and Hunchback are crucial for proper segmentation of the developing embryo(3, 4, 5) . In humans, mutations in a kidney zinc finger protein (WT1) result in Wilm's tumor(6, 7) . Recently, a zinc finger protein (REST) has been shown to repress neuronal gene expression in non-neuronal tissues(8, 9) .
Because retinoic acid receptor
(RAR) ()binds ineffectively to DNA response elements,
requesting a DNA binding co-regulator, expression screening was
performed with a
-retinoic acid response element (
RARE). This
proved to be a successful strategy for the cloning of a retinoic acid
receptor co-regulator which was identified as a member of the retinoid
X receptor family(10, 11, 12) . This
co-regulator binds DNA cooperatively as a heterodimer with the retinoic
acid receptor and other nuclear
receptors(11, 12, 13, 14, 15, 16) .
In order to investigate the mechanisms that underlie regulation of
the expression of the -retinoic acid receptor (RAR
) and Pit-1 genes further, we screened expression libraries based on
the detection of protein-DNA interactions using the
RARE and the
retinoic acid response element of the Pit-1 gene enhancer (17) . In this manuscript we report that, as a consequence of
these screens, we have identified a novel transcript encoding a
Cys-Cys, His-Cys zinc finger protein. Based on its restricted
expression pattern in the nervous system, this factor is referred to as
NZF-1 (neural zinc finger factor 1). Characterization of NZF-1 has
revealed the presence of two separate Cys-Cys, His-Cys type zinc finger
DNA binding domains, each of which can bind independently to similar
DNA sequences. The preferred consensus sequence is distinct from the
consensus retinoic acid receptor response element. The NZF-1 gene
exhibits a restricted pattern of expression in the nervous system,
testis, and pituitary gland. NZF-1 can serve as a transcription factor
and elicit synergistic activation with other factors, such as Pit-1.
Our results suggest that this novel zinc finger protein may exert roles
both in the development and function of the central nervous system and
the anterior pituitary gland.
For in situ hybridizations, cryosections of rat embryonic tissues were mounted
on poly-L-lysine-coated slides and air-dried. Pretreatment,
hybridizations, and washing conditions have been
described(20) . Briefly, sections were digested with proteinase
K (10 µg/ml, 37 °C, 30 min), acetylated, dehydrated, and dried.
80 µl of hybridization mixture containing S-labeled
probe (10
cpm/ml) was spotted on each slide. After
incubation at 55 °C for 16 h, slides were rinsed, digested with
ribonuclease A (20 µg/ml, 37 °C, 40 min), and washed in 0.1
SSC for 30 min at 65 °C. After dehydration, slides were
dipped in Kodak NTB2 autoradiographic emulsion (diluted 1:1 with
distilled water), dried in a humid chamber (2-4 h), exposed
desiccated in the dark at 4 °C for two weeks, and developed with
Kodak D-19, followed by Autoradiographic Fixer.
Mapping of the genes for Nzf-1 and Nzf-2 in mouse
was performed by linkage analysis of an interspecific backcross.
Progeny from a (C57BL/6J Mus spretus) F1
C57BL/6J backcross were typed for about 350 genetic markers spanning
the mouse genome as described previously(21) . To identify
restriction fragment length variants (RFLVs), DNAs from the parental
strains and F1 mice were digested with 10 different restriction enzymes
followed by Southern analysis. A 1060-bp XhoI-BamHI
fragment from the rat NZF-1 cDNA and a 1.3-kb XbaI fragment
from the mouse NZF-2 cDNA were utilized as probes. Blots were
hybridized with
P-labeled probes at 65 °C and washed
in 1.0
SSC, 0.1% SDS at 50 °C essentially as described
previously(21) .
Figure 1: Primary structure of NZF-1 protein. A, the predicted amino acid sequence of NZF-1. Six Cys-Cys, His-Cys zinc finger regions are boxed; a Glu/Asp-rich region and a Ser/Thr-rich region are indicated by a dashed line and a solid line, respectively. B, a schematic diagram of the NZF-1 cDNA generated by six overlapping cDNA clones. Each filled circle represents a zinc finger motif. Clones 12A and 3B are alternative splice variants, each lacking one zinc finger motif. C, amino acid sequence comparison of the six zinc fingers.
In electrophoretic
mobility shift assays, purified GST fusion proteins were preincubated
in 20 µl of binding buffer (50 mM HEPES, pH 7.9, 50 mM KCl, 5% glycerol, 2 mg/ml bovine serum albumin, 10 µM ZnSO) containing 1 µg of poly(dI-dC) and 0.1
µg of salmon sperm DNA for 5 min. Samples were incubated for an
additional 25 min at room temperature after adding
P-radiolabeled probe (0.1-0.5 ng). 4.5 µl of
each reaction was loaded onto a 5% nondenaturing 0.5
TBE
polyacrylamide gel and electrophoresed at 20 V/cm for 60 min. Gels were
dried and autoradiographed.
For methylation interference
experiments, oligonucleotides were labeled at the 5` end using T4
kinase and -[
P]ATP. After annealing,
double-stranded probes were gel-purified and methylated with dimethyl
sulfate. Binding reactions were carried out as described above. Free
and protein-bound DNAs were separated by electrophoresis on
nondenaturing polyacrylamide gels and subjected to piperidine cleavage
after elution. Cleaved products were separated on 12% polyacrylamide
gels containing 7 M urea.
The sequence predicted an 1187-amino acid protein that contained six Cys-Cys, His-Cys type zinc fingers, organized in clusters of two and three zinc fingers, separated by 329 amino acids, as well as a single zinc finger near the amino terminus (Fig. 1A). The amino acid sequences of the six zinc fingers share striking homology with each other (Fig. 1C). The NZF-1 protein contains a highly acidic region in the amino terminus (79% Asp or Glu, between amino acids 88 and 173), and a serine/threonine-rich sequence located between the two clusters of zinc fingers (35% Ser or Thr, between amino acids 625 and 715) (Fig. 1A).
We subsequently identified two alternatively spliced forms of NZF-1, which predict deletions of 63 and 135 nucleotides, removing coding information for the first and second zinc fingers, respectively. In an RNase protection assay, one of the splice forms is expressed at levels significant when compared to the predominant transcript encoding the larger 1187-amino acid protein (Fig. 1B and 2A and data not shown).
Figure 2: Expression of NZF-1 in various rat tissues and cell lines examined by RNase protection assays. A, using probe A (corresponding to amino acids 446-648), NZF-1 expression is restricted to the brain, PC12 cells, and pituitary cell lines. Open arrow indicates the protected NZF-1 messenger RNA, while the two lower bands indicate the presence of alternatively spliced forms of NZF-1. B, when using probe B (corresponding to amino acids 753-882), NZF-1 transcripts were detected in the adult testis, pituitary, and brain (indicated with open arrow). An actin probe was added as a control.
In order to determine the temporal and spatial expression patterns of NZF-1, we performed in situ hybridization experiments on both embryonic and adult rat tissues. As early as e11.5, NZF-1 expression was evident throughout the proliferating cortex neuroepithelium, the developing medulla, and in the spinal cord. At e12.5, NZF-1 mRNA was detected in the nasal epithelium as the nasal cavity forms (data not shown). At e13.5, NZF-1 expression was detected in the trigeminal ganglia, the dorsal root ganglia, and the ganglion cell layer of the retina (Fig. 3A). The expression of NZF-1 transcripts in the brain and the spinal cord reached the highest levels of detection at e14-e15 and subsequently decreased slightly ( Fig. 3and data not shown). Later in brain development, transcripts of NZF-1 were detected in the cerebellar neuroepithelium, choroid plexus primordium, and within the cephalic flexure. NZF-1 transcripts were widely expressed in the adult brain, at levels lower than that detected during development. In situ hybridization experiments revealed that in adult testis, NZF-1 was expressed in the periphery of a subset of seminiferous tubules, indicating that the mRNA was present at early stages of the developing germ cell, i.e. in spermatogonia and/or early phase of spermatocytes I (data not shown). Thus, expression of the NZF-1 gene is restricted to the nervous system, pituitary gland, and testis, exhibiting highest levels of expression in the developing central nervous system.
Figure 3:
In situ hybridization analysis of
NZF-1 gene expression during rat embryonic development. A and B, sagittal sections of e13.5 and e15.5 embryos, respectively,
were in situ hybridized with S-labeled NZF-1 cRNA
probe and visualized by photomicroscopy. NZF-1 expression was observed
in the dorsal root ganglia (DRG), the spinal cord (SC), the neuroepithelia, and the olfactory epithelia (OE). M, mesencephalon; T, telencephalon. C, D, and E, high magnification photos
showing sections of the dorsal root ganglia, the olfactory epithelia,
and the retina of an e15.5 embryo, respectively. PML,
postmitotic layer.
Figure 4: NZF-1 and NZF-2 are members of a gene family. A, NZF-1 and NZF-2 share extensive amino acid sequence homology, especially in the zinc finger regions. The amino acid sequence of zinc finger numbers 2 to 6 of NZF-1 were aligned with the sequence of zinc fingers 1, 2, 4, 5, and 6 of NZF-2, respectively, to obtain maximum homology. Identical amino acids in NZF-2 are indicated by dashed lines. B, genomic Southern blot experiments demonstrate distinct hybridization patterns for Nzf-1 and Nzf-2. Identical blots using the indicated enzymes were probed with a 400-bp NZF-1 cDNA fragment or with a NZF-2 cDNA 1.2-kb XbaI fragment. 1-kb DNA ladder was loaded on the first lane of each gel.
Genomic Southern blot analysis was employed to determine whether NZF-1 and NZF-2 were encoded by distinct genes. Duplicate Southern blots hybridized with cDNA probes encoding homologous regions of NZF-1 and NZF-2 revealed distinct patterns for the two probes, indicating they are encoded by two different genes (Fig. 4B). The chromosomal localization of the two NZF genes were determined in mouse by linkage analysis of a backcross between strains C57BL/6J and M. spretus, with C57BL/6J as the recurrent parent. A screen of restriction enzymes revealed a restriction fragment length variant (RFLV) for the NZF-1 gene, designated Nzf-1, with the enzyme PvuII. Following Southern analysis, DNA from strain C57BL/6J yielded hybridized bands of 8.0, 5.2, and 2.7 kb, DNA from M. spretus yielded bands of 9.8 and 8.0 kb, and DNA from F1 hybrids contained all these bands (data not shown). The RFLV was scored in 49 backcross mice and compared to the segregation of about 350 previously typed genetic markers. Linkage was observed with several genetic markers on proximal mouse chromosome 12 (Fig. 5). An RFLV for the Nzf-2 gene was identified using the enzyme SstI. Thus, DNA from C57BL/6J mice exhibited hybridizing bands of 7.6 and 3.3 kb, DNA from M. spretus mice exhibited bands of 10.5, 3.3, and 2.3 kb, and F1 mice exhibited all four bands (data not shown). The RFLV exhibited close linkage with the marker D2Mit25 at the distal end of mouse chromosome 2 (Fig. 5).
Figure 5:
Distinct chromosomal loci for Nzf-1 and Nzf-2. Using an interspecific backcross ((C57BL/6J
M. spretus) F1
C57BL/6J), Nzf-1 and Nzf-2 were mapped to chromosomes 12 and 2, respectively. The
ratios of the number of recombinations to the total number of mice and
the recombination frequencies ± S.E. (in centimorgans) for each
pair of loci, are indicated to the left of the chromosomes.
For pairs of loci that co-segregate, the upper 95% confidence interval
is shown in parentheses. All loci were linked with lod scores
greater than 8.0. Chromosomes are drawn to scale with the distance of
the most distal marker from the centromere (top) given below
each chromosome in centimorgans. Markers were reported in (21) or are unpublished data.
Figure 6:
DNA binding properties of the NZF-1
protein. A, electrophoretic mobility shift assays demonstrate
that specific binding of NZF-1 to DNA requires a minimum of two zinc
fingers. NZF-1 was expressed as GST fusion proteins containing either
one (1ZF), two (2ZF), three (3ZF), or five (5ZF) zinc fingers and incubated with a P-labeled
RARE probe. In the presence of competitor DNA, only 2ZF, 3ZF, and
5ZF bind
RARE specifically. B, 2ZF and 3ZF recognize
similar DNA sequences while 3ZF has more specificity. Only the bound
complexes are shown.
In order to determine the
specific nucleotide sequences that were critical for binding by these
two or three zinc finger regions, we utilized synthetic double-stranded
oligonucleotides encompassing wild-type and mutant RARE using
electrophoretic mobility shift assays. Systematic mutations altering
this element revealed the critical nucleotides of GAAAGTT motif for
binding by the 2ZF polypeptide. The region encompassing three zinc
fingers bound an additional GTT motif 4 bp 5` of the GAAAGTT core (Fig. 6B), perhaps due to the ability of an additional
finger to contact additional nucleotides, resulting in increased DNA
specificity. We therefore conclude that for all elements examined, the
core DNA element recognized by NZF-1 was GAAAGTT.
To further define
this core binding element, a series of mutant oligonucleotides were
tested for binding by the two-zinc finger segment. P-Labeled
RARE oligonucleotide was mixed with 3-,
10-, or 50-fold molar excess of unlabeled competitor oligonucleotides
before NZF-1 protein was added. These analysis identified that the
AAGTT sequence was crucial for optimal binding and that purines at the
two bases preceding this sequence are preferred for optimal binding by
the NZF-1 zinc fingers (Fig. 7).
Figure 7: Consensus DNA binding sequence for NZF-1. Four natural binding sites of NZF-1 are highly homologous. Oligonucleotides carrying mutations were used as competitors in the electrophoretic mobility shift assays using the 2ZF protein. Purines were found to be the preferred nucleotides 5` of the AGTT core (C4-C7).
Electrophoretic mobility
shift experiments confirmed that NZF-1 bound specifically to a series
of DNA sites: Pit-1 PSE, Pit-1 RDE, and the pyridoxal
phosphate promoter element (data not shown) in addition to the
RARE, and all four DNA elements used in expression cloning shared
a highly homologous core motif (Fig. 7). NZF-1 and NZF-2 share a
high degree of amino acid similarity in the zinc finger regions as well
as in the carboxyl terminus and bind to the same sequences.
Analysis
of methylation interference of binding by the methyl groups of the N-7
position of guanine and the N-3 position of adenines, indicative of
major and minor groove contacts, respectively, was then utilized to
investigate the specific contacts of the NZF-1 protein with target DNA
elements. P-Labeled probes (representing the
RARE and
the Pit-1 PSE) were methylated with dimethyl sulfate and
incubated with GST NZF-1. The pattern of inhibition of G residues in
the major groove of the consensus core motifs and of A residues showing
weaker signals in the bound complexes indicated that the core binding
sequence was consistent with that defined by mutagenesis,
electrophoretic mobility shift assays (Fig. 8).
Figure 8:
Methylation interference analysis.
Oligonucleotides were labeled at the 5` end using T4 kinase and
[P]ATP. After annealing, double-stranded probes
were gel-purified and methylated with dimethyl sulfate. Binding
reactions were carried out as described above. Free and protein-bound
DNAs were separated by electrophoresis on nondenaturing polyacrylamide
gels and subjected to piperidine cleavage after elution. Cleaved
products were separated on 12% polyacrylamide gels containing 7 M urea. Methylated guanine and adenine residues that interfere with
binding by NZF-1 are indicated by arrowheads. A, the sense and
antisense strands of
RARE. B, the sense strand of the Pit-1 PSE enhancer element.
Figure 9:
Transcriptional activity of NZF-1. A, human RAR promoter/luciferase reporter gene was
co-transfected into CV-1 cells with RSV vector or RSV NZF-1 expression
plasmids. Luciferase activity of the control RSV vector co-transfection
was set at 1. B, mouse Pit-1 enhancer/promoter/luciferase reporter was co-transfected into HeLa
cells with RSV vector, RSV NZF-1, or RSV NZF-1 and RSV Pit-1.
Luciferase activity for RSV vector co-transfection was set at
1.
We have identified a 4730-base pair cDNA encoding a novel 1187-amino acid protein, NZF-1, containing six Cys-Cys, His-Cys type zinc fingers, organized as internal clusters of two and three fingers, with one finger located in the amino terminus. The expression of this factor is highly restricted to the nervous system, pituitary gland, and testis with highest levels of expression in the developing nervous system.
The primary amino acid sequences of all component zinc fingers are very homologous. Because of the spacing between the Cys and His residues, the folding of the NZF-1 zinc finger motifs is likely to more closely resemble that of the Cys-Cys, His-His type of zinc fingers than the Cys-Cys, His-Cys fingers of retrovirus proteins, such as the nucleic acid-binding protein of Rauscher murine leukemia virus(28) .
Each zinc finger cluster forms an independent DNA binding domain and recognizes similar specific target sequences, although the three zinc finger domain demonstrates higher binding specificity. Crystallographic studies on the Cys-Cys, His-His class of zinc fingers has revealed that two zinc fingers make major groove contacts. The third and fourth zinc fingers in the same cluster make minor groove contacts(29) . A similar mechanism may operate to generate more selective binding site usage for the three zinc finger region.
Several transcription factors that contain two independent regions of zinc finger DNA binding domains have been described(28, 30) . In each case, the amino acid sequences of the two separate DNA binding domains are very homologous to each other, and the target sequences they recognize are also similar. Although the full biological significance of such dual DNA binding domains is not known, it raises the possibility that a single protein could simultaneously bind to two significantly spaced DNA sequences and approximate the DNA sequences.
Although NZF-1 is potentially expressed at low levels in embryonic pituitary, significant levels of NZF-1 transcripts are present in the adult anterior pituitary gland. In vitro, NZF-1 specifically binds the Pit-1 enhancer and transactivates the Pit-1 enhancer/promoter reporter genes, making it a candidate for modulation of Pit-1 gene regulation in vivo.
In the course of these studies, we isolated a related mouse gene, named NZF-2, which exhibits high homology to the human glia transcription factor MyT1(27) . Because of the Cys-Cys, His-Cys configuration, these two proteins form a subclass of the most common zinc finger transcription factors, and the extensive homology in both zinc finger regions and in the carboxyl terminus suggests that these factors might exert similar and overlapping functions, acting in distinct cell types in the central nervous system.
The genes for NZF-1 and NZF-2, designated Nzf-1 and Nzf-2, respectively, were mapped in the mouse by linkage analysis of an interspecific cross. Nzf-1 is located on mouse chromosome 12 in a region that appears to be syntenic with human chromosome 2p. Nzf-2 is located on distal mouse chromosome 2 in a region syntenic with human chromosome 20q. Neither region in the mouse contains mapped mutations that are likely to be relevant to these genes.
Thus, a new family of Cys-Cys, His-Cys proteins, expressed in the nervous and endocrine systems, has been identified, and these factors are likely to exert selective functions in development and homeostasis of the neuroendocrine system.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U48809[GenBank].