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The complete murine cDNA sequence of the transcription factor AP-2

November 1993
Nucleic Acids Research 21(20):4844

November 1993
21(20):4844

DOI:10.1093/nar/21.20.4844

Source
PubMed

Authors:

Markus Moser

Max Planck Institute of Biochemistry

Armin Pscherer

Reinhard Bauer

LIMES Institute

Axel Imhof

Ludwig-Maximilians-University of Munich

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4844

Nucleic

Acids

Research,

1993,

Vol.

21,

No.

The

complete

murine

cDNA

sequence

the

transcription

factor

AP-2

Markus

Moser,

Armin

Pscherer,

Reinhard

Bauer,

Axel

Imhof,

Silvia

Seegers,

Monika

Kerscher

and

Reinhard

Buettner*

Department

Pathology,

University

Regensburg

Medical

School,

D-93042

Regensburg,

Germany

Received

July

26,

1993;

Revised

and

Accepted

August

24,

1993

EMBL

accession

no.

X74216

Using

partial

human

AP-2

cDNA

clone

spanning

the

two

Pvull

sites

from

nucleic

acid

position

152

413

(1,

2),

have

isolated

four

independently

derived

overlapping

cDNA

clones

from

commercially

available

mouse

embryo

(day

13.5)

cDNA

library

(Novagen,

Madison,

WI).

The

complete

assembled

sequence

1596

length

with

the

ATG

start

codon

bps

71-73

and

the

stop

codon

bps

1382-1384.

comparison

the

human

and

murine

sequence

reveals

only

amino

acid

exchanges

and

number

different

codon

usages.

There

surprisingly

high

degree

nucleic

acid

sequence

homology

the

5-prime

and

3-prime

untranslated

murine

and

human

mRNA

suggesting

that

these

sequences

may

have

function

post-transcriptional

regululation

gene

expression.

partial

C-terminal

cDNA

has

been

reported

previously

(ref.

EMBL

databank

acession

number

X57012),

which

matches

our

sequence

perfectly,

but

misses

the

triplet

encoding

amino

acid

390.

compare

murine

and

human

RNA

transcripts

have

further

used

the

human

AP-2

cDNA

probe

hybridize

poly

(A)+-selected

RNA

from

murine

embryos

(day

13.5)

and

the

human

teratocarcinoma;

cell

line

PA-1

(ATCC

CRL

1572).

Northern

blots

were

hybridized

and

washed

under

conditions

high

stringency

with

the

final

wash

for

min

0.2

xSSC/0.01

SDS

65°C.

The

probe

detects

two

abundant

mRNAs

with

apparent

size

3.0

and

1.8

both

RNA

preparations

(Figure

pattern

that

equivalent

results

that

have

been

reported

for

HeLa

cells

(1).

Thus,

the

expression

pattern

AP-2

murine

and

human

embryonic

tissues

appears

very

similar.

The

EMBL

accession

number

for

the

murine

AP-2

cDNA

X74216.

ACKNOWLEDGEMENTS

are

indebted

Drs

Tjian

and

Liischer

for

providing

the

Pvull

cDNA

fragment

the

human

AP-2.

This

work

was

supported

research

grant

from

the

Deutsche

Forschungsgemeinschaft

R.B.

REFERENCES

Williams,T.,

Admon,A.,

Luscher,B.

and

Tjian,R.

(1988)

Genes

Dev.

1557-1569.

Buetter,R.,

Kannan,P.,

Imhof,A.,

Bauer,R.,

Yim,S.O.,

Glockshuber,R.,

VanDyke,M,W.

and

Tainsky,M.A.

(1993)

Mol.

Cell.

Biol.

13,4174-4185.

Mitchell,P.J.,

Timmons,P.M.,

Hebert,J.M.,

Rigby,P.W.

and

Tjian,R.

(1991)

Genes

Dev.

105-119.

Figure

Northern

blot

analysis

AP-2

mRNA

expression

poly

(A)I-selected

RNA

from

mouse

embryo

(day

13.5)

and

from

the

human

teratocarcinoma

cell

line

PA-1.

whom

correspondence

should

addressed

.-.

1993

Oxford

University

Press

M.musculus mRNA for transcription activator AP-2

Nucleotide Sequence

July 1993

R. Buettner

A Novel Alternative Spliced Variant of the Transcription Factor AP2α Is Expressed in the Murine Ocular Lens

Article

Full-text available

Nov 1998

The AP2alpha gene encodes a transcription factor containing a basic, helix-span-helix DNA-binding/dimerization domain, which is developmentally regulated and retinoic acid inducible. Recent reports about AP2alpha null mice indicate that AP2alpha plays an important role in embryogenesis, especially in craniofacial development and midline fusion. Ocular development is also affected in these null mice. As AP2alpha may be involved in transcriptional regulation in the lens, it was important to examine the expression of the AP2alpha gene in the lens. Four AP2alpha mRNA variants have been previously isolated from whole mouse embryos. Variants 1, 3, and 4 are transcriptional activators that are transcribed from different promoters and variant 2 is a repressor lacking the activation domain encoded by exon 2. Using in situ-PCR, we found that AP2alpha is expressed in the lens epithelia but not in the lens fibers. RT-PCR analysis of lens mRNA with amplimers specific for each variant revealed that AP2alpha variants 1, 2, and 3 are expressed in newborn mouse lenses. However, variant 4 is not expressed in the lens. In this report we characterized a novel isoform, which we named variant 5, expressed in the lens and kidney. Variant 5, which is generated by alternative splicing, may function as a repressor due to the partial deletion of the proline-rich transactivation domain encoded by exon 2. This is the first molecular characterization of AP2alpha gene expression in the lens. Our results indicate that two activator and two repressor AP2alpha isoforms may play a role in regulating gene expression in the lens.

Programming gene expression in developing epidermis

Article

Full-text available

Oct 1994

As the major proteins of adult keratinocytes, keratins provide biochemical markers for exploring mouse epidermal embryogenesis. Here, we used a modified method of whole-mount in situ hybridization to track skin-specific expression of endogenous keratin mRNAs through-out embryogenesis. To monitor transcriptional regulation, we coupled this with β-galactosidase expression of a human epidermal keratin promoter-driven transgene. These studies have radically changed our perception of how the program of gene expression becomes established during epidermal development. Specifically, we have discovered that (1) basal keratin (K5 and K14) genes are first detected at E9.5 in a highly regional fashion, and surprisingly as early as the single layered ectodermal stage; (2) the early patterns do not correlate with morphogenesis per se, but rather with regional variations in the embryonic origin of underlying mesenchyme, supporting morphogenetic criteria that early inductive cues are mesenchymal; (3) epidermal keratin genes are expressed in periderm, supporting the notion that this layer arises from ectodermal stratification, even though it is simple epithelial-like in morphology and is subsequently sloughed during development; (4) later embryonic patterns of K5 and K14 gene expression parallel proliferative capacity and not stratification; and (5) K1 and K10 mRNAs are first detected as early as E13.5, and their patterns correlate with differentiation and not stratification. These patterns of epidermal gene expression led us to explore whether potential transcriptional regulators of these genes are expressed similarly. We show that AP2 (but not Sp1) cRNAs hybridize in a pattern similar to, but preceding that of basal keratin cRNAs. Finally, using gene expression in cultured cells, we demonstrate that AP2 has a strong inductive effect on basal keratin expression in a cellular environment that does not normally possess AP2 activity.

Cloning and characterization of a second AP-2 transcription factor: AP-2β

Article

Full-text available

Oct 1995

AP-2 has been characterized previously as a unique 52 x 10(3) M(r) transcription activator encoded by a single gene that is expressed in a restricted pattern during embryonic morphogenesis of the peripheral nervous system, face, skin and nephric tissues. Here we report the isolation of genomic and cDNA clones encoding for a second AP-2 related transcription factor, designated AP-2 beta. AP-2 beta binds specifically to a series of well-characterized AP-2 binding sites, consensus to the sequence G/CCCN3GGC, and transactivates transcription from a reporter plasmid under the control of an AP-2-dependent promoter. A C-terminal domain known to mediate homodimerization of the previously cloned AP-2 alpha transcription activator is highly conserved and sufficient to mediate interaction between the two proteins. Northern blot and in situ hybridizations revealed that the two genes are expressed in murine embryos between days 9.5 and 19.5 p.c. Coexpression of both mRNAs was detected in many tissues at day 13.5 and 15.5 of embryogenesis but some regions of the developing brain and face including the primordium of midbrain and the facial mesenchyme differed in their expression pattern of AP-2 genes. AP-2 alpha and AP-2 beta signals in the central and peripheral nervous system overlapped with regions of developing sensory neurons. In adult tissues AP-2 alpha expression was found mainly in the skin, eye and prostate and AP-2 beta expression in the kidney. In summary, our analyses of embryonic and adult mice demonstrate that two different AP-2 transcription factors are specifically expressed during differentiation of many neural, epidermal and urogenital tissues.

Selective Transcription Factor Induction in Retinal Pigment Epithelial Cells during Photoreceptor Phagocytosis

Article

Full-text available

Dec 1996

Expression of early response genes during rod outer segment phagocytosis by normal Long Evans and Royal College of Surgeons-rdy+p+ rats and by dystrophic Royal College of Surgeons-p+ rat retinal pigment epithelial cells was studied in primary cell culture. Northern analysis revealed that the abundance of zif-268 (egr-1), c-fos, and tis-1 (NGF1-B) mRNA was rapidly and transiently increased in normal retinal pigment epithelial cells during rod outer segment phagocytosis but not during phagocytosis of latex particles. No increase in gene expression was found in Royal College of Surgeons-p+ dystrophic retinal pigment epithelial cells challenged with rod outer segments. As shown by electrophoretic mobility shift assay, a prominent short term increase in the intensity of the gel-shifted band was detected using nuclear protein extracts derived from rod outer segment-challenged, control retinal pigment epithelial cells and zif-268, AP-1, AP-2, or tis-1 consensus oligonucleotides. No such increase was detected when using nuclear factor kappaB consensus oligonucleotide or when the early response gene prostaglandin H synthase-2 mRNA was measured over the time course studied. The results suggest that in retinal pigment epithelial cells, rod outer segment-specific phagocytosis is accompanied by the selective expression of early response genes coding for transcription factors. The specific pattern of the induction of these transcription factors is predicted to modulate the expression of gene cascades.

Derepression of the C/EBP gene during adipogenesis: Identification of AP-2 as a repressor

Article

Full-text available

Mar 1998

During adipogenesis, CCAAT/enhancer binding protein alpha (C/EBPalpha) serves as a pleiotropic transcriptional activator of adipocyte genes. Previously, we identified dual repressive elements in the C/EBPalpha gene and a putative transacting factor (C/EBPalpha undifferentiated protein, or CUP) expressed by preadipocytes, but not adipocytes, that bind to these elements. In the present investigation, CUP was purified 17,000-fold from nuclear extracts of 3T3-L1 preadipocytes. Amino acid sequence and mass spectral analysis of tryptic peptides derived from purifed CUP (molecular mass approximately 50 kDa) revealed that the repressor is (or contains) an isoform of the transcription factor, AP-2alpha. Electrophoretic mobility shift and Western blot analysis on purified CUP and preadipocyte nuclear extracts confirmed the identity of CUP as AP-2alpha. Both AP-2alpha protein and CUP binding activity are expressed by preadipocytes and then decrease concomitantly during differentiation of 3T3-L1 preadipocytes into adipocytes. Consistent with a repressive role of AP-2alpha/CUP, an AP-2alpha1 expression vector, cotransfected with a C/EBPalpha promoter-reporter construct into 3T3-L1 adipocytes, inhibited reporter gene transcription. Taken together with previous results, these findings suggest that in preadipocytes the C/EBPalpha gene is repressed by AP-2alpha/CUP, which, upon induction of differentiation, is down-regulated, allowing expression of the gene.

The genomic structure of the human AP-2 gene

Article

Full-text available

May 1994

The transcription factor AP-2 is encoded by a gene located on chromosome 6 near the HLA locus. Here we describe the genomic organization of the AP-2 gene including an initial characterization of the promoter. We have mapped two mRNA initiation sites, the entire exon-intron structure and located two polyadenylation sites. The mature AP-2 mRNA is spliced from 7 exons distributed over a region of 18 kb genomic DNA. A recently cloned inhibitory AP-2 protein is generated by alternative usage of a C-terminal exon. The proline-rich transactivation motif is encoded by a single exon within the N-terminal region in contrast to the complex DNA binding and dimerization motif which involves amino acid residues located on four different exons. The sites of mRNA initiation are located 220 and 271 bases upstream from the ATG translation start site. Although the promoter contains no canonical sequence motifs for basal transcription factors, such as TATA-, CCAAT- or SP-1 boxes, it mediates cell-type-specific expression of a CAT reporter gene in PA-1 human teratocarcinoma cells and is inactive in murine F9 teratocarcinoma cells. We demonstrate that the promoter of the AP-2 gene is subject to positive autoregulation by its own gene product. A consensus AP-2 binding site is located at position -622 with respect to the ATG. This site binds specifically to bacterially expressed AP-2 as well as to multiple proteins, including AP-2, present in PA-1 and HeLa cell nuclear extracts. A partial AP-2 promoter fragment including the AP-2 consensus binding site is approximately 5-fold transactivated by cotransfection of an AP-2 expression plasmid.

Assoziationsstudien und Kandidatengenanalysen bei Neuralohrdefekten

Article

Karolin Stegmann

The 5′ enhancer of the mouse mammary tumor virus long terminal repeat contains a functional AP-2 element

Article

Full-text available

Jan 1995

The mouse mammary tumor virus (MMTV) retrovirus causes mammary adenocarcinomas in mice by proviral insertion near members of the wnt family of proto-oncogenes, leading to their deregulation and cellular transformation. The 5' end of the MMTV long terminal repeat (LTR) has been implicated in tissue-specific activation of these genes. In this study, we characterize an enhancer element (Ban2; -1075 to -978) at the 5' end of the MMTV LTR. We show that this enhancer is 5-fold more active in a murine mammary carcinoma cell line (34i) than in a fibroblast cell line (NIH3T3), and is inactive in the liver carcinoma cell line HepG2. Mutagenesis of the enhancer reveals four cis-acting elements that are required for maximal activity. DNA-binding proteins that interact with each of the four elements have been identified. One of these factors, designated mp5, is either identical to, or closely related to, the transcription factor AP-2. The mp5/AP-2 DNA binding activity co-migrates with recombinant AP-2 and is supershifted by anti-AP-2 antibodies. We also show that the lack of enhancer activity in HepG2 cells results from the absence of AP-2 protein in these cells. Co-transfection of an AP-2 expression vector restores the activity of this enhancer in HepG2 cells, and requires an intact mp5-binding site.

AP-2.2: A Novel AP-2-Related Transcription Factor Induced by Retinoic Acid during Differentiation of P19 Embryonal Carcinoma Cells

Article

Jul 1996

A 2.8-kb cDNA encoding a new transcription factor (AP-2.2) has been cloned from mouse P19 embryonal carcinoma cells, in which the corresponding mRNA begins to accumulate 30 min after retinoic acid (RA) addition. The predicted protein is 449 amino acids long and exhibits approximately 65% overall identity with other AP-2-related proteins (human AP-2, mouse AP-2alpha and beta). A 96-amino-acid-long sequence, which is almost fully conserved between all these proteins, corresponds to the previously characterized human AP-2 DNA binding domain. Expression of AP-2.2 in Escherichia coli generated a protein that formed a specific complex with the AP-2 recognition site GCCN3GGC. AP-2.2 activated transcription from a reporter gene containing an AP-2 DNA binding site and acted synergistically with RARalpha to activate transcription from the CRABPII gene promoter. Transcriptional activation required the AP-2.2 amino-terminal region that contains a domain rich in proline and glutamine residues. The pattern of AP-2.2 expression in adult tissues, which is distinct from that of AP-2alpha, is essentially restricted to male and female gonads, to most if not all the squamous epithelia, and to several exocrine glands.

Activation of the mouse inositol 1,4,5-trisphosphate receptor type 1 promoter by AP-2

Article

Apr 1999
GENE

Inositol 1,4,5-trisphosphate receptor (IP3R) functions as a Ca2+ channel that increases the intracellular Ca2+ upon binding to inositol trisphosphates. IP3R is expressed ubiquitously and consists of a multigene family. Since the type 1 IP3R (IP3R1) is highly expressed in the cerebellar Purkinje cells and moderately in hippocampus in the mammalian central nervous system (CNS), it is regarded as a neural member of this gene family. In this work, we investigated transcriptional regulation of the mouse ip3r1 gene. A DNaseI footprinting assay demonstrated that a sequence from -95 to -75, designated as box-II, was a binding site for a cerebellum-enriched factor. A consensus sequence for AP-2 was located in box-II. An electrophoretic mobility shift assay with anti-AP-2 antibody revealed that AP-2 is capable of binding to box-II. Deletion analysis of box-II showed that flanking sequences beside the box-II motif were required for the stable binding. We demonstrated by transient luciferase assay that exogenously expressed AP-2 activated box-II-dependent transcription. Moreover, we showed that endogenous AP-2 induced by retinoic acid also activated transcription via box-II in P19 cells. In-situ hybridization of the mouse brain revealed that AP-2 was predominantly expressed in the cerebellar Purkinje cells and hippocampal CA1 region, where IP3R1 is also highly expressed. From these observations, AP-2 binding to box-II is thought to be responsible for IP3R1 gene regulation in the CNS.

Transcription factor AP-2 is expressed in neural crest lineages during mouse embryogenesis

Article

Full-text available

Jan 1991
GENE DEV

We have analyzed the expression pattern of transcription factor AP-2 in mouse embryos to evaluate the potential of AP-2 as a regulator during vertebrate development. A partial cDNA encoding AP-2 was isolated from a mouse embryo cDNA library and used to prepare probes to measure AP-2 mRNA levels by RNase protection and RNA in situ hybridization. Between 10.5 and 15.5 days of embryogenesis, the relative abundance of AP-2 mRNA is greatest at 11.5 days and declines steadily thereafter. RNA in situ hybridization analysis of embryos between 8.5 and 12.5 days of gestation identified a novel expression pattern for AP-2. The principle part of this expression occurs in neural crest cells and their major derivatives, including cranial and spinal sensory ganglia and facial mesenchyme. AP-2 is also expressed in surface ectoderm and in a longitudinal column of the spinal cord and hindbrain that is contacted by neural crest-derived sensory ganglia. Additional expression of AP-2 occurs in limb bud mesenchyme and in meso-metanephric regions. This embryonic expression pattern is spatially and temporally consistent with a role for AP-2 in regulating transcription of genes involved in the morphogenesis of the peripheral nervous system, face, limbs, skin, and nephric tissues.

Mitchell PJ, Timmons PM, Hebert JM, Rigby PWJ & Tjian R. Transcription factor AP-2 is expressed in neural crest lineages during mouse embryogenesis. Genes Dev5: 105-119

Article

Full-text available

Feb 1991
GENE DEV

Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements

Article

Full-text available

Jan 1989
GENE DEV

Human AP-2 is a sequence-specific DNA-binding protein that interacts with inducible viral and cellular enhancer elements to stimulate transcription of selected genes. Here, we report the isolation and characterization of a human cDNA clone containing the entire protein-coding region of AP-2. The deduced primary amino acid sequence of AP-2 does not contain a domain resembling any previously identified DNA binding motif. However, an interesting feature of the AP-2 protein is a clustered arrangement of proline and glutamine residues that have been found recently within the activation domains of other transcription factors. Expression of the AP-2 clone in bacteria yields a protein that binds to DNA and activates transcription in vitro in a comparable manner to native human AP-2. Transfection of cDNA clones into Drosophila cells indicates that the AP-2 gene product can also activate gene expression in vivo in a DNA template-dependent manner. Expression of endogenous AP-2 is repressed in a hepatoma cell line and stimulated following retinoic-acid-induced differentiation of a human teratocarcinoma cell line. This indicates that AP-2 may be a transcription factor involved in the control of developmentally regulated gene expression.

Buettner R, Kannan P, Imhof A, Bauer R, Yim SO, Glockshuber R, Van Dyke MW, Tainsky MAAn alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by AP-2. Mol Cell Biol 13: 4174-4185

Article

Full-text available

Aug 1993

AP-2 is a retinoic acid-inducible and developmentally regulated activator of transcription. We have cloned an alternative AP-2 transcript (AP-2B) from the human teratocarcinoma cell line PA-1, which encodes a protein differing in the C terminus from the previously isolated AP-2 protein (AP-2A). This protein contains the activation domain of AP-2 and part of the DNA binding domain but lacks the dimerization domain which is necessary for DNA binding. Analysis of overlapping genomic clones spanning the entire AP-2 gene proves that AP-2A and AP-2B transcripts are alternatively spliced from the same gene. Both transient and stable transfection experiments show that AP-2B inhibits AP-2 transactivator function, as measured by an AP-2-responsive chloramphenicol acetyltransferase reporter plasmid. Furthermore, constitutive AP-2B expression in PA-1 cells causes a retinoic acid-resistant phenotype, anchorage-independent growth in soft agar, and tumorigenicity in nude mice, in a fashion similar to transformation of these cells by oncogenes. To determine the mechanism by which AP-2B exerts its inhibitory function, we purified bacterially expressed AP-2A and AP-2B proteins. While bacterial AP-2B does not bind an AP-2 consensus site, it strongly inhibits binding of the endogenous AP-2 present in PA-1 cell nuclear extracts. However, DNA sequence-specific binding of bacterially expressed AP-2A cannot be inhibited by bacterially expressed AP-2B. Therefore, inhibition of AP-2 activity by the protein AP-2B may require an additional factor or modification supplied by nuclear extracts.

Jan 1993
4174-4185

R Buetter
P Kannan
A Imhof
R Bauer
S O Yim
R Glockshuber
M Vandyke
W Tainsky

Buetter,R., Kannan,P., Imhof,A., Bauer,R., Yim,S.O., Glockshuber,R., VanDyke,M,W. and Tainsky,M.A. (1993) Mol. Cell. Biol. 13,4174-4185.

Jan 1988
1557-1569

T Williams
A Admon
B Luscher
R Tjian

Williams,T., Admon,A., Luscher,B. and Tjian,R. (1988) Genes Dev. 2, 1557-1569.

The complete murine cDNA sequence of the transcription factor AP-2

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