K+-ATPase α1-subunit gene

BIO('HIMICA ET BIOPHYSICA ACTA

ELSEVIER

Biochimica et Biophysica Acta 1309 (1996) 239-252

Negative transcriptional regulation of the chicken Na+/K+-ATPase oL1-subunit gene Hui-Ying Yu a, Saju Nettikadan a, Douglas M. Fambrough b, Kunio Takeyasu ~.c.. ~ Department of Medical Biochemistry and Neurobiotechnology Center, The Ohio State Universio~, Columbus, OH 43210, USA b Department of Biology, The Johns Hopkins Unil,ersiO~, Baltimore, MD 21218, USA c FaculO' of Integrated Human Studies, Kyoto Unil'ersio'. Kvoto 606-01, Japan

Received 22 April 1996; revised 17 June 1996; accepted 27 June 1996

Abstract Although the Na+/K+-ATPase c~l-subunit gene is ubiquitously expressed in vertebrates, its level of expression varies among tissue and cell types. In spite of similar mRNA distribution in tissues of mammals and birds, the 5'-flanking regions of al-subunit genes exhibit remarkable diversity; i.e., the core promoter activity of the TATA-Iess chicken c~l gene strongly depends upon multiple Spl-based regulation (six Spl sites), whereas the promoter activity of the TATA-like rat 1-subunit gene relies on the two Spl and additional positive regulatory factors. Further analysis of the regulatory regions of the Na+/K+-ATPase al-subunit genes revealed that the vertebrate etl-subunit genes may share common inhibitory mechanisms for subtle transcriptional regulation; the core promoter activities can be either enhanced or repressed depending on the availability of inhibitory factors. Two potential candidates for such inhibitory elements in both avian and mammalian Na+/K+-ATPase ecl-subunit genes are (1) a newly identified element, GCCCTC, and (2) a GCF-binding sequence, NN[G/c]CG[G/c][G/c][G/c]CN, or its reverse complement. Gel retardation assays using the inhibitory region of the chicken gene and crude nuclear extracts from tissue-cultured chicken and mouse cells showed the existence of a set of proteins that bind to this region. The amounts of individual regulatory proteins in different cell types seem to vary, resulting in differential formation of DNA/protein complexes in different cell types. Thus, the regulation of Na~/K+-ATPase o~l-subunit gene expression under different cellular environment as well as in different cell types can be achieved by a shared mechanism; modulation of the ratio of the abundance of individual inhibitory factors. Kevwords: Promoter activity; Negative regulatory element; Transcription factor; Spl: ATPase, N a + / K +-

1. Introduction Interactions between DNA and trans-acting factors may lead to either stimulation or repression of gene expression [15,35]. Considerable effort has been made to elucidate the regulatory mechanisms of tissue-

* Corresponding author. Fax: + 1 (614) 2925379.

specific and developmental-stage-specific gene expression, whereas very little has been known about the regulation of housekeeping gene expression. The N a + / K + - A T P a s e , an essential housekeeping enzyme in plasma membrane, is composed of a catalytic o~-subunit and a glycosylated [3,subunit. Multiple isoforms of the oL-subunit (e~l, oL2, c~3) as well as [3-subunit isoforms have been identified in vertebrates [13,14,46,53] (also see a review [50]). The

0167-4781/96/$15.00 Copyright © 1996 Published by Elsevier Science B.V. All rights reserved. PII S01 67 -4781 (96)001 30-3

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expression levels of the isoform genes vary in different tissues and developmental stages, and are subjected to hormonal regulation [14,32,43,50]. Within the c~-subunit gene family, the ~1 gene shows the most typical characteristics of housekeeping genes; i.e., the gene lacks a canonical TATA-box and carries clustered GC-rich domains [47,56] and is expressed in a broad range of tissues [14,33], while the genes for c~2- and ot3-subunits are regulated in a cell- and tissue-specific manner [14,33]. Studies of Na+/K+-ATPase otl-subunit expression have revealed that this gene is subjected to a variety of subtle regulation. During development, the c~1-subunit mRNA increases several fold in brain but remains constant in skeletal muscle, heart and kidney [33]. The level of c~1-mRNA in lung is significantly higher in the neonate than adult [33]. Expression level of the e~l-subunit was increased when myoblasts fused to form myotubes in tissue culture [54]. Thyroid hormone and aldosterone were shown to increase the o~I-mRNA level in rat heart [17,20]. cAMP has been shown to induce ~1 gene expression in renal cells [1]. A study of mechanisms regulating rat o~lsubunit gene expression showed that its positive regulation is caused by Spl and other trans-acting factors which interact with a positive regulatory element, ATP1A1 (ARE) [57]. In this report, we identify a set of positive and negative regulatory elements by structural and functional characterization of the chicken Na+/K+-ATPase e~l-subunit gene. Alignment of chicken (this study), rat [56], horse [21] and human otl-subunit gene sequences [47] revealed that many of the regulatory elements identified are conserved in these genes, indicating that the expression of the Na+/K+-ATPase c~l-subunit gene is likely regulated by mechanism shared among vertebrate species. Positive regulation has been assumed to be predominant in higher organisms, since most eukaryotic promoters require DNA-binding activator proteins to function in vivo [15]. However, it now appears that negative regulation is also quite common in eukaryotes. A Drosophila homeodomain protein (eve) has been shown to repress transcription of the Ultrabithorax gene [6]. A protein, GCF, which binds to GC-rich sequences, has been found to repress transcription of several genes including those encoding epidermal growth factor receptor, /3-actin and calcium-dependent protease [19]. These examples together with the

report presented in this study cast a new light on the importance of negative regulation in the expression of house-keeping genes in general.

2. Materials and methods

2.1. Promoter sequence and deletion constructs

The chicken Na+/K+-ATPase o~l-subunit gene was isolated by screening chicken genomic libraries [51] in )t-phage, EMBL4, with 32p-labeled cDNA encoding the chicken c~l-subunit [52]. The entire gene structure was elucidated by restriction enzyme digestion and partial DNA sequencing of 3 overlapping clones (Fig. 1). The nucleotide sequence of the promoter region was obtained by DNA-sequence analysis of the NcoI fragment (spanning from - 3453 to + 190 bp (ATG)) in a plasmid, pGEM5. This plasmid (NcoI-ATG/pGEM5) was then treated with exonuclease IlI to remove a portion of the 5'-untranslated region from + 4 6 to + 190 bp (ATG), and the resultant sequence from - 3 4 5 3 to +45 was cloned into the blunt-ended XbaI site of pCAT-Basic plasmid (p0CAT; Promega) yielding the plasmid p3.4kbCAT. A series of deletions of the p3.4kbCAT plasmid were made either by restriction-enzyme digestion (p900, p560, p163, p96, and p45CAT), by timed exonuclease III digestion (p3.2kb, pl.3kb, p836 and p210CAT), or by polymerase chain reaction (PCR) (p20, pl0, and p + 5CAT). Plasmids p20, pl0, p + 5CAT were constructed from the products generated by PCR using different 5' primers (5'-AAGCTTACGGGGCGGGGCGGCCG-3' (for p20CAT), 5'-AAGCTTGCGGCCGGGGCTTTATCCG-3' (for pl0CAT), 5'-AAGCTTAT CCGGCGGGAAGCTG-3' (for p + 5CAT)) and a common 3' primer (5'-CGTTATAA TATTTGCCCATGG-3'). The PCR products were cloned into HindIII-NcoI digested p0CAT after confirmation of their DNA sequences. 2.2. RNA isolation and Northern blot analysis

Total RNA was isolated from primary cultured chicken embryonic day-ll skeletal muscle, muscle fibroblast, cardiac myocyte and cardiac fibroblast cells using the method previously described [59]. For

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sages of cells in uncoated dishes [5,32]. Muscle cell cultures were fed with medium supplemented with 5 IxM cytosine arabino-furanoside to inhibit the proliferation of fibroblasts. These primary cultures of chicken cell types were established in Duibecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 - 5 % chicken embryo extract (CEE), penicillin (100 U / m l ) and streptomycin (100 ~ g / m l of culture medium). Twenty-four hours after final plating ( ~ 2 × 10 ~ cells/100 mm plate) of cells (36 h after final plating, in the case of skeletal muscle culture, just before fusion started), supercoiled plasmids of pCAT constructs (10 txg/100 mm dish) and pRSVI3gal (2.5-5 txg/100 mm dish) were transfected into cells by a standard calcium/phosphate-precipitation method, Mouse myoblasts (C ~_C~2) and fibroblasts (Ltk-) were seeded at a density of ~ 2 × 105 cells per plate and cultured for 12-24 h in the DMEM (with 10% FBS without CEE) before transfection.

Northern blot analysis, l0 g g / l a n e of total RNA was electrophoresed in denaturing formaldehyde !% agarose gels in a buffer containing 20 mM MOPS, 50 mM sodium acetate, 10 mM EDTA, blotted onto nitrocellulose, fixed with UV light, and hybridized with a random primed 3:P-DNA probe specific for 1-mRNA (0.7 kb E c o R I fragment from the 5' end of c~1-cDNA) [52]. 2.3. Tran,~fection o f t i s s u e - c u l t u r e d cells

Thigh muscles and hearts isolated from 10- to 1 l-day White Leghorn embryos (Department of Poultry Science, The Ohio State University) were used for preparing primary cell cultures. Following preplating of the mixture of cell types obtained by mechanical dissection of thigh muscles, the myoblast-enriched population was seeded onto collagen-coated dishes, and the fibroblast culture was prepared by additional serial passages of cells in uncoated dishes as described previously [54]. Similarly, following preplating, the mixture of cell types obtained by trypsin (0.025%) treatment of hearts, the myocyte-enriched population was seeded onto uncoated dishes, and the fibroblasts were prepared by additional serial pas-

N a , K - A T P a s e ~1 Subunlt Gene E

BEB

EE

I

III

II

Tissue-cultured cells were harvested by scraping, suspended in 100 p,1 of CAT buffer (250 mM Tris-

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2.4. E n z y m a t i c a s s a y s

1~

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EcoR1 Sacl HinclI

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[- RsrII

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300bp

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I Promoter F

50bp

ACGTCA(

"jI~"(GIc)CG(GIc)(GIc)(GIc)C AGCCCGGCGA

I

TT Tg:: I ICCGCCC [GGGCGG

AGGCGGGAACGGGGCA

Fig. 1. Organization of the chicken Na+/K+-ATPase e~1-subunit gene. The top line represents the entire e~I-subunit gene. The positions of the exons, depicted by solid bars, are shown relative to the restriction map. E: EcoRI; B: BamHI. The restriction map of the 5' 3.6 kb flanking Ncol fragment is showed in the middle. The defined cis-elements within 560 bp of the 5' flanking region are indicated at the bottom. The solid boxes in the middle and bottom lines represent the first exon.

242

H.- K Yu et al. / Biochimica et Biophysica Acta 1309 (1996) 239-252

CCATGGCTCTGAATGAAGGTAGGGC C A T T C A T G T G C A A ~ G G A T G G A G A C C T C _ A C G T T C A A A G G G C C T P T A A G G A C A ~ T ~

-3355

AAACAGTATTTGAATGACCTAGTTGAAAC43CPGC~GAAAAATOCAAAACACTGTCAGAAATGCAAAATG T T G C A T C C A T C A T ~ G ~ Octan~r R C~G/~ C~ P T A ~ C ~ T ~ ' ~ T " ~ GGT~ ~ P A ~ ~L"~--/~GCCACAG'I~~~ ~A~AT ~ Homeo API GCRE R GATC~CATTAAAGGCAGGGGACAAGGCAGCA~TGAGACCTGTAATTACAGCCAGGAGGTTGA~AGAAATAGTTACATACAGTCACCTGGGGTACT

-3256 ~

-3157

-3058

CTGTGTCCCCTGTTAAGTGAACCACCCTTCAGAAGGCATGAAq~PCAC~TT~GACT~TAAA~TITATATAGAGACTTCA~A~CAC

-2959

~-~FI~CTTTACCTGCACAGCT~TTTACTGTCTGCTTTAGTGTGCTG TGAGTAACTCCATCCTCTC~ TGACTGTAGTGATAAGGCATGC G TGGA APl R TAAGGAAAAATAGCTTGTTTGGGTCCAGCTCTGTPCCTTTCPCTTTAATTCTGAATAGGTGGGAGCI~PTGGCAG TTGGACTTGGTGAC C T T G A A G ~ C

-2860

ATCTTCCAATCTAGATAATTCTATGATCCTTC T C T G A T G C A A A A G G A C A T C A T A C T G T C C A C A A G C A A A C P G T C C C A A ~ G ~ T A ~ G ~ c- fosSRE R TGAAGCCAGACCCGTGCT~TCCAAAC43GAAGGGCAT~TTGCAACTCACCAAAAAACGGTGG~CAGAAGATCTCACAGAAAGAACAAA

- 2662

TAAGAGGGAGGTGATATTCTTTCCPGTTCTGGGTGGCATCCATTACCAGGATCTAACATCCTAACT•TCCAAACAAATGCAGCCCTAAGCTGTAGCAAT GRE NFI TGAAAAGGGATAAAGAq~fTGCTT~TCCATGTCAAGCTATATGCAGACTGCAAACAAAAACAGGAG TATACCTGAGCAGAAGCTGTTGCCTCTAA~CCA

-2464

AGAGCAGAGAGCTGTCAAAGCCTTGAATAGGTGAAATA TTAGGAAACATTTCITCPCAGAAGGAGTGCTAATGCATT G G C A C A G C ~ ~ T G

-2266

GTGGAGTTACTGTCC~AGGTGTTCAAGAACCGTGTGGACGCGGCACTGACAGACACAGCTTA~FGAGTGTC~TGGGGAT~GAT~T~GT

-2167

AGATGATCTTAAGGTCTTTTTCCAACCTTAATGACTCFGTGATTCTATGACACTCACCTGCATTGCCAAGTTTTCCACCTGC ~ ~ C ~ T GCRE API R GACACTCTCTTCT~CTAAAATAGCCATAATAAAACTCCCTGTCGTAGAAC GTCCTCAGGTTCTCAAACTGAAAAAAAAATGATACAGAAGGACTG

- 1969

-2761

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-2365

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TGTGATGTGCTAGTTATAAAAACAGCAGTTGGCACCCACATATTGCCCT~CACTACC~ACACAAGGCCCACTCCT~PCACT~CAGT~CCTA

-1870

CCCCPGGAATTCAACAGCTTTGCACCAGCCCACAT CTGGCTTCACAGTCAGGGTGAAAAACACCCTCTTGCTTCACATCTTCAAAAAGGATTCATGA~

- 1771

TPTI~CCGGGATGAGGTGAGAGGGACACACAAGTCAAAGAGCAGGAATTTTCAGGCA

-1672

AAAACCGACTTGGACCFPGATCCACTGGCAGGAGTGAAG

TGCCTGTATGTTATCTGCTTCTCCCAGTCACCATGCCAAGGTATCAAAAAGAGTTT~TAA~TAGATAC~GAAAACAGAGCTCTCTTTGG

~

CATAAGCTAATCTC~CTGATACCTATTAAGGGA~GAGAACACACGTACCTC~ATCCTCTGTCCTGGTTTTACGTAATACTATTTCCTTTGATCCGTATG

-1573 -1474

GRE

GATTTGCCCTCTTTTTATGTCACTGCCAGCAAGGGAACTCAGCCACGTTT~CTCCTGAAAGAGACAAAATAGCATCAGTTGATC~T~T~ACA TCAGGGAGAAACAGCAACTTGTCAACTATCCCAAAGCAGAAGATGGCPCACATTCG

TAGCATTCCAGCTCAGCTGTACTGTACAGATCACAGCTGTTAC

-1375 -1276

ACAAGGGGCAGAATGGCTTGGAAGCTGTCTGGAGGTCTTCTTTGAATGTTCTACTGATGATGCTTCACAGCCACACTGAAAGAAACCGAAGq•FTGACAT GRE NFI C43TGAA~PGTTGTTAAGGAGTGAAAATGAAACAAATAGTGATq'PCAGCCTCTGAGATTTCTACC C A G T T C C T A G T G C C T A C A A A G T T C ~ T C ~

-1177

CCTACTGCCATGAGCTAGCA~GGGAAAGAACACTTCAGAGATGAGTAACGGGAGCGAAATGACAGCCCTG~~~ API R v9O0 TFTTTACTTTAAA GTTCACTACGTCCTGTACTAGGAG TGCCTCAGGAGGACTTGOGACATGACCAGACTGAGC~CACGTGTGTGATGCTCCCCC~G V 836 ~AAGA~TGCCAT~PGTA~CAGCATCCCTTCCCATCACACACx~TCTGTGGACAAA~CGATGGCGCTACAGCTT~A~A~T

-979 -880

TAGGAGAAGGTTCCT C A C C A C A G G G C A G T G G G C A C G G C C C A G G C T G C C G G A G T T C A A G G A G C G T I ~ P T G G A C A G T G C T C T C A G A ~ T A ~ G T ~ A ~ AP2 TGGGTAGTGCPGTGTGGAGCCAGGGGTT GCACT CGATGATCCPTATGTGTCACCTACGGCTCGGGATATTCTGTGTTPCTGTCATCCTGTGATCCTC~ 560 API qL~TGCCCATCTCTCCCATGTGCTCGAGTGGGAAGGGACCTATGAGAC CATTGCCCTGCCAGGCACT G A G C A G C A G A ~ G A ~ C C ~

- 583

T~ACGA~CGCATACGCATCTCCTAACACACAGAAGCCCTGAACCCCGGAAGGTGCGCAGCTI~CGGACACCACGTCACT~~T CREB R AGGCAGGAAGGGACCCCTGGTGGTGCCCTCAGAGCAGGGTC CTGTG GCCGCGCCTGGCTTCACCC C G G T G C C T T T ~ G T ~ C A C G ~ C C G

-1078

-781 -682

-484 - 385 -286

T210 GAGCGCC~3CAGAGC-AGCC43GGGCCGCGCCCACTGCCC

CCT C A C G C X T T C G A C A T G G C G G C G G C G G C A G C ~

~ C

V 163

~ A ~

GCAGCC

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CTCAG~CGGCCTCACCC~CGGAAGGAGCCCGGCGACGCGCCGAGCCCTCCTCGG~GCC~T?GGC~CGGCCGAG~GA~CCCG~AC AP2

~45

20

Spl

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Spl

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ACGTGGGCAA~cCC43CACCAcCCCCCCC~CCCCAACCK3TCC~GGA~G~GCTA~TAC~CC~3CCGGG~ctttatcggcgg Spl R

-187

+12

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gaagccggtgccg~atcggagctgcgggtggttgcggcggcgaggagggcgcgagagagaggcgagcgg~aggaaagcggacgcgggcgc~gctattgt

+iii

tcggcacctgctgagccacgtggcgtcgttgagcgccgctcctcgctgccagccgcgtgtcccgcacgccggcaccatg

+190

Fig. 2. Nucleotide sequence of the 5' end of the chicken Na+/K+-ATPase c~ 1 gene. 5'-flanking sequences are shown by upper case letters; transcribed sequences occurring in the first exon are indicated by lower case. Sequences exibiting similarity to transcription factor or hormone receptor binding site consensus sequence are underlined. These sequences include five APl-like sites, two AP2 binding sites, an Octamer motif, a homeo box, two CTF/NF1 binding sites, a C R E B / A T F consensus binding site as well as six Spl sites. Three sequences which share homology to the core glucocorticoid response element TGTTCT were also found. Numbers to the right of the figure refer to nucleotide positions relative to the transcription initiation site [52]. The starting positions of different plasmid construct within the 900 bp promoter region are shown by arrowheads.

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H.-Y. Yu et al. / Biochimica et Biophysica Acta 1309 (1996) 239-252

HC1, pH 7.5), and subjected to three cycles of freezing and thawing. The cellular debris were removed by centrifugation, and the volume of the resultant cell extract was adjusted with CAT buffer. A portion of each extract was then used for CAT-activity assays as described [45]. The assay system was adjusted so that the highest CAT enzyme activity should fall within the range of less than 40% conversion. [3-Galactosidase activities in the same extract were used as internal standard for transfection efficiency. The background enzyme (CAT and [3-Galactosidase) activities in untransfected cells were null. 2.5. Gel-retardation assays

Four synthetic oligonucleotides were designed so that, after annealing in a solution containing 75 mM sodium chloride and 8.5 mM sodium citrate, two sets of double-stranded oligonucleotides could form as follows: oligo-NRRI

Oligo-NRR2

5

'

-CGGAAGGAGCCCGGCGACGCGCCGAGCCCTCCTCGGTGGGGCGCGG-3 3 '-GGGCCGCTGCGCGGCTCGGGAGGAGCCACCCCGCGCC-

5 ' -AGGCGGGAACGGGGCAGCGCAGCCCTCAGGCGGCCTCACCCCGG3 ' - TTGCCCCGTCGCGTCGGGAGTCCGCCGGAGTGGGGCCCGG-

2

3

pC CAT ~3.4kb CAT ~.2kb CAT ~t.9kb CAT ~900CAT CAT ~560CAT J210 CAT ,l&~ CAT ~96CAT ~45CAT p20 CAT

1000 bp

plO CAT p+5 CAT NcoI i

EcoRI t EcoRI

s=, i Hincn

I

, Xhol Ap'al[ i n , Rsrll

Fig. 4. Reporter gene constructs of 5' sequential deletion mutants. p3.2kb, pl.9kb, p836, p210CAT were constructed by exonuclease III deletion of p3.4kbCAT and pg00, p560, p163, p96 and p45CAT were constructed by cutting p3.4kbCAT with HindllISacI, HindIII-XhoI, HindIII-Apal, HindIII-BanII, HindIII-RsrII respectively, p20, pl0, p + 5 C A T were constructed from the products generated by polymerase chain reaction (PCR). The solid box represents transcribed sequence from the ~l-subunit gene.

'

5 ' 3 ' 5 '

These annealed oligonucleotides were labeled by Klenow fill-in reaction using [cx-32p]dCTP and used for gel-retardation assays. First, the DNA/protein interaction was initiated at room temperature by adding labeled oligonucleotides to the reaction mixture (final volume of 20 ~1) containing 1 p~g of

1

cFz-~f-q ~0CAT SV40 Promoter+Enhancer

4 8

Fig. 3. Expression of Na+/K+-ATPase a l-subunit mRNA in primary cultured chicken cells. Primary cultured cells from 5-day cultured myoblast, muscle fibroblast, cardiac myocyte and cardiac fibroblast were harvested in guanidine thiocyanate, and total RNA was purified through acid-phenol extraction. Ten p~g of total RNA were electrophoresed in denaturing formaldehyde gels and blotted onto nitrocellulose membranes. Filters were hybridized with the probe specific for the c~I-mRNA. Lanes: 1. muscle fibroblast; 2. 5-day myoblast; 3. cardiac myocyte; 4. cardiac fibroblast. The arrow corresponds to the location of 28S rRNA.

double-stranded poly (dI-dC), 0.5-1 ng of labeled DNA probe ( ~ 2 × 10 4 cpm) and 8-16 ~g of protein in crude nuclear extracts in a buffer solution (10 mM HEPES, 10% ( v / v ) glycerol, 500 mM KC1, 0.1 mM EDTA, 0.25 mM DTT, and 0.25 mM PMSF). Then, after 15 min, the resultant 32P-DNA/protein complexes were fractionated by polyacrylamide (5%) gel electrophoresis (at 200 V, 4°C for 3-5 h), and the radioactivity of the complexes was detected by conventional autoradiography a n d / o r quantitative phosphorimager analysis. The gel-retardation assay was repeated at least 3 times per cell type by using different nuclear extract preparationS. Nuclear extracts were prepared from tissue-cUltured skeletal muscle, muscle fibroblast cardiac myocyte, cardiac fibroblast, C2C u and Ltk by the me~hod of Dignam et al. [11]. 2.6. Nucleotide sequence accession number

The promoter sequence from - 3 4 5 3 to + 190 has been deposited in GenBank and given accession number L43603. 3. Results The relationship between the structure of the gene, 5'-flanking sequences and the promoter region is

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H.- Y. Yu et al. / Biochimica et Biophysica Acta 1309 (I 996) 239-252

depicted in Fig. 1, and the nucleotide sequence of the 5'-flanking region is shown in Fig. 2. Highly GC-rich sequences (80%) were found from - 2 8 7 bp to transcription initiation site in the 5'-flanking region (see Fig. 2 and figure legend). This region lacks a canonical TATA or C A A T box immediately upstream of the transcription initiation site, whereas it contains six potential binding sites for the transcription factor, Spl [9]. The TATA-Iike sequence, the ATP1A1 responsive element (ARE) [57], and a potential C R E B / A T F [39] binding site are highly conserved within the mammalian promoters. The chicken a l promoter contains an ARE-like sequence (see Fig. 9), but with only four out of eight matches to the ARE

I

3.1. Ouerall regulation o f the p r o m o t e r actizity in tissue-cultured cells

Northern blot analysis using a cDNA probe specific for the e~1-subunit showed that the tissue-cultured chicken cells (skeletal muscle, muscle fibroblast, cardiac myocyte and cardiac fibroblast) expressed o~1-subunit mRNA, the levels being in the

I

/ia / I[I

consensus sequence, A G G T T G C T [57]. The C R E B / A T F site [39] is located close to the TATAlike sequence in the mammalian promoters, but is located at - 4 1 2 , far from the transcription initiation site in the chicken promoter (Fig. 2).

i'll Ie2e,2

I

I

' /

~ i~-

I

Fig. 5. Reporter gene analysis of the activity of 5'-flanking sequences of the c~l-subunit gene revealed the existence of negative regulatory region. Four kinds of primary cultured chicken cells and two kinds of mouse cells were transfected with the sequential deletion mutants of the otl-gene promoter. SV40 promoter with enhancer was used as a positive control (pCCAT) and RSV driven 13-galactosidaseas an internal control for transfection efficiency. The activities of the deletion mutants were compared with the activity of p0CAT which was arbitrarily taken as l-fold. Although the relative overall levels of expression differ in different cell types, the pattern of activity distribution in the promoter region clearly remains constant. Chicken cells; H: cardiac myocyte; HF: cardiac fibroblast; M: myoblast; MF: muscle fibroblast. Mouse cells; C2C n2: myoblast; Ltk-: fibroblast.

H.- Y. Yu et al. / Biochimica et Biophysica Acta 1309 (1996) 239-252

following order: cardiac myocyte > skeletal muscle > fibroblasts (Fig. 3). The promoter activity of the c~1-subunit gene was assessed in various cell types by the activity level of the chloramphenicol acetyl-transferase (CAT) expressed under the control of various lengths of the 5'-flanking regions of the a l gene (Figs. 4 and 5). The distribution patterns of promoter activities of a 1-subunit gene are similar in different chicken cell types. It is interesting to note that the overall level of promoter activity in cardiac myocytes is lower than in skeletal muscle, whereas the endoge-

245

nous e~1-subunit mRNA level (Fig. 3) is higher in cardiac myocyte than in skeletal muscle. The stability of the endogenous mRNA encoding the oL1-subunit may differ in skeletal muscle and cardiac myocyte, or alternatively the regulation of transcription may be different between endogenous and transfected genes. The regulatory mechanism of the chicken Na+/K+-ATPase ~xl-subunit gene shows two major characteristics at the promoter level. First, the core promoter activity of the gene is apparently controlled by transcription factor Spl. The first 96 nucleotides,

Oligol

Oligo2

1~6 "

1 ~oligol linker oligo2

iigo2 linker oligol

;~

o o oxo -4----Cl-a -~---Cl-b

1

2

3

4

5

A

6

7

8

"1

2

3

4

5

6

7

8

B

Fig. 6. Gel retardation analysis of the negative regulatory region (NRR; - 2 1 0 to - 118 bp) of the chicken Na +/K+-ATPase a l-subunit gene. Sequence analysis shows that the NRRI contains a GCF consensus sequence in the middle and a Spl site at the 3' end, and that NRR2 contains a GCF site at its 3' end which overlaps with an AP2 consensus sequence. Since Spl has been observed to confer mainly positive transcriptional activity, it is likely that the negative regulation is caused by the remaining sequence in NRR1. A: 32p-labeled oligo-NRRl ( - 163 to - 118 bp) was incubated with 14 txg of nuclear extracts from skeletal muscle. DNA/protein complexes were separated by 5% polyacrylamide gel electrophoresis, and detected by autoradiography. Three complexes (C l-a, C l-b, C l-c) were formed with oligo-NNRl (lane 2), and the formation of the complexes was specifically blocked by the NNR sequences. Unlabeled oligo-NRR1 was used as a specific competitor (lanes 3 and 4), and 35bp XhoI-PstI polylinker sequence of pBluescript II was used as unspecific competitor (lanes 5 and 6). Unlabeled oligo-NRR2 was also used for the competition analysis (lanes 7 and 8). B: 32P-labeled oligo-NRR2 ( - 210 to - 164 bp) was incubated with nuclear extracts from skeletal muscle, separated by 5% polyacrylamide gel electrophoresis, and detected by autoradiography. Three specific complexes (C2-a, C2-b, C2-c) were formed with oligo-NNR2 (lane 2), and the formation of the complexes was specifically blocked by the NNR sequences. Competition experiments similar to oligo-NNRl were done by using unlabeled oligo-NRR2 (lanes 3 and 4), 35bp polylinker sequence of pBluescript II (lanes 5 and 6), and oligo-NRRl (lanes 7 and 8).

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which do not contain TATA and CAAT boxes but have 5 Spl sites (Figs. 1 and 2), are capable of directing 10- to 500-fold higher CAT activity than the promoterless p0CAT in all tested cells (Fig. 5). A deletion construct (p45CAT), in which the distal Spl consensus sequence of this core promoter was removed, exhibited 3 0 - 5 0 % lower promoter activity than p96CAT in most of the tested cells. An additional deletion of two 5'-end Spl binding sites from p45CAT (resulting in p20CAT) led to a 6 0 - 8 0 % decrease of promoter activity of p45CAT. Further deletion of two more Spl binding sites (resulting in pl0CAT) drastically reduced promoter activity in all tested cells. This proximal region (pl0) of the core promoter, which does not have any Spl binding sites, produced a background level of promoter activity similar to that produced by p0CAT. Second, it is apparent in all tested cell types that negative regulatory regions (NRR) are involved in the overall regulation of the expression of the N a + / K + - A T P a s e o~1-subunit gene (Fig. 5). When 67 bp (NRRI, - 1 6 3 to - 9 7 bp) of 5'-upstream sequence was added to the core promoter (forming p163CAT), the core promoter activities in chicken and mouse cells were inhibited by 4 0 - 7 0 % and 80-90%, respectively. Introduction of an additional 47 bp (NRR2, - 2 1 0 to - 1 6 4 bp) to this construct (resulting in p210CAT) further repressed the core promoter activity in chicken cells by 70-85%. It is interesting to note that the inhibitory effect of NRR1 is much stronger in fibroblasts (60-70% inhibition)

than in muscle cells (40-50% inhibition). These repressed promoter activities were moderately (2- to 10-fold) enhanced by further addition of a positive regulatory region (PRR: - 2 1 1 to - 5 6 0 bp). Similar patterns of the chicken promoter activity were observed in mouse C2C m cells (derived from skeletal muscle) and mouse fibroblast Ltk- cells. However, in mouse, the NRR1 inhibits the promoter activity to a large extent which is not further repressed by NRR2, whereas, in chicken cells, both copies of NRR repress expression. Thus, the high level of the core promoter activity of the chicken Na+/K+-ATPase oLl-subunit gene is strongly repressed by NRR, and the resulting overall activity is kept relatively low in both avian and mammalian cells. 3.2. Gel-retardation analysis of the negatiue regulatory region (NRR)

To examine the ability of the NRR to interact with trans-acting factors, complementary oligonucleotides that encompass - 163 to - 118 bp (oligo-NRR1; 46mer) and - 2 1 0 to - 164 bp (oligo-NRR2; 47mer) were synthesized and used in mobility shift assays. The nuclear extracts from cultured skeletal muscle cells formed three (Cl-a, Cl-b, Cl-c) and four complexes (C2-a, C2-b, C2-c, C2-d) with oligo-NRRl and oligo-NRR2, respectively (Fig. 6). Formation of the C l-x complexes (x = a, b, c) was effectively blocked by the addition of excess unlabeled oligoNRR1 (Fig. 6A, lanes 3 and 4) but not by an

Table 1 Relative quantity of DNA/protein complexes in the presence and absence of competitors, Oligo-NRRl and Oligo-NRR2 Relative amount in the presence and absence of competitors None Oligo-NRRI linker Oligo-NRR2 10 x 100 × 10 X 100 X 10 × 100 × 100 17.5+2.1 4.3+0.5 141.4+7.9 120.7+7.4 110.2+4.5 80.1+4.0 Oligo-NRR1 Complexes Cl-a Cl-b 100 15.3+ 1.3 4.5+0.6 140.9+8.9 110.3+7.5 22.1 +2.7 8.1 + 1.1 Cl-c 100 16.4+ 1.7 4.0+0.3 139.1__+6.4 124.5+ 6.5 49.1 _+5.8 7.1 _+ 1.2 Oligo-NRR2 Complexes C2-a 100 12.7_+ 1.1 1.5_+0.2 130.5_+8.9 129.7_+7.4 13.6_+0.8 1.5_+0.2 C2-b 100 80.7_+5.6 60.4_+4.4 132.3_+8.2 120.2_+6.9 10.2_+0.5 0.9_+0.1 C2-c 100 25.6+3.7 3.7-+0.5 135.1_+9.8 127.4_+7.3 29.1 +4.2 4.8+0.5 C2-d 100 112.4-+5.6 96.7-+6.4 129.4_+7.4 125.3_+6.5 115.2_+7.6 90.8_+7.1 32p-signals from each DNA/protein complex formed in gel-retardation assays using nuclear extracts from chicken skeletal muscle were quantified by using phosphorimager. The quantity of each complex formed in the absence of competitors was taken as 100%, and used as a standard to calculate the quantity of the complex formed in the absence of the competitors. Data (mean + standard deviation) were obtained from three different experiments.

H.- Y. Yu et al. / Biochimica et Biophysica Acta 1309 (1996) 239-252

unrelated oligonucleotide (e.g., 35 bp XhoI-Pstl pBluescript polylinker sequence (Fig. 6A, lanes 5 and 6)). Similarly, unlabeled oligo-NRR2, but not the 35 bp XhoI-PstI pBluescript polylinker sequence, competed for formation of the C2-x complexes (x = a, b, c) (Fig. 6B, lanes 3-6). On the other hand, the formation of the C2-d complex could not be blocked by either oligo-NRR2 or polylinker sequence, indicating that the formation of C2-d complex is nonspecific. Table 1 summarizes the quantitative aspect of the competition experiments assessed by using phosphoimager. The complexes C l-b and C l-c formed with oligoNRR1 migrated respectively with similar mobilities to the C2-a and C2-c formed with oligo-NRR2 (Figs. 6 and 7). In addition, the formation of these complexes was blocked respectively by oligo-NRR2 and oligo-NRR1 (Fig. 6A and B, lanes 7 and 8). Therefore, it is possible that both oligonucleotides may interact with common trans-acting factors, although the cross-competition data in Fig. 6 show that the

247

factors for each oligonucleotide prefer that oligonucleotide to the other oligonucleotide, possibly due to the diversity of the neighboring sequences around the factor-binding sites. On the other hand, the complexes, C l-a and C2-b, are respectively specific to oligo-NRR1 and oligo-NRR2. The nuclear extracts from other chicken cell types (cardiac myocytes and fibroblasts) exhibited a similar property in DNA/protein-complex formation to that from chicken skeletal muscles (Fig. 7), suggesting the existence of a common set of trans-acting factors in all tested chicken cells. The relative amount of each DNA/protein complex varies among different cell types, indicating that the amount of the trans-acting factors varies among different cell types. Quantification of the amount of all complexes formed with nuclear extracts from different chicken cell types (Fig. 8) shows that more of the complexes Cl-a, C l-b, C2-a, and C2-b, formed with skeletal muscle and cardiac myocyte extracts than with fibroblast extracts, whereas larger amounts of complexes Cl-c

Oligol

Oiigo2

Cl-a-~

Cl-b--~ CMI-a CMI-b

CM2-a

CMI-c

CM2-b =

CM 1-d

CM2-c CM2-d C2- c [CM2-e

CMI-e/CI-c---~

8

1

2

3

4

5

6

7

8

9 10 11 1;' 13 14

Fig. 7. Gel retardation analysis of the negatwe regulatory region (NRR) of the N a + / K + - A T P a s e a l-subunit gene revealed the existence of species-specific proteins that recognize the NRR. The target DNA (oligo-NRR1 (lanes 1-7) and oligo-NRR2 (lanes 8-14)) were incubated with 14 #xg of nuclear extracts from different cell types, and subjected to 5% polyacrylamide gel electroph0resis followed by autoradiography. Lanes: 1: free probe of oligo-NRR1 ; 2 and 9: with nuclear extract from muscle fibroblast; 3 and 10: with nuclear extract from muscle; 4 and 11: with nuclear extract from cardiac fibroblast; 5 and 12: with nuclear extract from cardiac myocyte; 6 and 13: with nuclear extract from Ltk-; 7 and 14: with nuclear extract from C2C i.~; 8. free probe of oligo-NRR2.

248

H.- E Yu et al. / Biochimica et Biophysica Acta 1309 (1996) 239-252

and C2-c formed with fibroblast extracts than in muscle extracts. The nuclear extracts from C2C J2 and L t k - cells formed several similar DNA/protein complexes with oligo-NRR1 (CMla-e) or oligo-NRR2 (CM2a-e) (Fig. 7). It is interesting to note that most of the complexes differ in mobility from those formed with the chicken nuclear extracts, with an exception of complexes CMI-e and CM2-e, which have mobilities similar to those of chicken C 1-c and C2-c.

4. D i s c u s s i o n

The avian and mammalian Na+/K+-ATPase a lsubunit genes contain 23 exons [21,51]. The protein coding sequences are highly conserved between mammalian and avian a 1-isoform genes, while the 5'-regulatory regions of these vertebrate genes are remarkably diverse. This study characterizes the structural and functional properties of the chicken a 1 gene promoter. The core promoter activity of the TATA-less chicken oL1 gene appears to depend strongly upon multiple Spl-based regulation in all tested chicken and mouse cell types (this study), Oligo-NRR

whereas the TATA-like mammalian otl gene relies on two (or three) Spl's and additional positive trans-factors [21,47,56]. The core promoter activity of the chicken oL1 gene varies greatly in the various tested cells, which is consistent with previous studies on variable Spl binding in different cell types [36]. This could be caused by the different levels of Spl expression [38] or by changes in the degree of Spl phosphorylation a n d / o r glycosylation [18,38,42]. In contrast to the different activation mechanisms of transcription, mammalian and avian Na+/K+-ATPase etl-subunit genes may share common inhibitory mechanisms for subtle transcriptional regulation. As discussed below, this study has identified two potential cis-acting inhibitory elements, GCCCTCCT and AGCCCTC, in the corresponding regions of avian and mammalian a l-gene promoters. 4.1. Negative regulatory regions (NRR's) of the Na + / K +-ATPase cel-subunit gene An alignment of oligo-NRRl and oligo-NRR2 revealed two common elements (identical residues are indicated by ( * )):

1

Oligo-NRR

2

ll~[J.

100

[] Cl-a

~

•

[]

[]

•

Cl-b

Cl-c

C2-a

C2-b

C2-c

80

~,

60

~1~ 60

40

~. 40

20

20

0 MF

M

HF

Cell Types

H

MF

M

HF

H

Cell Types

Fig. 8. Quantitativeanalysis of DNA/protein complexes revealed differentialexpression of DNA-bindingproteins in differentcell types. The target DNA included oligo-NRR1 and oligo-NRR2 correspondingto the negative regulatory region of the chicken Na+/K+-ATPase et l-subunit gene. 32p-signals from each DNA/protein complex identifiedin gel-retardationassays with nuclear extracts from chickencell types were quantifiedby using phosphorimager.The total quantityof all complexes formedwith the nuclear extract from a given cell type was taken as 100%. The percentage of each complex was calculated by using the corresponding total quantity. Data (mean + standard deviation) were obtained from three differentexperiments which were conductedwith differentpreparation of nuclear extracts of chicken cell types.

249

H.- E Yu et al. / Biochimica et Biophysica Acta 1309 (1996) 239-252 Oligo-NRRI

5'-CGGAAGGagcccggcgaCGCGCCGAGCCCTCCTCGGTGGGGCGCGG .w.. * ** ****it. *

NN[G/c]CG[G/c][G/c][G/c]CN

Oligo~NRR2

5'

p l e m e n t [8] (underlined). T h i s is c o n s i s t e n t w i t h the gel-retardation results s u g g e s t i n g the e x i s t e n c e o f t w o c o m m o n inhibitory factors that m a y bind to both s e q u e n c e s w i t h different affinities (Figs. 6 and 7). A l t h o u g h the s e q u e n c e s r e s p o n s i b l e for the o l i g o -

-aggcgggaacggggcaGCG~_I~CC'I~AGGCGGCCTCACCCCGGGC~

T h e first is a n e w l y identified e l e m e n t , A G C C C T C (bold). T h e s e c o n d is G C - r i c h s e q u e n c e , c o n s e n s u s to G C F binding s e q u e n c e

....

ATCCCTGA

....

.CGC$--T--T ....

....

Human

5'...AGCGCC.A.GCCCC.CT.CCAGC..

Horse

5'...-C-C--.-.C--.

Rat

5'...-C-....-

Chicken

5'...-C-C-GG-A-G...TG.CG

....... ......

or its r e v e r s e c o m -

--G..

TCT.G---... ....

TT

GTCC..CAG.AGG.AGA,TCGCCGAAGCGCT.GGAAAAGCCG.TTCCG.CCCACG.A.CTTC ,--.-.,---.---.--,.--,-,--,--,--.

.....

.-G-CCC-

T-AG-G-G

--AGG--CGGGCC-GGAT-T-C--..--.AAGAGT...CCCCTCGGAGT.CGCGC

.....

TCGG-CA--.ACGTCAC-CCA...-C---CT-TG-G--TGT--GC.A.-

AGC

...T-........

TG-

-GG-A-CCC-GGTGGTGC-CTCAGAG-

CRE/ATF TGGT..GCCGGGTCGGCGGCG...AC..TGGGGGAGCCCAGTTCCGAGA.CCGTA.CCGCACCCACTGCGGACCTCC. --TTTT...-

....

--T..CC.AACCC

C .... ....

A--GTCCTGT

. ....

-GAT-C

.................

A

A-.A-

T ..............

-C--CGCC.TGGCTTCACCCC--T-

TTTCAG-

ACG-A--.

......

G-

.T.GGGCCCTACATAGTAGGCGGGTCATGCAGGGC..AGCGGGAGGAGGGGCGTGGGAG G

.

---T-G

CC-TCG.-GA.CCCCC--CG-.TC-A..--A-A-G.--.-GC...TG-G...CA-G.-GGG

.-,,--..---,--,--,-,--,--,

......

..... . .......

C-A-CC--GACT..GCGCC---CCG--CC--CT--GG-C G ...............

- ........

C .....

...TTC.C-C-

--CGC--C

.....

...--.-A-..-G

--A--..

. ....

----.--CC

GGCCAAGGCCGCCG

..CT...G--.C-GGC--TCTACTT.CG--.-

T

-TC

C--CC...ACTGCCCCCTC-C-C-TCGAC boxl

AGT

......

CCCCGC

......

TaTTC.AGCCAG.CA.GGA...GCGCCCCG4AGceeTc~cGGGACACGGT i

G . . . . . .

- . . . . .

GGCCCCC-G...-

. . . . .

- -CAAA~A---G-.. . . . . . . . . -.-.

.....

C - . . - - T . . . -

. . . .

[

@--TGGGG~

C-CGGC--CCCGGGCGT

--GC-..--..C--- T --~". . . . . . .

T-CT-G

~A-A--CCGCGGCACTCGa

/

GG-G--GGCGGCA-CG-C---A

GC.G---ACG-G--AG

--~

/

. . . . .

hG-C-G-C...-CACCCCG

box2 GGACACGCTTGTCC --G

......

-T-

~TO

.......

CAGACC.TGGGC~CCTCC~CCGGGAGCCAGGACTGGCCGCAGCA

CGCG--CCGGGCC-G

....

. . . . . . . . . . . . . . . . . .

--C-CG-AAGGAG-CCGG

....

G

C.C

...........

cc

....

C---GCGCC-~

.....

~ ........

1

L. . . . . . . . 1

...........................

........

~GGT

GGCGC.

CAAG-A---G-GG--CGGCGC.GCMZC~C

1 1

..............

Gcccooc

G G - G C - - G G T T G C ~ C ~ C

Spl TG

....................

T C C G G G C C C G T . ~ C ~ . G G . T T T C G G G A G G G ~ C ~ I

--CTGG...GGCCGGAGTTTCG-TT---G..TGG

.....

T..AGG.CAGCGGCTAGCGTGG--C

......

......

AGCCAGGCCCAC

I

~-C..AA.-CC

...~C~7~]GAGGGGI

.C-C---TGTTGG

.....

C-.AGGTGAGCCCCG.CTTT..A-AC-TGGGCA~=CCG-ChCC...ACCCC.CCeCC.CCC.CAACG.ARE

core

A ...........

I ........

...........

~-c-

..........

CRE/ATF

~ ........

~ ....

- -G~G,~OCO~.

Spl

--MscAc

GC~.~C..

A-G.---

T...AGA.GGCGGAGGCGGAGGCCAGC.TGG

.....

TATA-Iike

J ......

~ ....

......

~,.~o~cd

T . . . . . . . . . . . .

box

i ........

[~_,~ o ~ . . . . . . . . . . . . . .

~

.

GAAGTGAGGAGGAGGCGCGGGCTggagctgcggc.ggggtctggggcgcagagcagcggegggaggaggcggacacgt ....

C ....

-

T . . . . . .

-

C .......

G---T-c AA

.................

....

.--gt.-gcact

.......

a .....

gt

............

g--cg-a

.............

.....

g---

ct

..........................

. ................

. . . . . . . . . . . . . . . . . . - .......

a-gc-

c

tat-

g

ggtagcagcccgggcggcggcagcaacagcggeggeggcatcggcccgag.ccgccggccgcc

---ageggc-gcggca-cg .....

a.gct

-C--GGcttta-.

ggc..aacagc

a--tgcgg

..........

.........

.-c

.........

agca

........

--t--g--ggc-a--ag--

g---gt---a ...-g

........

..........

t ....

t .....

.. . . . . . .

..ct.---t---g-g.

gcg-gagaga-gc-a-c...g

t-

-

t-

agga-a.g--ga.cg--gg

ctcccaccctcccgccccgcggcagccctagctcccrccac.ttggctcccctggtcccgctcgctcggccgggagct .....

t-g

.........

t.

t

. ...........

t--t--.g

c---..-g--t-.-c

.............

t---

-g..-tg-tattgtt-....----..--

.

--ga.g

........

g--t ....

.-c g-g-cg

.......

c--c-g

-

-a ...............

c--et--a---t-c-a

..............

.

---t-ga-

.

.

.

. ....

-

e

gctctgtgct.tttctctctgattctccagcgacaggacccggcgccgggcactgagcaccgceaccatg..3, • ..... ....

--c-t--

. -c---g---c

c-c--c

....

c .....

tcgctg

agtc

.... .....

c-a

.........................

- .....

......................

c ..............

a--c g--

--a-c---gt-t-c-gc

.............

-c-gc-

-g.

g---g

. .3' -

.......

..3' ..3"

Fig. 9. Alignment of nucleotide sequences of the 5' flanking region of a l-subunit genes of human [47], rat [56], mouse [21] and chicken Na+/K+-ATPase. The 5' flanking sequences end with translation initiation codons. The untranscdbed sequences are shown by upper case letters; transcribed sequences occurring in the first exon are indicated by lower case. The Spl consensus sequences are highlighted and the chicken CREB/ATF site is underlined. Homologues sequences present in three or more of the flanking regions are boxed. Gaps (.) are introduced to maximize the alignment. The residues identical to those in human sequences are shown by (-).

250

H.- Y. Yu et al. / Biochimica et Biophysica Acta 1309 (1996) 2 3 9 - 2 5 2

specific binding (Cl-a for oligo-NRR1; C2-b for oligo-NRR2) are unclear, it is likely that the imperfect dyad symmetry sequences (lower case letters; 10 nucleotides for NRR1 and 8 nucleotides for NRR2) might be the specific binding sites. The GCF-binding site and the sequence G C [ G / c ] [ G / c ] AGCCCTC were also found in the corresponding regions of the human and rat o~1-subunit genes (Figs. 9 and 10). In vertebrates, a few promoter elements with inhibitory activity have been characterized. Examples include elements of neuron restrictive silencer [29,30,34,40,44], DNA polymerase [3 gene silencer [58], BCR gene silencer [48], negative element of mouse alcohol dehydrogenase gene [27], and the GCF binding element [19]. Except the GCF binding site, none of the silencer elements defined in these studies shows any substantial sequence similarity with the chicken NRRs. However, a thorough search reveals that G C [ G / c ] [ G / c ] AGCCCTC shares some homology with other functional negative regulatory elements (Fig. 10) which were found in housekeeping genes (chicken lysozyme gene [3], and chicken vimentein gene [12]), hormone genes (rat growth hormone gene [24] and rat insulin 1 gene [23]) and mouse CD4 gene [41]. These homologous elements may play a role in the negative regulation of both housekeeping and tissue-specific gene expression. A search of the GenBank database revealed that 15 and 7 genes have homologous regions that are 4 6 67% identical to oligo-NRRl and oligo-NRR2 respectively, at the nucleic acid level. These genes include pseudorabies virus immmediate early gene (gene bank submission no. emblX15120l), herpes

virus type 1 immmediate early gene (no. gblJ043661), herpes simplex virus type 2 gene (no. dbjlD104711), human hepatitis virus gene (no. dbjID264301) and chicken TGF 133 gene (no. emblX581271). It is interesting that the viral genes share the highest homology to the chicken NRRs. Silencer function has not been demonstrated for these potential NRRs, the identification of potential NRRs therefore provides an opportunity to further understand the control of viral gene expression. The database search also revealed that some mRNAs also share homologous sequences to NRRs. A separate search with GC[C/c][G/c]AGCCCTC revealed that this element is widely dispersed in eucaryotic genes. This is consistent with previous findings by Lakshmikumaran et al. [22], who demonstrated that rat insulin silencer exists within a region of long interspersed rat repetitive DNA that is present in > 50 000 copies per cell in the rat genome.

4.2. Negati~,e regulatory mechanisms of different cell-types and species Negative regulation of the chicken o~1 promoter was observed in all tested cell types (Fig. 5). Moreover, DNA/protein complexes formed by the negative regulatory regions, NRR1 and NRR2, and the nuclear extracts from different cell-types exhibited highly similar mobilities on gel-retardation assays (Fig. 7). These results suggest that the negative regulation of the chicken Na+/K+-ATPase o~l-subunit gene in different cell types could be achieved by a shared mechanism involving common trans-acting regulatory proteins. However, the amounts of individ-

-161 CGG,~CCCGGCCACG~CCGAQC~eC~CG~r~G~

Chicken ~1 Chicken ~1 -202 GGCAC TCC~TACT~?._~C~ GGGA~C~CC Rat ~t -253 GTCCT~A~aCAG~CGCOC~C~'~A~A~CCGC~GCACTC~OGRat (zl -237 ccGcGccCC~C~CCC~GAC~C~¢~,,C'c~C~C~a~ Horse ~1 -214 G ~ T C C C A G A ~ C T G C ~ C ~ C CGGGA~CAGGACTGG Human ~I Human ~I Chicken vimentin -I.118 GCAGCC2V-.CPGCCAGCACCCTCACCACCCTCTCTGTGAAGAACTTTTCCCT Chicken lysozyme -I.009 GTCTACTCTTGTAAAAAGTTGATTCTCCTdC'~TTTGGAAGGTTC.-CAATG Chicken lysozyme +252 GTGGGGCTGCTGTGCTGGAACTGTGCCC~C~TCCCATCTCTGGGGGC Mouse CD4 +75 AACAtTCCTCC Rat insulin-i -153 GAAGCTCCTCC Rat growth hormone

Fig. 10. Sequences showing similarities to the chicken negative regulatory regions. The nucleotides identical to the newly identified sequence are shaded. The GCF consensus sequence [G/C]CG[G/C][G/C][G/C]C or its reverse complement G [C/G][C/G][C/G]CG[C/G] is underlined.Positions of the 5'-most nucleotides relative to their transcription start sites are indicated at left.

H.-K Yu et al./ Biochimica et Biophysica Acta 1309 (1996) 239-252

ual regulatory proteins in different cell types seem to vary, resulting in differential formation of D N A / p r o tein complexes in these cell types (Fig. 8). It is currently not clear whether all these factors are equally functional. Although the promoter region of the rat o~l-subunit gene ( - 155 to - 2 0 1 bp) contains the conserved negative regulatory element, GCCCTCCT, this element does not affect gene expression in the rat skeletal muscle L6 and fibroblast 3YI cells. Another region of the rat promoter ( - 2 0 2 to - 3 7 5 bp) contains the second conserved element, AGCCCTC [57], and this element inhibits the promoter activity about 40% in L6 cell but not in 3Y1 cell [57]. This mode of regulation may be related to the different activation mechanisms of the promoters; i.e., TATAdriven or Spl-driven activation in rat or chicken, respectively. Since multiple factors bind to NRR1 and NRR2 (Figs. 6-8), recruitment of different sets of protein factors may be critical in different modes of activation [16,31]. The importance of cooperative binding of a set of negative trans-acting factors in close proximity has been suggested for the regulation of human insulin gene [7] and chicken lysozyme gene [3,41.

251

shock transcriptional factor 2 have been directly imaged by using scanning force microscopy [55], and a family of transcriptional adapter (co-factor) proteins have been isolated and shown to associate with the adenoviral E1A oncoprotein [2,28] and with the TATA-binding protein [49]. Thus, these long-distant communication mechanisms (looping a n d / o r co-factor models) have established their molecular foundation and biological significance in general, although these mechanisms for the regulation of the N a + / K +ATPase a l-subunit gene have not been directly demonstrated. In summary, the activation of the N a + / K - - A T P a s e oLl-subunit gene appears to be under the control of multiple protein factors that bind to the negative regulatory regions. The level of the overall transcriptional activity appears to depend on the availability of inhibitory factors. This model could apply for gene regulation under different conditions as well as in different cell types/species. It will be interesting to explore the modulation of these factors as cellular environment changes during development and during hormonal regulation.

Acknowledgements 4.3. Possible regulatory mechanism o f NRRs

Negative regulation of gene expression may involve multiple mechanisms, as best exemplified in Drosophilia melanogaster embryogenesis [15,25,37]. Three molecular mechanisms proposed for transcriptional repression of eukaryotic promoters are direct competition, quenching, and squelching [25]. These mechanisms can be excluded in the negative regulation of the Na+/K+-ATPase c~l-subunit gene, because these three mechanisms explain how inhibitory thctors work directly at the transcriptional activation site, but not how regulatory factors exert their inhibitory actions by binding to the specific sites separate from the activation sites. However, two models to explain negative regulation, the looping model [26,35] and the co-factor model [10], would be applicable to the negative regulation of the oLl-subunit gene, in which there would need to be long-range communication over some 100-200 bp between the transcriptional machinery binding region and the NRRs. Recently, looped DNA complexes with heat-

This work was supported by grants from the American Heart Association National Center (901107 to K.T.) and the National Institute of Health (GM44373 to K.T. and NS23241 to D.M.F). H.Y. is a Postdoctoral Fellow of the American Heart Association Ohio Affiliate. K.T. is an Established Investigator of the American Heart Association.

References [1] Ahmad, M., Olliff, L., Weisberg, N. and Medford, R.M. (1994) Dr. D. Steinkopff Verlag, The Sodium pump. Germany. pp. 45-48. [2] Arany, Z., Newsome,D., Oldread, E., Livingston. D.M. and Eckner, R. (1995)Nature 374, 81-84, [3] Baniahmad, A., Muller, M., Steiner, C. and Renkawitz, R. (1987) EMBO J. 6, 2297-2307. [4] Baniahmad, A., Steiner, C., Kohne, A.C. and Renkawiz, R. (1990) Cell 57, 725-737. [5] Barry, W.H., Biedert+ S., Miura, D.S. and Smith, T.W. (1981) Circ. Res. 49, 141-149.

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