characterization of the bi-directional transcriptional control region between the human ufd1l and...

CCa

HI*a

R

l2cgsaocPldpoliaiwspwlrebf

Catgw

5

Biochemical and Biophysical Research Communications 283, 569–576 (2001)

doi:10.1006/bbrc.2001.4794, available online at http://www.idealibrary.com on

haracterization of the Bi-Directional Transcriptionalontrol Region between the Human UFD1Lnd CDC45L Genes

iroshi Igaki,*,† Keiichi Nakagawa,† Yukimasa Aoki,† Kuni Ohtomo,†wao Kukimoto,* and Tadahito Kanda*,1

Division of Molecular Genetics, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan;nd †Department of Radiology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

eceived March 15, 2001

(DGS/VCFS) suffering from cardiac defects, thymushmDtbVmfvrDiscgeh

astTgrppcfiIigtwtUb

The human UFD1L and CDC45L genes, adjacentlyocated in the head-to-head direction on chromosome2q11, are separated by a 884 base-pair (bp) segmentonstituting the putative transcriptional control re-ion. In this region we mapped one transcription startite at 69 bp upstream of UFD1L gene, and one majornd one minor start sites at 76 bp and 503 bp upstreamf CDC45L gene, which are to center in the putativeore promoters designated as PUFD1L, PCDC45L/major, andCDC45L/minor, respectively. The three core promoters

acked a TATA-motif and had a high GC-content. Toetermine the approximate ranges for the regulatoryromoters, the 884-bp fragment or those with a seriesf deletions were placed between firefly and renillauciferase genes present in the head-to-head directionn a single plasmid, and the resulting plasmids weressayed for the two transiently induced enzyme activ-ties. The PUFD1L and PCDC45L/major regulatory promotersere within 418 and 454 bp upstream of the respective

tart sites and their greater parts were not overlap-ing. The activity of PCDC45L/minor regulatory promoteras markedly enhanced when PCDC45L/major and its regu-

atory promoter were deleted. The deletion analysesevealed the basal activities of the three core promot-rs, which were enhanced by approximately twofoldy the respective regulatory promoters, on the trans-ected DNA templates. © 2001 Academic Press

Key Words: UFD1L; CDC45L; promoters.

Two human genes encoding proteins UFD1L andDC45L, respectively, are known to be closely locatednd adjacent to each other in the head-to-head direc-ion on chromosome 22q11.2 (1). Since the 22q11 re-ion is monoallelically deleted in many of the patientsith DiGeorge syndrome/velo-cardio-facial syndrome1 To whom reprint requests should be addressed. Fax: 11[81]-3-

285-1166. E-mail: [email protected].

569

ypoplasia, cleft palate, hypocalcemia, and T-cell im-une defects (2, 3), the region has been callediGeorge critical region. Thus, it has been suggested

hat the deletion of UFD1L and/or CDC45L genes maye a part of molecular mechanism underlying DGS/CFS (1). UFD1L is a human homologue of Saccharo-yces cerevisiae protein UFD1 involved in ubiquitin-

usion degradation pathway, which is essential for itsegetative growth (4, 5). CDC45L is involved in DNAeplication by binding to a prereplication complex onNA replication origin during G1 phase and by recruit-

ng DNA polymerase a through its association with p70ubunit of the polymerase (6, 7). Although it is notlear as yet how their abnormal expression is patho-enic to humans, these proteins (or one of these) arevidently required for maintenance of the body’sealth.Nucleotide sequence of the 59 regions of UFD1L

nd CDC45L genes in the AC000087 (GenBank)hows that the ATG codons in the first exons of thewo genes are separated by 884 base pairs (bp).herefore, the sequence between the two coding re-ions is expected to serve as a bi-directional controlegion, directing transcription from the respectiveromoters. Also, it is possible that closely locatedromoters within the region may be subject to theoordinated regulation by cellular transcriptionalactors. To address these questions we first need tosolate and characterize the putative control region.n this study we cloned the DNA fragment contain-ng the sequence between the two genes from humanenomic library and examined it for its bi-directionalranscriptional activities, using expression plasmidith one or two reporter genes placed in the head-

o-head direction. We identified the promoters forFD1L and CDC45L genes and characterized themy a series of deletion analyses.

0006-291X/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

MATERIALS AND METHODS

otfIppAwdCAqstSisp

CsebcmRAtaGTlt(aUpfpp

sgXl(HmSaw

isHTatleyabg

DNA fragment containing the sequence between the two genes wasablsfs

dwpbtbBamcr(ic

R

N

Uftt(stcaCbmaocaaTta(

M

tfmcHo

Vol. 283, No. 3, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cloning of genomic DNA fragment containing the promoter regionf CDC45L and UFD1L genes. Three DNA fragments containinghe promoter region of CDC45L and UFD1L genes were obtainedrom the human placenta genomic library constructed in lambda FIXI phage vector (Stratagene Co. Ltd., La Jolla, CA). The 32P-labeledrobe for the screening of 1 3 106 clones were generated by randomriming DNA labeling system (Rediprime II DNA labeling System,mersham Pharmacia, Uppsala, Sweden) and the DNA fragmenthich was amplified by PCR using DNA extracted from humaniploid WI38 cells and primers, 59-GAACTCACCTGAAGGAT-TTGCACGCACACAGAGCA (forward) and 59-CCACTCTTAG-ACCCCTGTGGGCCTATCTCACTCCT(reverse). The nucleotide se-uences of the primers were determined referring to the nucleotideequence of AC000087 (GenBank) which contains the sequences ofhe 59 regions of cDNAs of CDC45L (6) and UFD1L (8). The 7.0 kbcaI-fragment of the phage DNA hybridized with the probe was

solated and subcloned into pUC19 at the SmaI site. Nucleotideequence was analyzed with an ABI-PRISM 310 sequencer (PE Ap-lied Biosystems, Foster City, CA).

Identification of the 59-end of mRNAs transcribed from UFD1L andDC45L genes. The transcription start sites were examined byequencing of cDNA generated by 59-rapid amplification of cDNAnds method (59-RACE) or oligo-capping method (9). The cDNA li-rary used for 59-RACE was made from mRNAs extracted from HeLaells, a human cell line derived from cervical cancer, by QuickPrepRNA Purification Kit (Amersham Pharmacia Biotech) and SMARTACE cDNA Amplification Kit (Clontech Laboratories Inc., Palolto, CA). In addition to the adapter primer in the kit, primer specific

o UFD1L was 59-CATGATGGACACCACCTGGCAGACTCCGCTnd those to hCDC45 were 59-TGGCTCTGGACCACCTCGTA-AACTCTTTG and 59-CTCCCGCCAAATCACGGCTCCTCCGA-TGG (for nested PCR to clone minor species of cDNA). The cDNA

ibrary (Cap Site cDNA dT Human HeLa Cell), which was used forhe oligo-capping method, was purchased from Nippongene Co. Ltd.Toyama, Japan). cDNAs specific to UFD1L or CDC45L gene wasmplified by PCR using 1RDT primer in the kit and primer forFD1L (59-AATAGGGTGGTCgAACATGTTGAAAGAGAA) or

rimer for CDC45L (59-CTCTTCTAACCGTGTGCGCTTCTC) andurther amplified by nested PCR using 2RDT primer in the kit andrimers used for 59-RACE. The PCR products were cloned intoGEM-T Easy Vector (Promega Corp., Madison, WI) and sequenced.

Southern blot analysis. DNA samples obtained by 59-RACE wereubject to electrophoresis through 1.5% agarose gel, and DNA in theel was transferred to positively charged nylon membrane (Hybond-L; Amersham Pharmacia Biotech). DNA probes were prepared by

abeling of DNA fragments with 32P by random primer labeling kitRediprime II DNA labeling system; Amersham Pharmacia Biotech).ybridization was performed in Rapid-hyb Buffer (Amersham Phar-acia Biotech) at 65°C for 2 h. The membrane was washed in 23SC, 0.1% SDS at room temperature, in 13 SSC, 0.1% SDS at 65°C,nd in 0.13 SSC, 0.1% SDS at 65°C. X-ray film was exposed to theashed membrane at 280°C with a screen for 16 h.

Luciferase assay. Firefly luciferase expression plasmid contain-ng the promoter region of UFD1L and CDC45L genes were con-tructed by insertion of the DNA fragment, from nt 1 to 884, into theindIII site of pGL3-Basic (Promega) in both directions (Fig. 4A).he resultant plasmids were used to examine relative promoterctivities. A bi-directional expression plasmid, pRF(1/884), in whichhe coding region of first exon of UFD1L was replaced with fireflyuciferase gene and that of CDC45L was replaced with renilla lucif-rase gene, was constructed (Fig. 5A) and used for mutational anal-sis. A DNA fragment containing renilla luciferase gene, the HindIIInd BamHI fragment of pRL-null (Promega Corp.), was insertedetween the HindIII and BglII sites of pGL3-Basic (cohesive endsenerated by digestion with BamHI and BglII are identical). The

570

mplified by PCR with primers capable of adding HindIII sites tooth ends and inserted to the HindIII site between firefly and renillauciferase genes. A series of mutations, deletions caused by Bal31 orubstitutions generated by PCR, were introduced into the promoterragment of pRF(1/884). All of the mutated promoter regions wereequenced.HeLa cells, exponentially grown in Dulbecco’s modified Eagle me-

ium supplemented with 10% fetal bovine serum in 24-well plates,ere transfected with a mixture of 0.2 mg of luciferase expressionlasmid and 10 ng of pCMVb, an expression plasmid for-galactosidase (Clontech Laboratories Inc.) by Effectene Transfec-ion Reagent (Qiagen GmbH, Hilden, Germany). Cells were incu-ated for 24 h in a CO2 incubator at 37°C, and lysed in Passive Lysisuffer of PicaGene Dual Sea Pansy (Toyo Ink Co. Ltd., Tokyo, Japan)t 4°C for 1 h. The activities of luciferases and b-galactosidase wereeasured using PicaGene Dual Sea Pansy (Toyo Ink) and Lumines-

ent b-Galactosidase Detection Kit II (Clontech Laboratories Inc.),espectively, with MicroLumatPlus LB96V Microplate LuminometerBerthold, Bundoora, Australia). Luciferase activities were normal-zed to the b-galactosidase activities to correct transfection effi-iency.

ESULTS AND DISCUSSION

ucleotide Sequence of Newly Isolated PutativeTranscriptional Control Region for UFD1Land CDC45L Genes

To characterize the putative control region for theFD1L and CDC45L genes regarding its structure and

unctions, we molecularly cloned DNA fragments con-aining the separating sequence (884 bp) between thewo genes from a human placenta genomic libraryStratagene Co. Ltd.). Figure 1 shows the nucleotideequence, obtained with the two (cl#1 and cl#3) of ourhree clones, between the translational initiationodons of the first exons of the two genes. The uppernd lower strands represent the coding strands ofDC45L and UFD1L genes, respectively. Nucleotidesetween the two ATGs are numbered from the centro-eric end to the telomeric end. The 884-bp region hadGC-content of 55%, which is higher than the average

f the entire human genome of 40% (10). In our anotherlone (cl#2) T at nt 279 was replaced with C. Two (cl#1nd cl#3) of our three clones contained eight Gs (nt 150nd 156) in place of seven Gs in cl#2 and AC000087. AATA-motif and some binding sequences for knownranscriptional factors could be found within the regionnd are presented with the functional directionsFig. 1).

apping of the Transcription Start Sitesin the Putative Control Region

To locate transcription start sites in the 884-bp con-rol region we analyzed mRNA from HeLa cells andound that cDNAs generated by the 59-RACE from

RNAs of UFD1L and CDC45L genes seemed to beomposed of single and two species, respectively. AeLa cDNA library was constructed by the standard

ligo-dT priming reverse transcription method and

tsfieeeefh

paplpoi8b

frattu

sa8uw5wdpsmd

ootfa


hen cDNAs specific to 59-regions of mRNAs tran-cribed from the two genes in the library were ampli-ed by 59-RACE. The resultant DNA samples werelectrophoresed in agarose gel and stained withthidium bromide (Fig. 2A). A broad band with anstimated size of approximately 210 bp was found inach lane. Then, cDNAs in the agarose gel were trans-erred to a nylon membrane. When the membraneaving cDNA of UFD1L was incubated with the

32P-labeled probe, which were generated by the randomriming method using the DNA fragment of nt 1 to 884s a template, the radioactivity was detected at theosition of the broad band in Fig. 2A, lane 2 (Fig. 2B,ane 1). The cDNA of UFD1L did not hybridize with therobe generated from nt 97 to 884 (lane 2). The cDNAf CDC45L with a size of approximately 210 bp hybrid-zed with the radioactive probe generated from nt 1 to84 (lane 3) and that with a size of approximately 350p hybridized with the probe generated from DNA

FIG. 1. Nucleotide sequence of the common transcriptional contne (cl#1 or cl#3) of the three newly isolated clones containing the conf UFD1L gene is designated as nucleotide 1 and others are numberranscriptional factors are shown with arrows indicating the functionrom nucleotides in other clones; AC000087 (GenBank) and another inalyses of 59-end of mRNA from HeLa cells (Fig. 2). Genomic organ

571

ragment of nt 1 to 808 (lane 4). Thus, the cDNA of 59egion of UFD1L was suggested to be a single speciesnd those of CDC45L were composed of one major andhe other minor species. It was also suggested thatranscription of the minor species started from a sitepstream of the start site of the major species.Sequencing the cDNAs obtained by the 59-RACE

howed that the start sites of UFD1L, CDC45L-major,nd CDC45L-minor transcripts were G at nt 69, G at09, and A at 382, respectively, or at 69, 76, and 503 bppstream of the respective genes (Fig. 1). The cDNAsere cloned into pGEM-T Easy Vector and sequenced.9-ends of cDNAs with maximum size of plural clonesere considered as transcription start sites. The cDNAerived from the CDC45L-minor transcripts were am-lified by the nested PCR before cloning. These tran-cription start sites of UFD1L and CDC45L-majorRNAs were confirmed by sequencing cDNAs pro-

uced by the oligo-capping method (9). The minor spe-

region for UFD1L and CDC45L genes. Nucleotide sequence is fromregion. Two ATGs are boxed and the nucleotide adjacent to the ATG

toward CDC45L gene. A TATA-motif and the binding sequences forirection. Nucleotides marked with asterisks (*) and (**) are differentated clone (cl#2). Three transcription start sites were determined bytion is shown on the top.

roltroledal dsoliza

cowastc

CDC45L genes contains three RNA polymerase IIbnP

S

aotPlmrt6cmsettP6

l(Catcostaupbr

Ccac(mgrph8wt

sT


ies of the CDC45L cDNA was not amplified by theligo-capping method. The region from nt 488 to 741as deleted in the CDC45L minor transcript presum-bly by a splicing. The size of the cDNA with theplicing agreed with that of DNA fragment detected byhe Southern blotting (Fig. 2B, lane 4). These datalearly indicate that the region between UFD1L and

FIG. 2. Analyses of 59-ends of transcripts from UFD1L andDC45L genes. (A) cDNAs generated by 59-rapid amplification ofDNA ends method (59-RACE) were electrophoresed on agarose gelnd stained with ethidium bromide. DNA size markers (lane 1).DNA generated by 59-RACE using primers specific to UFD1L genelane 2) and CDC45L gene (lane 3). The cDNA library was made fromRNAs extracted from HeLa cells. (B) Southern blotting of cDNAs

enerated by 59-RACE. cDNA samples were transferred from aga-ose gel to nylon membrane and hybridized with 32P-labeled DNArobes. cDNAs amplified using the primer specific to UFD1L wereybridized with DNA fragments of nt 1 to 884 (lane 1) and nt 97 to84 (lane 2). cDNAs amplified using the primer specific to CDC45Lere hybridized with DNA fragments of nt 1 to 884 (lane 3) and nt 1

o 808 (lane 4).

FIG. 3. Structure of the core promoters. DNA sequences aroundhown. The sequence between approximately 40 upstream and 50 bFIIB recognition element, CGCCGCC and two initiator recognition

572

inding sites or core promoters, which were desig-ated as PUFD1L for transcription of UFD1L gene andCDC45L/major, and PCDC45L/minor for CDC45L gene.

tructure of the Putative Core Promoters

Based on the size of a typical core promoter, that ispproximately 40 bp upstream and 50 bp downstreamf the transcription start site (11), it was presumedhat three core promoters PUFD1L, PCDC45L/major, and

CDC45L/minor have structures shown in Fig. 3. These threeacked a TATA-motif and an apparent common pri-

ary structure. The only TATA-motif in the controlegion was at nt 276 to 267 (Fig. 1), which seems to beoo far from the transcription start site of UFD1L (nt9) to function as the binding site for the transcriptionomplex, whereas a TATA-motif in a typical core pro-oter is located 25 to 30 bp upstream of a transcription

tart site (11). It should be noted that two core promot-rs PUFD1L, and PCDC45L/major had a higher GC-contenthan that of the area outside of the core promoters inhe 884-bp control region. The GC-contents of PUFD1L,

CDC45L/major, PCDC45L/minor, and the outside area were 71,2, 53, and 52%, respectively.Among cis-elements characteristics of the TATA-

ess promoters, TFIIB recognition site (CGCCGCC)12) and two sites for the initiator element (CTA-ACT and CCAGACT) (13) are present in PCDC45L/major,nd PCDC45L/minor, respectively (Fig. 3). The location ofhe one site for the initiator element agrees with theonsensus that the initiator element-binding siteverlaps the transcription start site (14). Bindingites for Sp1, the ubiquitous mammalian transcrip-ion factor, are located at 137 bp upstream of PCDC45L/major

nd 233 bp upstream of PCDC45L/minor (Fig. 1). Since Sp1sually directs the formation of preinitiation com-lexes to a region 40 to 100 bp downstream of itsinding sites (13), functions of these Sp1-binding sitesemain to be investigated. E2F-binding motifs are

three transcription start sites for UFD1L and CDC45L mRNAs areownstream of a start site is defined as a typical core promoter (11).ements, CTACACT and CCAGACT, are indicated.

thep d

el

pm

B

rttpotgHftad4ap

moter (Fig. 4B). In addition, the 883-bp fragment fromccita8terct

D

crsbfrdsdgtsar

tmdfaTtratvpbdtsbuioat

mt

tSptFt(crbwtt


resent near the start sites of UFD1L and CDC45Lajor transcript (Fig. 1).

i-Directional Expression from the IsolatedControl Region

The 884-bp DNA fragment constituting the controlegion for the two genes was tested for its transcrip-ional activities to drive a firefly luciferase gene inransfected HeLa cells. In the constructed expressionlasmid the reporter gene was placed under the controlf the 884-bp DNA fragment in either of the two direc-ions, toward UFD1L and CDC45L genes in the humanenome (Fig. 4A). Measuring the enzyme activity witheLa cell lysates after transfection showed that the

ragment was capable of directing the reporter in ei-her direction, and that the activated transcriptionppeared to be approximately sixfold higher in theirection of UFD1L gene than of CDC45L gene (Fig.B). The level of expression toward UFD1L gene wasbout two-thirds of that obtained with the SV40 earlyromoter and enhancer, a highly efficient viral pro-

FIG. 4. Bi-directional transcription from the cloned DNA con-aining the control region of human CDC45L and UFD1L genes. (A)tructure of firefly luciferase expression plasmid used to measureromoter activity of the isolated DNA fragment containing the con-rol region. DNA regions of nt 1 to 884 of cl#1 and cl#2 (see legend forig. 1) were amplified by PCR using primers with HindIII site atheir 59 ends and inserted into the HindIII site of the pGL3-BasicPromega). (B) Relative luciferase activities of extracts from HeLaells transfected with the expression plasmids containing the controlegion. Cells were cotransfected with an expression plasmid for-galactosidase (Clontech Laboratories Inc.) and luciferase activitiesere normalized to the b-galactosidase activities to correct transfec-

ion efficiency. Data are presented with standard deviations (bars) inhree independent experiments.

573

lone cl#2 was as active as that from clone cl#1, indi-ating that T (cl#1 and cl#3) or C (cl#2) at nt 279 andnsertion of G between nt 150 and 156 were not impor-ant for the promoter activity. It was shown that thectivated transcription from the core promoters in the84-bp fragment can be measured in the context ofransfected DNA templates in vivo. Thus, the transientxpression system was used for characterization of theegulatory promoters adjacent to and upstream of theore promoters (9), as described in the following sec-ion.

eletion and Mutational Analyses of the RegulatoryPromoters

A dual expression plasmid pRF(1/884) (Fig. 5A) wasonstructed for determination of the approximateanges of regulatory promoters, which activate tran-cription from the respective core promoters capable ofinding basic transcription factors. The 884-bp DNAragment from cl#1 was placed between the firefly andenilla luciferase genes existing in the head-to-headirections in an expression plasmid, to measure tran-cription in the direction of UFD1L gene with the in-uced firefly luciferase and in the direction of CDC45Lene with the induced renilla luciferase after transfec-ion of HeLa cells. A series of deletions and some sub-titutions were introduced in the 884-bp DNA and ex-mined for their effects on transient expression of theeporter genes.Regarding the firefly luciferase activity (in the direc-

ion of UFD1L gene), it was possible to group theutant fragments with a series of deletions from the

istal end into three classes; one with a presumablyull activity, another with a reduced level (down topproximately 40%), and another with none (Fig. 5B).he enzyme activity was not affected much by dele-ions from nt 884 up to nt 488, but was markedlyeduced by deletions up to nt 328 and was totallybolished when deletion reached nt 109. The data seemo indicate that two boundaries exist in the area in-olved in transcription of the reporter gene. From itsosition and the induced enzyme activities, the firstoundary between nt 328 to 487 is presumed to be theistal end of PUFD1L regulatory promoter, suggestinghat the entire UFD1L promoter is within 418 bp up-tream of the start site (at nt 69). The second boundaryetween nt 109 and 133 can be presumed to be thepstream end of the putative core promoter, because it

s sufficiently close to the core, and because, like thene with a TATA-motif (15, 16), the core promoterlone is probably capable of directing a basal level ofranscription on transfected DNA templates in vivo.

Within the UFD1L promoter area some substitutionutations were introduced into the binding motifs for

ranscription factors to test their effects. Since substi-

tA1(ficr

twrewwnsPu

ua

ttw(nrAsh8Pt

hc

pClCiirabpe


utions in E2F (GC at 97 and 96 were replaced withA) (17), GATA-1 (GA at nt 404 and 403 with CT) (18,9), and AML-1a (GGT from nt 436 to 434 with TAG)20) (Fig. 1) in pRF(1/884) did not affect the level ofrefly luciferase activity (data not presented), it wasoncluded that these factors are not involved in theegulation of PUFD1L.

Comparison as to the renilla luciferase activity (inhe direction of CDC45L gene) of the mutant fragmentsith serial deletions from the distal end showed the

esults similar to those obtained as to the firefly lucif-rase activity (Fig. 5B). The renilla luciferase activityas not affected by deletions from nt 1 up to 354, butas reduced by half by deletion up to nt 487 and toone by deletion up to nt 803. Therefore, it was pre-umed that the distal end of regulatory promoter forCDC45L/major is between nt 355 and 487, or within 454 bppstream of the start site (at nt 809), and that the

FIG. 5. Deletion and mutational analyses of the regulatory promlasmid used for deletion and mutational analyses. The DNA fragmDC45L genes is placed as a promoter acting in the two directions to

uciferase activities of extracts from HeLa cells transfected with pRFells were cotransfected with an expression plasmid for b-galactosid

zed to the b-galactosidase activities to correct transfection efficiencyndependent experiments. (C) Schematic presentation of structure ofectangle represents a putative core promoter, which is defined as thtranscription start site. The actual size or range may differ from

etween A and B. A larger rectangle represents an approximate rangromoter is located between C and D, but the deletion analyses usednd. Directions of transcription are indicated by arrows from start s

574

pstream end of the core promoter is between nt 760nd 802.Within the PCDC45L/major promoter area some substitu-

ions were introduced in binding motifs for transcrip-ion factors. Substitutions in Staf (CCA from 366 to 368ere replaced with AAC) (21, 22), CHOP-C/EBP

TGCA from nt 372 to 375 with GCGC) (23), CP2 (C att 380, A at nt 382, and G at nt 383 with G, T, and C,espectively) (24) and E2F (GC at nt 815 and 816 withA) did not affect the transcription (data not pre-ented). Since the region from nt 276 to 884 inducedigher level of luciferase than the region from nt 1 to84, a suppressive element for the transcription fromCDC45L/miajor may be present within the region from nt 1

o 275.The expression from PCDC45L/minor was markedly en-

anced by removal of the region from nt 488 to 884 thatontains PCDC45L/major and the greater part of its regula-

ers. (A) Structure of a firefly and renilla luciferase dual expressiont containing the transcription control region between UFD1L andtrol expression of the luciferase genes. (B) Relative firefly and renilla/884) with or without mutations in the transcription control region.

(Clontech Laboratories Inc.) and luciferase activities were normal-ta are presented with standard deviations (bars) from at least threetranscription control region for UFD1L and CDC45L genes. A smallgion between nucleotides 40 bp upstream and 50 bp downstream ofe to core. The upstream end of a core promoter is presumed to bef a putative regulatory promoter. The upstream end of a regulatorythis study do not provide information on extent of the downstream

s.

oten

con(1

ase. Dathee recore oin

ite

tory promoter, suggesting that the activity induced bynimabutCfpPd

mmCmwpPptP4tPomrPwpsdtbmlasgsrUtad

A

THT

REFERENCES

1

1

1

1

1

1

1

1


t 1–487 represents the full activity of PCDC45L/minor andts regulatory promoter. The activity of 104-bp frag-

ent from nt 355 to 458 had half of the presumed fullctivity and probably represents the activity of the coreecause deletion of further 29 nucleotides from thepstream end abolished the expression. Since nucleo-ide substitutions in the binding motifs of Staf andHOP-C/EBP, mentioned above, abolished expression

rom the 104 bp region, Staf and CHOP-C/EBP may beositive cis-elements in the regulatory promoter forCDC45L/minor. The mutation in the binding motif for CP2id not affect the transcription.The results of this study are summarized and sche-atically presented in Fig. 5C. The short DNA seg-ent of 884 bp between first exons of UFD1L andDC45L genes was found by analyses of HeLa cellRNA to contain three transcription start sites, whichere to be centered in three putative TATA-less coreromoters; PUFD1L for UFD1L gene and PCDC45L/major andCDC45L/minor for CDC45L gene. The presumed size of coreromoter is based on the assumption that the core isypical. The deletion analyses have shown that the

UFD1L and PCDC45L/major regulatory promoters are within18 and 454 bp upstream of the respective transcrip-ion start sites. The greater part of the PUFD1L and

CDC45L/major regulatory promoters appeared not to beverlapping, although the distal ends of regulatory pro-oters are not yet defined. The activity of PCDC45L/minor

egulatory promoter, which was overlapping withUFD1L regulatory promoter, was markedly enhancedhen PCDC45L/major and the greater part of its regulatoryromoter were deleted. The deletion analyses have alsohown the three core promoters appear to be capable ofirecting basal transcription on the transfected DNAemplates, and that the basal activities are enhancedy twofold or more by the respective regulatory pro-oters. The promoters packaged in a compact space

ike the 884-bp segment studied here would have andvantage over the promoters placed distantly andeparately, for the coordinated expression of multipleenes controlled by cellular factors. The data in thistudy provide bases for further analyses of the possibleegulatory mechanisms for coordinated expression ofFD1L and CDC45L genes in the context of both on

he transfected DNA and on the chromosome and fornalyses of its relation to occurrence of the diseasesue to abnormal expression of these genes.

CKNOWLEDGMENTS

We thank Dr. K. Yoshiike for critical reading of the manuscript.his work was supported by a Grant-in-Aid from the Ministry ofealth and Welfare for the Research on Human Genome and Geneherapy.

575

1. Yamagishi, H., Garg, V., Matsuoka, R., Tiffani, T., and Sriv-astava, D. (1999) A molecular pathway revealing a geneticbasis for human cardiac and craniofacial defects. Science 263,1158 –1161.

2. Carey, A. H., Kelly, D., Halford, S., Wadey, R., Wilson, D., Good-ship, J., Burn, J., Paul, T., Sharkey, A., Dumanski, J., Nor-denskjold, M., Williamson, R., and Scambler, P. J. (1992)Molecular genetic study of the frequency of monosomy 22q11 inDiGeorge syndrome. Am. J. Hum. Genet. 51, 964–970.

3. Driscoll, D. A., Budarf, M. L., and Emanuel, B. S. (1992) Agenetic etiology for DiGeorge syndrome: Consistent deletionsand microdeletions of 22q11. Am. J. Hum. Genet. 50, 924–933.

4. Johnson, E. S., Ma, P. C., Ota, I. M., and Varshavsky, A. (1995)A proteolytic pathway that recognizes ubiquitin as a degradationsignal. J. Biol. Chem. 27029, 17442–17456.

5. Pizzuti, A., Novelli, G., Ratti, A., Amati, F., Mari, A., Calabrese,G., Nicolis, S., Silani, V., Marino, B., Scarlato, G., Ottolenghi, S.,and Dallapiccola, B. (1997) UFD1L, a developmentally expressedubiquitination gene, is deleted in CATCH 22 syndrome. Hum.Mol. Genet. 6, 259–265.

6. Kukimoto, I., Igaki, H., and Kanda, T. (1999) Human CDC45protein binds to minichromosome maintenance 7 protein and thep70 subunit of DNA polymerase a. Eur. J. Biochem. 265, 936–943.

7. Aparicio, O. M., Stout, A. M., and Bell, S. P. (1999) Differentialassembly of Cdc45p and DNA polymerases at early and lateorigins of DNA replication. Proc. Natl. Acad. Sci. USA 96, 9130–9135.

8. Novelli, G., Mari, A., Amati, F., Colosimo, A., Sangiuolo, F.,Bengala, M., Conti, E., Ratti, A., Bordoni, R., Pizzuti, A., Baldini,A., Crinelli, R., Pandolfi, F., Magnani, M., and Dallapiccola, B.(1998) Structure and expression of the human ubiquitin fusion-degradation gene (UFD1L). Biochim. Biophys. Acta 1396, 158–162.

9. Maruyama, K., and Sugano, S. (1994) Oligo-capping: A simplemethod to replace the cap structure of eukaryotic mRNA witholigonucleotides. Gene 138, 171–174.

0. Gardiner, K. (1996) Base composition and gene distribution:Critical patterns in mammalian genome organization. TrendsGenet. 12, 519–524.

1. Carey, M., and Smale, S. T. (1999) A primer on transcriptionalregulation in mammalian cells. In Transcriptional Regulation inEukaryotes (Carey, M., and Smale, S. T., Eds.), pp. 1–50, ColdSpring Harbor Laboratory Press, New York.

2. Lagrange, T., Kapanidis, A. N., Tang, H., Reinberg, D., andEbright, R. H. (1998) New core promoter element in RNA poly-merase II-dependent transcription: Sequence-specific DNA bind-ing by transcription factor IIB. Genes Dev. 12, 34–44.

3. Smale, S. T. (1994) Core promoter architecture for eukaryoticprotein-coding genes. In Transcription: Mechanisms and Regu-lation (Conaway, R. C., and Conaway, J. W., Eds.), pp. 63–81,Raven Press, New York.

4. Smale, S. T. (1997) Transcription initiation from TATA-less pro-moters within eukaryotic protein-coding genes. Biochim. Bio-phys. Acta 1351, 73–88.

5. Hernandez, N. (1993) TBP, a universal eukaryotic transcriptionfactor? Genes Dev. 7, 1291–1308.

6. Burley, S. K., and Roeder, R. G. (1996) Biochemistry and struc-tural biology of transcription factor IID (TFIID). Annu. Rev.Biochem. 65, 769–799.

7. Tao, Y., Kassatly, R. F., Cress, W. D., and Horowitz, J. M. (1997)Subunit composition determines E2F DNA-binding site specific-ity. Mol. Cell. Biol. 17, 6994–7007.

18. Merika, M., and Orkin, S. H. (1993) DNA-binding specificity of

1

2

2

promoter of the selenocysteine tRNA gene. EMBO J. 14, 3777–

2

2

2


GATA family transcription factors. Mol. Cell. Biol. 13, 3999–4010.

9. Ko, L. J., and Engel, J. D. (1993) DNA-binding specificities of theGATA transcription factor family. Mol. Cell. Biol. 13, 4011–4022.

0. Meyers, S., Downing, J. R., and Hiebert, S. W. (1993) Identifica-tion of AML-1 and the (8;21) translocation protein (AML-1/ETO)as sequence-specific DNA-binding proteins: The runt homologydomain is required for DNA binding and protein-protein inter-actions. Mol. Cell. Biol. 13, 6336–6345.

1. Schuster, C., Myslinski, E., Krol, A., and Carbon, P. (1995) Staf,a novel zinc finger protein that activates the RNA polymerase III

576

3787.2. Schaub, M., Myslinski, E, Schuster, C., Krol, A., and Carbon, P.

(1997) Staf, a promiscuous activator for enhanced transcriptionby RNA polymerases II and III. EMBO J. 16, 173–181.

3. Ubeda, M., Wang, X. Z., Zinszner, H., Wu, I., Habener, J. F., andRon, D. (1996) Stress-induced binding of the transcriptionalfactor CHOP to a novel DNA control element. Mol. Cell. Biol. 16,1479–1489.

4. Kim, C. G., Swendeman, S. L., Barnhart, K. M., and Sheffery, M.(1990) Promoter elements and erythroid cell nuclear factors thatregulate alpha-globin gene transcription in vitro. Mol. Cell. Biol.10, 5958–5966.

characterization of the bi-directional transcriptional control region between the human ufd1l and...

Documents