research report identification of nfi-binding sites and ...rgron/baumeis.pdffib and mfib were...

Ž .Molecular Brain Research 72 1999 65–79www.elsevier.comrlocaterbres

Research report

Identification of NFI-binding sites and cloning of NFI-cDNAs suggest aregulatory role for NFI transcription factors in olfactory neuron gene

expression 1

Hans Baumeister a, Richard M. Gronostajski b,c, Gary E. Lyons d, Frank L. Margolis e,)

a Deutsches Institut fur Ernahrungsforschung, Bergholz-Rehbrucke, 14558, Germany¨ ¨b Department of Cancer Biology, Research Institute, CleÕeland Clinic Foundation, CleÕeland, OH 44195, USA

c Department of Biochemistry, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106, USAd Department of Anatomy, UniÕersity of Wisconsin Medical School, Madison, WI 53706, USA

e Department of Anatomy and Neurobiology, UniÕersity of Maryland School of Medicine, HSF 280, 685 West Baltimore Street, Baltimore, MD 21201,USA

Accepted 22 June 1999

Abstract

Olfactory receptor neurons are responsible for the detection and signal transduction of odor ligands. Several genes associated with thisactivity are preferentially or exclusively expressed in these neurons. Among these genes are those coding for olfactory receptors, adenylyl

Ž . Ž .cyclase type III, the cyclic nucleotide gated olfactory channel 1 OcNC-1 , Ga and the olfactory marker protein OMP . Promoterolf

analyses of these genes identified a binding site for the new transcription factor family OrE whose initial member, Olf-1, is abundantlyexpressed in olfactory neurons. We report here that the proximal promoters of three of these genes, that are selectively expressed inolfactory neurons, each contains a functional NFI binding site and that the sites have different affinities for NFI proteins indicating aregulatory role for NFI proteins in olfactory gene expression. We further demonstrate, by cloning, that all four NFI genes are expressed inthe olfactory nasal mucosa. Analysis by in situ hybridization illustrates that at least three of these gene products are expressed in theneuroepithelium in which the olfactory neurons reside. NFI proteins are capable of functioning as positive or negative regulators oftranscription depending on the tissue, cell-type, age, and gene in question. These multivalent functions of NFI could be achieved bytemporally and spatially regulated expression of distinct subsets of NFI isoforms. It now remains to characterize the tissue and cellspecific patterns of expression of distinct NFI transcription factors during ontogeny and their roles in regulating gene expression. q 1999Elsevier Science B.V. All rights reserved.

Keywords: Upstream binding element; Olfactory gene promoter; Nuclear Factor I; Olfactory receptor neuron; Gene expression; Mobility shift assay

AbbreÕiations: UBE, Upstream binding element; Cyclase III, Adeny-lyl cyclase type III; UCY, Upstream binding element in the cyclase IIIgene; FIB, Consensus NFI binding site; mFIB, Mutated FIB; ORN,Olfactory receptor neuron; oCNC-1, Olfactory cyclic nucleotide-gatedchannel subunit 1; UC, Upstream binding element in the oCNC-1 gene;OR, Olfactory receptor gene; OMP, Olfactory marker protein; NFI,Nuclear factor I; Olf-1, Olf-1 transcription factor; EBF, Early B-cellfactor; NEM, N-ethyl maleimide; DTT, Dithiothreitol; EMSA, Elec-trophoretic mobility shift assay; Mash-1, Mouse homologue of theachaete-scute gene

) Corresponding author. Fax: q1-410-706-2512; E-mail:[email protected]

1 The sequences described here have been assigned Accession numbersAF112455–AF112459.

1. Introduction

The olfactory mucosa in the nasal vault contains theolfactory neuroepithelium, a tissue that is highly special-ized for odorant detection. Olfactory neurons in this strati-fied neuroepithelium express several genes, specifically orpredominantly, enabling them to respond to odorants andto transduce this information into action potentials directedto their terminals in the olfactory bulb. In mammals,several of these genes encode the components of a cyclicAMP-mediated pathway including 7TM-membrane recep-

w x w xtors 10,67 , G , a stimulatory G-protein 35 , type IIIa olf

0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0169-328X 99 00210-7

( )H. Baumeister et al.rMolecular Brain Research 72 1999 65–7966

w xadenylyl cyclase 6 and the olfactory cyclic nucleotide-Ž . w xgated ion channel 1 OcNC-1 19,37 . Together with

functional studies, these observations provide evidence forw xa model 9,12,13,31,75 in which odorant binding to puta-

tive membrane receptors stimulates the synthesis of cAMPthat in turn activates the ion channel resulting in depolar-ization of the sensory neuron. The diversity of odorants ismatched by a family of up to a thousand receptor genes in

w xmammals 11 . However, a single olfactory neuron ex-presses just one or a small subset of receptor genesw x61,62,68 assigning an important role in olfactory codingto the control of gene expression in olfactory neuronsw x17,22,44,68,76,80 .

An additional novel characteristic of mature olfactoryneurons, that is distinct from virtually all other differenti-ated neurons, is their ability to be replaced in a process of

w xcontinuous turnover throughout adult life 8,26 . The keyto this process is a population of globose basal cellslocated at the basal region of the neuroepithelium that actas progenitor cells. These globose basal cells undergomitosis and subsequent migration in the apical directionand progressive differentiation to mature olfactory neuronsreplacing those which degenerate and die. This process isassociated with the induction of olfactory neuron-specific

w xgene expression 49,55,56,74,77 . Mature olfactory neu-rons are identified by the presence of the olfactory marker

Ž . w xprotein OMP 50–52,77 , a 19-kDa cytoplasmic proteinw xthought to be a modulator of signal transduction 15

although a recent report suggests it may participate inw xregulating mitosis in olfactory neuronal precursors 20 .

Genes whose products are responsible for the differenti-ated properties of mature neurons, i.e., the olfactory recep-tors, Ga , type III cyclase, and OcNC-1, must be inducedolf

during the course of this differentiation in a coordinatedfashion.

The molecular mechanisms underlying olfactory neu-ron-specific differentiation remains unknown. An impor-tant regulator may be the mouse achaete-scute homolog-1Ž .Mash-1 , a transcription factor that was localized to earlystages of the developing mouse olfactory neuroepitheliumw x28 . Targeted deletion of the Mash-1 gene in mice re-

w xsulted in a loss of olfactory neuron precursors 29 . Butforced expression of the Mash-1 gene in P19 embryonalcarcinoma cells is insufficient to drive their neuronal dif-ferentiation, although these cells express endogenousMash-1 when induced to differentiate to neurons by retinoic

w xacid 34 . Therefore, Mash-1 may be a necessary but notw xsufficient factor for olfactory neuronal differentiation 34 .

A candidate to interact with Mash-1 is Olf-1, the firstw xmember of the OrE family 80 of transcription factors of

Ž .the helix–loop–helix HLH family like Mash-1, that islocalized to the nuclei of immature and mature olfactory

w xneurons 79 . Olf-1 binding sites were identified in severalŽgenes associated with the olfactory phenotype: OMP two

.sites , G , type III cyclase, OcNC-1, 50.06, and 50.11a olf

w x43,78 . The gene encoding Olf-1 is additionally expressedin early B-cells giving rise to the nearly identical transcrip-

Ž . w xtion factor EBF early B-cell factor 30 . Mice with apartial deletion of Olf-1rEBF are B-cell-deficient but do

w xnot suffer from deficits in olfactory gene expression 48 .This apparent discrepancy may reflect the presence ofmultiple members of the OrE family in olfactory neurons

w xbut not in the lymphoid system 80 or it may indicate thatadditional factors are important for olfactory gene expres-sion. One such factor may be Roaz, a zinc finger proteinthat interacts with Olf-1rEBF to regulate gene expressionw x76 .

In the present study, we demonstrate that members ofthe NFI family of transcription factors are present inolfactory neurons and that they bind specific sites inolfactory specific gene promoters. The upstream binding

Ž .element UBE within the OMP gene promoter and twoŽ .sites U-sites within the OcNC-1 and the type III cyclase

w xgene promoters were described previously 43,78 . Allthree sites were identified by footprint analyses but thetranscription factors that bind these sites remained un-known. The significance of the UBE site is emphasized bythe fact that it is conserved in the rat, mouse and human

w xOMP genes at nearly the same position 14 .

2. Materials and methods

( )2.1. Electrophoretic mobility shift assay EMSA

Nuclear extracts were prepared from the olfactory neu-Žroepithelium of 3-week-old CD rats Charles River Labo-

. w xratories as described previously 43 . Two microgramŽprotein of the nuclear extract 1 mgrml protein concentra-

.tion were incubated at room temperature for 15 min in abinding buffer containing 25 mM HEPES pH 7.5, 150 mM

Ž .NaCl, 5 mM MgCl , 4 mM DTT, 125 mgrml poly dI-dC2Ž . ŽPharmacia, Upsala, Sweden , 0.5 mgrml BSA Boeh-

.ringer-Mannheim, Germany and 0.5% Nonidet P-40. Af-ter the first incubation, samples were placed on ice, 1 ml of

Žthe radiolabeled DNA about 0.5 ng of DNA containing.100,000 c.p.m.rml was added to a final volume of 20 ml

and the samples were incubated for further 15 min at roomtemperature. After the second incubation, samples were

Žplaced on ice and 3 ml of dye 0.25% bromophenol blue,.0.25% xylene cyanol FF, 30% glycerol were added. The

samples were electrophoresed in a 5% nondenaturingŽacrylamide gel containing 0.5= TBE 44.5 mM Tris-Base,

.44.5 mM Boric acid, 1 mM EDTA, pH 8.3 as gel andrunning buffer. For autoradiography, the dried gels wereexposed at y808C for 2 h and overnight.

( )H. Baumeister et al.rMolecular Brain Research 72 1999 65–79 67

The DNA-probes were generated by annealing pairs ofsynthetic oligonucleotides as below:

w xFIB and mFIB were previously described 24 . Oligonu-cleotides were purified by electrophoresis in a 20% acryl-

Ž .amide gel before annealing. DNA 40 ng was labeled withw 32 xa P dCTP with the Klenow polymerase resulting in

Ž .100,000 c.p.m.r0.8 ng DNA UBE , 100,000 c.p.m.r0.5Ž . Žng DNA UCY and 100,000 c.p.m.r0.4 ng DNA FIB

.and UC , respectively.Supershift EMSAs were performed with 1 ml of the

Ž .rabbit antiserum or preimmune serum a8199 providedw xby N. Tanese 72 . Antiserum or preimmune serum were

added to the samples containing the DNA probes and wereincubated for an additional 7 min at room temperature.

To analyse the binding activity of NFI-C220 by EMSA,w xwe used the condition described by Novak et al. 63 ,

applying 5 ml of the enriched protein and about 1 ng DNAlabeled with 200,000 c.p.m., respectively. Fresh DTT andBSA were added separately.

To analyse the NEM-sensitivity of the UBE-bindingactivity, the nuclear extract was incubated with 10 mMNEM for 10 min at 48C. Excess NEM was then inactivatedby addition of 50 mM DTT and incubation for 10 minmore at 48C. Samples treated with NEMrDTT were anal-ysed by EMSA as described above except that 50,000c.p.m.rreaction were used. The sensitivity to NEM wasalso analysed by altering the order of addition of thevarious components.

2.2. Cross-linking of DNA-protein complexes

Ž .Nuclear extract 10 mg was incubated with 100,000c.p.m. of the radiolabeled DNA probe as described forEMSA. Competitors were added in 50-fold excess. Forcross-linking, the samples were exposed to short wave UV

Ž .light 256 nm for 5 min at room temperature. All sampleswere analysed on a 10% SDS polyacrylamide gel. 14C-

Žlabeled rainbow colored proteins Amersham, Arlington.Heights, USA were used as molecular weight standards.

Irradiating the protein standard with UV light under thesame conditions as used for cross-linking did not alter their

mobility pattern compared to an unirradiated protein stan-dard.

2.3. RT-PCR

To extract total RNA from olfactory neuroepithelium,we used RNAzol according to the manufacturer’s descrip-

Ž .tion AGS, Heidelberg, Germany . Synthesis of first strandcDNA was performed in the presence of 5 mg total RNA,

Ž . Ž . Ž100 pmol d N 6-primer Pharmacia , 1= buffer supplied. Žwith the enzyme , 10 mM DTT, 0.5 mM dNTP each

.nucleotide, respectively and 400 U of recombinantŽ .Moloney Murine Leukemia Virus MoMuLV reverse tran-

Ž .scriptase Superscript, BRL, Bethesda, USA in a totalvolume of 20 ml. After 1 h of incubation at 378C, theenzyme was inactivated by incubation at 528C for 30 min

Ž .followed by ethanol precipitation. One-tenth 5 ml of theredissolved precipitate was used for PCR which was per-

Ž .formed in the presence of 1= buffer Perkin-Elmer, USA ,Ž .200 mM dNTP each nucleotide, respectively , 2.5 U Taq

Ž .Polymerase Perkin-Elmer and 150 pmol of each primerŽ Ž . Ž .deg1 and deg2 deg1: TTCCGGATGA GrA TT CrT -

Ž . Ž . Ž . Ž .CA CrT CCITT CrT AT CrT GA GrA GC, deg2:Ž . Ž . Ž . Ž .AATCGAT GrA TG ArG TG CrTrG GGCTGIA CrT

. .GrA CAIAG in a final volume of 50 ml. These primerswere previously used to amplify fragments of NFI-cDNAs

w xfrom mouse 16 . The PCR amplification program was 1min at 948C, 2 min at 508C and 2.5 min at 728C for 30cycles. The same conditions were used to amplify NFIcDNA fragments from 1 ug of plasmid DNA from acDNA library of rat olfactory neuroepithelium. The PCR-products of 490 bp was isolated and cloned into pBlue-

Ž .script Stratagene at the SmaI site. Cloned PCR-productswere analysed by automated nucleotide sequencing using

Ž .the DNA sequencer A373 ABI .

2.4. Cloning of NFI-A2

A portion of a rat olfactory epithelium cDNA libraryŽ .50,000 clones at 5000 clones per plate subcloned into

Ž .pSport BRL was screened for full size cDNA-clonesencoding NFI of the A-type. As probe we used the NFI-AcDNA fragment which was amplified by PCR from thelibrary. Replica filters were prehybridized in 50% for-mamide, 20 mM HEPES pH 7.0, 5= SSC, 5= Denhardt’ssolution, 0.1% SDS and 50 ugrml salmon sperm DNA.For hybridization 50 ng of the NFI-A DNA fragment were

Ž .radiolabeled using the Ready-to-Go kit Pharmacia whichresulted in 1.4=108 c.p.m.rmg. The radiolabeled DNAfragment was added to 100 ml of prehybridization solutionŽ 4 .7=10 c.p.m.rml and hybridization was carried outover night at 428C. The replica filters were washed twicefor 5 min in 2= SSC, 0.1% SDS at room temperature andtwice for 10 min in 0.2= SSC, 0.1% SDS at 688C. Afterautoradiography overnight at y808C, three positive signals


were detected, two of which were verified on second roundscreening with the same probe. Nucleotide sequencingconfirmed that both clones contained the same cDNAencoding NFI-A2.

2.5. In situ hybridization

In situ hybridization was performed exactly as previ-w x 35ously described 16 . S-labeled antisense probes were

selected from the 3X coding and non-coding regions of NFIclones that differ between the four NFI transcripts.

3. Results

3.1. UBE, UC and UCY are binding sites for NFI tran-scription factors

On comparison of the nucleotide sequences of the threeUBE sites present in the human, mouse and rat OMPpromoter, respectively, with the nucleotide sequence of theUC-site, described within the promoter of the rat olfactory

Ž .cyclic nucleotide-gated channel OcNC we found a com-X Ž . Xmon palindromic motif of 5 -CTGG N 7-8CCAG-3 re-

sembling the palindromic motif present in the NFI bindingŽ .site FIB Table 1 . The UCY-site, described within the

promoter of the rat type III adenylyl cyclase, shares a 9-bpidentity with the UC-site and contains a palindromic motif

X Ž . Xof 5 -GGCA N 3TGCC-3 that is almost identical to theŽ .consensus sequence for NFI binding sites Table 1 . This

degree of homology prompted us to analyse whether NFIproteins bind to UBE andror the U-sites.

The EMSA was used to analyse the binding activity ofnuclear proteins of rat olfactory neuroepithelium towardsynthetic double-stranded DNA probes containing the highaffinity NFI binding site FIB, the rat UBE, the UC and the

Ž .UCY sites, respectively Figs. 1 and 2 . Nuclear proteinsŽof the olfactory neuroepithelium are able to bind FIB Fig.

.1A illustrating that NFI transcription factors are presentwithin this tissue. DNA-protein complexes formed withFIB, UBE, UC and UCY, respectively, all migrate with thesame electrophoretic mobility. To confirm that NFI tran-scription factors bind to UBE, UC and UCY, weperformed EMSAs in the presence of a specific NFI

Ž .antiserum Fig. 1B , in the presence and absence of com-Ž .petitors Fig. 1C , and in the presence of NEM, an alkylat-Ž .ing agent Fig. 2A . The addition of unlabelled FIB totally

abolished binding to all the sites. By contrast, mFIB, themutated NFI binding site with a single base alterationŽ .Table 1 and Section 2 that is devoid of any NFI-bindingactivity, was ineffective in altering binding to FIB, UBE,UC or UCY. A polyclonal antiserum that recognizes the

Table 1Sequence homologies between UBE, UC and UCY and the NFI-consensus sequence

aUBE, UC and UCY were described elsewere.b Bold characters mark matching nucleotides between UBE, UC, UCY, FIB and the NFI-consensus sequence. The point mutation in mFIB is marked by anarrow head.c Ž . Ž .The position refers to the transcription start site UC and UCY and the translation start site UBE , respectively. The UCY-site is located within the5X UTR on the noncoding DNA strand.d w x w xFootprint and EMSA analyses were published elsewhere 14,44,78 . FIB and mFIB do not represent genomic sites for protein binding 63 . The

w xNFI-consensus sequence was published 63 .


amino-terminal portion of NFI-CrCTF1, a human NFItranscription factor, was analysed for its ability to form

supershifts with the UBE-, UC- or UCY-binding proteinsin rat olfactory epithelial extracts. The amino-terminalregion contains the DNA binding domain known to beconserved among all NFI transcription factors so far identi-

w x Ž .fied 41 . The results of this experiment Fig. 1B demon-strate that the NFI-antiserum specifically recognizes pro-teins bound to UBE, UC and UCY, respectively, resulting

Ž .in ternary complexes with reduced mobility supershiftsŽ .of the respective DNA, the bound protein s and the

antibody. Preimmune serum does not form any supershift,nor is the antiserum able to bind on its own to any DNAused in these experiments. Thus, the immunochemicalresults and binding characteristics indicate that UBE-, UC-and UCY-binding proteins belong to the family of NFItranscription factors. Finally, the demonstration that the

Ž .Fig. 1. A Binding of rat olfactory epithelial nuclear proteins withputative NFI-binding sites from genes expressed abundantly within theolfactory epithelium. EMSAs were performed as described with 2 ug ofnuclear extract of rat olfactory epithelium and radiolabeled synthetic

ŽDNA as probe 100,000 c.p.m. per binding reaction, for nucleotide.sequence, see Section 2 . Protein-bound DNA is visible in lanes 1–6. In

all cases, the band representing the rapidly migrating unbound DNA hasbeen trimmed from the Figures. Lane 1: binding to FIB containing the

w xNFI-binding site described previously 24 . Lanes 2–4: binding to puta-Ž .tive NFI-binding sites, UC lane 2 contains the U-site from the OcNC

w x Ž . w xgene 78 , UBE lane 3 contains the UBE-site of the OMP gene 43 , andŽ .UCY lane 4 contains the U-site from the adenylate cyclase type III

gene, respectively. To demonstrate binding to UBE and UCY, the sameŽ . Ž .gel was exposed for 2 h lanes 1–4 and overnight lanes 5 and 6 . As

negative controls, the binding reactions were performed in the absence ofnuclear proteins. Lane 7 represents the control experiment with UCY.

Ž .The same results were obtained with all four probes. B Recognition ofUBE-, UC-, and the UCY-binding proteins by an antiserum directedagainst NFI protein. The binding reaction with the radiolabeled DNA-probe, the nuclear extract of rat olfactory epithelium and the NFI-anti-serum or preimmune serum were performed as described in Section 2.

Ž .The location of protein-bound DNA bound DNA and protein-boundŽ . w32 xDNA including antibodies supershift are indicated. Lane 1: P FIB-

DNA incubated with nuclear extract of rat olfactory epithelium in theabsence of any serum; lane 2: same as lane 1 but in the presence of

w32 xNFI-antiserum; lane 3: same as lane 2 but with P UC-DNA as probe;w32 xlane 4: same as lane 2 but with P UCY-DNA as probe; lane 5: same as

w32 x w32 xlane 2 but with P UBE-DNA as probe; lane 6: P FIB-DNA incu-bated with NFI-antiserum in the absence of nuclear extract; lane 7: same

Ž .as lane 1 but in the presence of preimmune serum. C Efficacy of FIBand mFIB as competitors. EMSAs were performed as described for this

Ž . Ž . Ž .figure. FIB lanes 1–3 , UC lanes 4–6 , UBE lanes 7–9 and UCYŽ . Žlanes 10–12 , respectively. The competitors FIB or mFIB 2.5 ng or

.about five-fold excess over the amount of probe present in each reactionwere each added to the binding reactions presented in lanes 2, 5, 8 and 11Ž . Ž .FIB and in lanes 3, 6, 9 and 12 mFIB . The DNA mFIB contains the

Ž .NFI-binding site with one point mutation see Section 2 that abolishesthe ability of NFI to bind to this site. This property of mFIB is shown in

w32 xlanes 1–3: lane 1 shows binding of the nuclear proteins to P FIBŽ .without competitor. mFIB has no effect on this binding activity lane 3

but FIB as competitor reduces greatly the amount of protein-DNAw32 x Ž .complexes with P FIB lane 2 . Only the bands containing the protein

bound DNA are shown. Lane 13 gives the result of one binding reactionwithout nuclear extract as a negative control.


UBE-binding activity is sensitive to the thiol-specific alky-Ž .lating reagent NEM Fig. 2A offers additional strong

support that these observations reflect the presence of NFI,as it is known that the site-specific DNA binding activity

w xof NFI is abolished by NEM 63 .To obtain independent confirmation of these findings,

Žwe evaluated the ability of a recombinant protein ex-.pressed in Escherichia coli representing the amino-termi-

nal DNA-binding domain of NFI-CrCTF1 to bind UBE,Ž .UC and UCY. Analysis by EMSA shows Fig. 2B that

this protein forms complexes of identical electrophoreticmobilities with FIB and with all three DNAs. The differingintensities of the shifted complexes correspond to thoseobserved when nuclear proteins of the olfactory epitheliumwere used.

The difference in the intensities of the shifted DNA-pro-tein complexes shown in Fig. 1A indicated that NFI pro-teins bind to the three sites with different affinities. Toanalyse this in more detail, we evaluated the abilities ofUBE, UC and UCY to compete in EMSA with FIB as

Ž .radiolabeled probe. The results Fig. 2C demonstrate thatUC is most effective in competing with FIB for proteinbinding and is as efficient as FIB competing with itselfŽ .result not shown . By contrast, UBE and UCY are at least10-fold less effective as competitors compared to UC butare quite similar to each other. This indicates that NFIproteins bind UC with high affinity comparably to theaffinity of NFI proteins to its canonical binding site FIB.UBE and UCY are bound by NFI proteins with lower butvery similar affinity.


3.2. NFI transcription factors are present within the olfac-tory neuroepithelium

The presence of NFI transcription factors within theolfactory neuroepithelium is strongly indicated by theFIB-binding activity of the nuclear extract shown in Fig.1A. However, NFI comprises a family of transcriptionfactors consisting of four gene products NFI-A, -B, -C, and

Ž-X and multiple alternative splice variants for more detail,.see Section 4 . To determine whether one or several

isoforms of NFI proteins are present within the olfactoryneuroepithelium, we analysed the nuclear extract after UVcross-linking with radiolabeled FIB-DNA as probe by

Ž .SDS-polyacrylamide gel electrophoresis Fig. 2D . At leastfour different DNA-protein complexes could be detectedwith an apparent molecular weight of 36–40 kDa. Cross-linking with radiolabeled UBE-DNA resulted in detectionof two DNA-protein complexes of 40 and 43 kDa. Thespecificity of these cross-linked products is demonstratedby the ability of non-radioactively labeled FIB-DNA butnot mFIB-DNA, to compete and by the absence of anycross-linked products in the absence of nuclear proteins.Taken together, these results indicate, but do not prove,that several NFI isoforms are present in this tissue that arecapable of binding to UBE.

Compelling evidence for the presence of NFI isoformsin the olfactory neuroepithelium would derive fromdemonstration of appropriate PCR products and isolationand sequencing of NFI cDNA clones from this tissue.

Ž .Degenerate primers see Section 2 that anneal within theconserved, amino-terminal region of all NFI-cDNAs, wereused to identify and amplify transcripts of all four genesknown to encode NFI proteins. Analysis of RT-PCR prod-ucts by electrophoresis in 1% agarose gels showed a singleband, slightly smaller than 500 bp, corresponding to the

Ž .expected size of 486 bp Fig. 3 . DNA products of thesame size could be amplified directly by PCR of a rat

olfactory neuroepithelium cDNA library. The RT-PCRproducts could only be detected after reverse transcriptionof RNA confirming the absence of contamination by ge-nomic DNA.

Ž .Both the RT-PCR products 12 clones , and the PCR-Žproducts directly amplified from the cDNA library 21

.clones , were cloned into pBluescript and sequenced. These33 clones revealed four different nucleotide sequencesŽ .Fig. 4 which were identified as NFI-like sequences,because of their high homology with nucleotide sequencesof the corresponding NFI-cDNA fragments described in

w x Ž w x .chicken 40,71 and mouse Ref. 16 , Fig. 4 and Table 2 .One nucleotide sequence is identical with the correspond-

Ž .ing sequence of the rat NFI encoding cDNA rNFI-Lcloned from liver, identifying it as an NFI-A type se-quence. The identification of the remaining three NFI likenucleotide sequences as products of the NFI-B, -C, or -Xgene, respectively, is based on sequence comparisons with

Ž .corresponding sequences of mouse Fig. 4 and chickenŽ .NFI-cDNAs Table 2 . The mouse and rat nucleotide

Žsequences are highly homologous 97% identical nu-cleotides for NFI-A, 96% for NFI-B, 95% for NFI-C, and

.99% for NFI-X, Fig. 6 . In contrast to the high inter-speciesŽ .mouse vs. rat sequence identity for each NFI isoform thenucleotide sequence homology between the four rat cDNA

Ž .fragments is significantly lower 79%–80% . ComparisonŽ .with the chicken sequences Table 2 revealed the highest

Ž .homology among the NFI-B sequences 91% identity .Whereas the homology between the NFI-C, and -X se-quences range between 83%–86% identity. In total, 33cDNA clones were sequenced. Sequences of the NFI-A, B,-C and -X type were present in 13, six, seven and sevenclones, respectively. In summary, the olfactory neuroep-ithelium of 3-week-old rats express all four NFI genes A,

Ž .B, C and X as they are classified in chicken and mouse .Using the rNFI-A probe to screen a cDNA library from

the olfactory neuroepithelium of adult rats, we isolated two

Ž .Fig. 2. A Inactivation of the UBE binding activity by NEM. EMSA was performed as described except that 50,000 c.p.m. of radiolabeled DNA wereused per reaction. The order of addition of components for each reaction is indicated by numbers above each lane. Free sulfhydryl residues wereinactivated by incubation of the nuclear extract of rat olfactory epithelium with 10 mM NEM for 10 min at 48C. Excess NEM was then inactivated by

Ž .incubation with 50 mM DTT for 10 min at 48C before the UBE probe was added for further incubation lane e . To ensure that the activity of NEM isessential for this effect, we inactivated 10 mM NEM by pre-incubation with 50 mM DTT for 10 min at 48C before addition of the nuclear extract and the

Ž . Ž . Ž . w32 xUBE probe lane c . Further, the inactivation by NEM occurred after the protein s were bound to DNA lanes d and f . Lane a: P UBE probe alone;w32 xlane b: nuclear extract with P UBE; lane c: NEM and DTT added prior to nuclear extract; lane d: NEM and DTT added after incubation of nuclear

w32 x w32 x Ž .extract with P UBE; lane f is lane d exposed longer; lane e: NEM and DTT added to the nuclear extract prior to P UBE. B Binding of recombinantNF-I C220 to UC, UCY and UBE. EMSA was performed as described except that 5 ml of the NF-I C220 extract were incubated with 200,000 c.p.m. of the

w32 x w32 x w32 x w32 xradiolabeled DNA-probe at 48C for 30 min. Lanes 1–4: EMSA with NF-I C220 and P FIB, P UC, P UCY and P UBE, respectively. Lane 5:w32 x Ž . Ž . Ž .P FIB without protein. The exposure time was 2 h lanes 1–3 or overnight lanes 4 and 5 . C Binding affinity of UC-, UCY- and UBE-binding

w32 xproteins. The binding affinity of NF-I to UC, UCY and UBE, respectively, was analysed by their ability to compete with P FIB as probe. EMSA wasŽ . Ž .performed as described with 2 ng of competitor DNA five-fold excess, lanes 2, 4 and 6 or 40 ng of competitor DNA 100-fold excess, lanes 3, 5 and 7 .

w32 xLane 1: P FIB with the nuclear extract; lanes 2 and 3: with UC-DNA as competitor; lanes 4 and 5: with UCY-DNA as competitor; lanes 6 and 7: withw32 x Ž .UBE-DNA as competitor; lane 8: P FIB alone in the absence of extract protein. D Cross-linking of UBE- and FIB-binding proteins. Nuclear extract

Ž . w32 x w32 x Ž . Ž .proteins 10 ug were cross-linked with P FIB and P UBE 100,000 c.p.m.rreaction , respectively, see Section 2 . The cross-linked products wereŽanalysed on a 10% SDS-polyacrylamide gel. Competitors were present at 50-fold excess. The relative molecular weights of protein standards lanes M1

. w32 x Ž .and M2, where M1 was treated as were a–h for cross-linking are indicated. Lanes a–d, cross-linking with P UBE without nuclear extract a ; withŽ . Ž . Ž . w32 x Ž .nuclear extract d ; with additional competitors FIB c and mFIB b . Lanes e–h, cross-linking with P FIB without nuclear extract e ; with nuclear

Ž . Ž . Ž .extract h ; with additional competitors FIB g and mFIB f . Specific cross-linking products are marked with arrow heads.


clones representing the same rNFI-A cDNA of 3389 bp.We designate the new cDNA rNFI-A2 because of its high

Ž .homology to mNFI-B2 Fig. 5 , which is an NFI cDNAisolated from mouse brain and classified as a product of

w xalternative splicing of the NFI gene A transcript 32 . TheŽ .open reading frame ORF chosen corresponds to the ORF

described in NFI-B2 and predicts a protein of 532 aminoacids with a calculated molecular weight of 58.6 kDa. Theamino acid sequence derived from NFI-A2 contains withinthe amino-terminal region the highly negatively chargedmotif as well as the four conserved cysteines found in allNFI proteins. The carboxy-terminal 100 amino acids are

Ž .very proline-rich 24% including one stretch of sevenprolines. The proline-rich C-terminus of CTF-1, a humanhomolog of NFI-C, was reported to activate transcription

ŽFig. 3. RT-PCR with primers for NFI encoding cDNAs. Total RNA 5.ug isolated from olfactory epithelium of 3-week-old rats was reverse

transcribed with random hexamers as primers. The first strand cDNAŽ . Ž .ssDNA as well as double-stranded cDNA dsDNA from an olfactoryepithelial cDNA-library were used as templates for PCR with degenerateprimers. These primers were designed from a conserved 486 bp domain

Ž .common to all four NFI encoding see Section 2 . The PCR-productswere analysed on a 2% agarose gel containing ethidium bromide. Lanes 1

Ž .and 2: PCR-products derived from the dsDNA 1 ug of the library asw x Ž .template 1 , from the ssDNA 10% of the synthesized ssDNA asŽ .template RT-PCR, 2 . Lane 3: to ensure that no genomic DNA was

amplified, the RNA that was used for RT-PCR was also used for PCRŽ .directly in the absence of RT 0.5 ug . Lane 4: no nucleic acids added.

Ž . Ž .DNA-standard M : 1 kb ladder Boehringer-Mannheim with fragmentsof 506 and 396 bp marked.

w x38,57,82 . The nucleotide sequence of rNFI-L is identicalŽ .to rNFI-A2 rNFI-A2 is about 1.7 kb longer than rNFI-L

except for a 16-bp oligonucleotide at the 5X end of rNFI-Lwhere there is only about 20% nucleotide identity. Thebreak point matches exactly with the location of an exon–intron boundary described in the rat and porcine genesw x3,54 and with alternative splicing products described in

w xchicken and mouse 16,41 . Thus, rNFI-A2 seems to beanother splicing variant of rNFI-L derived from the NFI-Agene.

3.3. NFI transcripts are present in olfactory receptorneurons

Our ability to isolate transcripts of all four of the NFIgenes demonstrates their presence in olfactory mucosa butdoes not prove their presence in ORNs. To confirm thepresence of NFI transcripts in ORNs, in situ hybridizationwas performed on prenatal nasal tissue at embryonic day

Ž .16.5 Fig. 6 . Transcripts of all four NFI genes, A, B, XŽ .and C are all present in the nasal mucosa Fig. 6 . The

most abundant expression is observed in nasal glandulartissue, a site of active protein synthesis and secretion. Atthis age, transcripts of NFI-A, -B and -X are clearlyassociated with the olfactory neuroepithelium where theORNs reside. Transcripts of all four NFI genes are ex-pressed in olfactory neurons, although with differing de-

Ž .velopmental profiles Behrens et al., ms. submitted . Theseobservations provide further support for the role of NFI inolfactory neuron gene transcription.

4. Discussion

Ž .We report here 1 the identification of three functionalbinding sites of NFI transcription factors in three distinctpromoters that drive gene expression specifically or prefer-

Ž .entially in olfactory neurons; 2 expression of four NFIŽ .genes within the rat olfactory epithelium; and 3 cloning

of a new full length rat NFI cDNA from a rat olfactoryepithelium cDNA library. Together with our preliminary insitu localization data, these results suggest that NFI tran-scription factors participate in olfactory neuron gene ex-pression.

Genes that are preferentially, or even specifically, ex-pressed within the olfactory neuroepithelium include the

w xputative olfactory receptors 10,67 , the GTP binding pro-w x w xtein G 35 , the adenylyl cyclase type III 6 , thea olf

w x w xOcNC-1 19,37 , the OMP 50–52,77 , and additional genesŽ . w xof unknown function 50.06, 50.11 78 . Promoter analy-

ses of six of the rat genes by footprint assays and EMSArevealed four protein binding sites that might function ascis-regulatory elements in olfactory gene expression. These

Ž . Žsites are: 1 Olf-1rEBF present in the G , type IIIa olf.cyclase, OcNC, OMP, 50.06, and 50.11 genes , the bind-


Fig. 4. Expression of NFI genes in rat olfactory epithelium. RT-PCR products and PCR products derived from the cDNA-library were subcloned intoŽ .pBluescript and sequenced. Of 12 clones derived from RT-PCR, six contained the sequence designated as rNFI-X see text , while two each contained the

sequences designated as rNFI-A, -B and -C. Of 21 clones derived from the cDNA-library 11, 5, 4 and 1 clones contained the sequences designated asw xrNFI-A, -C, -B and -X, respectively. The sequence of each rat isoform was aligned with the corresponding mouse NFI sequence 16 . For each isoform,

nucleotides that are not conserved between mouse and rat are in bold.

ing site of the transcription factor Olf-1rEBF, that hasw xbeen confirmed as a transcriptional activator 30,43,78–80 ;

Ž . w x Ž .2 the UBE localized in the OMP gene 14,43 , 3 theŽ . w xU-site localized in the OcN-channel gene UC 78 ; and

Ž . Ž .4 the U-site localized in the adenylyl cyclase gene UCYw x78 . The identity of the proteins that interact at the U andUBE sites, and the function of these sites was unknown.We have now demonstrated that these sites bind to mem-bers of the ubiquitously expressed NFI family of transcrip-tional regulators. Further, we illustrate that these bindingproteins are members of a complex set since distinctDNA-protein complexes are formed with nuclear extracts

w xfrom different tissues 43,78 .The UBE site is conserved in nucleotide sequence and

position in the promoters of the cloned human, rat andŽ .mouse OMP genes Table 1 . Furthermore, sequence com-

parison of these and the rat U-sites identified a homolo-gous, palindromic motif that corresponds to a consensussequence for binding sites of NFI transcription factorsŽ .Table 1 . Five lines of evidence from this study converge

to confirm that the UBE, UC, and UCY-binding proteinsof the rat olfactory neuroepithelium are indeed members of

Ž .the NFI family of transcription factors. They are: 1 theDNA-protein complexes formed with FIB-DNA, contain-

Ž .ing a previously described NFI-binding site Table 1 , andwith UBE, UC and UCY-DNA, migrate identically in a

Ž . Ž .native EMSA gel Fig. 1A ; 2 an antiserum directed

Table 2Comparison of nucleotide sequences of NFI-cDNA fragments derived

Ž . Ž .from chicken c- and rat r-a% Identity Chicken

cNFI-A cNFI-B cNFI-C cNFI-X

Rat rNFI-A 85 78 78 77rNFI-B 76 91 79 78rNFI-C 74 77 85 83rNFI-X 79 79 86 85

a The nucleotide sequences shown in Fig. 4 were compared using thePearson and Lipman algorithm which also provides the percentage ofidentical nucleotides.


against NFI specifically recognizes the DNA-protein com-plex formed between olfactory tissue extracts and FIB,

Ž . Ž .UBE, UC, and UCY-DNA Fig. 1B ; 3 FIB-DNA, butnot the mutated mFIB DNA, is a very efficient competitor


Fig. 6. Localization of NFI transcripts in E16.5 rat nasal mucosa by in situ hybridization. Micrographs of parasagittal sections of nasal mucosa hybridized35 Ž . Ž .with S-antisense probes to NFI-A, NFI-B, NFI-X and NFI-C. Strong signal is seen in the nasal glands g and in the olfactory mucosa om , the site of

Ž .the olfactory neuroepithelium where olfactory receptor neurons reside. Signal is also evident in the olfactory bulb b that is separated from the nasal sideŽ .by the portion of the skull identified as the cribriform plate cp . The scale bar is 300 mm. At this age, NFI-B expression seems most robust in the

neuroepithelium.

Ž . Ž .for UBE, UC, and UCY-binding proteins Fig. 1C ; 4 theŽ .UBE-binding activity is NEM sensitive Fig. 2A , a char-

w x Ž .acteristic of NFI proteins 63 ; and 5 the recombinantDNA-binding domain of the human NFIrCTF expressedin E. coli interacts with FIB, UBE, UC, and UCY-DNA inEMSA to give shifts of indistinguishable electrophoretic

Ž .mobility Fig. 2B .These results demonstrate that all three sites, UBE, UC,

and UCY, are recognized by NFI proteins. The role thistranscription factor plays in regulating the expression of

Žthe corresponding genes OMP, OcNC-1, and type III.cyclase remains to be elucidated. Recently, Glusman et al.

w x Ž22 reported the presence of an NFI binding motif Table.1 in the putative promoter region of an olfactory receptor

gene. The fact that four promoters driving olfactory neuronspecific gene expression contain an NFI binding site sug-gests that NFI is an important regulator of olfactory neu-rons gene expression. All three sites, UBE, UCY, and UC

contain a CCAG motif, while in many NFI-binding sites, aw xCCAA motif is conserved 36 . This latter motif is the

basis for the alternative identification of NFI as CAATŽ . w xbinding transcription factor CTF 72 . Recently, a slow

cAMP response element was localized to an NFI-bindingŽ . w xsite TGGGCGCCTTGCCAG 83 that also contains the

ŽCCAG motif and that is almost identical 12 of 15 nu-.cleotides with the UCY-site. This is intriguing because

cAMP plays a major role as a second messenger in olfac-w xtory signal transduction 9,31 , suggesting that it could be

involved in regulating gene expression of some signalŽ .transduction components type III cyclase and OcNC-1

distinct from the well-documented transcriptional regula-tion by the cAMP response element CRE.

NFI proteins bind the three U-sites with varying affini-Ž .ties Fig. 1A,C . Evaluation of UBE, UC, and UCY-DNA,

Ž .as competitors for NFI binding to FIB-DNA Fig. 2Cdemonstrated that UC has the highest affinity comparable

Fig. 5. Nucleotide sequence of the full length rat cDNA NFI-A2. The nucleotide sequence of rNFI-A2 was generated by independently sequencing twoŽ .identical clones obtained by screening a rat olfactory neuroepithelial cDNA-library see Section 2 . The homology between the nucleotide sequences of

rNFI-A2, rNFI-L, and mNFI-B2 is illustrated and identical nucleotides are marked by a dot. Three amino acid exchanges between mNFI-B2 and rNFI-A2are indicated above the translated rNFI-A2 ORF. The positions of the degenerate primers used for PCR are underlined. Note that although long polyA runsare present at the 3X end no appropriate polyadenylation signal sequence was found indicating that this 3X end may not represent the 3X end of thecorresponding mRNA.


Ž .to FIB in its binding strength data not shown . In contrast,UBE and UCY are of lower affinity by about a factor of10. The binding affinity of NFI proteins to different sites

8 11 y1 w xare reported to range from 10 to 10 M 53 . Aw xwell-known site of relative low affinity 54,64 is the half

site of the canonical NFI binding site, TGGCA, that hasbeen shown to be an active cis-regulating element in

w xseveral promoters 65,69,71 . Nevertheless, confirmationof the activity of the three U-sites as functional cis-regulat-ing elements in olfactory gene expression remains to beelucidated. Interestingly, the high affinity site UC and thelow affinity site UCY differ by just 1 bp from the NFIconsensus sequence. In the case of UCY, a contact point

w xfor NFI binding 18 , the first T of the consensus sequence,is replaced by a G. In UC, the fourth bp is exchanged as is

w xcommon in NFI binding sites 18 . All three UBE sitesŽ .contain a stretched linker sequence 4 instead of 3 bp

compared to the other NFI binding sites presented hereŽ . w xTable 1 . Gronostajski 27 reported a slightly different

Ž .consensus sequence TGGGCrANCrGNNGrTGCCAAŽ .for NFI binding sites in HeLa cell extracts with a stretched

linker that matches very well with all three UBE se-quences. In agreement with our results, these sites werefound to have a lower binding affinity to NFI proteins.

NFI is a family of structurally related proteins that arew xinvolved in regulation of viral replication 23,58,59 and

w xtranscription of certain viral 4,46 and cellular genesw x21,25,32,47,69,70,83 . The diversity of the NFI familywas first discovered in chicken when cloning of severalNFI encoding cDNAs indicated the presence of four closelyrelated but distinct genes cNFI-A, cNFI-B, cNFI-C, and

w xcNFI-X 40,71 and alternative splicing products of eachw xgene 5,41 . Additionally, post-translational modification

w x w xby glycosylation 33 and oxidation 7,39 as well asheterodimer formation between individual members of thefamily provides mechanisms for further modulating the

w xactivities of these DNA-binding proteins 42 . The NFIw x w xencoding cDNAs cloned from frog 66 , porcine 54 ,

w x w x w x w xmouse 16,32 , hamster 21 , rat 65 and human 5,72,81libraries give rise to corresponding genes and splice vari-

w xants between vertebrate species 41 . Sequence comparisonof NFI encoding cDNAs revealed a highly conservedregion of about 600 bp that is located 5X to very divergentsequences that are characteristic for each gene and splicing

w xvariant 71 . The conserved region codes for the amino-terminal domains within NFI proteins that are necessaryfor DNA-binding, regulation of the viral replication and

w xprotein dimerization 23 , while some but not all NFIproteins contain a carboxy-terminal proline-rich region thatis considered to contain the transcriptional activator do-

w xmain 2,38,57,82 . Although NFI expression is consideredto be ubiquitous, RNA blot, RNase protection assays, insitu hybridization and biochemical analyses in mouse, ratand human suggest a tissue, cell-type and developmentallyregulated distribution of distinct NFI transcripts and pro-

w xteins 16,32,45,65 . Indeed such a distribution seems essen-

tial to explain the role NFI proteins play in regulating thetissue and cell-type specific expression of several target

w xgenes 1,5,25,32,45,47,73 .Electrophoretic analysis of UV-cross-linked products

Žformed between the radioactive FIB-DNA containing one.NFI binding site and nuclear proteins of the rat olfactory

neuroepithelium revealed the presence of multiple NFIŽ .proteins within this tissue Fig. 2D . This was confirmed

when we analysed the number and identity of NFI genesthat are expressed in the rat olfactory neuroepithelium.Using both the RT-PCR and conventional PCR techniqueswith degenerate primers, we amplified cDNA-fragmentsfrom common to the conserved region of all NFI cDNAs.Cloning and sequencing of the PCR products revealed four

Ž .distinct nucleotide sequences of 486 bp Fig. 4 . Identitiesranging from 74%–91% and 95%–99% compared to the

Ž .corresponding regions of the cNFI genes Table 2 andmNFI cDNAs, respectively, prove that all four genes,NFI-A, -B, -C and -X, are expressed within the rat olfac-tory epithelium.

In our search for rNFI encoding cDNA fragments, wemostly detected sequences of the NFI-A type suggestingthat among the four NFI genes, the NFI-A gene is pre-dominantly expressed in the rat olfactory neuroepithelium.

w xInterestingly, Osada et al. 64 reported a DNA-bindingsite for expressed mouse NFI-A that resembles the UBEand U-sites. Therefore, we screened a cDNA library de-rived from rat olfactory epithelium for a full length cDNAusing the PCR product identified as a fragment of arNFI-A cDNA. Two identical cDNA clones were obtained

Ž . Xof 3389 bp Fig. 5 containing an ORF of 1596 bp, and 5X Ž .and 3 untranslated regions UTR of 195 and 1595 bp.

This represents the longest NFI encoding cDNA so faridentified. The ORF encodes for a protein of 532 aminoacids. Both, the nucleotide sequence and the amino acidsequence are highly homologous with NFI encoding se-

Ž . Žquences Fig. 5 : The sequence of the rNFI-L cDNA 1712.bp is identical over 1689 bp and the sequence of mNFI-B2

w x32 , one of six cDNA clones from mouse brain, shows100, 97.7, and 92.4% identity to the 5X UTR, ORF, and3X UTR, respectively. This includes AT rich sequenceswithin the two 3X UTRs that have been suggested as signals

w xfor controlled degradation of NFI transcripts 60 . Theamino acid sequences derived from the two cDNAs are

Ž .identical except for three residues Fig. 5 . The highsequence identity with mNFI-B2 indicates that we havecloned the rat homologue of the mNFI-B2 splicing productof the NFI-A gene, which we designate rNFI-A2. rNFI-L,

X X w xthat lacks the 5 UTR and the 5 end of the ORF 65 ,differs from rNFI-A2 by 23 nucleotides at the 5X end of

Ž .rNFI-L. The first amino acid aspartate that is encoded byboth sequences matches exactly with the first amino acid

w xencoded by exon 2 of the rNFI-L gene 3 , indicating thatrNFI-A2 transcripts contain an alternative exon 1 com-pared to rNFI-L. Thus, the NFI-A gene is expressed in theolfactory epithelium as well as in liver but distinct tran-


scripts are generated in each tissue by the mechanism ofalternative splicing and possibly by the use of alternatepromoters. Differential use of two promoters could explaina tissue specific distribution of NFI transcripts. This resultsin a protein that is 24 residues longer at the amino-terminusthan rNFI-L. The functional difference, if any, between thetwo proteins derived from rNFI-A2 and rNFI-L is un-known.

In summary, we have shown that the proximal promot-ers of three genes that are selectively expressed in olfac-tory neurons each contain an NFI binding site and that NFIproteins are present in olfactory tissue extracts. We havedemonstrated, by cloning and in situ hybridization, that allfour NFI genes are expressed in olfactory tissue and that atleast three of these are expressed in the neuroepithelium inwhich the olfactory neurons reside. The functional rela-tionships in olfactory neuron gene expression among theseelements remains to be elucidated. NFI proteins are capa-ble of functioning as positive or negative regulators oftranscription depending on the tissue, cell-type, age, and

w xgene investigated 1,16,32,84 . These multivalent functionsof NFI could be achieved by temporally and spatiallyregulated expression of distinct subsets of NFI isoforms as

w xrecently demonstrated 16 . All four NFI genes are ex-pressed in the olfactory neuroepithelium. It now remains tocharacterize the tissue and cell specific patterns of expres-sion of distinct NFI transcription factors during ontogenyand their roles in regulating gene expression. In the olfac-tory epithelium NFI binding to the U-sites could modulatethe function of Olf-1 as a transcriptional activator or couldbe involved in induced regulation of transcription by cAMP

w xor the growth factor TGFb 69,78 .

Acknowledgements

We thank N. Tanese for the generous gift of anti-NFIantiserum, R. Wurzburger for DNA sequencing and MaikBehrens for valuable discussions. Supported in part byHD34908 to RMG and DC03112 to FLM.

References

w x1 A.D. Adams, D.M. Choate, M.A. Thompson, NF1-L is the DNA-bi-nding component of the protein complex at the peripherin negative

Ž .regulatory element, J. Biol. Chem. 270 1995 6975–6983.w x2 H. Altmann, W. Wendler, E.-L. Winnacker, Transcriptional activa-

tion by CTF proteins is mediated by a bipartite low-proline domain,Ž .Proc. Natl. Acad. Sci. U.S.A. 91 1994 3901–3905.

w x3 R. Ammendola, F. Gounari, G. Piaggio, V. De Simone, R. Cortese,Transcription of the promoter of the rat NF-1 gene depends on the

Ž .integrity of an Sp1 recognition site, Mol. Cell. Biol. 10 1990387–390.

w x4 D. Apt, T. Chong, Y. Liu, H.-U. Bernard, Nuclear factor 1 andepithelial cell-specific transcription of human papillomavirus type

Ž .16, J. Virol. 67 1993 4455–4463.w x5 D. Apt, Y. Liu, H.-U. Bernard, Cloning and functional analysis of

spliced isoforms of human nuclear factor I-X: interference withtranscriptional activation by NFIrCTF in a cell-type specific man-

Ž .ner, Nucleic Acids Res. 22 1994 3825–3833.w x6 H.A. Bakalyar, R.R. Reed, Identification of a specialized adenylyl

Ž .cyclase that may mediate odorant detection, Science 250 19901403–1406.

w x7 S. Bandyopadhyay, R.M. Gronostajski, Identification of a conservedoxidation-sensitive cysteine residue in the NFI family of DNA-bind-

Ž .ing proteins, J. Biol. Chem. 269 1994 29949–29955.w x Ž .8 W. Breipohl Ed. , Ontogeny of Olfaction, Springer, Berlin, 1986.w x9 L.J. Brunet, G.H. Gold, J. Ngai, General anosmia caused by a

targeted disruption of the mouse olfactory cyclic nucleotide-gatedŽ .cation channel, Neuron 17 1996 681–693.

w x10 L. Buck, R. Axel, A novel multigene family may encode odorantŽ .receptors: a molecular basis for odorant recognition, Cell 66 1991

175–187.w x11 L. Buck, Identification and analysis of a multigene family encoding

odorant receptors: implications for mechanisms underlying olfactoryŽ .information processing, Chem. Sens. 18 1993 203–208.

w x12 L.B. Buck, S. Firestein, R.F. Margolskee, Olfaction and taste invertebrate: molecular and organizational strategies underlyingchemosensory perception, in: G.J. Siegel, B.W. Agranoff, W. Al-

Ž .bers, P.B. Molinoff Eds. , Basic Neurochemistry, Raven Press, NewYork, 1994, pp. 157–177.

w x13 L.B. Buck, Information coding in the vertebrate olfactory system,Ž .Annu. Rev. Neurosci. 19 1996 517–544.

w x14 O.I. Buiakova, N.S.R. Krishna, T.V. Getchell, F.L. Margolis, Hu-man and rodent OMP genes: conservation of structural and regula-

Ž .tory motifs and cellular localization, Genomics 20 1994 452–462.w x15 O.I. Buiakova, H. Baker, J.W. Scott, A. Farbman, R. Kream, M.

Grillo, L. Franzen, M. Richman, L.M. Davis, S. Abbondanzo, C.L.Ž .Stewart, F.L. Margolis, Olfactory marker protein OMP gene dele-

tion alters physiological activity of olfactory neurons, Proc. Natl.Ž .Acad. Sci. U.S.A. 93 1996 9858–9863.

w x16 A.Z. Chaudhry, G.E. Lyons, R.M. Gronostajski, Expression patternsof the four nuclear factor I genes during mouse embryogenesis

Ž .indicate a potential role in development, Dev. Dyn. 208 1997313–325.

w x17 A. Chess, I. Simon, H. Cedar, R. Axel, Allelic inactivation regulatesŽ .olfactory receptor gene expression, Cell 78 1994 823–834.

w x18 E. De Vries, W. van Driel, S.J.L. van den Heuvel, P.C. van derVliet, Contactpoint analysis of the HeLa nuclear factor I recognitionsite reveals symmetrical binding at one side of the DNA helix,

Ž .EMBO J. 6 1987 161–168.w x19 R.S. Dhallan, K.-W. Yau, K.A. Schrader, R.R. Reed, Primary

structure and functional expression of a cyclic nucleotide-activatedŽ .channel from olfactory neurons, Nature 347 1990 184–187.

w x20 A.I. Farbman, J.A. Buchholz, E. Walters, F.L. Margolis, Doesolfactory marker protein participate in olfactory neurogenesis?, Ann.

Ž .N.Y. Acad. Sci. 855 1998 248–251.w x21 G. Gil, J.R. Smith, J.L. Goldstein, C.A. Slaughter, K. Orth, M.S.

Brown, T.F. Osborne, Multiple genes encode nuclear factor 1-likeproteins that bind to the promoter for 3-hydroxy-3-methylglutaryl-

Ž .coenzyme A reductase, Proc. Natl. Acad. Sci. U.S.A. 85 19888963–8967.

w x22 G. Glusman, S. Clifton, B. Roe, D. Lancet, Sequence analysis in theolfactory receptor gene cluster on human chromosome 17: recombi-

Ž .natorial events affecting receptor diversity, Genomics 37 1996147–160.

w x23 F. Gounari, R. De Francesco, J. Schmitt, P.C. van der Vliet, R.Cortese, H. Stunnenberg, Amino-terminal domain of NFI binds toDNA as a dimer and activates adenovirus DNA replication, EMBO

Ž .J. 9 1990 559–566.w x24 N. Goyal, J. Knox, R.M. Gronostajski, Analysis of multiple forms of

nuclear factor I in human and murine cell lines, Mol. Cell. Biol. 10Ž .1990 1041–1048.

w x25 R.A. Graves, P. Tontonoz, S.R. Ross, B.M. Spiegelman, Identifica-


tion of a potent adipocyte-specific enhancer: involvement of anŽ .NF-1-like factor, Genes Dev. 5 1991 428–437.

w x26 P.P.C. Graziadei, G.A. Monti-Graziadei, Continuous nerve cell re-Ž .newal in the olfactory system, in: M. Jacobson Ed. , Handbook of

Sensory Physiology: IX. Development of Sensory Systems, Springer,Berlin, 1978, pp. 55–83.

w x27 R.M. Gronostajski, Site-specific DNA binding of nuclear factor I:Ž .effect of the spacer region, Nucleic Acids Res. 15 1987 5545–5559.

w x28 F. Guillemot, A.L. Joyner, Dynamic expression of the murineachaete-scute homologue Mash-1 in the developing nervous system,

Ž .Mech. Dev. 42 1993 171–185.w x29 F. Guillemot, L.C. Lo, J.E. Johnson, A. Auerbach, A.L. Joyner,

Mammalian achaete-scute homolog 1 is required for the earlyŽ .development of olfactory and autonomic neurons, Cell 75 1993

463–476.w x30 J. Hagman, C. Belanger, A. Travis, W. Turck, R. Grosschedl,

Cloning and functional characterization of early B-cell factor, aregulatory of lymphocyte-specific gene expression, Genes Dev. 7Ž .1993 760–773.

w x31 J.G. Hildebrand, G.M. Shepherd, Mechanisms of olfactory discrimi-nation: converging evidence for common principles across phyla,

Ž .Annu. Rev. Neurosci. 20 1997 595–631.w x32 T. Inoue, T. Tamura, T. Furuichi, K. Mikoshiba, Isolation of com-

plementary DNAs encoding a cerebellum-enriched nuclear factor Ifamily that activates transcription from mouse Myelin basic protein

Ž .promoter, J. Biol. Chem. 265 1990 19065–19070.w x33 S.P. Jackson, R. Tjian, O-glycosation of eukariotic transcription

factors: implications of mechanisms of transcriptional regulation,Ž .Cell 55 1988 125–133.

w x34 J.E. Johnson, K. Zimmerman, T. Saito, D.J. Anderson, InductionŽ .and repression of mammalian achaete-scute homolog MASH gene

expression during neuronal differentiation of P19 embryonal carci-Ž .noma cells, Development 114 1992 75–87.

w x35 D.T. Jones, R.R. Reed, G : an olfactory neuron-specific G proteinolfŽ .involved in odorant signal transduction, Science 244 1989 790–795.

w x36 K.A. Jones, J.T. Kadonaga, P.J. Rosenfeld, T.J. Kelly, R. Tjian, Acellular DNA-binding protein that activates eukariotic transcription

Ž .and DNA replication, Cell 48 1987 79–89.w x37 U.B. Kaupp, The cyclic nucleotide-gated channels of vertebrate

Ž .photoreception and olfactory epithelium, Trends Neurosci. 14 1991150–157.

w x38 T.K. Kim, R.G. Roeder, Proline-rich activator CTF1 targets theTFIIB assembly step during transcriptional activation, Proc. Natl.

Ž .Acad. Sci. U.S.A. 91 1994 4170–4174.w x39 L. Knoepfel, C. Steinkuhler, M.-T. Carrı, G. Rotilio, Role of zinc-¨ `

coordination and of the glutathione redox couple in the redoxsusceptibility of human transcription factor Sp1, Biochem. Biophys.

Ž .Res. Commun. 201 1994 871–877.w x40 U. Kruse, F. Qian, A.E. Sippel, Identification of a forth nuclear

factor I gene in chicken by cDNA cloning: NFI-X, Nucleic AcidsŽ .Res. 19 1991 6641.

w x41 U. Kruse, A.E. Sippel, The genes for transcription factor nuclearfactor I give rise to corresponding splice variants between vertebrate

Ž .species, J. Mol. Biol. 238 1994 860–865.w x42 U. Kruse, A.E. Sippel, Transcription factor nuclear factor I proteins

Ž .form stable homo- and heterodimers, FEBS Lett. 348 1994 46–50.w x43 K. Kudrycki, C. Stein-Izsak, C. Behn, M. Grillo, R. Akeson, F.L.

Margolis, Olf-1-binding site: characterization of an olfactoryŽ .neuron-specific promoter motif, Mol. Cell. Biol. 13 1993 3002–

3014.w x44 K. Kudrycki, O.I. Buiakova, G. Tarazzo, E. Walters, F.L. Margolis,

Effects of mutation of the Olf-1 motif on transgene expression inŽ .olfactory receptor neurons, J. Neurosci. Res. 52 1998 159–172.

w x45 S. Kulkarni, R.M. Gronostajski, Altered expression of the develop-mentally regulated NFI gene family during phorbol ester-induceddifferentiation of human leukemic cells, Cell Growth Differ. 7Ž .1996 501–510.

w x46 K.U. Kumar, A. Pater, M.M. Pater, Human JC virus perfect palin-dromic nuclear factor 1-binding sequences important for glial cell-specific expression in differentiating embryonal carcinoma cells, J.

Ž .Virol. 67 1993 572–576.w x47 S. Li, J.M. Rosen, Nuclear factor I and mammary gland factor

Ž .STAT5 play a critical role in regulating rat whey acidic proteinŽ .gene expression in transgenic mice, Mol. Cell. Biol. 15 1995

2063–2070.w x48 H. Lin, R. Grosschedl, Failure of B-cell differentiation in mice

Ž .lacking the transcription factor EBF, Nature 376 1995 263–267.w x49 T. Margalit, D. Lancet, Expression of olfactory receptor and trans-

Ž .duction genes during rat development, Dev. Brain Res. 73 19937–16.

w x50 F.L. Margolis, A brain protein unique to the olfactory bulb, Proc.Ž .Natl. Acad. Sci. U.S.A. 69 1972 1221–1224.

w x51 F.L. Margolis, A marker protein for the olfactory chemo-receptorŽ .neuron, in: R.A. Bradshaw, D.M. Schneider Eds. , Proteins of the

Nervous System, Raven Press, New York, 1980, pp. 59–84.w x52 F.L. Margolis, Molecular cloning of olfactory specific gene prod-

Ž .ucts, in: F.L. Margolis, T.V. Getchell Eds. , Molecular Neurobiol-ogy of the Olfactory System, Plenum, New York, 1988, pp. 237–265.

w x53 M. Meisterernst, I. Gander, L. Rogge, E.-L. Winnacker, A quantita-tive analysis of nuclear factor IrDNA interactions, Nucleic Acids

Ž .Res. 16 1988 4419–4435.w x54 M. Meisterernst, L. Rogge, R. Foeckler, M. Karaghiosoff, E.-L.

Winnacker, Structural and functional organization of a porcine geneŽ .coding for nuclear factor I, Biochemistry 28 1989 8191–8200.

w x55 B.P. Menco, F.D. Tekula, A.L. Farbman, W. Danho, Developmentalexpression of G-proteins and adenylyl cyclase in peripheral olfactorysystems. Light microscopic and freeze-substitution electron micro-

Ž .scopic immunocytochemistry, J. Neurocytol. 23 1994 708–727.w x56 B.P.M. Menco, Ultrastructural aspects of olfactory signaling, Chem.

Ž .Sens. 22 1997 295–311.w x57 N. Mermod, E.A. O’Neill, T.J. Kelly, R. Tjian, The proline-rich

transcriptional activator of CTFrNF-I is distinct from the replicationŽ .and DNA binding domain, Cell 58 1989 741–753.

w x58 A. Monaghan, A. Webster, R.T. Hay, Adenovirus DNA bindingŽ .protein: helix destabilising properties, Nucleic Acids Res. 22 1994

742–748.w x59 K. Nagata, R.A. Guggenheim, T. Enomoto, J.H. Lichy, J. Hurwitz,

Adenovirus DNA replication in vitro: identification of a host factorthat stimulates synthesis of the preterminal protein-dCMP complex,

Ž .Proc. Natl. Acad. Sci. U.S.A. 79 1982 6438–6442.w x60 G. Nebl, N. Mermod, A.C.B. Cato, Post-transcriptional down-regu-

lation of expression of transcription factor NF1 by Ha-ras onco-Ž .gene, J. Biol. Chem. 269 1994 7371–7378.

w x61 P. Nef, I. Hermans-Borgmeyer, H. Artieres-Pin, L. Beasley, V.E.Dionne, S.F. Heinemann, Spatial pattern of receptor expression in

Ž .the olfactory epithelium, Proc. Natl. Acad. Sci. U.S.A. 89 19928948–8952.

w x62 J. Ngai, A. Chess, M.M. Dowling, N. Necles, E.R. Macagno, R.Axel, Coding of olfactory information: topography of odorant recep-

Ž .tion expression in the catfish olfactory epithelium, Cell 72 1993667–680.

w x63 A. Novak, N. Goyal, R.M. Gronostajski, Four conserved Cysteineresidues are required for the DNA binding activity of nuclear factor

Ž .I, J. Biol. Chem. 267 1992 12986–12990.w x64 S. Osada, S. Daimon, T. Nishihara, M. Imagawa, Identification of

DNA binding-site preferences for nuclear factor I-A, FEBS Lett. 390Ž .1996 44–46.

w x65 G. Paonessa, F. Gounari, R. Frank, R. Cortese, Purification of aNF1-like DNA-binding protein from rat liver and cloning of the

Ž .corresponding cDNA, EMBO J. 7 1988 3115–3123.w x66 M. Puzianowska-Kuznicka, Y.-B. Shi, Nuclear factor I as a potential

regulator during postembryonic organ development, J. Biol. Chem.Ž .271 1996 6273–6282.

w x67 K. Raming, J. Krieger, J. Strotmann, I. Boekhoff, S. Kubick, C.


Baumstark, H. Breer, Cloning and expression of odorant receptors,Ž .Nature 361 1993 353–356.

w x68 K.J. Ressler, S.L. Sullivan, L.B. Buck, Information coding in theolfactory system: evidence for a stereotyped and highly organized

Ž .epitope map in the olfactory bulb, Cell 79 1994 1245–1255.w x69 P. Rossi, G. Karsenty, A. Roberts, N.S. Roche, M.B. Sporn, B. de

Crombrugghe, A nuclear factor 1 binding site mediates the transcrip-tional activation of a type I collagen promoter by transformation

Ž .growth factor-b, Cell 52 1988 405–414.w x70 R.J. Roy, S.L. Guerin, The 30-kDa rat liver transcription factor´

nuclear factor 1 binds the rat-growth hormone proximal silencer,Ž .Eur. J. Biochem. 219 1994 799–806.

w x71 R.A.W. Rupp, U. Kruse, G. Multhaup, U. Gobel, K. Beyreuther,¨A.E. Sippel, Chicken NFIrTGGCA proteins are encoded by at leastthree independent genes: NFI-A, NFI-B and NFI-C with homologues

Ž .in mammalian genomes, Nucleic Acids Res. 18 1990 2607–2616.w x72 C. Santoro, N. Mermod, P.C. Andrews, R. Tjian, A family of human

CCAAT-box-binding proteins active in transcription and DNA repli-cation: cloning and expression of multiple cDNAs, Nature 334Ž .1988 218–224.

w x73 G. Schweizer-Groyer, A. Groyer, F. Cadepond, T. Grange, E.-E.Baulieu, R. Pictet, Expression from the tyrosine aminotransferase

Ž .promoter nt y350 to q1 is liver-specific and dependent on thebinding of both liver-enriched and ubiquitous trans-acting factors,

Ž .Nucleic Acids Res. 22 1994 1529–1583.w x74 J. Schwob, E. Mieleszko, K.E. Szumowski, A. A Stasky, Olfactory

sensory neurons are trophically dependent on the olfactory bulb forŽ .their prolonged survival, J. Neurosci. 12 1992 3896–3919.

w x75 G.M. Shepherd, Current issues in the molecular biology of olfaction,Ž .Chem. Sens. 18 1993 191–198.

w x76 R.Y.L. Tsai, R.R. Reed, Cloning and functional characterization ofRoaz, a zinc finger protein that interacts with Olf-1rEBF to regulate

gene expression: implications for olfactory neuronal development, J.Ž .Neurosci. 17 1997 4159–4169.

w x77 J. Verhagen, A.B. Oestreicher, M. Grillo, Y.-S. Khew-Goodall,W.H. Gispen, F.L. Margolis, Neuroplasticity in the olfactory system:differential effects of central and peripheral lesions of the primaryolfactory pathway on the expression of B50rGAP43 and the olfac-

Ž .tory marker protein, J. Neurosci. Res. 26 1990 31–44.w x78 M.M. Wang, R.Y.L. Tsai, K.A. Schrader, R.R. Reed, Genes encod-

ing components of the olfactory signal transduction cascade containa DNA binding site that may direct neuronal expression, Mol. Cell.

Ž .Biol. 13 1993 5805–5813.w x79 M.M. Wang, R.R. Reed, Molecular cloning of the olfactory neuronal

transcription factor Olf-1 by genetic selection in yeast, Nature 364Ž .1993 121–126.

w x80 S.S. Wang, R.Y.L. Tsai, R.R. Reed, The characterization of theOlf-1rEBF-like HLH transcription factor family: implications inolfactory gene regulation and neuronal development, J. Neurosci. 17Ž .1997 4149–4158.

w x81 S. Wenzelides, H. Altmann, W. Wendler, E.-L. Winnacker, CTF5— a new transcriptional activator of the NFIrCTF family, Nucleic

Ž .Acids Res. 24 1996 2416–2421.w x82 H. Xiao, J.T. Lis, H. Xiao, J. Greenblatt, J.D. Friesen, The upstream

activator CTFrNF1 and RNA polymerase II share a common ele-ment involved in transcriptional activation, Nucleic Acids Res. 22Ž .1994 1966–1973.

w x83 X. Zhang, R. Miskimins, Binding at an NFI site is modulated bycyclic AMP-dependent activation of myelin basic protein gene ex-

Ž .pression, J. Neurochem. 60 1993 2010–2017.w x84 L. Zou, R.K. Menon, A member of the CTFrNF-1 transcription

factor family regulates murine growth hormone receptor gene pro-Ž .moter activity, Endocrinology 136 1995 5236–5239.

research report identification of nfi-binding sites and ...rgron/baumeis.pdffib and mfib were...

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