research article : a novel primate gene with possible

Research ArticleELFN1-AS1 A Novel Primate Gene with Possible MicroRNAFunction Expressed Predominantly in Human Tumors

Dmitrii E Polev12 Iuliia K Karnaukhova1 Larisa L Krukovskaya1 and Andrei P Kozlov1

1 Biomedical Center 8 Vyborgskaja Street Saint Petersburg 194044 Russia2 Department of Genetics and Breeding Saint Petersburg State University 79 Universitetskaya EmbankmentSaint Petersburg 199034 Russia

Correspondence should be addressed to Andrei P Kozlov contactbiomedspbru

Received 23 September 2013 Revised 24 December 2013 Accepted 27 December 2013 Published 24 February 2014

Academic Editor Franco M Buonaguro

Copyright copy 2014 Dmitrii E Polev et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Human gene LOC100505644 uncharacterized LOC100505644 [Homo sapiens] (Entrez Gene ID 100505644) is abundantly expressedin tumors but weakly expressed in few normal tissues Till now the function of this gene remains unknown Here we identifiedthe chromosomal borders of the transcribed region and the major splice form of the LOC100505644-specific transcript Wecharacterised the major regulatory motifs of the gene and its splice sites Analysis of the secondary structure of the major transcriptvariant revealed a hairpin-like structure characteristic for precursormicroRNAs Comparative genomic analysis of the locus showedthat it originated in primates de novo Taken together our data indicate that human gene LOC100505644 encodes some non-proteincoding RNA likely a microRNA It was assigned a gene symbol ELFN1-AS1 (ELFN1 antisense RNA 1 (non-protein coding)) Thisgene combines features of evolutionary novelty and predominant expression in tumors

1 Introduction

UniGene cluster Hs633957 (Ref UGID2140851) corre-sponds to the human gene LOC100505644 uncharacter-ized LOC100505644 [Homo sapiens] (Entrez Gene ID100505644) which resides in the 7th chromosome in theintron of ELFN1 gene It was found to be expressed primarilyin human tumors by in silico analysis [1] and by experimentaldetection of LOC100505644 transcripts in tumors of varioushistological origin In general transcripts were absent frommost normal tissues but some weak expression signals couldbe detected in adult heart and digestive system [2ndash4]

The broad spectrumof positive tumors and only few posi-tive normal tissues make this gene potentially meaningful foroncology however the question whether the LOC100505644transcripts are functional or just a tumor-related transcrip-tional noise remained unanswered Here we confirm thetumor-associated expression pattern of the gene using anindependent set of samples and present experimental and insilico evidence that LOC100505644 shows alternative splicing

possesses its own promoter and is likely to code for amiRNAwhich is the evidence of a functional gene Interestingly thisgene seems to have originated in primates de novo from anintronic region of the ELFN1 gene It was assigned a genesymbol ELFN1-AS1 (ELFN1 antisense RNA 1 (non-proteincoding))

2 Material and Methods

21 RNA and cDNA Total RNA samples from human pla-centa and ovary carcinoma were obtained fromAmbion TXUSA Total RNA samples from humanmoderately differenti-ated endometrium adenocarcinoma and lung squamous cellcarcinoma were kindly provided by Dr Kolyubaeva KirovMilitary Medical Academy St Petersburg Russia Appropri-ate informed consent was obtained cDNA preparations fromhuman healthy tissues were purchased from Clontech CAUSA (Human MTC Panels) Human tumor cDNA prepara-tions were purchased from BioChain CA USA (catalog nosC8235544 C8235545 C8235546 C8235547 and C8235549)

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 398097 10 pageshttpdxdoiorg1011552014398097

2 BioMed Research International

22 PCR The quality of cDNA panels was assessed byPCR with primers for the GAPDH gene (forward 51015840-TGAAGGTCGGAGTCAACGGATTTGGT-31015840 and reverse51015840-CATGTGGGCCATGAGGTCCACCAC-31015840) Annealingtemperature was 68∘C and amplicon size was 983 bpTwenty-five cycles of PCR were performed Gene-specificprimers were as follows 51015840-GGTCTTTACTCCCATTCAA-31015840 and 51015840-CTCCTGTCATTCACTCCG-31015840 The reaction wasconducted in 25 120583L of a solution containing 25 ng of cDNAPCR buffer (67mM Tris HCl pH 89 4mM MgCl

2 16mM

(NH4)2SO4 10mM 2-mercaptoethanol and 01mgmL

BSA) dNTPs (200120583M each) 04 120583M each primer and 1Uof Taq DNA polymerase PCR program denaturation at95∘C for 1min followed by 35 cycles 95∘C for 30 s 56∘Cfor 30 s and 72∘C for 30 s Postextension was carried out at72∘C for 5min The resulting amplificates were resolved byelectrophoresis in 2 agarose gel and stained with ethidiumbromide The presence of the mRNA under study in thesample was indicated by the presence of the 322 bp longamplification product The gels were photographed underUV illumination

23 RACE We used SMARTRACE cDNAAmplification Kit(Clontech) with ovary carcinoma and placenta RNA samplesand Marathon cDNA Amplification Kit (Clontech) withuterus adenocarcinoma and lung carcinomaRNA samples forRACE experiments cDNA was prepared as recommendedby the manufacturer Gene-specific primers 5gsp1 5gsp1n5gsp2 5gsp2n 5gsp3 5gsp3n 3gsp1 and 3gsp1n for theamplification of the 51015840- and 31015840-cDNA ends are presented inTable 1

cDNA ends were amplified using Advantage 2 PCR Kit(Clontech) with gene-specific andMarathon adaptor primersunder the following conditions 1 cycle of 94∘C for 2min 5cycles of 94∘C for 30 sec 72∘C for 3min 5 cycles of 94∘C for30 sec 70∘C for 3min 35 cycles of 94∘C for 30 sec 68∘C for3min and 1 cycle of 68∘C for 5min A 100-fold diluted 1mklaliquote from the 1st round of amplification was used for the2nd round of amplification with nested primers under thesame conditions PCR products were further cloned into thepGEM-T Easy Vector (Promega WI USA) propagated in Ecoli and sequenced using conventional techniques

Primers for identification of the major splice variant arepresented in Table 1 Amplifications were performed underthe following conditions 1 cycle of 95∘C for 2min 15 cyclesof 95∘C for 30 sec 58∘C for 30 sec and 72∘C for 1min and 1cycle of 72∘C for 5min A 1mkl aliquote from the 1st roundof amplification was used for the 2nd round of amplificationwith nested primers Cycling conditions were as above but 35cycles of amplification were used

24 Software and Databases We used BioEdit software [5]for basic manipulations with nucleic and amino acidssequences Resources of the NCBI databases (httpwwwncbinlmnihgov) and UCSC Genome Browser (GB) [6 7](httpgenomeucscedu) were used extensively RNAfolding was done using Mfold [8] (httpwwwbioinforpieduapplicationsmfold) and Vienna RNA Websuite [9]

online software (httprnatbiunivieacatcgi-binRNAfoldcgi)with default settings PROMO3web software [10] (httpalggenlsiupcescgi-binpromo v3promopromoinitcgidirDB=TF 83) was used for identification of putative TFBSNetGene2 Server [11] (httpwwwcbsdtudkservicesNetGene2) was used for identification of potential splicesites in nucleotide sequences

25 Genomic Sequences We used the following genomicsequences for the comparative analysis Otolemur garnettii(GI 393210271 positions 4461840-4466746) Callithrixjacchus (GI 290467407 positions 56153639-56159372)Saimiri boliviensis (GI 395721681 positions 473433-479005) Macaca mulatta (GI 109156890 positions39676154-39682121) Papio anubis (GI 395728659 positions34224856-34230574) Nomascus leucogenys (GI 328833306positions 1257019-1262659) Pongo abelii (GI 241864935positions 1664355-1670047) Pan paniscus (GI 393728162positions 1586174-1591896) Pan troglodytes (GI 319999821positions 417230-422955) Homo sapiens (hg19 chr71777236-1782938) and Gorilla gorilla (gorGor31gorGor3chr7 1684515-1691410 gaps excluded)

3 Results

31 Expression of the Gene in Human Normal Tissues andTumors The specificity of expression of our gene was studiedusing PCR with gene-specific primers and panels fromnormal and tumor tissues

The results are presented in Figure 1 They are in agree-ment with our previous data and show that ELFN1-AS1 ismore abundant in tumors with week expression in normaltissues for example in liver (Figure 1(a) 04) and in heart(Figure 1(a) 02)

32 Primary Structure of the Hs633957-Specific TranscriptTo identify the borders of the transcribed region we con-ducted a series of 51015840- and 31015840-RACE experiments using RNAfrom lung uterus and ovarian tumors and human normalplacenta We obtained 7 different RACE ragments 3 ofthem corresponded to the spliced 51015840-end of RNA (GenBankaccession nos HO663743 HO663744 HO663747) the other3 corresponded to the unspliced 51015840-end of RNA (GenBankaccession nos HO663745 HO663746 HO663748) and 1 tothe 31015840-end of the RNA (GenBank accession no HO663742)

Based on the alignment of the Hs633957-specificESTs with the human genome (human genome assemblyNCBI36hg18) 3 alternative splice acceptor sites (SA) and 2polyadenylation sites could be predicted for the ELFN1-AS1RNA (Figure 2(a)) To identify the major transcript spliceform we conducted nested PCR with the primers UP times DDPand nested primers NUP times NDDP (position from 39 to3642) which border the transcribed region (Figure 2(a)) onthe cDNA template from various tumors with known locusexpression Only the products with distal polyadenilationsites could be detected (Figure 2(b)) In each case a majoramplicon corresponding to the transcript with the 1st intronsplicing out at SA3 site was observed To verify this result

BioMed Research International 3

Table 1 Gene-specific amplification primers

No Primer name Primer sequence1 5gsp1 GCTGAGAGTGAATTCGGGGTGCAG2 5gsp1n GCTGCTGCGTCTCGGAGTGAATG3 5gsp2 GACTGGGAGTGAGAAGGAGC4 5gsp2n GGAGTGAGAAGGAGCGGAG5 5gsp3 GGTCTTTACTCCCATTCAACGGAAGAG6 5gsp3n CATTCAACGGAAGAGGAAGCAGAGC7 3gsp1 CCTCCTGTCATTCACTCCGAGACGC8 3gsp1n CATTCACTCCGAGACGCAGCAGC9 Upstream primer (UP) GTGGCGCCTCAGCCACAATC10 Nested upstream primer (NUP) GCCTCAGCCACAATCGTAAT11 Distal downstream primer (DDP) GTGAGAAACCACAAGCTCCCTG12 Nested distal downstream primer (NDDP) GGTCTTTACTCCCATTCAA13 Proximal downstream primer (PDP) CAGGTTCTTCAGCCAGGAAG14 Nested proximal downstream primer (NPDP) GGAGTGAGAAGGAGCGGAG

M 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 PC MNTC

322bp

983bp

(a)

322bp

983bp

M 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 PC MNTC

(b)

Figure 1 Expression of the ELFN1-AS1 gene in human normal tissues and tumors (a) Upper pane PCR with ELFN1-AS1-specific primers(a) Lower pane GAPDH control (a) Lanes M DNA size marker 1 normal brain 2 normal heart 3 normal kidney 4 normal liver 5normal lung 6 normal pancreas 7 normal placenta 8 normal skeletal muscle 9 brain malignant meningioma moderately differentiated10 lung nonsmall cell carcinoma 11 kidney transitional cell carcinoma papillary moderately differentiated 12 kidney renal cell carcinoma13 liver hepatocellular carcinoma well differentiated 14 liver adenocarcinoma moderately differentiated 15 gallbladder adenocarcinoma16 esophagus squamous cell carcinoma ulcer well differentiated 17 stomach adenocarcinoma ulcer well differentiated 18 small intestinemesenchymoma moderately differentiated NTC no template control PC positive control (b) Upper pane PCR with ELFN1-AS1-specificprimers (b) Lower pane GAPDH control (b) Lanes M DNA size marker 1 normal colon 2 normal ovary 3 normal peripheral bloodleukocytes 4 normal prostate 5 normal small intestine 6 normal spleen 7 normal testis 8 normal thymus 9 colon adenocarcinomamoderately differentiated 10 rectum adenocarcinoma moderatelypoorly differentiated 11 ovary adenocarcinoma ulcer moderatelydifferentiated 12 fallopian tubemedullary carcinoma poorly differentiated 13 uterus adenocarcinoma 14 ureter moderately differentiatedtransitional cell carcinoma 15 bladder transitional cell carcinoma papillary 16 testis germinoma 17 parotid squamous cell carcinomamixed NTC no template control PC positive control

we conducted nested PCR with primers UP times PDP andnested primers NUP times NPDP specific for a shorter regionof the gene (positions 39-3289) on the template of the samecDNA samples The selected position of the primers allowedamplification of cDNAs corresponding to the RNAs withboth distal and proximal polyadenilation sites Again the

major amplicon corresponded to the transcript with splicingat SA3 (Figure 2(c)) Faint signals for amplicons about380 bp might correspond to the transcript with splicingat SA2 (expected size 378 bp) however no validation wasperformed Thus SA3 was the major splice acceptor site ofthe 1st intron


96 2270 3103 3431 3619 3676SA1 SA2 SA3

2967

UP PDP DDP

AAAAAA AAAAAA

(a)

SA3

1 2 3 654 NTCM

1000

800700600500400

300

200

100

900

Fragment size (bp)

(b)

900700

SA3

1 2 3 654 NTCM

1000800

600500400

300

200

100

Fragment size (bp)

(c)

Figure 2 Identification of the primary structure of the Hs633957-specific RNA (a) Scheme of the transcript variants for the locus Hs633957Splice sites and polyadenylation sites are marked and their positions are given according to the leftmost TSS Arrows indicate position of theprimers which were used for identification of the major splice form BX119057-like variant is filled in dark grey (b) Results of cDNA PCRamplification with primers bordering the 39 to 3642 gene region (c) Results of cDNA PCR amplification with primers bordering the 39ndash3289gene region cDNA samples 1 gallbladder adenocarcinoma 2 rectum well differentiated adenocarcinoma 3 ureter papillary transitionalcell carcinoma 4 T-cell Hodgkinrsquos lymphoma 5 kidney 6 liver NTC no template control

We used RepeatMasker ver 327 integrated into the GBand identified several repeat units within the gene SINEsLINE simple (TG)n DNA repeat and a DNA transposon(Figure 3 B) However the LINE L2b repeat was not recog-nized by the more recent RepeatMasker version open-330The (TG)n repeat is of particular interest It is located 129 bpdownstream from the splice donor site (SD) (CAAGandGTAA)of the 1st intron in the positive DNA strand It is 136 bplong and holds 33 TG dinucleotides Its divergence from theconsensus repeat sequence is 36

33 Promoter Region of the Putative Gene We usedldquoENCODE Enhancer and Promoter Histone Marksrdquo andldquoENCODE Transcription Factor ChIP-seqrdquo tracks of theGB to identify the promoter region of the putative geneand detected promoter and enhancer epigenetic markswithin the minus1000 to +500 bp region relative to the TSSas well as association of various TFs with this region(Figure 3) We searched for the most common eukaryoticcore promoter motifs such as TATA-box CCAAT-box

and GC-box within the region and verified them using theBucherrsquos position-specific weight matrices [12] A GC-boxlocated at the position from minus13 to +1 satisfied the selectioncriteria (Figure 3(C) Supplementary File 1 avaliable onlineat httpdxdoiorg1011552014398097)

We analysed the data available from the ldquoENCODE UnivWashington DNaseI Hypersensitivity by Digital DNaseIrdquotrack of the GB and noticed that peaks of DNaseI hypersen-sitivity signal in various cell lines were concentrated aroundtwo sites (ldquoHS1rdquo and ldquoHS2rdquo) within the region under study(Figure 3(C)) The GC-box coincided with the center of HS2site (Figure 3 ldquoHS2rdquo and ldquoGC-boxrdquo) which supports the ideathat this GC-box is an active promoter element

Analysis of ChIP-seq data from various GB tracksrevealed association of Ini1 c-Myc Max BAF155 BAF170STAT1 TAF1 HEY GABP TCF12 FOXP2 USF1 SETDB1PolII E2F-1 E2F-4 E2F-6 YY1 and GATA-2 with thechromatin region from minus1000 to +500 in certain cell types(Figure 3(E) Supplementary Figures S1ndashS5) Association of c-Myc and Max with the chromatin region was of particular


SA2 SA3SA1 PolyA PolyA

Window position chr7

Hs633957 transcript variants

RepMask 327

GC-box

HS sites

NRSFIni1c-MycMaxBAF155BAF170STAT1TAF1HEY1GABPTCF12FOXP2USF-1BAF155

1746000174700017480001749000

Repeating elements by RepeatMasker version 327

ENCODE enhancer- and promoter-associated histone mark (H3K27Ac) on 8 cell lines

ENCODE enhancer- and promoter-associated histone mark (H3K4Me1) on 8 cell lines

ENCODE promoter-associated histone mark (H3K4Me3) on 9 cell lines

ENCODE transcription factor ChIP-seq

GC-box

HS1 HS2

A

B

C

D

E

F

G

ENCODE TFBS YaleUCDHarvard ChIP-seq peaks (Pol2 in HeLa-S3 cells)

ENCODE TFBS YaleUCDHarvard ChIP-seq peaks (Pol2 in K562 cells)

ENCODE TFBS YaleUCDHarvard ChIP-seq peaks (Pol2 in HCT-116 cells)

ENCODE TFBS YaleUCDHarvard ChIP-seq peaks (Pol2 in NB4 cells)

ENCODE transcription levels assayed by RNA-seq on 6 cell lines

pH

HKHhK

HH

HgHK

LKHLK

GP

KH

Enhanced H3K27Ac

Layered H3K4Me1

Layered H3K4Me3

Transcription

SSA2SA2SA2SA222 SA3AAASA1AAAA PolyPoPo AAAAA Polyllll A

17 4644464 0000000017777474477 0000000000 0711777774888 000000000 000017111 777449999 00000000

RReRepeee eateeateate ttiningnnggg ele emeeemeemem ntsnn byyyyyy ReRRR peapppeappeapeapppppp tMaMMMaMMaaaskekkk r vrrr vverseeersee sionooonn 33333 272272 777

ENCNCNCNCNCNCODEOODDDDD enennnhanaaana cercerccer- aand nnnn propprprroopppppp motmooooter-rrrr assaassssociccccc atettt d hddd hd hhiststtttonennnn mammmmm rk rrrrr (H(H3HHHH K27KKK2K2K2 Ac)AAAc)Ac)Ac))) onooo 88 celcccelc lll ll ll lineiineii ees

ENCNCNCNCNCNCODEOODDDD eneennnhanaaaa cercerccerr- aaaaand ndd proppproproooomotmotttter-rrrr assaa ooocoocicciiatettt d hddd hd hhisttttttonennnn mammmark rrkkk (H3((HHHH K4MKKK4MK4MK MMe1)ee1e1e1) onooo 8 celcccelc llll lllliineees

ENCEEENENENNCNCCODEODODEDEE prppp omoooomommm terttterterrrr-asassocooooo iiatiiiatteeded ddd hishhhii tontttonnnne mmmaarkaaarkkkk (H((H3K3K3K3K4KK Me3MMeeeee ) o))) o) o) on 999 ceccccc ll lll liniii eseeee

ENCEEENCEENNNNCODEDEDEDDEE trtt ansaannsnsscricriccriiptitttt onoo ffacfffacccctorooo ChCCChChChhhIP-PP seqssseqseqeqqq

GC-GGCGCGCC boxbbboxbb

HS1HHH 1111 HS2HHHS2HS2222

A

B

C

D

E

F

G

ENCENCNNNCNCCCODEOOO EEEE TFTFTTFFBSSSSS YaYYaYaYaleleleUCDUCDDDDDHaHaHarvavvvv rd rrrrr ChIhhhh P-sP-sPP-sssseq qqqq peapeapeap aaaks sssss (PoPPoooool2 innnn HeLHHHeLHeLHeLLLa-S--- 3 c333 c3 cccelllllls))

ENCEEENENENNCNCCODEODODEDEE TFTTTFFFFBSSSSS YaYYYY lllellleUCDCCDCDCCDDHaHHaaaarvavvvvv rd rrr CChCChIhh P-sPPP-sPP ssseq qqq peapppeapeapeaaaksss (PoPPPPoPollll2 in in nn K56KKK56KK5662 cccellllll s))))

ENCENCENNCNCNCCODEODEO EEE TFTFTTFFBSSSSS YaYYaYaY lellleleUCDUCDCDDDDHaHaHHarvavvvv rd rrrrr ChIhhhh P-sP-sPP-sssseq qqqq peapeapeap aaaks sssss (PoPPoool2 l2 l2 in nnn HCTHHCTHCHCHCT-11-1111116 ccccellllll s)))))

ENCNNNCNNCCODEOOODOD TFTTFFFFBSSSS YaYaYaaaaleUCDUUCDUCDUU DDHaHaHaarvavvvvv rrrrdd ChIChhIP-PPP-ssseq qqq peapppeapeapeaaaks ks (Po((PoPoPoool2 22 in nnnnn NB4NNNBNNBB cecccc llsllll )))))

ENCNNNCCCCODEODOODD ttttransaansansa sscrirrrrr ptip on ooooo levevvelselll aaaassaysssayyyyedddd by bbbbb RNARRRNANANAAA-sesssss q oqq ooon 6n 6n n 666 ceccc ll lllll liniiiii eseee

pppH

HKHHHHHHHHhhhKhhhh

HHHHHHH

HHHggggggHHHHHggKKKKHH

LLLKKLLHHHLKKKKLL

GGGPPPP

KKKKKHH

(GT)n AluSx MIR3 MER3 L2b AluJo

Figure 3 Transcribed locus Hs633957 overview A sketch of the chromosome 7 region containing transcribed locus Hs633957 is shownaccording to the human genome assembly NCBI35hg18 (A) Transcript variants are given to show the location of the region (B) Repeatingelements by RepeatMasker version 327 track indicates the location of repeating elements (C) GC-box marks the GC-box motif identified incurrent study HS sites mark position of the DNase I hypersensitivity sites Location of the sites was estimated as averages of the left and rightpeak boundaries coordinates among the overlapping peaks from independent experiments for which data is available from UCSC GenomeBrowser (24 for HS1 and 17 for HS2) (D) Enhanced H3K27Ac H3K4Me1 and Promoter H3K4Me3 tracks indicate association of the specifichistone marks with the genome in certain cell lines Overlaid data for several cell lines is shown (E) ENCODE transcription factor ChIP-seq track provides a summary for association of various TFs with chromatin in various cell lines Cell lines are indicated by letter code (HHeLa-S3 K K-562 h HUVEC g GM12891 L HepG2 G GM12878) (F) Association of Pol 2 with chromatin in various cell lines is shownaccording to ENCODEChIP-seq data (G) ENCODE data on transcription levels in various cell lines are obtained by RNA-seq Overlaid datais shown

interest since the binding signal was the highest for theseproteins as indicated by the darkest color in Figure 3 Wesearched for canonical and noncanonical c-Myc-binding sites[13] (E-boxes) within the chromosome region and identifieda CACGTG ldquoE-box 1rdquo and a CGCGTG ldquoE-box 2rdquo motifs280 bp and 75 bp upstream of the TSS respectively (Supple-mentary Figure S1 Supplementary File 1) However the most

intriguing was the 85 bp region located 210 bp downstreamof the genersquos TSS because it contained 6 MycMax-bindingmotifs (ldquoE-boxesrdquo 3ndash8 Supplementary Figure S1) Matchingthe chromosomal location of these MycMax sites with allthe ChIP-seq data for MycMax available from GB revealedthat c-Myc and Max bind the whole region surrounding the


C-T-G-TG-T

G-T-C-C-T-G-G-TC-A-C

T-G-C-T-G

T

G-G-C-A-T-CA-G

G

G

C-C

G-A-C-A

G-G-T

C-A-G-G-A-C-C-GC-C

G-G

C-G-C

A-C-G-A-C C-C-G-T-G-G

3502

3569

(a)

t tggc tc

ag

tcACc t tgtg gg cac

a acac cc gtga accg agGgg

5 155998400

3998400

5998400

3998400

25

(b)

A-C

C-C-C-T-G T-G-G-A-G-C-G-

g

a-c-c-ag

A-a-a-g

C-A-G-G

G-T-C-C

C-C

C-C

CA

A

-C-G-T-G-T-GT

g-g-g-a-c a-c-a-c-c-g-t-

1670

1680

C-T

G-A

-G-C-A-C-A-C

(c)

Figure 4 Transcribed locus Hs633957 may code for a microRNA (a) A hairpin-like fragment of the secondary structure was predictedfor the BX119057 sequence by Mfold Nucleotide positions are given according to the genome sequence starting from the leftmost TSS (b)The potential interaction of the predicted mature miRNA with its target site in DPYS mRNA (c) Fragment of the DPYS mRNA secondarystructure The target site for the miRNA is in lower case The miRNA seed site-interacting region is shaded Nucleotide positions are givenaccording to the mRNA (acc No NM 0013852)

TSS with the highest signal rate around the E-boxes 2ndash8(Supplementary Figure S1)

For the other E-box-binding proteins USF1 binding sig-nal overlapped with the E-boxes 1ndash8 (Supplementary FigureS1) Transcription factors TCF12 and HEY1 were associatedwith the TSS rather than with the E-boxes (SupplementaryFigure S1)

We analysed the minus1000ndash+500 region of the gene for thepresence of putative TFBS using PROMO3 web softwarewhich utilizes TRANSFAC ver 83 TFBS matrixes Of allthe TFs which were associated with the promoter proximalregion of the gene E2Fs YY1 GABP and GATA2 are knownto bind specific DNA motifs other than E-boxes UsingPROMO3 we identified multiple putative TFBS for E2F andYY1 proteins within the chromatin region associated withthese proteins (Supplementary Figures S2-S3 SupplementaryFile 1) A single GABP-binding site was identified at positionfrom minus17 to minus6 (Supplementary File 1) within the chro-mosome region associated with GABP in HeLa-S3 HepG2and K-562 cells (Supplementary Figure S4) Using PROMO3we failed to identify any GATA2-binding sites within theregion under study however there was a GATA1-bindingsite there according to the ldquoHMR Conserved TranscriptionFactor Binding Sitesrdquo track which holds location and scorefor the transcription factor binding sites conserved in thehumanmouserat alignment This site coincides with thepeak point ofGATA-2-binding inK-562 cells (SupplementaryFigure S5)

34 Analysis of the ORFs We identified two ORFs cod-ing for 53 and 62 aa proteins Amino acid sequence ofthe first ORF was MLELLLPLQEQELCVLVTAVASQGR-RGAQQPGLDRLGPRCARAGMRLLCFLFR and that of the

second ORF was MGLRSFSLPVLWCMPPSWLKNLHQP-PLRLGSSLLSFTPRRSSVAPRALPALHPEFTLSPHLV

We searched the NCBI database for homologs amongavailable proteins using BLAST algorithm [14] but did notdetect any significant similarities The nucleotide contextof the AUG codons also appeared to be suboptimal fortranslation initiation [15]

35 RNA Secondary Structure To check the possibility thatthe putative gene under study codes for a microRNA wemodelled secondary structure for the BX119057-like RNAisoform using Mfold software [8] and identified a 68 nthairpin-like structure (Figure 4(a)) This hairpin was sup-ported by 7 predicted secondary structure models with thelowest free energy (data not shown) It was formed by thenucleotides 3502ndash3569 of the putative gene and resembledhairpins of known pre-miRNAs The 51015840-strand of the hairpinstem holded a 22 nt fragment which was complementary to aregion of DPYS (dihydropyrimidinase) mRNA (Figure 4(b))This 22 nt site was located in the coding region of the mRNAnear the 31015840UTR Interestingly theDPYSmRNA region 1666ndash1671 complementary to the seed site of the potential miRNAis single stranded which makes it easier to interact withthe putative miRNA (Figure 4(c)) These data support thepossibility that the transcribed locus Hs633957 may code fora miRNA and DPYS mRNA is its target

36 Phylogenetic Analysis of the Gene We used repeat-masked DNA sequence for transcribed region under studyand 1 kb upstream region as a query to searchNCBI databasesfor its homologs in available genomes using BLAST algo-rithm Homology regions covering the whole gene weredetected only in primates They were all single copied Two


GABP GC 34 5678

GABP GC 34 56

GABP GC1 34 567

Human

Chimp(Pan troglodytes)

Gorilla (Gorilla gorilla gorilla)

Orangutan (Pongo pygmaeus abelii)

Baboon (Papio anubis)

Marmoset (Callithrix jacchus)

Black-capped squirrel monkey(Saimiri boliviensis boliviensis)

Greater Galago

GABP GC2 4 5678

GABP GC 3

Exon1 Exon2GABP GC

21

SA1 SA2 SA3SD

34 5678

GABP GC2 34 5678 Gibbon

(Nomascus leucogenys)

1

2

2

GABP GC 3

5

5

6

8

(Otolemur garnettii)

Homo sapiens

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowast

lowast

lowast lowast

lowastlowast

lowastlowast

lowast

lowast

Figure 5 Emergence pattern of functional regions around ELFN1-AS1The changes of the functional regions in the context of the phylogenyare shown E-boxes are shown as grey boxes numbered 1ndash8 Lighter colour with ldquolowastrdquo sign indicates an alternative E-box sequence at the pointGABP and GC represent the GABP binding site and the GC-box SD represents the splice donor site SA1-SA3 the acceptor splice sites Blackcrosses indicate the absence of a splice signal The ldquolowastrdquo indicates presence of an alternative splice signal Exons and introns are not to scaleData for the nonprimate vertebrates are not shown

major conserved fragments were detected in nonprimatemammals The first one was about 200 bp long and it waslocated about 500 bp upstream of the TSS The second onewas about 100 bp with its center located near the SD site Theoverviewof the gene conservation is presented in Supplemen-tary Figure S6 It shows the ldquoVertebrate Multiz Alignmentamp Conservation (44 Species)rdquo track of GB which providessequence conservation level within synthetic chromosomeregions as calculated by PhastCons algorithm In accordancewith the data above only short conserved regions can beobserved

Using the results of BLAST-search we extracted ortholo-gous gene sequences from the most recent assemblies of pri-mate genomes and made multiple alignment using ClustalWalgorithm [16] The alignment was then used to constructan average distance tree with Jalview v27 software [17](Supplementary Figure S7)The tree topology correlates withprimate phylogeny [18] Galagidae (Otolemur garnettii) formsan outgroup indicating a distinct evolutionary pattern for thegene We analysed the TSS region of the multiple alignmentfor the presence of core promoter elements correspondingto human counterparts The GC-box [12] and GABP-binding

[19] motifs were present in all primates with an exception ofO garnettii (Figure 5) The SD site was intact in all primateswhile the SA sites varied between species Promoter regionand the 51015840-end of the intron were enriched with E-boxes inmost of the primates

4 Discussion

We have characterised LOC100505644 uncharacterizedLOC100505644 [Homo sapiens] gene corresponding to thehuman transcribed locus Hs633957 as a putative gene withtumor-specific expression by bioinformatic approach [1]It was experimentally demonstrated to be transcribed invarious tumors but in normal tissues its expression seemsto be weak and specific to liver heart and stomach [2ndash4]Because transcription of the gene was tumor-related and veryweak in normal tissues we were unsure if it was functionalAlternatively it could be a result of transcriptional noiseThus the status of this gene was uncertain The datapresented here confirm that LOC100505644 is expressedmostly in tumors and indicate that it is a novel human geneIn accordance with Human Gene Nomenclature Committee


regulations we propose to designate it ELFN1-AS1 ELFN1antisense RNA 1 (non-protein coding)

We used an independent set of cDNA samples to recheckthe expression of the gene in human tissues and tumorswith PCR We obtained results similar to our previous data[2ndash4] which show that ELFN1-AS1 is a tumor-related gene(Figure 1) We used RACE and genetic database analysis todefine the borders of the ELFN1-AS1 transcript and identifyits possible splice variants Although a considerable diversityof transcript variants was predicted only one splice variantpredominated over others in healthy tissues and tumorsas defined by PCR Such stability would not be expectedfor ldquojunkrdquo RNA In fact there was some variation in SAof the 1st intron (Figure 2) but the SD site was invariantsuggesting the presence of a strong splicing signal The GT-repeat downstream of the SD site (Figure 3) is the primarycandidate for this role as GTCA repeats are known toregulate activity of nearby splice sites [20 21] and are widelyspread in introns of human genes [22]

Knowing the TSS we could analyse the promoter regionof the gene We found that the TSS fell into a chromatinregion rich in promoter- and enhancer-specific histone mod-ifications A GC-box which is a common core promoterelement [12] was identified right before the TSS in a DNaseIhypersensitive site suggesting a core promoter responsive toSp1 transcription factors family to be there About 20 tran-scription factors were found to be specifically associated withthe promoter region of the gene (Figures 3 S1ndashS5) in differentcells several of the DNA-binding TFs had putative bindingsites within the regionThree cell linesmdashK-562 HeLa-S3 andGM12878mdashwere best characterised in genetic databases inrespect of TFs association with chromatin Of those threeTFs binding to the promoter were observed in K-562 andHeLa-S3 cell lines (Figures 3 S1ndashS5) In agreement with thisELFN1-AS1 expression in K-562 cells was supported by datafrom Transcription Track of GB (Figure 3) and expressionin HeLa-S3 cells was supported by 4 HeLa-S3-specific ESTs(GenBank acc nos AA190879 AA190847 AA488351 andAA488481) These data suggest that ELFN1-AS1 has its ownpromoter

c-MycMax and PolII association with the chromosomeregion is of particular interest c-Myc is one of the majorregulators of cell cycle Its upregulation in normal cells isassociated with cell proliferation and protein synthesis [23]c-Myc may act through the regulation of transcription pauserelease [24] or by attracting chromatin-modifying complexesin combination with Max [25] We found 8 E-boxes inpromoter proximal region and all of them rest in chromatinregions with which c-Myc and Max are associated in specificcells Though the individual impact of each E-box into thegene regulation is a subject of further studies the datapresented here clearly indicate that ELFN1-AS1 is a c-Myc-responsive gene

Another noteworthy fact is the association of E2F familytranscription factors with the ELFN1-AS1 promoter Tran-scription factors of E2F family regulate cell cycle through thegenes involved in DNA synthesis [26] They are also knownto be able to work synergistically with Sp1 for gene activation[27] Thus regulation of the gene by E2Fs supports the idea

that ELFN1-AS1must be normally active in proliferating cellsSince c-Myc overexpression is a common event for manymalignancies [23] this may explain why we detect ELFN1-AS1expression in various kinds of tumors [2ndash4]

In general the current data on TF association withchromatin which is available from GB is not sufficient tospeculate about the details of ELFN1-AS1 regulation but itclearly indicates that ELFN1-AS1 possesses its own promoterand is a subject of a complex regulation by various TFs c-MycMax and E2F family proteins in particular With regardto the tissue-specific transcription and reproducible RNApri-mary structure the human transcribed locus Hs633957 maybe considered ldquoa complete chromosomal segment responsiblefor making a functional productrdquo which is a definition of agene according to Snyder and Gerstein [28] The functionof this gene is yet to be identified however we considerit unlikely that ELFN1-AS1 is translated and functions as aprotein Although there are 2 ORFs in ELFN1-AS1 mRNAneither of the corresponding proteins is present in proteindatabases Also the nucleotide context of the AUG codons[15] is suboptimal for translation initiation

Based on the RNA secondary structure we speculate thatELFN1-AS1 codes for a miRNA and its function is related toregulation of gene expression with DPYS mRNA being itsmost probable target (Figure 4) This prediction is indirectlysupported by the incomplete complementarity of the putativemiRNA and its target site with unpaired nucleotides inposition 9 and presence of adenine in the first position ofthe target site (Figure 4(c)) Such features are favourablefor miRNAs action as supressors at RNA level [29 30] Itwas also supposed that the seed sites of the miRNAs serveas initiators for the miRNA-target interaction through fasttarget recognition [31] In compliance with this idea theputative miRNA seed site counterpart in the DPYS mRNAis single stranded (Figure 4(c)) while it is surrounded withdouble-stranded RNA regions It is also known that DPYSparticipates in pyrimidine catabolism [32] and is active inliver and in solid tumors [33] According to our data liver isone of the few normal tissues where weak expression of thegene is observed [2ndash4] These data give indirect evidence infavour of the miRNA function of ELFN1-AS1 andDPYS beingits primary target This is also in good correspondence withthe idea that the gene is normally activated in proliferatingcells We are currently working on experimental verificationof this prediction

Another possibility is that ELFN1-AS1 somehow interactswith ELFN1 function which is located on the oppositeDNA strand Elfn1 protein is selectively expressed by O-LM interneurons and directs the formation of highly facil-itating pyramidal-O-LM synapses [34] An antisense insideof ELFN1 gene may also participate in regulation of thisfunction however the only evidence of such a possibility sofar is expression of ELFN1-AS1 in fetal brain

The origin of the gene is of particular interest Accordingto our data it originated from an intronic region of aconserved gene ELFN1 (NCBI Ref Seq NM 0011286362)in primate lineage Nonprimate mammals had only smallregions of homology with human ELFN1-AS1 Homologoussequences for the gene were identified in all primates but


DNA sequence from the representative of suborder Strep-sirrhini Otolemur garnettii had more than 50 differencesfrom its human counterpart and formed an outgroup on thephylogenetic tree It was also different from other primatesin that it had no GC-box GABP and E2F binding sites inthe region corresponding to human TSS (Figure 5) ThusELFN1-AS1 could become transcriptionally active after thedivergence of Strepsirrhini and Haplorhini primates It isnoteworthy that all theHaplorhini primates had a regionwith5 or more E-boxes downstream of the DS site This indicatesthat this gene could always be c-Myc-responsive This genecombines features of predominant expression in tumors andevolutionary novelty

5 Conclusion

Here we describe a novel human non-protein coding geneELFN1-AS1 We give a description of its splice forms andmajor regulatory elements and present evidence that it maycode for a miRNA We also show that this gene originated inprimates de novo Considering the tumor-related expressionof the gene we suppose that it might be a regulatory geneplaying some important role in carcinogenesis The furtherdetailed studies of its function and regulation would beinteresting

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to Dr A Masharsky for technicalassistance The authors are grateful to Dr Christian TschudiYale School of Public Health for the possibility to conducta part of RACE experiments in his laboratory This researchwas partly supported by the Fogarty International CenterGrant no D43TW1028 DP was supported by the Ministry ofEducation and Science of Russian Federation and by the grantfor young PhDs from the president of Russian Federation(MK-115220134) Selected figures were preparedwith the useof equipment from the research resource center Molecularand Cell Technologies Saint Petersburg State University

References

[1] A V Baranova A V Lobashev D V Ivanov L L KrukovskayaN K Yankovsky and A P Kozlov ldquoIn silico screening fortumour-specific expressed sequences in human genomerdquo FEBSLetters vol 508 no 1 pp 143ndash148 2001

[2] L L Krukovskaja A Baranova T Tyezelova D Polev and AP Kozlov ldquoExperimental study of human expressed sequencesnewly identified in silico as tumor specificrdquo Tumor Biology vol26 no 1 pp 17ndash24 2005

[3] D E Polev Y KNosova L L Krukovskaya AV Baranova andA P Kozlov ldquoExpression of transcripts corresponding to clusterHs633957 in human healthy and tumor tissuesrdquo MolecularBiology vol 43 no 1 pp 88ndash92 2009

[4] D Y Polev A P Krukovskaya and K Kozlov ldquoExpression ofthe locus Hs633957 in human digestive system and tumorsrdquoVoprosy Onkologii vol 57 no 1 pp 48ndash49 2011

[5] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[6] W James Kent C W Sugnet T S Furey et al ldquoThe humangenome browser at UCSCrdquo Genome Research vol 12 no 6 pp996ndash1006 2002

[7] B Rhead D Karolchik R M Kuhn et al ldquoThe UCSC genomebrowser database update 2010rdquo Nucleic Acids Research vol 38no 1 pp D613ndashD619 2009

[8] M Zuker ldquoMfold web server for nucleic acid folding andhybridization predictionrdquoNucleic Acids Research vol 31 no 13pp 3406ndash3415 2003

[9] A R Gruber R Lorenz S H Bernhart R Neubock and I LHofacker ldquoThe Vienna RNA websuiterdquo Nucleic Acids Researchvol 36 pp W70ndashW74 2008

[10] X Messeguer R Escudero D Farre O Nunez J Martınez andM M Alba ldquoPROMO detection of known transcription regu-latory elements using species-tailored searchesrdquo Bioinformaticsvol 18 no 2 pp 333ndash334 2002

[11] S Brunak J Engelbrecht and S Knudsen ldquoPrediction ofhuman mRNA donor and acceptor sites from the DNAsequencerdquo Journal of Molecular Biology vol 220 no 1 pp 49ndash65 1991

[12] P Bucher ldquoWeight matrix descriptions of four eukaryotic RNApolymerase II promoter elements derived from 502 unrelatedpromoter sequencesrdquo Journal of Molecular Biology vol 212 no4 pp 563ndash578 1990

[13] T K Blackwell J Huang A Ma et al ldquoBinding of Myc proteinsto canonical and noncanonical DNA sequencesrdquoMolecular andCellular Biology vol 13 no 9 pp 5216ndash5224 1993

[14] S F Altschul J CWootton EM Gertz et al ldquoProtein databasesearches using compositionally adjusted substitution matricesrdquoFEBS Journal vol 272 no 20 pp 5101ndash5109 2005

[15] M Kozak ldquoPoint mutations define a sequence flanking theAUG initiator codon that modulates translation by eukaryoticribosomesrdquo Cell vol 44 no 2 pp 283ndash292 1986

[16] M A Larkin G Blackshields N P Brown et al ldquoClustalW andclustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[17] AMWaterhouse J B Procter DM AMartinM Clamp andG J Barton ldquoJalview version 2mdashamultiple sequence alignmenteditor and analysis workbenchrdquo Bioinformatics vol 25 no 9pp 1189ndash1191 2009

[18] P Perelman W E Johnson C Roos et al ldquoA molecularphylogeny of living primatesrdquoPLoSGenetics vol 7 no 3 ArticleID e1001342 2011

[19] D Alamanova P Stegmaier and A Kel ldquoCreating PWMs oftranscription factors using 3D structure-based computation ofprotein-DNA free binding energiesrdquo BMC Bioinformatics vol11 article 225 2010

[20] N Gabellini ldquoA polymorphic GT repeat from the human car-diac Na+Ca2+ exchanger intron 2 activates splicingrdquo EuropeanJournal of Biochemistry vol 268 no 4 pp 1076ndash1083 2001

[21] J Hui K Stangl W S Lane and A Bindereiff ldquoHnRNP Lstimulates splicing of the eNOS gene by binding to variable-length CA repeatsrdquo Nature Structural Biology vol 10 no 1 pp33ndash37 2003


[22] V K Sharma S K Brahmachari and S Ramachandranldquo(TGCA)n repeats in human gene families abundance andselective patterns of distribution according to function and genelengthrdquo BMC Genomics vol 6 article 83 2005

[23] L Soucek and G I Evan ldquoThe ups and downs of Myc biologyrdquoCurrent Opinion in Genetics and Development vol 20 no 1 pp91ndash95 2010

[24] P B Rahl C Y Lin A C Seila et al ldquoC-Myc regulates tran-scriptional pause releaserdquo Cell vol 141 no 3 pp 432ndash445 2010

[25] N Meyer and L Z Penn ldquoReflecting on 25 years with MYCrdquoNature Reviews Cancer vol 8 no 12 pp 976ndash990 2008

[26] JM Trimarchi and J A Lees ldquoSibling rivalry in the E2F familyrdquoNature Reviews Molecular Cell Biology vol 3 no 1 pp 11ndash202002

[27] J Karlseder H Rotheneder and E Wintersberger ldquoInteractionof Sp1 with the growth- and cell cycle-regulated transcriptionfactor E2Frdquo Molecular and Cellular Biology vol 16 no 4 pp1659ndash1667 1996

[28] M Snyder and M Gerstein ldquoGenomics defining genes in thegenomics erardquo Science vol 300 no 5617 pp 258ndash260 2003

[29] B P Lewis C B Burge and D P Bartel ldquoConserved seedpairing often flanked by adenosines indicates that thousandsof human genes are microRNA targetsrdquo Cell vol 120 no 1 pp15ndash20 2005

[30] M Selbach B Schwanhausser N Thierfelder Z Fang RKhanin and N Rajewsky ldquoWidespread changes in proteinsynthesis induced by microRNAsrdquo Nature vol 455 no 7209pp 58ndash63 2008

[31] N Rajewsky ldquomicroRNA target predictions in animalsrdquoNatureGenetics vol 38 no 1 pp S8ndashS13 2006

[32] A B P vanKuilenburgDDobritzsch JMeijer et al ldquoDihydro-pyrimidinase deficiency phenotype genotype and structuralconsequences in 17 patientsrdquo Biochimica et Biophysica Acta vol1802 no 7-8 pp 639ndash648 2010

[33] F NM NaguibM H El Kouni and S Cha ldquoEnzymes of uracilcatabolism in normal and neoplastic human tissuesrdquo CancerResearch vol 45 no 11 pp 5405ndash5412 1985

[34] E L Sylwestrak and A Ghosh ldquoElfn1 regulates target-specificrelease probability at CA1-interneuron synapsesrdquo Science vol338 no 6106 pp 536ndash540 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


22 PCR The quality of cDNA panels was assessed byPCR with primers for the GAPDH gene (forward 51015840-TGAAGGTCGGAGTCAACGGATTTGGT-31015840 and reverse51015840-CATGTGGGCCATGAGGTCCACCAC-31015840) Annealingtemperature was 68∘C and amplicon size was 983 bpTwenty-five cycles of PCR were performed Gene-specificprimers were as follows 51015840-GGTCTTTACTCCCATTCAA-31015840 and 51015840-CTCCTGTCATTCACTCCG-31015840 The reaction wasconducted in 25 120583L of a solution containing 25 ng of cDNAPCR buffer (67mM Tris HCl pH 89 4mM MgCl

2 16mM

(NH4)2SO4 10mM 2-mercaptoethanol and 01mgmL

BSA) dNTPs (200120583M each) 04 120583M each primer and 1Uof Taq DNA polymerase PCR program denaturation at95∘C for 1min followed by 35 cycles 95∘C for 30 s 56∘Cfor 30 s and 72∘C for 30 s Postextension was carried out at72∘C for 5min The resulting amplificates were resolved byelectrophoresis in 2 agarose gel and stained with ethidiumbromide The presence of the mRNA under study in thesample was indicated by the presence of the 322 bp longamplification product The gels were photographed underUV illumination

23 RACE We used SMARTRACE cDNAAmplification Kit(Clontech) with ovary carcinoma and placenta RNA samplesand Marathon cDNA Amplification Kit (Clontech) withuterus adenocarcinoma and lung carcinomaRNA samples forRACE experiments cDNA was prepared as recommendedby the manufacturer Gene-specific primers 5gsp1 5gsp1n5gsp2 5gsp2n 5gsp3 5gsp3n 3gsp1 and 3gsp1n for theamplification of the 51015840- and 31015840-cDNA ends are presented inTable 1

cDNA ends were amplified using Advantage 2 PCR Kit(Clontech) with gene-specific andMarathon adaptor primersunder the following conditions 1 cycle of 94∘C for 2min 5cycles of 94∘C for 30 sec 72∘C for 3min 5 cycles of 94∘C for30 sec 70∘C for 3min 35 cycles of 94∘C for 30 sec 68∘C for3min and 1 cycle of 68∘C for 5min A 100-fold diluted 1mklaliquote from the 1st round of amplification was used for the2nd round of amplification with nested primers under thesame conditions PCR products were further cloned into thepGEM-T Easy Vector (Promega WI USA) propagated in Ecoli and sequenced using conventional techniques

Primers for identification of the major splice variant arepresented in Table 1 Amplifications were performed underthe following conditions 1 cycle of 95∘C for 2min 15 cyclesof 95∘C for 30 sec 58∘C for 30 sec and 72∘C for 1min and 1cycle of 72∘C for 5min A 1mkl aliquote from the 1st roundof amplification was used for the 2nd round of amplificationwith nested primers Cycling conditions were as above but 35cycles of amplification were used

24 Software and Databases We used BioEdit software [5]for basic manipulations with nucleic and amino acidssequences Resources of the NCBI databases (httpwwwncbinlmnihgov) and UCSC Genome Browser (GB) [6 7](httpgenomeucscedu) were used extensively RNAfolding was done using Mfold [8] (httpwwwbioinforpieduapplicationsmfold) and Vienna RNA Websuite [9]

online software (httprnatbiunivieacatcgi-binRNAfoldcgi)with default settings PROMO3web software [10] (httpalggenlsiupcescgi-binpromo v3promopromoinitcgidirDB=TF 83) was used for identification of putative TFBSNetGene2 Server [11] (httpwwwcbsdtudkservicesNetGene2) was used for identification of potential splicesites in nucleotide sequences

25 Genomic Sequences We used the following genomicsequences for the comparative analysis Otolemur garnettii(GI 393210271 positions 4461840-4466746) Callithrixjacchus (GI 290467407 positions 56153639-56159372)Saimiri boliviensis (GI 395721681 positions 473433-479005) Macaca mulatta (GI 109156890 positions39676154-39682121) Papio anubis (GI 395728659 positions34224856-34230574) Nomascus leucogenys (GI 328833306positions 1257019-1262659) Pongo abelii (GI 241864935positions 1664355-1670047) Pan paniscus (GI 393728162positions 1586174-1591896) Pan troglodytes (GI 319999821positions 417230-422955) Homo sapiens (hg19 chr71777236-1782938) and Gorilla gorilla (gorGor31gorGor3chr7 1684515-1691410 gaps excluded)

3 Results

31 Expression of the Gene in Human Normal Tissues andTumors The specificity of expression of our gene was studiedusing PCR with gene-specific primers and panels fromnormal and tumor tissues

The results are presented in Figure 1 They are in agree-ment with our previous data and show that ELFN1-AS1 ismore abundant in tumors with week expression in normaltissues for example in liver (Figure 1(a) 04) and in heart(Figure 1(a) 02)

32 Primary Structure of the Hs633957-Specific TranscriptTo identify the borders of the transcribed region we con-ducted a series of 51015840- and 31015840-RACE experiments using RNAfrom lung uterus and ovarian tumors and human normalplacenta We obtained 7 different RACE ragments 3 ofthem corresponded to the spliced 51015840-end of RNA (GenBankaccession nos HO663743 HO663744 HO663747) the other3 corresponded to the unspliced 51015840-end of RNA (GenBankaccession nos HO663745 HO663746 HO663748) and 1 tothe 31015840-end of the RNA (GenBank accession no HO663742)

Based on the alignment of the Hs633957-specificESTs with the human genome (human genome assemblyNCBI36hg18) 3 alternative splice acceptor sites (SA) and 2polyadenylation sites could be predicted for the ELFN1-AS1RNA (Figure 2(a)) To identify the major transcript spliceform we conducted nested PCR with the primers UP times DDPand nested primers NUP times NDDP (position from 39 to3642) which border the transcribed region (Figure 2(a)) onthe cDNA template from various tumors with known locusexpression Only the products with distal polyadenilationsites could be detected (Figure 2(b)) In each case a majoramplicon corresponding to the transcript with the 1st intronsplicing out at SA3 site was observed To verify this result




M 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 PC MNTC

322bp

983bp

(a)

322bp

983bp

M 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 PC MNTC

(b)





96 2270 3103 3431 3619 3676SA1 SA2 SA3

2967

UP PDP DDP

AAAAAA AAAAAA

(a)

SA3

1 2 3 654 NTCM

1000

800700600500400

300

200

100

900

Fragment size (bp)

(b)

900700

SA3

1 2 3 654 NTCM

1000800

600500400

300

200

100

Fragment size (bp)

(c)











RepMask 327

GC-box

HS sites


1746000174700017480001749000






GC-box

HS1 HS2

A

B

C

D

E

F

G






pH

HKHhK

HH

HgHK

LKHLK

GP

KH

Enhanced H3K27Ac

Layered H3K4Me1

Layered H3K4Me3

Transcription


17 4644464 0000000017777474477 0000000000 0711777774888 000000000 000017111 777449999 00000000








A

B

C

D

E

F

G






pppH

HKHHHHHHHHhhhKhhhh

HHHHHHH


LLLKKLLHHHLKKKKLL

GGGPPPP

KKKKKHH






C-T-G-TG-T


T-G-C-T-G

T

G-G-C-A-T-CA-G

G

G

C-C

G-A-C-A

G-G-T

C-A-G-G-A-C-C-GC-C

G-G

C-G-C


3502

3569

(a)

t tggc tc

ag

tcACc t tgtg gg cac


5 155998400

3998400

5998400

3998400

25

(b)

A-C


g

a-c-c-ag

A-a-a-g

C-A-G-G

G-T-C-C

C-C

C-C

CA

A

-C-G-T-G-T-GT


1670

1680

C-T

G-A

-G-C-A-C-A-C

(c)











GABP GC 34 5678

GABP GC 34 56

GABP GC1 34 567

Human







Greater Galago

GABP GC2 4 5678

GABP GC 3

Exon1 Exon2GABP GC

21

SA1 SA2 SA3SD

34 5678



1

2

2

GABP GC 3

5

5

6

8


Homo sapiens

lowast

lowastlowastlowast


lowast

lowast

lowast lowast

lowastlowast

lowastlowast

lowast

lowast





4 Discussion















5 Conclusion




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















M 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 PC MNTC

322bp

983bp

(a)

322bp

983bp

M 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 PC MNTC

(b)





96 2270 3103 3431 3619 3676SA1 SA2 SA3

2967

UP PDP DDP

AAAAAA AAAAAA

(a)

SA3

1 2 3 654 NTCM

1000

800700600500400

300

200

100

900

Fragment size (bp)

(b)

900700

SA3

1 2 3 654 NTCM

1000800

600500400

300

200

100

Fragment size (bp)

(c)











RepMask 327

GC-box

HS sites


1746000174700017480001749000






GC-box

HS1 HS2

A

B

C

D

E

F

G






pH

HKHhK

HH

HgHK

LKHLK

GP

KH

Enhanced H3K27Ac

Layered H3K4Me1

Layered H3K4Me3

Transcription


17 4644464 0000000017777474477 0000000000 0711777774888 000000000 000017111 777449999 00000000








A

B

C

D

E

F

G






pppH

HKHHHHHHHHhhhKhhhh

HHHHHHH


LLLKKLLHHHLKKKKLL

GGGPPPP

KKKKKHH






C-T-G-TG-T


T-G-C-T-G

T

G-G-C-A-T-CA-G

G

G

C-C

G-A-C-A

G-G-T

C-A-G-G-A-C-C-GC-C

G-G

C-G-C


3502

3569

(a)

t tggc tc

ag

tcACc t tgtg gg cac


5 155998400

3998400

5998400

3998400

25

(b)

A-C


g

a-c-c-ag

A-a-a-g

C-A-G-G

G-T-C-C

C-C

C-C

CA

A

-C-G-T-G-T-GT


1670

1680

C-T

G-A

-G-C-A-C-A-C

(c)











GABP GC 34 5678

GABP GC 34 56

GABP GC1 34 567

Human







Greater Galago

GABP GC2 4 5678

GABP GC 3

Exon1 Exon2GABP GC

21

SA1 SA2 SA3SD

34 5678



1

2

2

GABP GC 3

5

5

6

8


Homo sapiens

lowast

lowastlowastlowast


lowast

lowast

lowast lowast

lowastlowast

lowastlowast

lowast

lowast





4 Discussion















5 Conclusion




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















96 2270 3103 3431 3619 3676SA1 SA2 SA3

2967

UP PDP DDP

AAAAAA AAAAAA

(a)

SA3

1 2 3 654 NTCM

1000

800700600500400

300

200

100

900

Fragment size (bp)

(b)

900700

SA3

1 2 3 654 NTCM

1000800

600500400

300

200

100

Fragment size (bp)

(c)











RepMask 327

GC-box

HS sites


1746000174700017480001749000






GC-box

HS1 HS2

A

B

C

D

E

F

G






pH

HKHhK

HH

HgHK

LKHLK

GP

KH

Enhanced H3K27Ac

Layered H3K4Me1

Layered H3K4Me3

Transcription


17 4644464 0000000017777474477 0000000000 0711777774888 000000000 000017111 777449999 00000000








A

B

C

D

E

F

G






pppH

HKHHHHHHHHhhhKhhhh

HHHHHHH


LLLKKLLHHHLKKKKLL

GGGPPPP

KKKKKHH






C-T-G-TG-T


T-G-C-T-G

T

G-G-C-A-T-CA-G

G

G

C-C

G-A-C-A

G-G-T

C-A-G-G-A-C-C-GC-C

G-G

C-G-C


3502

3569

(a)

t tggc tc

ag

tcACc t tgtg gg cac


5 155998400

3998400

5998400

3998400

25

(b)

A-C


g

a-c-c-ag

A-a-a-g

C-A-G-G

G-T-C-C

C-C

C-C

CA

A

-C-G-T-G-T-GT


1670

1680

C-T

G-A

-G-C-A-C-A-C

(c)











GABP GC 34 5678

GABP GC 34 56

GABP GC1 34 567

Human







Greater Galago

GABP GC2 4 5678

GABP GC 3

Exon1 Exon2GABP GC

21

SA1 SA2 SA3SD

34 5678



1

2

2

GABP GC 3

5

5

6

8


Homo sapiens

lowast

lowastlowastlowast


lowast

lowast

lowast lowast

lowastlowast

lowastlowast

lowast

lowast





4 Discussion















5 Conclusion




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















RepMask 327

GC-box

HS sites


1746000174700017480001749000






GC-box

HS1 HS2

A

B

C

D

E

F

G






pH

HKHhK

HH

HgHK

LKHLK

GP

KH

Enhanced H3K27Ac

Layered H3K4Me1

Layered H3K4Me3

Transcription


17 4644464 0000000017777474477 0000000000 0711777774888 000000000 000017111 777449999 00000000








A

B

C

D

E

F

G






pppH

HKHHHHHHHHhhhKhhhh

HHHHHHH


LLLKKLLHHHLKKKKLL

GGGPPPP

KKKKKHH






C-T-G-TG-T


T-G-C-T-G

T

G-G-C-A-T-CA-G

G

G

C-C

G-A-C-A

G-G-T

C-A-G-G-A-C-C-GC-C

G-G

C-G-C


3502

3569

(a)

t tggc tc

ag

tcACc t tgtg gg cac


5 155998400

3998400

5998400

3998400

25

(b)

A-C


g

a-c-c-ag

A-a-a-g

C-A-G-G

G-T-C-C

C-C

C-C

CA

A

-C-G-T-G-T-GT


1670

1680

C-T

G-A

-G-C-A-C-A-C

(c)











GABP GC 34 5678

GABP GC 34 56

GABP GC1 34 567

Human







Greater Galago

GABP GC2 4 5678

GABP GC 3

Exon1 Exon2GABP GC

21

SA1 SA2 SA3SD

34 5678



1

2

2

GABP GC 3

5

5

6

8


Homo sapiens

lowast

lowastlowastlowast


lowast

lowast

lowast lowast

lowastlowast

lowastlowast

lowast

lowast





4 Discussion















5 Conclusion




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C-T-G-TG-T


T-G-C-T-G

T

G-G-C-A-T-CA-G

G

G

C-C

G-A-C-A

G-G-T

C-A-G-G-A-C-C-GC-C

G-G

C-G-C


3502

3569

(a)

t tggc tc

ag

tcACc t tgtg gg cac


5 155998400

3998400

5998400

3998400

25

(b)

A-C


g

a-c-c-ag

A-a-a-g

C-A-G-G

G-T-C-C

C-C

C-C

CA

A

-C-G-T-G-T-GT


1670

1680

C-T

G-A

-G-C-A-C-A-C

(c)











GABP GC 34 5678

GABP GC 34 56

GABP GC1 34 567

Human







Greater Galago

GABP GC2 4 5678

GABP GC 3

Exon1 Exon2GABP GC

21

SA1 SA2 SA3SD

34 5678



1

2

2

GABP GC 3

5

5

6

8


Homo sapiens

lowast

lowastlowastlowast


lowast

lowast

lowast lowast

lowastlowast

lowastlowast

lowast

lowast





4 Discussion















5 Conclusion




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GABP GC 34 5678

GABP GC 34 56

GABP GC1 34 567

Human







Greater Galago

GABP GC2 4 5678

GABP GC 3

Exon1 Exon2GABP GC

21

SA1 SA2 SA3SD

34 5678



1

2

2

GABP GC 3

5

5

6

8


Homo sapiens

lowast

lowastlowastlowast


lowast

lowast

lowast lowast

lowastlowast

lowastlowast

lowast

lowast





4 Discussion















5 Conclusion




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























5 Conclusion




Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article : a novel primate gene with possible

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