The Journal of Farm Animal in Infectious Disease
Vol.1 No.1 2012

GENOME-WIDE MAPPING OF TSS IN BMEC

ORIGINAL ARTICLE

Genome wide mapping of transcriptional start sites
in bovine mammary epithelial cells using cap analysisof gene expression (CAGE)
Tomoko Matsubara1), Yoshio Kiku2), Tomomi Ozawa1), Shigeki Inumaru1), Hisashi Aso3),
Takahiro Yamaguchi3), David Reynolds4), Toshiro Arai5) and Tomohito Hayashi2)＊
1) Pathology and Pathophysiology Research Division, National Institute of Animal Health (NIAH),
National Agriculture and Food Research Organization (NARO)(3-1-5 Kan-nondai, Tsukuba, Ibaraki 305-0856, Japan)
2) Dairy Hygiene Research Division, NIAH, NARO
(4 Hitsujigaoka, Toyohira, Sapporo, Hokkaido 062-0045, Japan)
3) Cellular Biology Laboratory, Graduate School of Agricultural Science, Tohoku University(1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Miyagi 981-8555, Japan)
4) Genomics Core, Albert Einstein College of Medicine(1301 Morris Park Avenue, Bronx, New York 10461-1602, USA)
5) Department of Veterinary Science, School of Veterinary Medicine,
Nippon Veterinary and Life Science University(1-7-1 Kyonancho, Musashino, Tokyo 180-8602, Japan)＊To whom correspondence should be addressed: Tomohito Hayashi.

Tel: +81-11-851-2175

Fax: +81-11-853-0767

E-mail: hayatomo@affrc.go.jp[Abstract]
Mastitis is the most frequent infectious disease in dairy cattle and causes serious economic damage.
The identification of a gene regulatory network including signal pathways involved in immune response enabled us to explain a pathogenic mechanism of mastitis. However, studies on bovine mastitis
fall behind human and mouse studies, especially expression analysis studies. Recent works revealed
that the majority of the genome is transcribed into non-coding RNA (ncRNA) and alternative promoters play important roles in controlling gene expression, thus it is important to profile the bovine transcriptome. Cap analysis of gene expression (CAGE) makes it possible to identify transcriptional startsites (TSS) and quantify the expression levels at each individual TSS. Precise analysis of TSS distribution enables us to predict promoter regions and identify novel genes. It was our goal to obtain unsupervised genome-wide expression data for transcript and promoter identification in the bovine genome. As a model system for this study we used bovine mammary epithelial cells (BMEC), because
they are commonly used for studying mastitis. In this study, we show that there were huge amountsof active TSS in BMEC as identified by CAGE. These TSS clusters located in 4,623 intergenic regions
Received: 21 December 2011
Accepted: 2 February 2012

－1－家畜感染症学会誌
1 巻 1 号 2012

ウシ乳腺上皮細胞における CAGE 解析法を用いたゲノム全域にわたる転写開始点の解析

suggested the existence of candidate novel promoters. These results confirm the necessity of global
analysis of transcripts to uncover the precise molecular mechanisms of mastitis using BMEC. This
study is the first comprehensive analysis of TSS in BMEC by a genome wide scan using CAGE.
Key words: Bovine mammary epithelial cell, cap analysis of gene expression, transcriptional start site,transcriptome.

ncRNA is the subject of many studies today.

[Introduction]

Recent studies reveal the transcriptional regu-Mastitis, the most frequent infectious dis-latory network that drives the expression ofease in dairy cattle, has detrimental effects ongenes and ncRNA [17]. We can predict thatthe quantity and quality of milk productionthere may be a large number of TSS in bovine

and causes serious economic damage [14]. In

mammary epithelial cells (BMEC) similarly tocurrent research on mastitis, gene expression

human and mouse cells. For a better under-

analysis has been done to understand inflam-standing of transcriptional regulation, it is im-mation mechanisms and immune response. Re-

portant to identify TSS in BMEC.

cently, studies of bovine genetic research us-

CAGE makes it possible to identify TSS anding microarrays led to the construction of aquantify the expression levels at each individ-

gene network including pathways involved inual TSS. The main step in CAGE is construct-immune response [8, 11]. The annotation of theing and sequencing DNA tags derived frombovine genome falls behind the human andthe initial 27 nucleotides at the 5ʼ ends ofmouse genome because the bovine transcrip-mRNAs. By detecting TSSs, we can obtain thetome has not been studied to the same extent

transcriptional profile, identify novel genes andas the human or mouse counterparts. There-

predict promoter regions including alternative

fore, currently available cow expression ar-promoters and binding sites of transcription

rays are most likely falling short on coveringfactors. CAGE-tag based expression measure-

all genes, and further unsupervised experi-ments provide insight into the regulation ofmental data are needed to improve cow ge-

transcription initiation. From CAGE databases,

nome annotations. Genomic sequences require

recent studies discovered transcription prod-annotation based on experimental evidence foructs (e.g., ncRNA and alternatively spliced

their use in data analysis and interpretation.

transcripts) and the functional domains on theContrary to previous notions, recent studies

genome, which are involved in transcriptional

have shown that more than 72% of the mouseregulation (e.g., promoter and enhancer). More-genome is transcribed as RNA [12]. Genome

over, analysis of CAGE data eventually allows

scale analysis of the human and mouse tran-

the inference of transcriptional regulatory net-

scriptomes using cap analysis of gene expres-works [5].

sion (CAGE), revealed huge numbers of tran-

Focusing on expression analysis for a better

scription start sites (TSSs) that are far more

understanding of biological processes in bovine

abundant than previously expected [4]. Theremastitis, it was our goal to obtain unsuper-

are numerous transcripts derived not only

vised genome-wide expression data for tran-

from coding regions but also from non-coding

script and promoter identification in the bo-regions. Recent evidence suggests roles ofvine genome. As a model system for thisnon-coding RNAs (ncRNAs) in regulating tran-

study we used BMEC, because the transcrip-

scription [10] and involvement in disease onset

tome of the cell has not yet been identified al-[16]. Hence, the transcriptional regulation ofthough it is a commonly used cell line for－2－

The Journal of Farm Animal in Infectious Disease
Vol.1 No.1 2012

GENOME-WIDE MAPPING OF TSS IN BMEC

studying mastitis. This study is the first com-duce the CAGE tags. Next, a linker was at-prehensive analysis of TSS in BMEC. Our re-tached to the 3ʼ end of the tag sequence forsults show that TSSs in BMEC localize to sev-

amplification [Fig. 1(1)].

eral regions including intron and intergenic
Sequencing and mappingregions.

Sequencing of the CAGE library was per-[Materials and methods]

formed on a Genome Analyzer II (Illumina,Bovine mammary epithelial cells (BMEC)

San Diego, CA, USA) [Fig. 1(2)]. Mapping ofWe used lined BMEC isolated from bovine

CAGE-tag sequences to bovine genome (NCBI

mammary epithelium. BMEC was established

Btau 4.0) was done at Genomatix (Genomatix

from the mammary gland isolated from a 200-Software, München, Germany). Up to two mis-day pregnant Holstein cow [13]. The cells

matches (mutations, insertion and/or deletion)were cultured in Dulbeccoʼs Modified Eagleʼs

were permitted during mapping of the CAGE

Medium (Invitrogen, Carlsbad, U.S.A.), supple-tags. We used cluster analysis for genome

mented with 20% inactivated fetal bovine se-wide identification of local enrichments ofrum, apo-transfferin (10μg/ml), sodium acetate

CAGE tags representing TSSs obtained from(5 mM), penicillin (10 IU/ml), and streptomycin

Genomatix (Genomatix Software). Neighboring(10 μg/ml) at 37°C in air plus 5% CO 2. WeTSSs with a high probability of expression inseeded 1×10 BMEC into 75 cm flask (BDa constant proportion were joined into clusters

Biosciences, San Jose, USA) and the cells were

[Fig. 1(3)]. All TSS clusters were correlated

grown to confluence in culture medium. The

with transcripts annotated in the ElDorado

cells had typical morphological features: adatabase (Genomatix, NCBI Btau 4.0 Version

monolayer, cobblestone, epithelial-like morphol-07-2009) and assigned to these region types:

ogy, with close contact between cells. Then,exons, introns or intergenic regions. Regions

adherent cells were washed with phosphate-

which included an exon and neighboring in-buffered saline (PBS) and released using su-tron or intergenic region were categorized as

crose buffer and 0.025% trypsin (Invitrogen).

“partial”.

62

CAGE library preparation[Results]

Total RNA was extracted from the BMEC

Definition of TSS by CAGE tags

using TRIzol (Invitrogen) and used to preparea CAGE library. The CAGE library was pur-

CAGE tags are 27-nt sequence tags derived

chased from DNAFORM (Yokohama, Japan),from the 5ʼ end of mRNA in the proximity of

who prepared the CAGE library following thethe cap site. Mapping CAGE tags onto genom-

protocol described by Kodzius et al., [9] modi-

ic regions identifies TSSs. From sequencing

fied by using adaptors suitable for direct se-

CAGE library, we obtained 3,775,288 CAGE

quencing on an Illumina GAII platform. Intags from BMEC (Fig. 2). We mapped tags onbrief, cDNA complementary strands were

the bovine genome according to tag sequenc-

synthesized from total RNA by using a mix-es, of these CAGE tags, 1,108,153 tags (29.4%)

ture of random and oligo-dT primers. The 5ʼ

were uniquely hit to one position in the ge-

end of cDNA was selected by using the cap-nome (unique-hit), 253,192 tags (6.7%) weretrapper method and cDNA was ligated to amapped to more than two regions on the ge-

linker containing a recognition site for Eco-nome [207,938 tags (5.5%) were less than 50

P15I. After the second strand was synthe-regions (multi-hit) and 45,254 tags (1.2%) weresized, EcoP15I cleaved cDNAs at a sequence

more than 50 regions (ambiguous)], 2,413,910

27 nucleotides away from the 5ʼ end to pro-tags (63.9%) were insufficiently mapped and 33－3－

家畜感染症学会誌
1 巻 1 号 2012

Fig. 1

ウシ乳腺上皮細胞における CAGE 解析法を用いたゲノム全域にわたる転写開始点の解析

 AGE tag preparation, mapping to bovine genome and distribution by region of CAGE tags. (1) CAGE tags areC
27bp sequence tags derived from the mRNA sequenced in the proximity of the cap site. 5ʼ linker and 3ʼ linker
were ligated to CAGE tags. (2) CAGE library was sequenced on a Genome Analyzer II. (3) CAGE tags weremapped to the bovine genome and nearby CAGE tags were clustered into a single TSS cluster.

and the genetic diversity within breeds [1]. Al-tags (0.0%) were ignored.

ternative splicing has been noted as a factor
Identifying promoters by CAGE tags (uniqueof the diversity of gene expression [2]. Consid-hit tags)

ering these things, it is necessary to examine

We clustered nearby CAGE tags into TSS

TSS for better understanding of transcription-cluster. The TSS clusters mapped not only toal regulation. Thus, we identified complete set

known promoter sites but also unconventionalof TSSs in BMEC.

sites such as introns and intergenic regions.

Recent studies revealed that large propor-

Of the total 19,197 regions which TSS clusters

tions of the mouse and human genome aremapped into, 6,949 regions (36.2%) map in exon

transcribed into RNA [4]. In this study, simi-

sites, 5,766 regions (30.0%) partially overlap anlarly, we show that there are large amounts ofexon and an intron/intergenic region, 1,859 re-CAGE tag clusters in bovine genome. Further-gions (9.7%) map in intron sites and 4,623 re-more, we found TSS not only in already-gions (24.1%) map in intergenic regions (Fig. 3).

known promoter regions but also in intergenic

Based on the bovine genome annotation, 6,681

regions. Thus, it was revealed that there were

CAGE regions (34.8%) were annotated as4623 regions as candidate novel transcripts

known promoters.

and suggested the existence of novel promoters.

[Discussion]

It was reported that ncRNA is a major com-

ncRNA has been implicated in control of ge-ponent of the transcriptome in mouse [3]. Re-netic networks. Many studies demonstrated

cent work suggests that ncRNA can play criti-

the existence of tissue-specific transcripts [7]cal roles in a wide range of cellular processes,－4－The Journal of Farm Animal in Infectious Disease
Vol.1 No.1 2012

Fig. 2

GENOME-WIDE MAPPING OF TSS IN BMEC

 apping CAGE tags to bovine genome. TheM
upper value is the absolute number of tags
and the bottom value is the percentage of thetype of tag to total tags.

Fig. 3

Region distribution of TSS clusters to genomicelements. The upper value is the number ofregions overlapped with genomic elements
and the bottom value is the percentage of thetype of region to total regions.

from protein secretion to gene regulation [15].
Furthermore, because ncRNA is considered toin BMEC. This TSS data potentially indicates

be related to disease onset [16], understanding

the existence of novel transcripts and promot-ncRNA is very important in the development

ers. Further studies are needed to identifyof therapy and drug. However, in the bovine

promoters from these TSSs. This data set will

genome, ncRNA have scarcely been studied.

be useful for understanding the diversity with-In this study, there was a multitude of TSSs

in breeds and epigenetic control of transcrip-

mapped to introns and intergenic regions,tion. With advances in annotation of bovine

which are possibly ncRNAs. Our data will begenome, this data set will allow us to better

useful for identifying ncRNAs in the cow ge-

understand the biological processes in bovinenome.

mastitis.

In mouse, the CAGE tags mapped to single[Acknowledgements]

regions were approximately 61.8%, multiregion were 14.4% and the other 23.8% of tags

This work was supported in part by acould not be mapped [6]. Contrary to these re-grant-in-Aid from mastitis treatment project

ports, this study found that uniquely mappedof the Ministry of Agriculture, Forestry andtags were only 29.3% of the CAGE tags, multi-

Fisheries of Japan and the Strategic Researchple mapped tags were 5.5% and 64.9% wereBase Development Program for Private Uni-

not mapped in this study. It is possible thatversities from the Ministry of Education, Cul-

this low efficiency of mapping to genome is at-ture, Sports, Science and Technology of Japan

tributed to insufficiency of the available bovine(MEXT), 2008-2012.

genome and diversity within breeds. Similarly,[References]

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1 巻 1 号 2012

ウシ乳腺上皮細胞における CAGE 解析法を用いたゲノム全域にわたる転写開始点の解析

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Hayashizaki, Y., Carninci, P., 2006. CAGE: cap

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Lewin, H. A., Loor, J. J., 2009. Gene networkand pathway analysis of bovine mammary
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The Journal of Farm Animal in Infectious Disease
Vol.1 No.1 2012

GENOME-WIDE MAPPING OF TSS IN BMEC

ウシ乳腺上皮細胞における CAGE 解析法を用いたゲノム全域にわたる転写開始点の解析
松原朋子 1） 菊 佳男 2） 尾澤知美 1） 犬丸茂樹 1） 麻生 久 3）
山口高弘 3） David Reynolds4） 新井敏郎 5） 林 智人 2）＊1）動物衛生研究所 病態研究領域（〒305-0856 茨城県つくば市観音台 3-1-5）2）動物衛生研究所 寒地酪農衛生研究領域（〒062-0015 北海道札幌市豊平区羊ヶ丘 4）3）東北大学 大学院 農学研究科 動物機能科学講座（〒981-8555 宮城県仙台市青葉区堤通雨宮町 1-1）
4）Genomics Core, Albert Einstein College of Medicine（1301 Morris Park Avenue, Bronx, New York 10461-1602, USA）
5）日本獣医生命科学大学 獣医学部 獣医生理化学教室（〒180-8602 東京都武蔵野市境南町 1-7-1）＊連絡者：林 智人
Tel：011-851-2175 Fax：011-853-0767 E-mail：hayatomo@affrc.go.jp［要約］

乳房炎は病原微生物の乳房内感染によって引き起こされ、乳質ならびに泌乳量の低下を招く疾病である。その経済的損失は大きく、酪農経営を脅かす問題である。免疫応答に関わる遺伝子制御機構や細胞内シグナル伝達経路が同定されることによって、近年様々な病気の発症機序の解明が進みつつある。しかし、乳房炎研究におけるアプローチは遺伝子研究の観点からすると遅れを取っている。一方、ノンコー
ディング RNA（ncRNA）や選択的プロモーターが遺伝子発現の調節に重要な役割を果たすことが明らかになってきており、転写産物の網羅的解析が必要になってきている。遺伝子発現プロファイルやプロモーターを特定する新しいアプローチ方法として最近開発された Cap Analysis Gene Expression（CAGE）法は、転写開始点（TSS）の位置と発現量を解析することができ、TSS の正確な分布情報はプロモーター領域の予測や新規遺伝子の発見を可能にしている。本研究では乳房炎発症に関与するウシゲノムの転写産物とプロモーターの同定のためのゲノム全域にわたる発現データを得ることを目的とし、乳房炎の研究に一般的に用いられているウシ乳腺上皮細胞（BMEC）を用いた CAGE 解析を行った。
その結果、膨大な数の転写開始点がゲノム上に特定され、これらの TSS クラスターが 4623 の遺伝子間領域に位置していることが明らかになり、さらに新規プロモーターが存在することが示唆された。本研
究は CAGE 解析により BMEC の TSS を網羅的に解析した初めての研究である。今後さらに BMEC を用いて乳房炎の発症機序に関与する遺伝子動態を理解するためには、個々の転写産物におけるより詳細な解析が必要になると考えている。
［方法］

CAGE 解析の概略：①全 RNA から相補鎖 cDNA の合成、②キャップトラッピング法による 5ʼ 末端の選別、③ EcoP151 を含むビオチン化したリンカーの結合、④第二鎖 cDNA の合成、⑤ 5ʼ 末端から 27塩基の切り出しの後に、⑥この配列の 3ʼ 側へのリンカーの結合を行い、⑦ビオチン化した cDNA タグ（CAGE タグ）のみ抽出して精製した［Fig. 1（1）］。得られた CAGE タグの配列をシークエンシングにより決定し［Fig. 1（2）］、ゲノム上にマップした［Fig. 1（3）］。
［結果］

BMEC から全部で 3,775,288 の CAGE タグが得られた。そのうち、29.4％はゲノム上の 1 ヶ所（ユニークヒット）、6.7％は 2 ヶ所以上（マルチヒット）にマッピングされ、63.9％はマッピングできなかった（Fig. 2）
。また、ゲノム上で隣接する CAGE タグを TSS クラスターとしてクラスター化したところ、クラスターがマッピングされたゲノム上の領域は 19197 ヶ所あった。そのうち、36.2％はエクソン、9.7％はイントロン、30.0％はエクソン－イントロン／遺伝子間領域に跨いだ領域、24.1％は遺伝子間領域であった。ウシゲノムのアノテーションに基づくと、34.8％の領域がプロモーターとして知られている領域であった。

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家畜感染症学会誌
1 巻 1 号 2012

［考

ウシ乳腺上皮細胞における CAGE 解析法を用いたゲノム全域にわたる転写開始点の解析

察］

今回、BMEC において膨大な数の TSS が見付かった。多数の TSS がイントロンや遺伝子間領域にマッピングされたことから、本解析により、新規プロモーターや ncRNA の同定に役立つデータが得られたと考えている。マウスに比べて、マッピング効率が低かったが、その原因として、ウシゲノムの情報が不十分なことや品種間の違いが考えられる。今後、ウシゲノムのアノテーションが進むのにつれ、このデータセットはより詳細な生物学的プロセスを理解するために有用な情報源となりうると考えている。
以上の結果から、BMEC を用いて乳房炎の発症機序に関与する遺伝子動態を理解するためには、個々の転写産物における詳細な解析が必要となると考えられる。

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