Methyl-Typing: An improved and visualized COBRA software for epigenomic studies

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Methyl-Typing: An improved and visualized COBRA software for epigenomic studies

Cheng-Hong Yang

a,b

, Li-Yeh Chuang

c,*

, Yu-Huei Cheng

a

, De-Leung Gu

f

, Chung-Ho Chen

d,e

,

Hsueh-Wei Chang

f,g,h,* a

Department of Electronic Engineering, National Kaohsiung University of Applied Sciences, Kaohsiung, Taiwan

b

Department of Network Systems, Toko University, Chiayi, Taiwan

c_{Department of Chemical Engineering, I-Shou University, Kaohsiung, Taiwan} d

Department of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

e

Department of Oral and Maxillofacial Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

f

Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan

g

Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan

h

Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

a r t i c l e

i n f o

Article history:

Received 14 October 2009 Revised 14 December 2009 Accepted 16 December 2009 Available online 22 December 2009

Edited by Paul Bertone

Keywords: Methylation COBRA Restriction enzyme Software Promoter

a b s t r a c t

Combined bisulfite restriction analysis (COBRA) is one of the most commonly used methylation quantification methods. However, it focuses on relatively few restriction enzymes. Here, we present Methyl-Typing, a web-based software that provides restriction enzyme mining data for methyl-cyto-sine-containing sequences following bisulfite-conversion. Gene names, accession numbers, sequences, PCR primers, and file upload are accessible for input. Promoter sequences and restriction enzymes for CpG- and GpC-containing recognition sites are retrieved. Four representative enzymes were tested successfully by COBRA on the experimental work. Therefore, the Methyl-Typing tool provides a comprehensive COBRA-restriction enzyme mining. It is freely available athttp://bio.kuas. edu.tw/methyl-typing.

1. Introduction

Currently, combined bisulﬁte restriction analysis (COBRA)

[1]

is

one of the most commonly used methylation methods in

laborato-ries

[2]

. In principle, a technique using any kind of restriction

enzymes to distinguish between the methylated- and the

unme-thylated-sequences with bisulﬁte-conversion are regarded as the

COBRA method. However, the traditional COBRA approach only

re-lies on a few restriction enzymes, such as BstUI (5

0

_-CG

;

CG-3

0

₎

_[2]

and Taq

a

I (5

0

_-T

;

CGA-3

0

₎

_[1]

_{. Other restriction enzymes available}

for COBRA are less frequently mentioned, such as HinP1I (5

0

-G

;

CGC-3

0

_{), HpyCH4IV (5}

0

_-A

;

CGT-3

0

_{), and AciI (5}

0

_-G

;

CGG-3

0

_{). This}

may in part be due to the fact that restriction enzyme mining tools

for possible methylation sequences are poorly developed.

More-over, the traditional COBRA approach is not naturally speciﬁc to

CpG islands in the promoter region but it depends on the

user-de-ﬁned sequence. Therefore, the integration for COBRA-restriction

enzyme mining, CpG island searching, and promoter prediction is

still challenging.

In order to circumvent this constraint, we have developed a

novel visualization software, named Methyl-Typing, which

pro-vides comprehensive restriction enzymes for

methyl-cytosine-con-taining sequences after bisulﬁte-conversion, i.e. unmethylated

cytosine converts to uracil (regarded as thymine for PCR

ampliﬁca-tion) while 5-methylcytosine remains unchanged. Moreover, the

insulators’ CCCTC-binding factor (CTCF)-binding site database

(CTCFBSDB)

[3]

is implemented in Methyl-Typing. The insulators

of chromatin, such as CTCF, can block the activity of a down-stream

enhancer and are neutralized by methylation

[4]

, thereby

contrib-uting to gene regulation. In conclusion, Methyl-Typing is a fast and

efﬁcient tool for providing all possible methylation sites of

restric-tion enzymes.

Abbreviations: COBRA, combined bisulﬁte restriction analysis; CTCF, CCCTC-binding factor; CTCFBSDB, CCCTC-CCCTC-binding factor-CCCTC-binding site database; RFLP, random fragment length polymorphism

* Corresponding authors. Address: Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan (H.-W. Chang). Fax: +886 7 312 5339.

E-mail addresses:chuang@isu.edu.tw (L.-Y. Chuang), changhw@kmu.edu.tw

(H.-W. Chang).

FEBS Letters 584 (2010) 739–744

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gest that the restriction enzymes provided by Methyl-Typing are

informative in determining the methylation status and in

applica-tion to epigenomic study. However, the possible methylaapplica-tion role

for oral oncogenesis still needs further investigation due to our

limited sample size.

4. Discussion

Several approaches are available to predict the location of CpG

islands

[8,12]

, provide promoter sequences

[6]

, and determine

DNA methylation status

[2]

. For example, some CpG island search

tools have been developed, but these tools do not provide mining

functions for COBRA-restriction enzymes and promoter

identiﬁca-tion, such as CpG Island Searcher

[8]

and CpG analyzer

[12]

, which

are web server and Window-based programs, respectively.

More-over, many software tools have been developed only to analyze

the bisulﬁte sequencing data. For example, BiQ Analyzer

[13]

and

BDPC web server

[2]

provide visualization and quality control for

DNA methylation data from bisulﬁte sequencing. MethTools

[14]

and CyMATE

[15]

are web-based methylation analyzers for input

and email output. CyMATE and CpG PatternFinder

[16]

require

aligned sequences as input data. QUMA provides quantiﬁcation

for methylation analysis by bisulﬁte sequencing

[17]

. However,

bisulﬁte sequencing-based methods are limited by the occurrence

of continuous nucleotides, such as poly Ts. The sequences behind

the region of poly T sequences are frequently frame-shifted or

ap-pear as multiple nucleotides at the same locus (data not shown),

leading to misrepresentation by computation. This potential poly

T problem can be detected in silico in Methyl-Typing (

Fig. 2

G).

Another disadvantage for those software tools is that the

inte-gration of CpG island search, promoter sequence retrieval, and

methylation analysis is unavailable. In contrast, our proposed

Methyl-Typing tool provides such an integrated system. Gene

name, accession number, sequence and ﬁle inputs as well as

pri-mer input for ePCR-generating sequence are all acceptable for

Methyl-Typing analysis. Recently, the MethMarker tool

[18]

was

developed to implement the COBRA assay and ﬁve other widely

used experimental techniques, providing the optimization of

gene-speciﬁc DNA methylation assays. However, only 35 different

kinds of COBRA enzymes were included in the MethMarker while

all restriction enzymes in REBASE version 806 (total 3961) are

available in our proposed Methyl-Typing.

While the majority (about 90%) of methylated cytosine residues

in mammals are found at CpG dinucleotides

[19,20]

, the GpC can

still be slowly, although not fully, methylated

[21]

. Methylations

in non-CpG sequences were found in plants

[15]

, ﬁshes

[22]

, and

mammals

[23]

as well as in human breast cancer

[24]

. A high

per-centage of non-CpG DNA methylation in mammals was also found

in embryonic stem (ES) cells in contrast to somatic cells

[25]

. It is

consistent with our results that some oral cancer samples were

methylated at both CpG (

Fig. 3

A–C) and GpC sites (

Fig. 3

D).

Fur-thermore, DNA methylation on human repetitive sequences was

recently reported including AluI (5

0

_-AG

;

_CT-3

0

₎

_[26]

_{. Therefore,}

non-CpG methylation may play an important role in gene

expres-sion

[26]

and cancer oncogenesis

[27]

.

While most methylation software tools focus nearly exclusively

on CpG sites, few tools are designed for DNA with non-CpG

meth-ylation sites. In contrast, Methyl-Typing can provide all the

restric-tion enzymes for the recognirestric-tion sequences for all possible CpG

sites, e.g., Taq

a

I (5

0

_-T

;

_CGA-3

0

_{), HinP1I (5}

0

_-G

;

_CGC-3

0

_{), HpyCH4IV}

(5

0

_-A

;

CGT-3

0

_{), AciI (5}

0

_-G

;

CGG-3

0

_{) and for all possible GpC sites,}

e.g., HaeIII (5

0

_-GG

;

CC-3

0

_{), Cac8I (5}

0

_-GCN

;

NGC-3

0

_{), and AluI (5}

0

-AG

;

_CT-3

0

_{). Therefore, the Methyl-Typing provides a more}

compre-hensive source of restriction enzymes for COBRA assay for both

CpG and GpC methylations than the traditional COBRA.

In summary, we describe an improved COBRA software

provid-ing restriction enzyme minprovid-ing and visualization of

methyl-cyto-sine-containing sequences after bisulﬁte-conversion.

Methyl-Typing improves the scope of the entire analysis of traditional

CO-BRA methylation typing and provides visualization platforms

across different computing environments.

Acknowledgements

This work was partly supported by the National Science Council

in Taiwan under grant NSC98-2221-E-151-040,

98-2622-E-151-024-CC3, NSC97-2311-B-037-003-MY3,

NSC97-2622-E-151-008-CC2, and by the grants KMU-EM-97-1.1b and KMU-EM-98-1.4.

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