초록 ▼

$\circ$ 인간배아줄기세포를 안정적으로 대량 배양할 수 있는 시스템을 중심으로 genome-wide DNA 메칠화 및 히스톤 단백질의 수식, 염색체 변이 등의 후성유전체 및 유전체 수준에서의 정보를 도출하여 후성유전자지도를 작성하는 한편 분화 관련 유전자를 동정하여 기전 연구를 통해 배아줄기세포의 분화-역분화에 중요한 후성유전학적 인자를 발굴함
$\circ$ 인간배아줄기세포의 후성유전체적 특성을 효과적으로 분석하기 위해서 DNA methylation과 함께 염색체에서 발생할 것으로 예

$\circ$ 인간배아줄기세포를 안정적으로 대량 배양할 수 있는 시스템을 중심으로 genome-wide DNA 메칠화 및 히스톤 단백질의 수식, 염색체 변이 등의 후성유전체 및 유전체 수준에서의 정보를 도출하여 후성유전자지도를 작성하는 한편 분화 관련 유전자를 동정하여 기전 연구를 통해 배아줄기세포의 분화-역분화에 중요한 후성유전학적 인자를 발굴함
$\circ$ 인간배아줄기세포의 후성유전체적 특성을 효과적으로 분석하기 위해서 DNA methylation과 함께 염색체에서 발생할 것으로 예측되는 aberration과 methylation에 의한 유전자 발현 양상 및 chromatin 구조연구를 동시에 수행함
$\circ$ 따라서 본 연구에서는 인간배아줄기세포의 후성유전체적 특성 규명을 위해 (1) Genome-wide 메틸화 분석, (2) Genome-wide 염색체 구조이상 분석, (3) 유전체 전장의 유전자 발현 패턴, (4) 후성유전체지도 작성, (5) 분화-역분화에 중요한 후성유전학적 인자를 발굴 등 크게 다섯가지 연구분야를 통합하는 연구를 수행함

Abstract ▼

1. Human embryonic stem cells (hESC)
Four human embryonic stem cell lines were analyzed for this study. Retinoic acid (RA) was treated to each embryonic stem cells, and the DNA were extracted for genome-wide and whole-genome screening.
2. Development of genome-wide epigenetic analysis techniqu

1. Human embryonic stem cells (hESC)
Four human embryonic stem cell lines were analyzed for this study. Retinoic acid (RA) was treated to each embryonic stem cells, and the DNA were extracted for genome-wide and whole-genome screening.
2. Development of genome-wide epigenetic analysis technique
MeDIP (Methylated-DNA Immuno Precipitation) based on high density oligo chip, such as Affymetrix's Promoter Array and Illumina's Methylation27 BeadChip, were used to profile the epigenome of human embryonic stem cells and to determine which system can produce a good reproducibility.
3. Genome-wide epigenetic profiling of hESC
Genomic DNA from RA-treated hESC and nontreated hESC were used for epigenome analysis. For Promoter assay, methylated DNA was isolated by MeDIP procedure and hybridized with Promoter Array. For Methylation27 BeadChip assay, genomic DNA was converted by bisulfite treatment and then amplified. Amplified DNA was hybridized to the Illumina Infinium methylation 27 array and the arrays were imaged using a Bead Array Reader. The result of genome-wide epigenetic analysis was combined with SNP and gene expression data to identify genes associated with the differentiation of hESCs.
4. Combined analysis of DNA methylation and gene expression
Differentially methylated CpG sites (n=166) and differentially expressed genes (n=2,013) were combined based on gene name represented on both data sets. Pearson correlation coefficients were calculated between the expression signal values and methylation $\beta$ values, and we collected significantly correlated sites/genes.
5. Analysis of allele-specific expression pattern of imprinted genes in hES cells differentiation
To determine allele-specific expression pattern of imprinted genes following hES cells differentiation into DA neurons, direct sequencing of RT-PCR products containing heterozygous polymorphisms are carried in undifferentiated hES cells, hES-NP, and DA neurons derived from hES-NP. The epigenetic mechanisms responsible for imprinting were also investigated. Clonal bisulfite sequencing was used to analyze the methylation status of several imprinting control regions (H19, KCNQ10T1, SNRPN, PEG3, PEG10, PEGl and NESP55 DMR).
6. Mechanistic study of chromatin structure of regulatory genes required for stem cell pluripotency
Since the maintenance of stemness is important for developing mass production of stem cell to cure human disease, we want to study mechanistic role of epigenetic control processes which should be involved in self-renewal or differentiation of stem cells. To do this, we will try to the following experiments. First, we want to understand chromatin structure and histone modification patterns of pluripotency-related genes using chromatin IP and conventional biochemistry. Second, we will demonstrate transcription mechanism for regulating pluripotency-related genes using biochemical approaches. Third, we will define stem cell markers and chromatin factors which regulate the pluripotency of stem cells. Fourth, we will try to understand how pluripotency-related factors such as Nanog, Oct4, Sox2, Klf4, and Lin28 can be regulated to establish stemness in human stem cells.
7. Establishment of Bioinformatics Pipeline for Stem Cell Epigenomics
We aimed to construct database for epigenetic profiles in human embryo stem cell, and develop a new algorithm to predict modification level of DNA methylation.

목차 Contents

• 제 1 장 연구개발과제의 개요 ...13
• 제 2 장 국내외 기술개발 현황 ...16
• 제 3 장 연구개발수행 내용 및 결과 ...18
• 제 4 장 목표달성도 및 관련분야에의 기여도 ...27
• 제 5 장 연구개발결과의 활용계획 ...35
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ...37
• 제 7 장 참고문헌 ...41
• 인간배아줄기세포의 Genome-wide 후성유전체 분석 ...44
• 줄기세포의 유전체 구조이상, 유전자 발현 및 유전자 특이 메틸화 규명 ...85
• 후성유전적 조절을 통한 민간배아줄기세포의 분화연구 ...182
• 스템세포의 전분화능에 중요한 유전자들의 크로마틴 구조분석 및 기능 연구 ...282
• 줄기세포 후성유전체학을 위한 생물정보학 파이프라인 개발 ...309