A polymorphic minisatellite region of BORIS regulates gene expression and its rare variants correlate with lung cancer susceptibility

윤세련,노윤길,추인선,허정훈,김승일,장희경,강태홍,정진웅,고상석,Vladimir Larionov,임선희 생화학분자생물학회 2016 Experimental and molecular medicine Vol.48 No.-

Aberrant expression of BORIS/CTCFL (Brother of the Regulator of Imprinted Sites/CTCF-like protein) is reported in different malignancies. In this study, we characterized the entire promoter region of BORIS/CTCFL, including the CpG islands, to assess the relationship between BORIS expression and lung cancer. To simplify the construction of luciferase reporter cassettes with various-sized portions of the upstream region, genomic copies of BORIS were isolated using TAR cloning technology. We analyzed three promoter blocks: the GATA/CCAAT box, the CpG islands and the minisatellite region BORIS-MS2. Polymorphic minisatellite sequences were isolated from genomic DNA prepared from the blood of controls and cases. Of the three promoter blocks, the GATA/CCAAT box was determined to be a critical element of the core promoter, while the CpG islands and the BORIS-MS2 minisatellite region were found to act as regulators. Interestingly, the polymorphic minisatellite region BORIS-MS2 was identified as a negative regulator that repressed the expression levels of luciferase reporter cassettes less effectively in cancer cells compared with normal cells. We also examined the association between the size of BORIS-MS2 and lung cancer in a case–control study with 590 controls and 206 lung cancer cases. Rare alleles of BORIS-MS2 were associated with a statistically significantly increased risk of lung cancer (odds ratio, 2.04; 95% confidence interval, 1.02–4.08; and P=0.039). To conclude, our data provide information on the organization of the BORIS promoter region and gene regulation in normal and cancer cells. In addition, we propose that specific alleles of the BORIS-MS2 region could be used to identify the risk for lung cancer.

Characterization of unstable minisatellites in the human SCK1/SLIgene

정윤희,윤세련,김우연,김승일,김재우,선우양일,임선희 한국유전학회 2006 Genes & Genomics Vol.28 No.4

The hTERT‑VNTR2‑2nd alleles are involved in genomic stability in gastrointestinal cancer

권정아,정미소,윤세련,문정연,김민혜,양기은,박성환,정진웅,최영현,차희재,임선희 한국유전학회 2019 Genes & Genomics Vol.41 No.12

Background hTERT contains a high density of minisatellites, of which rare alleles of hTERT-VNTR2-2nd have been reported to be associated with prostate cancer. This shows an association between VNTR and cancer, but this repeat sequence is likely to be associated with genomic instability. Therefore, we investigated the effects of hTERT-VNTR2-2nd on gastrointestinal cancer and the relationship between repeated sequence and chromosome instability. Methods A case–control study was performed using DNA from 818 cancer-free controls, 539 cases with gastric cancer, 275 cases with colon cancer and 274 cases with rectal cancer. To determine whether minisatellites affect gene expression, expression levels were examined using TERT-reporter vectors in cell lines. In addition, the length of the hTERT-VNTR2-2nd alleles were determined in blood and cancer tissues from 107 gastric cancers, 112 colon cancers and 76 rectal cancers patients to determine whether the repeat sequence was associated with genomic instability during cancer development. Results No statistically significant association between hTERT-VNTR2-2nd and risk of gastrointestinal cancer was detected. However, it has been shown that VNTRs inserted into the enhancer region can regulate the expression of TERT in gastrointestinal cancer cells. Moreover, hTERT-VNTR2-2nd was analyzed in matched blood and cancer tissue from patients with gastrointestinal cancer and in seven among 294 subjects, and hTERT-VNTR2-2nd was found to be rearranged. Conclusions We suggest that minisatellites are associated with genomic instability in cancer and that the hTERT-VNTRs region may increase hTERT expression in gastrointestinal cancer cells.

Target DNA 염기서열 내에 존재하는 비상동성 간격이 상동성재조합을 이용한 클로닝 빈도에 미치는 영향

김재우,도은주,윤세련,정윤희,윤영호,임선희,선우양일,박인호,Kim Jae-Woo,Do Eun-Ju,Yoon Se-Lyun,Jeong Yun-Hee,Yoon Young-Ho,Leem Sun-Hee,Sunwoo Yangil,Park In-Ho 한국미생물학회 2005 미생물학회지 Vol.41 No.4

Transformation-associated recombination (TAR) 클로닝 법은 복잡한 게놈으로부터 염색체 내의 특정부위나 유전자를 선택적으로 분리할 수 있다. 이 방법은 목적 유전자에 근접한 작은 게놈DNA 염기서열 정보를 필요로 한다. 이 기술은 효모의 spheroplast transformation을 시키는 동안 목적으로 하는 유전자의 5' 또는 3' 서열을 포함하고 있는 TAR vector와 게놈DNA사이에서 일어나는 상동성재조합에 의해 이루어진다. 본 연구에서는 plasmid 모델시스템을 이용하여 target hooks 내에 존재하는 비상동성 염기서 열이 상동성재조합에 미치는 영향을 조사하였다. plasmid에 존재하는HIS3유전자와 변형시킨 his3-TRP1-his3 단편 사이의 상동성재조합의 효율은 $Ura^+$ 형질전환체의 형질분석에 의해 이루어졌다. $Ura^+$ 형질전환체의 수는 7종류의 서로 달리 변형된 his3-TRP1-his3 단편들을 사용하였을 매 거의 동일하게 나타났다. 그러나 $Trp^+His^+$ positive recombinants의 빈도는 변형된 his3-TRP1-his3 단편 내에 비상동성 영역에 부정확한 간격을 지닐 때 현저한 감소를 나타내었다. 이러한 결과로서, 부정확한 간격이 target hook과 substrate DNA 사이에 일어나는 상동성재조합을 방해하는 것으로 사료된다. 그러므로 이종간의 상동유전자를 클로닝 할 때에는 target hook내의 비상동성 염기서열이 존재한다면 이것이 정확한 간격을 지니는지 여부를 중요란 요인으로 고려해야 한다. Transformation-Associated Recombination (TAR) cloning technique allows selective isolation of chromosomal regions and genes from complex genomes. The procedure requires knowledge of relatively small genomic sequences that reside adjacent to the chromosomal region of interest. This technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences. In this study, we examined the effect of non-homologous spacing sequence in target hooks on homologous recombination using a plasmid model system. The efficiency of homologous recombination between the modified his3-TRP1-his3 fragments and HlS3 gene on plasmid were analyzed by the characterization of $Ura^+$ transformants. The numbers of $Ura^+$ transformant showed same level when seven different modified his3-TRP1-his3 fragments were used. But the percentage of positive recombinants. $Trp^+His^-$, dramatically decreased when used the modified his3-TRP1-his3 fragments contained incorrect spacing of nonhomologous region. As a result, we suggest that incorrect spacing inhibits the homologous recombination between target hook and substrate DNA. Therefore, we should consider the correct spacing in target hook when the target hook are used for cloning of orthologue gene.

대장균 내에서 불안정한 Minisatellite DNA 영역의 클론닝 및 DNA 염기서열 결정

임선희,김재우,김광섭,정윤희,윤세련,배호정,안태진,선우양일 한국미생물학회 2004 미생물학회지 Vol.40 No.2

진핵생물의 특정 염기배열을 원핵생물 내에서 증폭시킬 때 불안정성이 비교적 빈번히 관찰되어진다. 특히 long inverted repeats나 AT-rich sequences그리고 Z-DNA와 같은 구조를 지닌 염기배열은 대장균 내에서 매우 불안정하다. 이러한 염기서열은 대장균 내에서 부분적으로 결실되거나 완전히 손실된다. 본 연구실에서 human SCKI 유전자에 존재하는 몇 개의 tandem repeat (TR)에 대하여 다형성을 조사하였을 때, 어떤 TR 부분은 플라스미드로부터 빈번히 결실되어 그에 대한 염기서열 결정이 어려웠다. 그 결과 이러한 부분은 클론닝 될 수 없는 염기서열로 남게 되었다. 본 연구에서는 클론닝이 어려운 두 개의 TR 영역을 저온에서 클론닝하고 nebulizer나 sonicator를 이용하여 두 개의 library를 만들어 DNA 염기서열을 결정하였다. 이러한 연구는 복잡한 고등생물의 게놈연구에서 불안정한 게놈부분의 염기서열을 결정하는데 도움을 줄 것으로 사료된다. Instability of some eukaryotic sequence propagated in prokaryotic hosts is a frequently observed phenomenon. It is well documented that long inverted repeats, AT-rich sequences with structures like Z-DNA are extremely unstable in E. coli. These sequences may either be under-represented or even lost when cloned in E. coli. When we analyzed the polymorphic pattern for several tandom repeat (TR) in human SCKI gene, we found some TR regions were frequently deleted from plasmids and had difficult problem for their sequencing. These regions may result in non-clonability of the DNA sequence. Here we have cloned two difficult TR regions under low temperature and made two library for DNA sequencing using a nebulizer or sonicator. This study will help to determine the unstable genomic elements in complex mammalian genome.

출아효모에서 TAR 클론닝법을 이용한 고등동물의 게놈으로부터 특정 염색체 부위의 분리

박정은,윤영호,이윤주,김광섭,윤세련,안태진,임선희,선우양일 동아대학교 기초과학연구소 2003 基礎科學硏究論文集 Vol.20 No.1

복잡한 게놈 분석에 용이하도록 효모의 인공 염색체(YAC) 클론닝 시스템은 발달되어왔다. YAC은 박테리아의 인공 염색체(BAC)보다 더 큰 단편을 클론닝할 수 있고, 또한 클론된 단편을 쉽게 변형시킬 수 있다. 형질전환과 연계된 재조합법(Transformation-Associated Recombination; TAR)은 게놈 라이브러리를 만들지 않고 직접 게놈 DNA로부터 분리하고자 하는 유전자나 특정 염색체 부분을 클론닝 할 수 있는 방법이다. 이 방법은 spheroplast transformation을 수행하는 동안, 목적으로 하는 유전자의 5' 그리고 3' 염기 배열(hooks)을 지닌 TAR 벡터와 게놈 DNA 사이에서 일어나는 상동성 재조합에 의해 이루어진다. 효모 내의 in vivo 재조합을 이용한 TAR 클론닝법은 복잡한 게놈으로부터 목적의 염색체 부분을 원형 YAC의 형태로서 선택적으로 분리할 수 있다. 그러므로 TAR 클론닝 법은 특정 염색체로부터 YACs을 만드는데 매우 유용하여, 전체 게놈으로부터 특이적 유전자나 유전자의 family를 분리하는데 효과적인 방법으로 사료된다. Yeast artificial chromosome (YAC) cloning systems have advanced the analysis of complex genomes considerably. They permit the cloning of larger fragments than do bacterial artificial chromosome systems, and the cloned material is more easily modified. Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences (hooks). Using in vivo recombination in yeast, TAR cloning selectively isolates, as circular YACs, desired chromosome segments or entire genes from complex genomes. We propose that TAR cloning can provide an efficient means for generating YACs from specific chromosomes and that TAR cloning may be useful for isolating families of genes and specific genes from total genome DNA.