In Depth Locus Specific Protein Interaction Characterized by Ultrasensitive Genome Scale Proteome Quantification (GSPQ) Platform
Abstract
It is now well accepted that the gene regulation plays an important roles in evolution, development and pathological transitions. Nowadays, the chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing (SEQ) is well established to study the cis-regulatory elements. It provides the in-depth information for the specific genomic loci binding to the given proteins. However, due to the lack of effective methodology, the vast majority of proteins binding to the given genome loci remains largely unkonown. Here, we describe a CRISPR affinity purification method, which utilizes an in vivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs to purify the regulatory protein complex binding to the given genomic loci. With the integration of ultrahigh sensitive nano scale three dimensional (RP-SAX-RP) chromatography platform, we can successfully characterize the protein complex binding to a single copy genomic loci. The hierarchical organization of the proteins binding to the enhancer cluster controlling human b-globin genes is established with the in depth profiling results. Futhermore, we also analyze the protein complex associating to the disease related super enhancer, which allow us to discover the mechanism to control the gene transcription. Therefore, the powerful method we develop provides the power to unbiasly characterize the cis-regulatory element for the given genes in development and disease.
A study of transposable element-associated structural variations (TASVs) using a de novo assembled Korean genome
Abstract
Advancements in next-generation sequencing (NGS) technology has made personal genome sequencing possible, and indeed many human genomes have now been sequenced. Comparisons of these individual genomes has revealed the substantial genomic differences found between human populations and even between individuals from closely related ethnic groups. There are many causative agents known to contribute to these genomic variations. Transposable elements (TEs) are known to be one of the major sources of these variations through various mechanisms including de novo insertion, insertion-mediated deletion, and TE-TE recombination-mediated deletion. In this study, we carried out de novo whole-genome sequencing of one Korean individual (KPGP9), via multiple insert-size libraries and phasing analysis using statistically aided, long-read haplotyping (SLRH). The de novo whole-genome assembly results in 31,305 scaffolds with a scaffold N50 size of 13.23 Mb. With these sequencing results, we performed variant annotations of the KPGP9 genome and compared non-synonymous SNPs to six other individual human genomes (AK1, SJK, YH, Yoruba, Venter, and Watson). We identified 97 non-synonymous SNPs detected only in three Korean genomes (AK1, SJK, and KPGP9). Further, through computational data analysis and experimental verification, we revealed that 182 TE-associated structural variation (TASV) insertions and 89 TASV deletions contributed 64,232 bp (NCAI: -3369 bp) in sequence gain and 82,772 bp in sequence loss in the KPGP9 genome, respectively. Biological function and clinical implication were annotated for SNPs and TASVs identified. Here, we release another Korean de novo whole-genome and propose common Korean genetic variations through comparison to several ethnic groups. Our finding highlights again the role of TEs as one of major drivers creating structural variations in human individuals.
College of Veterinary Medicine, Seoul National University, Korea
Title
Comparative analysis of proteins profiles of canine plasma
Abstract
Compararive medicine is to Identify similarities and differences in bilogy among animals and to understand the mechanisms of human and animal disease. Many studies demonstrated that increased risk for various types of human diseases such as cancer and heart disease are clearly affected by life environment.
Using concept of compatative medicine, we tried to identify useful breast cancer markers for both human and dog. We first analyzed canine plasma samples with LC-MS/MS (Q exactive) to get a raw data and data were further analyzed with Maxquant and Persues software. Total 57 plasma samples obtaidned from dogs with breast benign tumors (n=14), breast tumors (n=28), and normal (n=15) were analysed and differentilly expressed proteins in each sample were identified. Total 190 proteins were identified and enriched proteins in each disease were sorted and compared among groups. Haptoglobin, Serum albumin, Immunoglobulin heavy constant mu, and Complement C3 were idendified (p<0.05, 2 fold) as enriched proteins in tumor samples as compared normal. Haptoglobin and Serum albumin were also identified as enriched proteins (p<0.05, 2 fold) in benign tumor sample as compared normal. Interestingly, Haptoglobin and Serum albumin were significantly increased in both tumor and benign tumor as compared normal, indicating that these proteins could be diagnostic markers of breast tumor for dog and human.
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