| Embryologic study shows that vertebrate animals such as fish, reptiles, amphibians, birds and mammals look much the same at a particular developmental stage named phylotypic period in their embryos. It has been generally accepted that gene regulation is dedicated to the property of animals. Proteins involved in the early embryonic development are always homologous. Their coding genes are evolutionally conserved, so are the flanking regulatory sequences. So we proposed that the conserved embryonic development plan of vertebrate may be controlled by the somewhat conserved (epi-) genetic regulation networks.We commenced with highly conserved regulation elements, such as UCEs (Ultra-conserved elements), to dig up this conserved networks. UCEs, which always located nearby of key developmental regulatory genes, are clustered in the human genome. Parts of UCEs were verified as enhancers in mouse embryos. We inferred that conserved elements with functional relationships may interact on each other to form conserved genetic or epi-genetic networks. For the cognition of the compartment structure of the nucleus and the dimensional structure of chromatins in the process of gene regulation, the conserved networks may be picked out by tracing the interactions among chromosomes carrying these conserved elements, which is the objective of our research.Human embryonic stem cell of pluripotency is a good model in the embryonic development study. Unique gene expression patterns of human embryonic stem cells were elucidated recently. Genes involved in the developmental process are poised. As cells induced differentiation, specific genes evoked swift activation to differentiate into specialized cell types. Besides, poised (Class II) enhancers, involved in orchestrating early steps in embryogenesis were identified in hESCs. The forgoing study showed us that developmental patterns of human embryos have been deposited in the human embryonic stem cells, which can be used as the model in the study of embryonic development.SOX2 is a key transcription factor in the pluripotency maintenance of human embryonic stem cells. In our study, we started with SOX2 gene and its conserved regulatory region,SOX2 Bait, to investigate the inter-chromatin and intra-chromatin interactions and find out the connections with pluripotency. We will expatiate as follows.We established the stable culture of human embryonic stem cells and harvested enough cells combined manual dissociation and 0.5% trypsin digestion assays in short term to offer sufficient stuff of experiment subsequently. We followed methods of human embryonic stem cells differentiation into embryonic body (EB) cells spontaneously and into NPCs directionally and succeeded. Attentions were attracted to gene expression changes from human embryonic stem cells to EB cells, and we found that human embryonic cell line, H9, was shown the characteristic of non-genetic heterogeneity in the culture conditions.4C related assays should be performed to find out interaction fragments with SOX2 Bait genome-wide. First of all,we constructed the plasmid named pDNA to mimic the genuine sample of 4C and established the quality control of 4C-Screening assay. PCR amplification conditions of SOX2 Bait were optimized and the 4C-Screening assay was proved to be efficient and feasible, which could guarantee the quality of sample for 4C-clone screening and 4C-seq.Limited 4C-clone screening had been used to find out some intro-chromatin interactions of SOX2 gene locus in human embryonic stem cells. Farther, we detected the cross-link frequency variation in the process of human embryonic stem cells differentiation into EB cells by 3C assay and constructed the three-dimensional chromosomal organization model of SOX2 gene regulation. We investigated the relationship between the chromosome structure and SOX2 expression. It was found that interactions among promoter and two reported enhances, SRR1 and SRR2 were variation followed the regulation of SOX2 expression. Besides, the way of SRR1 and SRR2 regulating promoter of SOX2 are totally different in two kinds of cells, such as hESC and EB cells. Region011, which contacted with SOX2 promoter in 20 days of EB cells, was verified the enhancer activity in 293A cells by luciferase reporter assay. We presumed that Region011 may be chosen as a potential enhancer to regulate the expression of SOX2 gene in EB cells.Then, 4C-seq assay was used to analyze the single-round amplified production of 4C sample (4C-DpnII) related to SOX2 Bait. After sequencing and mapping, the captured sequences were positioned over 135 individual sites in human genome, which were defined target sequences (fragments). These fragments, which were distributed over almost chromosomes in the genome, were predicted the binding sites of transcription factors of p300 and Elk1. And one fourth of them were highly conserved. GO annotation inferred that some of the fragment-related genes were involved in transmembrane receptor protein kinase activity, flavin-containing monooxygenase activity, calcium ion binding, as well as process such as cell adhesion and cell growth. Some genes were annotated in lung and genitalia development process while SOX2 is related with lung squamous cell carcinomas, ovarian cancer and breast cancer. Sixteen target genes may have the function of calcium ion binding, which may correlate with the functional mechanism of SOX2 in tumorigenesis and anti-apoptotic property of prostate cancer cells. So, fragments interacting with SOX2 may have some similar functions. We provided the elementarily data of SOX2 gene related conserved genetic or epi-genetic networks.3D-FISH assay was used to detect the interactions in single cell level. We improved the data manipulation and tried to estimate the interactions of two interested genes by comparing significance of distance distribution with that of reference sites. We verified the interactions in hESCs and NPCs between SOX2 and the 4C target sequences, 4C-8 and 4C-9, which were located in the introns of HMCN1. We found that SOX2 approached HMCN1 specifically in hESCs. After hESCs differentiated to NPCs, chromosomes of the two genes separated from each other. HMCN1 and SOX2 are involved in eye development, which may have some similar functions. Bioinformatics analysis indicated that there are potential enhancer signatures in 4C-8/9 regions, which may belong to the poised Class II enhancers with the enhancer of SOX2,SOX2 Bait. So, we found the co-localization of poised (Class II)enhancers in hESCs for the first time. These results inferred us that function related fragments may be located intensively because of their biological functions, which may offered us the experimental data to support our hypothesis of co-localization of function related fragments.At last,we further study the biological significance and mechanisms of co-localization. We detected genes in the same core regulation pathway of hESCs by 3D-FISH assay, including Nanog, SOX2 and Oct4.We found that Oct4 and Nanog are near each other. Four genomic positions, R1, R2, C3 and C4, with highly conservation and binding sites of nine transcription factors in hESCs were also detected. Chromosomes of R1 and C4 were found gathered together, too. The observation showed us that chromosomal interactions of genes in the same regulation pathway or of functional-related conserved fragments may truly exist. It should be more reasonable that conserved fragments can interact with each other because of their functional pertinence. |
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