Multiple control elements are required for expression of the human CD34 gene

Two cis regulatory elements of the human CD34 gene, the promoter and a 3′ enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3′ enhancer was active only in CD34⁺ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34⁺ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3′ enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160 kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.     

血清反应因子结合位点在小鼠及人基因组中保守性、 多样性及进化的研究

目的:CArG元件因其为血清反应因子识别的结合位点近年来备受关注。然而迄今为止尚未见到有关CArG元件的序列特征及进化模式的研究。方法:本研究应用生物信息学方法结合遗传学方法对小鼠及人基因组中CArG元件的位置分布序列类型、多样性及保守性进行深入研究。结果:多样性研究结果显示,CArG元件的序列在小鼠及人类基因组存在大量的不同类型。但是,小鼠和人基因组中CArG元件的主要类型又存在明显差异。同源性分析结果表明人类和小鼠中的CArG元件存在两种进化历程,一部分CArG元件拥有共同的祖先,一部分是在物种分化以后突变产生的。结论:上述研究结果将为更为深入阐述SRF的调控模式奠定理论基础,同时为更清楚的阐释CArG元件序列变化对下游基因的表达影响提供理论支持。     

CT-FOCS: a novel method for inferring cell type-specific enhancer–promoter maps

Spatiotemporal gene expression patterns are governed to a large extent by the activity of enhancer elements, which engage in physical contacts with their target genes. Identification of enhancer–promoter (EP) links that are functional only in a specific subset of cell types is a key challenge in understanding gene regulation. We introduce CT-FOCS (cell type FOCS), a statistical inference method that uses linear mixed effect models to infer EP links that show marked activity only in a single or a small subset of cell types out of a large panel of probed cell types. Analyzing 808 samples from FANTOM5, covering 472 cell lines, primary cells and tissues, CT-FOCS inferred such EP links more accurately than recent state-of-the-art methods. Furthermore, we show that strictly cell type-specific EP links are very uncommon in the human genome.     

A Brief Review on the Human Encyclopedia of DNA Elements (ENCODE) Project

The ENCyclopedia Of DNA Elements (ENCODE) project is an international research consortium that aims to identify all functional elements in the human genome sequence. The second phase of the project comprised 1640 datasets from 147 different cell types, yielding a set of 30 publications across several journals. These data revealed that 80.4% of the human genome displays some functionality in at least one cell type. Many of these regulatory elements are physically associated with one another and further form a network or three-dimensional conformation to affect gene expression. These elements are also related to sequence variants associated with diseases or traits. All these findings provide us new insights into the organization and regulation of genes and genome, and serve as an expansive resource for understanding human health and disease.     

DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

Mapping DNase I hypersensitive sites is an accurate method of identifying the location of gene regulatory elements, including promoters, enhancers, silencers and locus control regions. Although Southern blots are the traditional method of identifying DNase I hypersensitive sites, the conventional manual method is not readily scalable to studying large chromosomal regions, much less the entire genome. Here we describe DNase-chip, an approach that can rapidly identify DNase I hypersensitive sites for any region of interest, or potentially for the entire genome, by using tiled microarrays. We used DNase-chip to identify DNase I hypersensitive sites accurately from a representative 1% of the human genome in both primary and immortalized cell types. We found that although most DNase I hypersensitive sites were present in both cell types studied, some of them were cell-type specific. This method can be applied globally or in a targeted fashion to any tissue from any species with a sequenced genome.     

T cell-specific expression of the human TNF-alpha gene involves a functional and highly conserved chromatin signature in intron 3

Using a phylogenetic approach, we identified highly conserved sequences within intron 3 of the human TNF-alpha gene. These sequences form cell type-specific DNase I hypersensitivity sites and display cell type-specific DNA-protein contacts in in vivo genomic footprints. Consistent with these results, intron 3 confers specific activity upon a TNF-alpha reporter gene in Jurkat T cells, but not THP-1 monocytic cells. Thus, using a combinatorial approach of phylogenetic analysis, DNase I hypersensitivity analysis, in vivo footprinting, and transfection analysis, we demonstrate that intronic regulatory elements are involved in the cell type-specific regulation of TNF-alpha gene expression.     

Cutting edge: Ig heavy chain 3' HS1-4 directs correct spatial position-independent expression of a linked transgene to B lineage cells.

The Ig H chain locus is regulated by a set of cis-acting elements. Hypersensitive sites (HS) located 3' of the IgH, HS1-4, has been suggested to act as a locus control region (LCR) in cell lines. To assess the proposed role of HS1-4 acting as an LCR, we generated transgenic mice harboring a VH promoter-beta-globin reporter gene linked to the Ig H chain HS1-4 3'regulatory sequences. Transgene expression is strictly confined to B lymphocytes, with no detectable expression outside the B cell lineage in all transgenic founder lines. Furthermore, reporter gene activity is integration independent but not copy number dependent. Thus, additional sequences are required to allow the HS1-4 regulatory region to act as a classical LCR in mice. Our data are discussed in the context of tissue-specific gene expression in B lineage cells.     

Identification of regulatory motifs in the CHO genome for stable monoclonal antibody production

Chinese hamster ovary (CHO) cell lines are widely used for therapeutic protein production. When a transgene is integrated into the genome of a CHO cell, the expression level is highly dependent on the site of integration because of positional effects such as gene silencing. To overcome negative positional effects and establish stable CHO cell lines with high productivity, several regulatory DNA elements are used in vector construction. Previously, we established the CHO DR1000L-4N cell line, a stable and high copy number Dhfr gene-amplified cell line. It was hypothesized that the chromosomal location of the exogenous gene-amplified region in the CHO DR1000L-4N genome contains regulatory motifs for stable protein production. Therefore, we isolated DNA regulatory motifs from the CHO DR1000L-4N cell line and determined whether these motifs act as an insulator. Our results suggest that stable expression of a transgene can be promoted by the CHO genome sequence, and it would be a powerful tool for therapeutic protein manufacturing.