We developed a method to co-express protein pairs from collections of otherwise identical Escherichia coli plasmids expressing different ORFs by incorporating a 61-nucleotide sequence (LINK) into the plasmid to allow generation of tandem plasmids. Tandem plasmids are formed in a ligation-independent manner, propagate efficiently, and produce protein pairs in high quantities. This greatly facilitates co-expression for structural genomics projects that produce thousands of clones bearing identical origins and antibiotic markers.
Active gene promoters are associated with covalent histone modifications, such as hyperacetylation, which can modulate chromatin structure and stabilize binding of transcription factors that recognize these modifications. At the -globin locus and several other loci, however, histone hyperacetylation extends beyond the promoter, over tens of kilobases; we term such patterns of histone modifications "hyperacetylated domains." Little is known of either the mechanism by which these domains form or their function. Here, we show that domain formation within the murine -globin locus occurs before either high-level gene expression or erythroid commitment. Analysis of -globin alleles harboring deletions of promoters or the locus control region demonstrates that these sequences are not required for domain formation, suggesting the existence of additional regulatory sequences within the locus. Deletion of embryonic globin gene promoters, however, resulted in the formation of a hyperacetylated domain over these genes in definitive erythroid cells, where they are otherwise inactive. Finally, sequences within -globin domains exhibit hyperacetylation in a context-dependent manner, and domains are maintained when transcriptional elongation is inhibited. These data narrow the range of possible mechanisms by which hyperacetylated domains form. (Blood. 2009;114: 3479-3488) IntroductionA crucial feature of gene activation is the interaction between transcription factors and chromatin. All eukaryotic genomic DNA, with limited exceptions, is packaged with core histones to form chromatin. The fundamental subunit of chromatin is the nucleosome, consisting of approximately 147 bp of DNA wrapped in approximately 1.75 turns about an octamer of core histones; a variable length of linker DNA extends between nucleosomes and can in turn be partially sequestered by interactions with core histone amino-terminal "tail" regions and/or linker histones, such as histone H1. The resulting structure, when observed on low-salt spreads by electron microscopy, has been termed "beads on a string" based on its appearance. 1,2 At physiologically relevant salt concentrations, however, this structure spontaneously condenses, first to a helical array of nucleosomes termed the 30-nm fiber, then through additional levels of higher-order structure, which are not well understood. 3 Nevertheless, this packaging renders the eukaryotic DNA relatively inaccessible to transcription factors. [4][5][6] The transcriptional machinery possesses mechanisms for modulating chromatin structure. One of these is covalent modification of core histones, including acetylation, methylation, phosphorylation, ubiquitylation, SUMOylation, and ADP ribosylation, in short, the gamut of modifications known to occur on cellular proteins. Different modifications can lead to different functional consequences. Histone acetylation is associated with transcriptional activation; indeed, core histones proximal to active gene promoters are universally hyperacetylated. 7,8 Histone methylation can...
In mammalian nuclei, a select number of tissue-specific gene loci exhibit broadly distributed patterns of histone modifications, such as histone hyperacetylation, that are normally associated with active gene promoters. Previously, we characterized such hyperacetylated domains within mammalian -globin gene loci, and determined that within the murine locus, neither the -globin locus control region nor the gene promoters were required for domain formation. Here, we identify a developmentally specific erythroid enhancer, hypersensitive site-embryonic 1 (HS-E1), located within the embryonic -globin domain in mouse, which is homologous to a region located downstream of the human embryonic ⑀-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-offunction assays. Deletion of HS-E1 from the endogenous murine -globin locus results in significant decrease in the expression of the embryonic -globin genes and loss of the domain-wide pattern of histone hyperacetylation. The data suggest that HS-E1 is an enhancer that is uniquely required for -like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domainwide modulation of chromatin structure. (Blood. 2011;117(19):5207-5214)
In mammals, the complex tissue- and developmental-specific expression of genes within the β-globin cluster is known to be subject to control by the gene promoters, by a locus control region (LCR) located upstream of the cluster, and by sequence elements located across the intergenic regions. Despite extensive investigation, however, the complement of sequences that is required for normal regulation of chromatin structure and gene expression within the cluster is not fully defined. To further elucidate regulation of the adult β-globin genes, we investigate the effects of two deletions engineered within the endogenous murine β-globin locus. First, we find that deletion of the β2-globin gene promoter, while eliminating β2-globin gene expression, results in no additional effects on chromatin structure or gene expression within the cluster. Notably, our observations are not consistent with competition among the β-globin genes for LCR activity. Second, we characterize a novel enhancer located 3′ of the β2-globin gene, but find that deletion of this sequence has no effect whatsoever on gene expression or chromatin structure. This observation highlights the difficulty in assigning function to enhancer sequences identified by the chromatin “landscape” or even by functional assays.
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