An embryonic stage-specific enhancer within the murine ß-globin locus mediates domain-wide histone hyperacetylation.

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 ß-globin gene loci, and determined that within the murine locus, neither the ß-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 ß-globin domain in mouse, which is homologous to a region located downstream of the human embryonic ε-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-of-function assays. Deletion of HS-E1 from the endogenous murine ß-globin locus results in significant decrease in the expression of the embryonic ß-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 ß-like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domain-wide modulation of chromatin structure.

Histone hyperacetylation within the beta-globin locus is context-dependent and precedes high-level gene expression.

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 beta-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 beta-globin locus occurs before either high-level gene expression or erythroid commitment. Analysis of beta-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 beta-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.

The chromatin "landscape" of a murine adult ß-globin gene is unaffected by deletion of either the gene promoter or a downstream enhancer.

In mammals, the complex tissue- and developmental-specific expression of genes within the ß-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 ß-globin genes, we investigate the effects of two deletions engineered within the endogenous murine ß-globin locus. First, we find that deletion of the ß2-globin gene promoter, while eliminating ß2-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 ß-globin genes for LCR activity. Second, we characterize a novel enhancer located 3' of the ß2-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.

A facile method for high-throughput co-expression of protein pairs.

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.