Abstract
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.
Highlights
The transcriptional regulation of protein-coding genes often results from the combined regulatory inputs of multiple cis-acting DNA sequences, which together comprise the transcriptional control region
To evaluate the contribution of the adult b-globin gene promoters to chromatin modifications associated with this region, we engineered the deletion of the b2-globin gene promoter by homologous recombination in embryonic stem (ES) cells (Figure 2A)
Mice were derived from these ES cells and the neo marker removed by mating with a Creexpressing mouse line
Summary
The transcriptional regulation of protein-coding genes often results from the combined regulatory inputs of multiple cis-acting DNA sequences, which together comprise the transcriptional control region. For most genes in prokaryotes and unicellular eukaryotes, the transcriptional control region is limited entirely to sequences located in the region just upstream of the site of transcription initiation. Metazoan genes are often regulated by distal sequences, such as enhancers, which are capable of influencing transcriptional events at the promoter and during transcriptional elongation despite being located large distances (tens to hundreds of kilobases) away [1], [2]. This property makes the complete identification and characterization of the transcriptional control regions of metazoan genes a difficult task. The locations of enhancers can be predicted via several features of chromatin structure – DNase I hypersensitive sites, specific histone modification ‘‘signatures’’ like histone H3 K4 monomethylation, noncoding sequence conservation, etc. – and functional enhancer sequences can be characterized via gain-of-function assays like the transient reporter gene or colony assays, but the assignment of a distal enhancer to a given gene depends, in the end, upon genetic assays that manipulate endogenous gene loci
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