The murine beta-globin locus control region regulates the rate of transcription but not the hyperacetylation of histones at the active genes

Abstract

Locus control regions (LCRs) are defined by their ability to confer high-level tissue-specific expression to linked genes in transgenic assays. Previously, we reported that, at its native site, the murine beta-globin LCR is required for high-level beta-globin gene expression, but is not required to initiate an open chromatin conformation of the locus. To further investigate the mechanism of LCR-mediated transcriptional enhancement, we have analyzed allele-specific beta-globin expression and the pattern of histone acetylation in the presence and absence of the LCR. In single cells from mice heterozygous for a deletion of the LCR, beta-globin expression from the LCR-deleted allele is consistently low ( approximately 1-4% of wild type). Thus, the endogenous LCR enhances globin gene expression by increasing the rate of transcription from each linked allele rather than by increasing the probability of establishing transcription per se. Furthermore, in erythroid cells from mice homozygous for the highly expressing wild-type beta-globin locus, hyperacetylation of histones H3 and H4 is localized to the LCR and active genes. In mice homozygous for the LCR deletion reduced histone hyperacetylation is observed in LCR proximal sequences; however, deletion of the LCR has no effect on the localized hyperacetylation of the genes. Together, our results suggest that, in its native genomic context, the LCR follows the rate model of enhancer function, and that the developmentally specific hyperacetylation of the globin genes is independent of both the rate of transcription and the presence of the LCR.

Figure 1

Transcription from individual β-globin alleles in ΔLCR and wt mice. RNA from mice heterozygotic for the Hbb^D allele (D), and the Hbb^S allele (S) was analyzed from individual reticulocytes by RT-PCR. The targeted deletion of the LCR deletion is on the D allele, whereas the S allele is wt. Representative samples of 10-cell pools (10), single cells (1), and no-cell controls (0) from wt D/S mice and ΔLCR-D/S mice are shown. WT D/S, wt D/S animals; ΔLCR-D/S, mutant ΔLCR-D/S mice. S and D mark the RT-PCR products from the S and D alleles, respectively.

Figure 2

Quantitation of transcription from individual β-globin alleles in ΔLCR and wt mice. The ratio of expression from the D allele to that from the S allele for wt D/S animals (WT) or mutant ΔLCR-D/S mice (ΔLCR). Data from 10-cell pools and individual cells are shown.

Figure 3

Analysis of histones H3 and H4 acetylation across the wt and ΔLCR mouse β-globin locus. Chromatin was immunoprecipitated with an Ab that detects all acetylated variants of histone H4 [aH4-Ac] and an Ab that detects acetylated lysines 9 and 14 of histone H3 [aH3-Ac]. Abundance of the mouse globin sequence was detected relative to the mouse amylase gene, which is hypoacetylated in erythroid cells (12). Duplex PCR was performed on the input and Ab-bound fraction from the chromatin IPs by using amylase- and βglobin-specific primers. In each set, the amylase amplification product is marked with an asterisk. The β-globin amplicons are numbered in 5′ to 3′ direction of the locus. Locations of the amplicons in the locus are shown in Fig. 4 and primer sequences are listed in Table 1. Quantification of the ratio of globin to amylase is pictured in Fig. 4 and reveals up to 25-fold enrichment of globin sequences in the bound fraction. The figure shows the amplification products from one of three independent experiments.

Figure 4

Quantification of duplex PCR results. Position of amplicons in the β-globin locus and quantification of chromatin IP results. The ratios of products obtained with the β-globin and amylase primers were determined for each input and Ab-bound sample for the wt and ΔLCR locus. The ratio for the bound fraction is normalized to the input to determine the relative level of enrichment. Enrichment of the globin sequences is reflected by a number >1. The x axis is drawn at 1, reflecting no enrichment. Shown are the averages of enrichment with standard deviation from three independent IPs.