Flanking HS-62.5 and 3' HS1, and regions upstream of the LCR, are not required for beta-globin transcription

Abstract

The locus control region (LCR) was thought to be necessary and sufficient for establishing and maintaining an open beta-globin locus chromatin domain in the repressive environment of the developing erythrocyte. However, deletion of the LCR from the endogenous locus had no significant effect on chromatin structure and did not silence transcription. Thus, the cis-regulatory elements that confer the open domain remain unidentified. The conserved DNaseI hypersensitivity sites (HSs) HS-62.5 and 3'HS1 that flank the locus, and the region upstream of the LCR have been implicated in globin gene regulation. The flanking HSs bind CCCTC binding factor (CTCF) and are thought to interact with the LCR to form a "chromatin hub" involved in beta-globin gene activation. Hispanic thalassemia, a deletion of the LCR and 27 kb upstream, leads to heterochromatinization and silencing of the locus. Thus, the region upstream of the LCR deleted in Hispanic thalassemia (upstream Hispanic region [UHR]) may be required for expression. To determine the importance of the UHR and flanking HSs for beta-globin expression, we generated and analyzed mice with targeted deletions of these elements. We demonstrate deletion of these regions alone, and in combination, do not affect transcription, bringing into question current models for the regulation of the beta-globin locus.

Figure 1.

Map of mutant D alleles generated by homologous recombination. (A) Map of the mouse β-globin locus. Arrowheads represent genes. Vertical arrows represent HSs. ORG indicates olfactory receptor gene. The extent of the DNaseI-sensitive domain is diagrammed below the locus map. (B) Diagrammatic representations of the targeted deletions generated. Striped region designates the UHR, the region upstream of the LCR that has homology to the region deleted in the Hispanic deletion.

Figure 2.

Expression of the adult β-globin genes in mice heterozygous for targeted deletions. The Hbb^D allele (D) carries the targeted mutation, while the Hbb^S allele (S) is wild-type. Pools of 100 reticulocytes were isolated from peripheral blood by flow cytometry and analyzed by RT-PCR with primers that amplify both adult genes from both alleles. Analysis of 2 representative independent pools is shown for each mouse strain. WT indicates wild-type D/S animals. Other strain designations are described in “Results.” S and D mark the RT-PCR product from the S and D alleles, respectively.

Figure 3.

Mapping of DNaseI HSs in the HS-62.5 and 3′HS1 regions of WT and ΔHS-62.5, Δ3′HS1 mice. DNaseI series from WT and homozygous mutant mice were digested with EcoRV and hybridized with probes to the 3′ end of each restriction fragment. After hybridization to HS-62.5 or 3′HS1 region probes, blots from mutant mice were stripped and rehybridized to a probe to the 5′HS4 region to demonstrate the quality of the DNase series. The edge of a 1-kb marker lane is observed on the far left. DNaseI digestion increases from left to right. The characteristic doublet of HSs for the HS-62.5 and 3′HS1 regions in WT samples are marked by arrows (left blots). The characteristic doublet of 5′HS4 is marked by arrows (right blots). An asterisk marks the size of expected fragments if a HS formed at the site of the targeted deletions in mutant samples (middle blots). Left blots are from WT mice. Middle and right blots are from homozygous ΔHS-62.5, Δ3′HS1 mice. Probes used are indicated above each panel.