Mutation of gene-proximal regulatory elements disrupts human epsilon-, gamma-, and beta-globin expression in yeast artificial chromosome transgenic mice

Abstract

Previous studies have defined transcriptional control elements, in addition to the promoters, that both lie near individual human beta-globin locus genes and have been implicated in their differential stage-specific regulation during development (i.e., are believed to directly participate in hemoglobin switching). We have reinvestigated the activities during erythropoiesis that might be conferred by two of the more intensively analyzed of these elements, the epsilon-globin gene 5' silencer and the beta-globin gene 3' enhancer, by deleting them from a yeast artificial chromosome that spans the human beta-globin locus, and then analyzing transgenic mice for expression of all of the human genes. These studies show that sequences within the epsilon-globin "silencer" are not only required for silencing but are also required for activation of epsilon-globin transcription; furthermore, deletion of the silencer simultaneously reduced gamma-globin transcription during the yolk sac stage of erythroid development. Analysis of the adult beta-globin gene 3' enhancer deletion showed that its deletion affects only that gene.

Figure 1

Structure of the wild-type and mutant human β-globin YACs. (A) YAC clone A201F4 (22) was modified as described (26) to permit ease of manipulation for targeted mutagenesis in yeast. Expanded diagrams depicting the two targeting constructs generated here are shown above (the ɛ-globin silencer deletion mutant) or below (the β-globin enhancer deletion mutant) the diagram of the YAC clone (the position of the deleted elements is indicated by a break in the contiguity of the subclone). Note the position of the SfiI and SalI sites in the locus that were used in the mapping experiments described in Fig. 2. (B Upper) An ethidium bromide-stained pulse field gel of the wild-type, ɛ-silencer, and β-enhancer deletion mutant YACs. (B Lower) Autoradiographic exposure of the blots of the gels shown in the Upper panel after hybridization to probes corresponding to the ɛ-globin gene silencer (Left) or β-globin gene enhancer (Right). (C) Southern blots of both wild-type and mutant YAC DNAs depicting the size and integrity of the EcoRI fragments bearing the LCR and the ɛ-, γ-, and β-globin genes.

Figure 2

Copy number and integrity of YAC transgenes. Lanes: 1–3, ɛ-globin silencer deletion mutant transgenic lines 1–3, respectively; 4, wild type β-globin YAC transgenic line HS4321b (26); 5 and 6, β-globin enhancer deletion mutant lines 1 and 2, respectively (numbering at the top of each panel). (A) Tail DNA from F₁ or F₂ pups representing each of the transgenic lines was digested with PstI and electrophoresed and blotted normally. A 1.7-kbp EarI fragment isolated from plasmid vector pYAC4 was radiolabeled and then hybridized to the Southern blot, and finally exposed for autoradiography. Each lane displays a (common) internal fragment band from the pYAC4 vector (1.58 kbp) as well as other bands, depending on the transgene copy number and integration site. Head-to-head tandem integrations would be expected to yield a 4.5-kbp band, whereas head-to-tail tandem integrations are predicted to yield a 5.3-kbp band. (B) Spleen cells were isolated from each transgenic line and digested with SfiI plus SalI as described prior to electrophoresis on pulse field gels. The gel was then blotted to nylon and hybridized to probes corresponding to either LCR HS4 or the Gγ-globin gene.

Figure 3

Expression of human β-globin YAC mutants during erythropoiesis. (A Upper). Typical reverse transcriptase–PCR expression data for the human ɛ-, γ-, and β-globin genes as compared with the murine α-globin used as the internal control for each RNA sample. The transgenic line examined is abbreviated at the top of each lane, while the developmental stage and sites are shown below each panel. A +1 refers to RNA isolated from the single copy wild-type YAC transgenic line, HS4321b (26). (B) Quantification of the expression data shown in A after PhosphorImager analysis and correction for gene copy number (Fig. 2). Open rectangles represent ɛ-globin expression, shaded boxes represent γ-globin expression, and filled boxes represent β-globin expression levels.