Targeted inactivation of the major positive regulatory element (HS-40) of the human alpha-globin gene locus

Abstract

We have examined the role of the major positive upstream regulatory element of the human alpha-globin gene locus (HS-40) in its natural chromosomal context. Using homologous recombination, HS-40 was replaced by a neo marker gene in a mouse erythroleukemia hybrid cell line containing a single copy of human chromosome 16. In clones from which HS-40 had been deleted, human alpha-globin gene expression was severely reduced, although basal levels of alpha 1 and alpha 2-globin mRNA expression representing less than 3% of the level in control cell lines were detected. Deletion of the neo marker gene, by using FLP recombinase/FLP recombinase target system, proved that the phenotype observed was not caused by the regulatory elements of this marker gene. In the targeted clones, deletion of HS-40 apparently does not affect long-range or local chromatin structure at the alpha promoters. Therefore, these results indicate that, in the experimental system used, HS-40 behaves as a strong inducible enhancer of human alpha- globin gene expression.

Long-range chromosomal interactions regulate the timing of the transition between poised and active gene expression

“…D Vernimmen et al recruitment of Pol II was severely reduced. As before, the level of a globin mRNA expression was considerably reduced (B3% of normal; Bernet et al, 1995, and data not shown). These findings show that recruitment of the complete PIC at the a globin promoter is dependent on the combined effect of the upstream elements.…”

“…Grey boxes refer to previously defined multi-species conserved elements (MCS). Previously described deletions from the human a globin cluster (Bernet et al, 1995;Craddock et al, 1995) are shown as annotated (C40, IJ and MC) boxes. therefore, good candidates to be involved in triggering the transition from a poised to an active transcriptional state.…”

“…To determine if the PIC is recruited independently at the promoter and the regulatory elements, we analysed interspecific hybrids derived from normal individuals or those with previously characterised, natural mutations of the a globin cluster. We analysed two types of mutation: those in which the remote upstream elements had been fully (IJ) or partially (C40) deleted but the a promoters remained intact ( Figure 1B; Bernet et al, 1995;Craddock et al, 1995), and another in which all a-like genes were deleted (MC) but the upstream elements were still present ( Figure 1B; Craddock et al, 1995).…”

“…Using these hybrids, we have previously shown that the formation of DHSs occurs independently at the a globin promoters and the upstream elements (Bernet et al, 1995;Craddock et al, 1995). Here, we have shown that key, tissuerestricted TFs (NF-E2 and SCL) still bind the upstream elements when the a globin promoters are deleted (MC hybrid; Supplementary Figure S3) and when the major a-regulatory element is missing (HS-40, C40 hybrid; Supplementary Figure Figure 3 Recruitment of the pre-initiation complex at the mouse a globin core promoter during erythroid maturation.…”

“…MEL cell line 585 and interspecific MEL hybrids were grown and induced as described (Higgs et al, 1990;Bernet et al, 1995;Craddock et al, 1995). ES cells were grown as reported (Anguita et al, 2004).…”