Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression.

Abstract

The human beta‐globin dominant control region (DCR) was previously identified as a region from the 5′ end of the human beta‐globin locus which directs high level, site of integration‐independent, copy number‐dependent expression on a linked human beta‐globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a beta‐globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of beta‐globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3′ enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron.

Transcriptional activation by hypersensitive site three of the human β-globin locus control region in murine erythroleukemia cells

“…Addition of the HS3 NF-E2 site did not appear to have any effect on the expression of constructs containing footprints 1-6 and 1-3. This would argue that NF-E2 is not important in the context of HS3, and is consistent with the observation that HS2 and HS3 do not synergize when tested in cis [17,29]. However, addition of the HS2 NF-E2 dimer site to the otherwise inactive construct containing footprints 3-6 does result in a synergistic enhancement of transcription.…”

“…In this paper, we have focussed our attention on the core fragment of HS3, which is transcriptionally the most active individual site of the LCR [15,17]. It is a 225 bp fragment which we have functionally defined in both transgenic mice and murine erythroleukemia (MEL) cells.…”

Modeling Oculopharyngeal Muscular Dystrophy in Myotube Cultures Reveals Reduced Accumulation of Soluble Mutant PABPN1 Protein

“…The human PABPN1 transgene was expressed under the control of the human Des-LCR and promoter 31 ( Figure 1A), and mRNA was stabilized with the use of the 3= half of the HBB gene. [43][44][45][46] Stable clones of ImmortoMouse myoblasts (clone IM2) 33 were generated, and the transgene mRNA expression level was determined using RT-qPCR. The D7E and WTA clones, expressing the Ala17 or Ala10 alleles, respectively (Figure 1, A and B), were selected for further study.…”

Intergenic transcription and transinduction of the human β-globin locus

“…The ␤-globin LCR contains five erythroid-specific, developmentally stable hypersensitive sites, HS1-HS5, situated upstream of the ⑀-globin gene (Grosveld et al 1987;Zafarana et al 1996). Transcription has been detected both in a microlocus fragment containing HS1-HS4 linked together and in HS2 as part of a chimeric construct, raising the possibility that the ␤-globin LCR is transcribed (Collis et al 1990;Tuan et al 1992). In addition, the existence of large transcripts from the avian and murine ␤-globin clusters, up to seven times longer than the globin mRNAs, was described over two decades ago (Imaizumi et al 1973;Bastos and Aviv 1977).…”