Developmental Regulation of Human γ-Globin Genes in Transgenic Mice

Abstract

We report results showing that several gamma gene promoter elements participate in the developmental control of gamma-globin genes. Four gamma gene constructs with 5' truncated at -141, -201, -382, and -730 of the A gamma gene promoter linked to a micro locus control region (microLCR) cassette were used for production of transgenic mice and analysis of gamma gene expression during development. Mice carrying a microLCR -141 A gamma construct displayed downregulation of gamma gene expression in the adult stage of development, indicating that the proximal promoter contains elements participating in gamma gene silencing. Mice carrying a microLCR -201 A gamma or a microLCR -382 A gamma construct displayed high gamma gene expression in the fetal stage of development and complete loss of gamma gene downregulation in the adult stage, suggesting that the -141 to -201 gamma gene sequence contains elements which upregulate gamma gene expression and are dominant over the negative element 3' to -141. Extension of the promoter to -730 resulted in reappearance of gamma gene downregulation, suggesting that the -382 to -730 sequences contain an adult-stage-specific silencer. gamma gene expression in the microLCR -201 A gamma and the microLCR -382 A gamma transgenic mice was copy number dependent. All the microLCR -730 A gamma transgenic mice expressed gamma mRNA; however, gamma gene expression was copy number independent, indicating that levels of gamma gene expression were modulated by the surrounding chromatin. Our results suggest that multiple elements participate in gamma gene silencing. The findings in the microLCR-201 A gamma and microLCR -382 A gamma transgenic mice are interpreted to indicate that the LCR interacts not only with the minimal gamma gene promoter but also with sequences of the upstream promoter. We postulate that gamma gene downregulation is achieved when the interaction between LCR and the upstream promoter is disturbed by the silencer located in the -382 to -730 region. We propose that gamma gene silencing is achieved by the combined effect of negative elements located 3' to -141, the negative element located between -382 and -730, and the competition by the beta gene promoter during the adult stage of development.

Analysis of the mechanism of action of non-deletion hereditary persistence of fetal hemoglobin mutants in transgenic mice

“…day 10.5, the day 12 blood and yolk sac are composed predominantly of embryonic nucleated red cells and therefore provide information on globin gene expression in embryonic erythropoiesis. Results of g-globin expression during the development of the mLCR ±382 A g mice were published before (Stamatoyannopoulos et al, 1993). As in all transgenic lines carrying human g-globin genes, the highest g mRNA levels were observed in the day 12 embryonic blood and yolk sac of the mLCR ±382 A g mice.…”

“…Mean values are shown in bold. Data in mLCR ±382 A g control mice are from Stamatoyannopoulos et al (1993). b Days post-coitus.…”

“…This mutation results in the phenotype of Greek HPFH, which is characterized by production of 10±20% HbF in adult carriers (Stamatoyannopoulos and Grosveld, 2001). Since a truncation of the A g promoter to position ±141 A g destroys the gCACCC box (Stamatoyannopoulos et al, 1993), the ±117 G®A HPFH mutant was introduced into the mLCR ±141 A g construct. Results in the four transgenic mouse lines we established are shown in Table III and Figure 4.…”

Major erythrocyte membrane protein genes in EKLF-deficient mice

“…Prior attempts to rescue the EKLF-deficient mice with a human A g-globin transgene have failed. Perkins et al [22] mated EKLF-deficient mice to mice expressing a deregulated human A g-globin transgene ( A g-globin gene linked to a micro locus control region, mLCR 2201A g) [23]. Although improvement in globin chain balance was observed, abnormal morphology of circulating erythroid cells of fetal liver origin persisted and EKLF-deficient/ g-globin mice had no significant survival advantage over littermates.…”

An autonomous TCR signal-sensing switch influences CD4/CD8 lineage choice in mice

“…For this purpose, we carried out a converse silencer swap approach in which the Sil ThPOK was replaced by an exogenous silencer element. We first used the human adult-specific γ-globin silencer 21 . However, this silencer failed to repress ThPOK expression in DP thymocytes or in any other thymocyte stage (Supplementary Fig.…”