Role of the duplicated CCAAT box region in gamma-globin gene regulation and hereditary persistence of fetal haemoglobin.

Hereditary persistence of fetal haemoglobin (HPFH) is a clinically important condition in which a change in the developmental specificity of the gamma-globin genes results in varying levels of expression of fetal haemoglobin in the adult. The condition is benign and can significantly alleviate the symptoms of thalassaemia or sickle cell anaemia when co-inherited with these disorders. We have examined structure-function relationships in the -117 HPFH gamma promoter by analysing the effect of mutating specific promoter elements on the functioning of the wild-type and HPFH promoters. We find that CCAAT box mutants dramatically affect expression from the HPFH promoter in adult blood but have little effect on embryonic/fetal expression from the wild-type promoter. Our results suggest that there are substantial differences in the structure of the wild-type gamma promoter expressed early in development and the adult HPFH promoter. Together with previous results, this suggests that gamma silencing is a complex multifactorial phenomenon rather than being the result of a simple repressor binding to the promoter. We present a model for gamma-globin gene silencing that has significant implications for attempts to reactivate the gamma promoters in human adults by pharmacological means.     

The deletion of the distal CCAAT box region of the A gamma-globin gene in black HPFH abolishes the binding of the erythroid specific protein NFE3 and of the CCAAT displacement protein.

Non-deletion Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by great elevation of the synthesis, in adult age, of fetal hemoglobin (HbF), of either the A gamma or G gamma type. Strong genetic evidence indicates point mutations in the G gamma- or A gamma-globin promoter as responsible for overexpression of the mutated gene. Here we report that a 13 nucleotides deletion in the CCAAT box region of the A gamma-globin promoter, associated with greater than 100 fold overexpression of the gene, abolishes the in vitro binding of the ubiquitous factors CP1 and CDP (CCAAT displacement protein) and of the erythroid specific protein NFE3. Loss of NFE3 binding is consistent with a similar effect of the -117 G greater than A HPFH mutation, suggesting a possible role of NFE3 as a negatively acting factor. In addition, loss of CDP binding indicates that this alteration might also contribute to the HPFH phenotype in this particular case, suggesting possible heterogeneity of the mechanisms causing HPFH.     

Developmentally regulated expression of specific tau sequences

Tau protein undergoes a shift in its molecular mass and its electrophoretic complexity during early postnatal development. We have sequenced a tau cDNA from an adult rat brain expression library and have found two inserted sequences. One of these inserts predicts a fourth repeated sequence homologous to the other three in the carboxyl end of tau that have the property of microtubule binding. Oligonucleotide probes directed against the insert hybridized only to tau mRNA at postnatal time points, even though tau is first expressed as early as embryonic day 13. A probe directed against the junction revealed expression of non-insert-containing tau mRNA from embryonic day 14 until postnatal day 8, after which time there was an abrupt decline in the expression of this immature form. Comparison of the developmentally expressed tau sequences with those sequences obtained directly from Alzheimer paired helical filaments revealed the presence of both the mature and the immature tau mRNA sequences.     

Developmentally dependent expression of tissue specific amylases in maize

Summary The mapping of three genes (speA, speB and metK) involved in the biosynthesis of spermidine is described. They are located between minutes 56 and 57 on the linkage map of Escherichia coli. The clustering of these genes suggests the possibility of an operon for spermidine synthesis. Two other genes, argP, controlling the production of an arginine permease, and can, conferring resistance to canavanine, were found to be located in the same region.     

Developmental regulation of human gamma-globin genes in transgenic mice.

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.     

Tissue specific hormonal regulation of the rat angiotensinogen gene expression

Angiotensinogen is the precursor of the most potent pressor substance, angiotensin. Angiotensinogen levels are increased in some forms of human hypertension. Its levels are modulated by various factors including glucocorticoids, estrogens, and prostaglandins. We have recently reported the isolation of a human angiotensinogen cDNA clone and provided evidence for the presence of its mRNA in rat liver, brain, and heart. In this communication we report the effect of dexamethasone and estradiol on angiotensinogen mRNA levels in rat liver, brain, and heart. Our results indicate that angiotensinogen levels are increased to different extents in these three tissues as a result of glucocorticoid or estrogen administration.     

Developmentally regulated and erythroid-specific expression of the human embryonic beta-globin gene in transgenic mice.

Transgenic mice have proven to be an effective expression system for studying developmental control of the human fetal and adult beta-globin genes. In the current work we are interested in developing the transgenic mouse system for the study of the human embryonic beta-globin gene, epsilon. An epsilon-globin gene construction (HSII,I epsilon) containing the human epsilon-globin gene with 0.2 kb of 3' flanking sequence and 13.7 kb of extended 5' flanking region including the erythroid-specific DNase I super-hypersensitive sites HSI and HSII was made. This construction was injected into fertilized mouse ova, and its expression was analyzed in peripheral blood, brain, and liver samples of 13.5 day transgenic fetuses. Fetuses carrying intact copies of the transgene expressed human epsilon-globin mRNA in their peripheral blood. Levels of expression of human epsilon-globin mRNA in these transgenic mice ranged from 2% to 26% per gene copy of the endogenous mouse embryonic epsilon y-globin mRNA level. Furthermore, the human epsilon-globin transgene was expressed specifically in peripheral blood but not in brain or in liver which is an adult erythroid tissue at this stage. Thus, the HSII,I, epsilon transgene was expressed in an erythroid-specific and embryonic stage-specific manner in the transgenic mice. A human epsilon-globin gene construction that did not contain the distal upstream flanking region which includes the HSI and HSII sites, was not expressed in the embryos of transgenic mice. These data indicate that the human epsilon-globin gene with 5' flanking region extending to include DNase I super-hypersensitive sites HSI and HSII is sufficient for the developmentally specific activation of the human epsilon-globin gene in erythroid tissue of transgenic mice.