The characterization of atypical mutations in loci associated with diseases is a powerful tool to discover novel regulatory elements. We previously identified a dinucleotide deletion in the human ankyrin-1 gene (ANK-1) promoter that underlies ankyrin-deficient hereditary spherocytosis. The presence of the deletion was associated with a decrease in promoter function both in vitro and in vivo establishing it as a causative hereditary spherocytosis mutation. The dinucleotide deletion is located in the 5' untranslated region of the ANK-1 gene and disrupts the binding of TATA binding protein and TFIID, components of the preinitiation complex. We hypothesized that the nucleotides surrounding the mutation define an uncharacterized regulatory sequence. To test this hypothesis, we generated a library of more than 16,000 ANK-1 promoters with degenerate sequence around the mutation and cloned the functional promoter sequences after cell-free transcription. We identified the wild type and three additional sequences, from which we derived a consensus. The sequences were shown to be functional in cell-free transcription, transient-transfection, and transgenic mouse assays. One sequence increased ANK-1 promoter function 5-fold, while randomly chosen sequences decreased ANK-1 promoter function. Our results demonstrate a novel functional motif in the ANK-1 promoter.

Diamond Blackfan anemia (DBA) is an inherited erythroblastopenia associated with mutations in at least 8 different ribosomal protein genes. Mutations in the gene encoding ribosomal protein S19 (RPS19) have been identified in approximately 25% of DBA families. Most of these mutations disrupt either the translation or stability of the RPS19 protein and are predicted to cause DBA by haploinsufficiency. However, approximately 30% of RPS19 mutations are missense mutations that do not alter the stability of the RPS19 protein and are hypothesized to act by a dominant negative mechanism. To formally test this hypothesis, we generated a transgenic mouse model expressing an RPS19 mutation in which an arginine residue is replaced with a tryptophan residue at codon 62 (RPS19R62W). Constitutive expression of RPS19R62W in developing mice was lethal. Conditional expression of RPS19R62W resulted in growth retardation, a mild anemia with reduced numbers of erythroid progenitors, and significant inhibition of terminal erythroid maturation, similar to DBA. RNA profiling demonstrated more than 700 dysregulated genes belonging to the same pathways that are disrupted in RNA profiles of DBA patient cells. We conclude that RPS19R62W is a dominant negative DBA mutation.

The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3-ankyrin-beta-spectrin and glycophorin C-protein 4.1-beta-spectrin.(1-7) Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C-protein 4.1 interaction has little effect on membrane stability.(8) We demonstrate the existence of a novel band 3-adducin-spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3-adducin-spectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane.

Diamond-Blackfan Anemia (DBA) is associated with mutations in several ribosomal protein genes, including Ribosomal Protein S19 (RPS19), which is mutated in approximately 25% of patients. Most RPS19 mutations are deletions of all or part of the RPS19 gene and are predicted to cause DBA by a haploinsufficiency mechanism. However, approximately 30% of RPS19 mutations are missense mutations in the RPS19 coding sequence, which we hypothesize act through a dominant negative mechanism. To test for a dominant negative effect, we generated a transgenic mouse model expressing a common and penetrant mutation at codon 62 that replaces an Arginine with a Tryptophan (R62W). The constructs contain the ubiquitous actin promoter linked to the wild-type or R62W human RPS19 cDNA followed by the 3′ region of the Gamma globin gene to provide RNA stability and intron splicing to facilitate RNA transport to the cytoplasm. The constructs are flanked by chicken HS4 barrier elements to ensure transgene expression regardless of the location in the genome. Eight lines of wild-type RPS19 transgenic mice were fertile, expressed RPS19 in all tissues, and had normal hematology. Twelve RPS19R62W founder animals were generated, six of which died before they reached 2 months of age. Two of these animals were analyzed and found to have a macrocytic anemia. None of the other 6 founder animals transmitted the RPS19R62W transgene to F1 pups or d13.5 embryos, suggesting either that the RPS19R62W transgene was not present in the germ line and/or that expression of the RPS19R62W protein may cause early lethality. Supporting this hypothesis, embryonic stem cells (ES) expressing wild-type RPS19 were viable, while ES cells expressing RPS19R62W were not viable. To circumvent potential embryonic lethality, we generated conditional RPS19R62W transgenic mice with stop sequences flanked by lox P sites inserted between the promoter and the RPS19 gene. In the presence of Cre recombinase, lox P sites are combined, excising the sequences between them. Adult mice carrying the conditional RPS19R62W transgene and the interferon inducible Mx1-Cre gene were treated with poly (I:C) to induce excision of the stop sequence. Following poly (I:C) administration, hematocrits dropped significantly in RPS19R62W/Mx1-Cre animals compared to controls, but rebounded to normal within two weeks, due to incomplete stop sequence excision and expansion of unexcised cells in the bone marrow. Colony-forming cell assays indicate that RPS19R62W-expressing bone marrow contains 2 to 3 fold fewer BFU-E and CFU-E (p<0.05) and similar numbers of CFU-GM compared to wild-type animals. The decrease in erythroid progenitors was variable, indicating different levels of excision as well as penetrance. When RPS19R62W mice were crossed to Prion-Cre mice, which express Cre at the early embryonic stage, small, anemic d13.5 embryos and occasional small, adult animals with macrocytic anemia were observed. Day 13.5 RPS19R62W/Prion-Cre fetal livers had reduced overall numbers of erythroid cells, and reduced numbers of BFU-E and CFU-E. The decrease in erythroid progenitors was variable, especially in the line carrying 1 copy of the transgene compared to the line carrying 4 copies of the transgene. FACS analysis of d13.5 fetal liver and adult RPS19R62W/Prion-Cre erythroid cells revealed a relative accumulation of erythroid progenitor cells and a relative decrease in the number of terminally differentiating erythroid cells, suggesting that terminal erythroid differentiation is delayed. These findings are consistent with the reticulocytopenia observed in adult RPS19R62W/Prion-Cre mice. In summary we have successfully generated a mouse model of DBA caused by ectopic expression of mutant human RPS19R62W. The development of a severe anemia following conditional expression of mutant RPS19 suggests that the R62W missense mutation has a dominant negative effect that delays erythropoiesis causing an overall reduction in erythroid cells.

Hereditary persistence of fetal hemoglobin (HPFH) is characterized by increased levels of Hb F during adult life. Nondeletional forms of HPFH are characterized by single base mutations in the Aγ and Gγ promoters, resulting in an increase of Hb F ranging from 3 to 20% in heterozygotes. Many point mutations in this region have been described, including the Aγ −195 (C>G) mutation that causes the Brazilian type of HPFH (HPFH-B). To better understand this mechanism, we have developed HPFH-B transgenic mice. mRNA levels of human γ-globin of −195 transgenic mice were clearly higher when compared with control transgenic mice bearing a wild type sequence of the γ promoter. Thus, our data indicate that the −195 mutation is the unique cause of elevation of Hb F in Brazilian HPFH. These results could provide us with an opportunity to study the modifying effects of the Hb F in the phenotype of sickle cell disease and β-thalassemia (β-thal).

Insulator elements are found at the boundary between euchromatin and heterochromatin, and are responsible for maintaining the correct chromatin configuration for a locus. The best characterized vertebrate insulator element, 5′ Hypersensitive Site (HS) 4 from the chicken β-globin locus (ch5′HS4), has two separable activities: enhancer blocking, which requires binding of the transcription factor CTCF, and barrier, which prevents transgene silencing. We have previously reported that transgenic mice carrying a wild-type erythrocyte ankyrin promoter (ANK-1E)/γ-globin gene showed uniform (γ-globin in 100% of red cells), position-independent (32/32 lines express), copy number-dependent (p=0.0005) expression of γ-globin mRNA and protein. Mutations in the ANK-1E promoter at positions −108 and −153 cause ankyrin-deficient Hereditary Spherocytosis. Transgenic mice with the −108/−153 ANK-1E/γ-globin transgene showed variegated (γ-globin in 0–80% of red cells), position-dependent (8/14 lines express), copy number-independent (p=0.27) expression of γ-globin. Flanking the −108/−153 ANK-1E/γ-globin transgene with the ch5′HS4 insulator restored uniform, position-independent (9/9 lines), copy number-dependent expression (p=0.003) at levels identical to the wild-type ANK-1E promoter. We hypothesized that we could test sequences for barrier activity by assaying their ability to restore normal expression to the −108/−153 ANK-1E/γ-globin gene in transgenic mice. In mammalian β-globin loci, human 5′HS5 and mouse 3′HS1 have been proposed to be insulator elements, similar to chicken 5′HS4, based on their ability to block enhancer element function. To test barrier function, we generated transgenic mice containing the −108/−153 ANK-1E/γ-globin transgene flanked by human 5′HS5, mouse wild-type 3′HS1, or mouse 3′HS1 with mutations that disrupt the binding of CTCF (×CTCF). A total of 5 lines of transgenic mice were generated containing the 5′HS5/−108/−153 ANK-1E/γ-globin transgene. γ-globin mRNA and protein were undetectable in 3/5 lines, indicating that expression was position-dependent, and in the two positive lines, mRNA levels did not correlate with copy number. A total of 9 lines of 3′HS1-flanked transgenic mice were generated, 3 of which did not express γ-globin mRNA and protein, demonstrating position-dependent expression. Among the 6 expressing lines, two lines showed variegated expression and the correlation between γ-globin mRNA level and copy number was significant (p=0.0117). In contrast, 3′HS1×CTCF transgenic mice expressed γ-globin in a uniform, position-independent (7/7 lines express), copy number-dependent (p=0.0005) manner. The levels of γ-globin mRNA in both the 3′HS1 and 3′HS1×CTCF transgenic mice were 2-fold greater than the levels measured in transgenic mice with the wild-type ANK-1E promoter (p=0.019; 0.0003 respectively), suggesting that 3′HS1 may contain an enhancer element. Our results indicate that while human 5′HS5 and mouse 3′HS1 block the effects of enhancer elements, neither are barrier elements as defined by the ability to prevent gene silencing. We hypothesize that the mutation of the CTCF binding sites allows the ANK-1E promoter to take greater advantage of the 3′HS1 enhancer, leading to a more uniform, position-independent, and copy number-dependent pattern of expression, as has been described for other enhancer elements in the β-globin Locus Control Region.