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The production of hemoglobin by RPE suggests the potential involvement of this cell type in the etiology of SR. Monomethylfumarate influences multiple parameters consistent with improved retinal health in SCD and may therefore be of therapeutic potential in SR treatment.

SRY, an architectural transcription factor encoded by the sex-determining region of the Y chromosome, initiates testicular differentiation in mammalian embryogenesis. The protein contains a high-mobility group (HMG) box, a DNA-bending motif conserved among a broad class of nuclear proteins. Mutations causing human sex reversal (46, XY pure gonadal dysgenesis) are clustered in this domain. Basic N-and C-terminal regions of the HMG box are each proposed to provide nuclear localization signals. The significance of the Cterminal basic cluster (SRY residues 130 -134) is uncertain, however, as its activity in cell culture varies with assay conditions. To test its importance, we have investigated a C-terminal sex-reversal mutation (R133W, position 78 of the HMG box). This de novo mutation impairs nuclear localization but not specific DNA binding or sharp DNA bending. Correlation between these properties and the phenotype of the patient suggests that nuclear localization of SRY is required for testicular differentiation and directed in part by the C-terminal basic cluster. To our knowledge, these results provide the first example of impaired organogenesis due to a nuclear localization signal mutation.SRY, the testes-determining factor encoded by the human Y chromosome (1), contains a high-mobility group (HMG) 1 box (2-4), a conserved motif of DNA bending (Fig. 1, A and C, and Ref. 5). Mutations in SRY are associated with 46, XY pure gonadal dysgenesis leading to failure of testicular differentiation and female somatic phenotype (XY sex reversal; Refs. 3 and 6 -8). Clinical mutations cluster in the HMG box 2 and most commonly impair specific DNA binding (7,9,10). SRY is a nuclear protein (11) expressed in the primordial Sertoli cells of the differentiating gonadal ridge (12-14). Although SRY is presumed to function as an architectural transcription factor (9, 15, 16), its downstream genetic pathway is not well characterized (for a review, see Ref. 17).Immunohistochemical studies of murine and human embryos have demonstrated that SRY is a nuclear protein (11,18). Nuclear localization signals (NLSs) in human SRY have been defined in cell culture. Berta and colleagues (11), using microinjection of proteins in adult human fibroblastic cells, identified an NLS in the N-terminal region of the human HMG box 3 ( Fig. 2A; SRY residues 59 -75). This NLS comprises two sets of basic amino acids separated by 12 residues (Fig. 2B), features characteristic of bipartite NLS motifs in diverse proteins (19,20). An isolated N-terminal SRY peptide (residues 58 -78) was shown to be sufficient to direct nuclear translocation of coupled rabbit IgG (protein SRY21 in Fig. 2A). By contrast the remainder of the HMG box (residues 74 -137) was unable to direct nuclear translocation of coupled rabbit IgG (protein SRY64 in Fig. 2A). Although these findings appear to exclude a second NLS in SRY, Sü dbeck and Scherer (21) subsequently used a complementary methodology (transient transfection of SRY-␤-galactosidase fusion genes in COS-7 cells; Fig. 2B)...

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The receptor-binding surface of insulin is broadly conserved, reflecting its evolutionary optimization. Neighboring positions nevertheless offer an opportunity to enhance activity, through either transmitted structural changes or introduction of novel contacts. Nonconserved residue A8 is of particular interest as Thr(A8) --> His substitution (a species variant in birds and fish) augments the potency of human insulin. Diverse A8 substitutions are well tolerated, suggesting that the hormone-receptor interface is not tightly packed at this site. To resolve whether enhanced activity is directly or indirectly mediated by the variant A8 side chain, we have determined the crystal structure of His(A8)-insulin and investigated the photo-cross-linking properties of an A8 analogue containing p-azidophenylalanine. The structure, characterized as a T(3)R(3)(f) zinc hexamer at 1.8 A resolution, is essentially identical to that of native insulin. The photoactivatable analogue exhibits efficient cross-linking to the insulin receptor. The site of cross-linking lies within a 14 kDa C-terminal domain of the alpha-subunit. This contact, to our knowledge the first to be demonstrated from the A chain, is inconsistent with a recent model of the hormone-receptor complex derived from electron microscopy. Optimizing the binding interaction of a nonconserved side chain on the surface of insulin may thus enhance its activity.

Inherited genetic modifiers and pharmacologic agents that enhance fetal hemoglobin (HbF) expression reverse the clinical severity of sickle cell disease (SCD). Recent efforts to develop novel strategies of HbF induction include discovery of molecular targets that regulate γ-globin gene transcription and translation. The purpose of this study was to perform genome-wide microRNA (miRNA) analysis to identify genes associated with HbF expression in patients with SCD. We isolated RNA from purified reticulocytes for microarray-based miRNA expression profiling. Using samples from patients with contrasting HbF levels, we observed an eightfold upregulation of miR-144–3p (miR-144) and miR-144–5p in the low-HbF group compared with those with high HbF. Additional analysis by reverse transcription quantitative polymerase chain reaction confirmed individual miR-144 expression levels of subjects in the two groups. Subsequent functional studies in normal and sickle erythroid progenitors showed NRF2 gene silencing by miR-144 and concomitant repression of γ-globin transcription; by contrast, treatment with miR-144 antagomir reversed its silencing effects in a dose-dependent manner. Because NRF2 regulates reactive oxygen species levels, additional studies investigated mechanisms of HbF regulation using a hemin-induced oxidative stress model. Treatment of KU812 cells with hemin produced an increase in NRF2 expression and HbF induction that reversed with miR-144 pretreatment. Chromatin immunoprecipitation assay confirmed NRF2 binding to the γ-globin antioxidant response element, which was inhibited by miR-144 mimic treatment. The genome-wide miRNA microarray and primary erythroid progenitor data support a miR-144/NRF2-mediated mechanism of γ-globin gene regulation in SCD.

Sickle cell anemia is a common genetic disorder caused by a point mutation in the sixth codon of the b-globin gene affecting people of African descent worldwide. A wide variety of clinical phenotypes ranging from mild to severe symptoms and complications occur due to hemoglobin S polymerization, red blood cell sickling, and vaso-occlusion. Research efforts are ongoing to develop strategies of fetal hemoglobin (HbF; a 2 g 2 ) induction to inhibit sickle hemoglobin polymerization and improve clinical outcomes. Insights have been gained from investigating mutations in the b-globin locus or transcription factors involved in the mechanisms of hemoglobin switching. Recent efforts to expand molecular targets that modulate g-globin expression involve microRNAs that work through posttranscriptional gene regulation. Therefore, the goal of our study was to identify novel microRNA genes involved in fetal hemoglobin expression. Using in silico analysis, we identified a miR-34a binding site in the g-globin mRNA which was tested for functional relevance. Stable expression of the shMIMIC miR-34a lentivirus vector increased fetal hemoglobin levels in single cell K562 clones consistent with silencing of a g-globin gene repressor. Furthermore, miR-34a promoted cell differentiation supported by increased expression of KLF1, glycophorin A, and the erythropoietin receptor. Western blot analysis of known negative regulators of g-globin including YY1, histone deacetylase 1, and STAT3, which are regulated by miR-34a showed no change in YY1 and histone deacetylase 1 levels; however, total-and phosphorylated-STAT3 levels were decreased in single cell miR-34a K562 clones. These data support a mechanism of fetal hemoglobin activation by miR-34a involving STAT3 gene silencing.