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.

Membrane trafficking is essential to eukaryotic life and is controlled by a complex network of proteins that regulate movement of proteins and lipids between organelles. The GBF1/GEA family of Guanine nucleotide Exchange Factors (GEFs) regulates trafficking between the endoplasmic reticulum and Golgi by catalyzing the exchange of GDP for GTP on ADP Ribosylation Factors (Arfs). Activated Arfs recruit coat protein complex 1 (COP-I) to form vesicles that ferry cargo between these organelles. To further explore the function of the GBF1/GEA family, we have characterized a fission yeast mutant lacking one copy of the essential gene gea1 (gea1+/−), the Schizosaccharomyces pombe ortholog of GBF1. The haploinsufficient gea1+/− strain was shown to be sensitive to the GBF1 inhibitor brefeldin A (BFA) and was rescued from BFA sensitivity by gea1p overexpression. No overt defects in localization of arf1p or arf6p were observed in gea1+/− cells, but the fission yeast homolog of the COP-I cargo sac1 was mislocalized, consistent with impaired COP-I trafficking. Although Golgi morphology appeared normal, a slight increase in vacuolar size was observed in the gea1+/− mutant strain. Importantly, gea1+/− cells exhibited dramatic cytokinesis-related defects, including disorganized contractile rings, an increased septation index, and alterations in septum morphology. Septation defects appear to result from altered secretion of enzymes required for septum dynamics, as decreased secretion of eng1p, a β-glucanase required for septum breakdown, was observed in gea1+/− cells, and overexpression of eng1p suppressed the increased septation phenotype. These observations implicate gea1 in regulation of septum breakdown and establish S. pombe as a model system to explore GBF1/GEA function in cytokinesis.

The yeast vacuole plays key roles in cellular stress responses. Here, we show that deletion of lvs1, the fission yeast homolog of the Chediak-Higashi Syndrome CHS1/LYST gene, increases vacuolar size, similar to deletion of the Rab4 homolog ypt4. Overexpression of lvs1-YFP rescued vacuolar size in ypt4Δ cells, but ypt4-YFP did not rescue lvs1Δ, suggesting that lvs1 may act downstream of ypt4. Vacuoles were capable of hypotonic shock-induced fusion and recovery in both ypt4Δ and lvs1Δ cells, although recovery may be slightly delayed in ypt4Δ. Endocytic and secretory trafficking were not affected, but ypt4Δ and lvs1Δ strains were sensitive to neutral pH and CaCl2, consistent with vacuolar dysfunction. In addition to changes in vacuolar size, deletion of ypt4 also dramatically increased cell size, similar to tor1 mutants. These results implicate ypt4 and lvs1 in maintenance of vacuolar size and suggest that ypt4 may link vacuolar homeostasis to cell cycle progression.

Children with hemoglobin SC (HbSC) disease suffer a significant incidence of silent cerebral infarcts but stroke is rare. A 2-year-old African American boy with HbSC disease presented with focal neurologic deficits associated with magnetic resonance imaging evidence of cerebral infarction with vascular abnormalities. After the acute episode he was treated with monthly transfusions and subsequently transitioned to hydroxyurea therapy. The benefits of hydroxyurea as a fetal hemoglobin inducer in HbSC disease, to ameliorate clinical symptoms are supported by retrospective studies. This case highlights the rare occurrence of stroke in a child with HbSC disease and the use of hydroxyurea therapy.