Zhenning He

A human embryonic hemoglobin inhibits Hb S polymerization in vitro and restores a normal phenotype to mouse models of sickle cell disease

The principle that developmentally silenced globin genes can be reactivated in adults with defects in β-globin gene expression has been well established both in vitro and in vivo. In practice, levels of developmental stage-discordant fetal γ globin that can be achieved by using currently approved therapies are generally insufficient to fully resolve typical clincopathological features of sickle cell disease. The therapeutic potential of another developmentally silenced globin—embryonic ɛ globin—has been difficult to evaluate in the absence of a convenient expression system or an appropriate experimental model. The current work analyzes the antisickling properties of an ɛ-globin-containing heterotetramer (Hb Gower-2) both in vitro as well as in vivo in a well-established mouse model of sickle cell anemia. These animals, expressing 100% human Hb S, display a chronic hemolytic anemia with compensatory marrow and extramedullary erythropoiesis, abundant circulating sickled erythrocytes, and chronic tissue damage evidenced by parallel histopathological and functional deficits. By comparison, related mice that coexpress Hb S as well as Hb Gower-2 exhibit normal physiological, morphological, histological, and functional attributes. Subsequent in vitro analyses substantiate results from whole-animal studies, indicating that the polymerization of deoxygenated Hb S can be significantly slowed by relatively small quantities of Hb Gower-2. Together, the in vivo and in vitro analyses suggest that reactivation of ɛ-globin gene expression would be therapeutically beneficial to adults with sickle phenotypes, and provide a rationale for detailed investigations into the molecular basis for its developmental silencing.

Antisickling effects of an endogenous human alpha-like globin.

Gene replacement or gene reactivation therapies for sickle-cell disease (SCD) typically target the mutant βS-globin subunits of hemoglobin-S (α2βS2) for substitution by nonpathological β-like globins. Here we show, in vitro and in vivo in a transgenic mouse model of SCD, that the adverse properties of hemoglobin-S can be reversed by exchanging its normal α-globin subunits for ζ-globin, an endogenous, developmentally silenced, non-β-like globin.

Functional effects of replacing human α‐ and β‐globins with their embryonic globin homologues in defined haemoglobin heterotetramers

Embryonic‐ and adult‐stage globin subunits assemble into haemoglobin (Hb) heterotetramers that are expressed at low levels throughout human intrauterine development. These haemoglobins differ from adult Hb A (α2β2) by the substitution of embryonic ζ for adult α globin (Hb ζ2β2), or embryonic ε for adult β globin (Hb α2ε2). Several key physiological properties of these ‘semiembryonic’ haemoglobins remain undefined, as ethical and methodological considerations have limited their availability from both human sources and conventional expression systems. The current study attempts to estimate how the physiological properties of semiembryonic and adult haemoglobins may differ, by determining whether the O2‐binding characteristics of hybrid human/mouse haemoglobins change when human α‐ or β‐globin subunits are replaced by human embryonic ζ‐ or ε‐globin subunits respectively. Each of the four human globins is expressed in transgenic mice that are nullizygous for either the endogenous mouse α‐ or β‐globin genes, resulting in the high‐level expression of haemoglobins that can be studied either in situ in intact erythrocytes or in vitro. We showed that the exchange of human ζ‐globin for human α‐globin chains increased haemoglobin O2 affinity, both in the presence and in the absence of 2,3‐bisphosphoglycerate (2,3‐BPG), and reduced the pH‐dependent shift in its oxygen equilibrium curve (Bohr effect). By comparison, hybrid haemoglobins containing either human ε‐globin or human β‐globin exhibited nearly identical O2‐binding properties, both in situ and in vitro, regardless of 2,3‐BPG levels or ambient pH. Neither the ζ‐for‐α nor the ε‐for‐β substitutions substantially altered binding affinity for 2,3‐BPG or cooperativity between globin subunits. These studies suggest that semiembryonic haemoglobins that assemble entirely from human subunits may exhibit properties that are similar to those of human Hb A.

Structure of fully liganded Hb ζ2β2s trapped in a tense conformation.

A variant Hb ζ2β2(s) that is formed from sickle hemoglobin (Hb S; α2β2(s)) by exchanging adult α-globin with embryonic ζ-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb ζ2β2(s) inhibits the polymerization of deoxygenated Hb S in vitro and reverses characteristic features of SCD in vivo in mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (α2β2), Hb ζ2β2(s) exhibits atypical properties that include a high oxygen affinity, reduced cooperativity, a weak Bohr effect and blunted 2,3-diphosphoglycerate allostery. Here, the 1.95 Å resolution crystal structure of human Hb ζ2β2(s) that was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb ζ2β2(s) displays a central water cavity, a ζ1-β(s)2 (or ζ2-β(s)1) interface, intersubunit salt-bridge/hydrogen-bond interactions, C-terminal βHis146 salt-bridge interactions, and a β-cleft, that are highly unusual for a relaxed hemoglobin structure and are more typical of a tense conformation. These quaternary tense-like features contrast with the tertiary relaxed-like conformations of the ζ1β(s)1 dimer and the CD and FG corners, as well as the overall structures of the heme cavities. This crystallographic study provides insights into the altered oxygen-transport properties of Hb ζ2β2(s) and, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.

Structural determinants of human ζ-globin mRNA stability

BackgroundThe normal accumulation of adult α and β globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells. The impact of these post-transcriptional regulatory events on the expression of embryonic ζ globin is not known, as its encoding mRNA is not normally transcribed during adult erythropoiesis. Recently, though, ζ globin has been recognized as a potential therapeutic for α thalassemia and sickle-cell disease, raising practical questions about constitutive post-transcriptional processes that may enhance, or possibly prohibit, the expression of exogenous or derepresssed endogenous ζ-globin genes in definitive erythroid progenitors.MethodsThe present study assesses mRNA half-life in intact cells using a pulse-chase approach; identifies cis-acting determinants of ζ-globin mRNA stability using a saturation mutagenesis strategy; establishes critical 3′UTR secondary structures using an in vitro enzymatic mapping method; and identifies trans-acting effector factors using an affinity chromatographical procedure.ResultsWe specify a tetranucleotide 3′UTR motif that is required for the high-level accumulation of ζ-globin transcripts in cultured cells, and formally demonstrate that it prolongs their cytoplasmic half-lives. Surprisingly, the ζ-globin mRNA stability motif does not function autonomously, predicting an activity that is subject to structural constraints that we subsequently specify. Additional studies demonstrate that the ζ-globin mRNA stability motif is targeted by AUF1, a ubiquitous RNA-binding protein that enhances the half-life of adult β-globin mRNA, suggesting commonalities in post-transcriptional processes that regulate globin transcripts at all stages of mammalian development.ConclusionsThese data demonstrate a mechanism for ζ-globin mRNA stability that exists in definitive erythropoiesis and is available for therapeutic manipulation in α thalassemia and sickle-cell disease.