Generation of a Synthetic Transcription Activator Protein Targeting the Human Fetal Γ-Globin Gene
Total Page：16
File Type：pdf, Size：1020Kb
Generation of a synthetic transcription activator protein targeting the human fetal γ-globin gene By Maclean Bassett An honors thesis presented to the college of Liberal Arts and Sciences University of Florida 2019 0 Table of Contents 1. Abstract – Page 2 2. Introduction – Page 3 3. Materials and Methods – Page 8 4. Results – Page 12 5. Discussion and Perspective – Page 15 6. Figures – Page 17 7. Acknowledgements – Page 29 8. Abbreviations – Page 30 9. References – Page 31 1 1. Abstract: The human β-globin gene locus is located on chromosome 11 and constitutes five β-type globin genes that are organized in a manner reflecting their expression during development: an embryonic ɛ-globin gene located at the 5’ end, followed by the two fetal γ-globin genes, and the adult β- and δ- globin genes at the 3’ end. Elevated expression of fetal hemoglobin in the adult ameliorates the symptoms associated with β- hemoglobinopathies like sickle cell disease. A newly discovered DNase I hypersensitive site (HS) located 4 kb upstream of the γ-globin gene (HBG-4kb HS) was targeted as a candidate for affecting expression of γ-globin. Previous work conducted by our research group demonstrated that introduction of a synthetic zinc finger DNA-binding domain (ZF-DBD) to this site blocked recruitment of transcription factors, thus affecting expression of γ-globin. Considering this, a 6 ZF ZF-DBD was generated with a VP64 activator domain joined to the C terminus. The VP64 domain consists of 4 VP16 domains each of which encode viral proteins involved in activation of transcription by recruitment of co-activators. The synthetic protein was shown to interact with the target DNA sequence. Direct delivery of the generated ZF-DBD to mouse erythroleukemia cells revealed that the presence of the VP64 activation domain may not impede direct protein delivery to erythroid cells. 2 2. Introduction: Hemoglobin Hemoglobin is the basic protein component of human red blood cells, and functions as a respiratory carrier for transport of oxygen from the lungs to the tissues and return of carbon dioxide from the tissues to the lungs. Adult human hemoglobin possesses a heterotetrametric quaternary structure composed of four globular subunits: two α subunits and two β subunits. Each globin subunit consists of eight alpha helices which form a unique ‘globin fold’ and a non-protein heme group. Each heme group is bound to a gap between the helices and is composed of an Iron cation complexed in a porphyrin plane that forms four stable Fe-N bonds (Fig. 2.1). It is attached to the globin subunit via the central iron coordinating to the nitrogen(τ) of a proximal histidine residue found on one of the alpha helices.1 When the hemoglobin is oxygenated in the lung the oxygen becomes bound to the vacant sixth coordination position. When the Heme group is deoxygenated, the porphyrin ring sits in a nonplanar orientation to the iron atom, which is pulled toward the histidine residue. When an oxygen molecule binds, the porphyrin ring adopts a configuration planar with the iron atom, which draws the histidine residue towards the ring complex. This change in conformation induces a cooperativity effect, whereby the alpha helix becomes shifted, which alters the shape of congruent alpha helices that allows higher affinity for oxygen binding.2 Human expression of hemoglobin follows a specific timing of subunit expression constituted by a series of hemoglobin switching events. The first eight weeks of human development constitute the embryonic stage, during which embryonic hemoglobin (HbE) is composed of two ζ subunits and two ε subunits. Following embryonic development fetal development occurs. Fetal hemoglobin (HbF) is constituted of two α subunits and two γ subunits. HbF is the primary form of hemoglobin for approximately six months until shortly after birth when a second hemoglobin switching event occurs. Hemoglobin switching is characterized by a marked decrease in γ subunit production and activation of β globin subunits. The adult hemoglobin (HbA) is now composed of two α subunits and two β subunits (Fig. 2.1), which will remain the primary form of hemoglobin throughout adult life.3 This shift in hemoglobin expression is dictated by the physical proximity of the globin genes on chromosome 11, whereby their placement order follows their expression according to the general structure of 5’ ---- 3’. Control of the timing of gene activation is done by gene proximal regulatory DNA elements and the Locus Control Region (LCR). The LCR is a cis regulatory element composed of five DNAse I Hypersensitive Sites (HS). These HS elements contain multiple transcription factor motifs. The HS elements work cooperatively at the globin gene loci to mediate high levels of globin expression in time with developmental stages.3 The actual mechanism of 3 the switch is in part mediated by competition for the LCR by the and gene loci4, but primarily by autonomous silencing of the -globin gene locus.5 Hemoglobinopathies and Hereditary Persistence of Fetal Hemoglobin (HPFH) Diseases that affect the synthesis of functional hemoglobin are extremely common globally, there are more than 1,000 naturally occurring hemoglobin mutants that occur in humans6 which alter structure and biochemical properties of the hemoglobin. Six hundred diseases have hence been defined by the American College of Medical Genetics, notably including Sickle Cell Anemia and Beta Thalassemia, which affect the ability of hemoglobin to carry oxygen effectively.7 These diseases are congenital, as hemoglobinopathies are linked to genetic heritage, most notoriously Sickle Cell Anemia resulting from the inheritance of an abnormal hemoglobin S gene from both parents. An estimated 7% of the world’s population is affected by a form of hemoglobin mutation.8 In rare instances, usually due to a point mutation in the γ-globin gene promoter or deletions in the -gene loci9, HbF production continues into adulthood. This condition, known as Hereditary Persistence of Fetal Hemoglobin (HPFH), is harmless to the carrier and is characterized by elevated levels of HbF in the blood in addition to HbA. Increased levels of HbF in the blood have been shown to ameliorate complications associated with β-globin disorders10, especially so in the case of Sickle Cell Anemia where high levels of HbF were demonstrated to prevent basic mechanisms that lead to the development of Sickle Cell Anemia. Sickle cell anemia is the pathological culmination of conditions caused by the polymerization of a mutant form of -hemoglobin subunits named Hemoglobin S (HbS). Hemoglobin S subunits polymerize together upon deoxygenation, causing the cell’s shape to distort into that iconic sickle shape. HbF directly disrupts the polymerization of HbS.11 Furthermore, it decreases the relative concentration of HbS in the blood and is capable of binding oxygen more tightly than HbA, both of which effectively compensates for the defective HbS.12 In patients where HbS makes up over 50% of their total hemoglobin, clinical Sickle Cell Anemia is observed, however when blood hemoglobin is composed of at least 10% the severity of Sickle Cell Anemia is highly diminished.13 The occurrence of HPFH is genetically linked and there is a clinical pedigree whereby an individual inherits HPFH from one parent and Sickle Cell Anemia from the other parent (HbS/HPFH) which leads to a concentration of HbF in the blood well above this threshold. Although, increasing levels of HbF in the blood is by no means a cure, and even with heightened levels of HbF life threatening complications can still arise in patients.14 However, finding methods of increasing HbF concentration levels in 4 the blood represents the most promising strategy for developing treatments for Sickle Cell Anemia due to its ability to impact the physiological symptoms of the disease. Fetal globin gene silencing and reactivation The mechanisms that modulate the silencing of the -globin genes during hemoglobin switching have remained largely elusive, however efforts to understand the proteins and pathways that comprise the “big picture” have shed light on the role of BCL11A in - globin gene silencing. The BCL11A gene, located on chromosome 2, is a highly conserved gene sequence containing multiple Single Nucleotide Polymorphisms (SNPs) associated with elevated fetal hemoglobin expression. BCL11A contains three C2H2-type zinc finger (ZF) motifs, a proline rich region, and an acidic domain. The BCL11A zinc fingers bind directly to GG-rich sequences and BCL11A functions as transcriptional repressor.15 The SNPs are highly associated with F-cell levels and the expression of HbF16 as well as prediction of pain crises in sickle cell disease.17 BCL11A acts within the β-globin locus to control the silencing of -globin gene18, however the exact mechanism that modulates this silencing is unknown. Mutations in the BCL11A gene have thus been associated with HPFH.19 20 Furthermore, the silencing of an enhancer associated GATA motif present in the BCL11A locus results in elevated expression of HbF.21 Synthetic transcription activators Synthetic transcription activators or artificial transcription factors are proteins developed to target and modulate the transcription of specific genes in the genome. Coupling a transcription activation domain with a zinc finger DNA-binding domain (DBD) allows for targeted delivery of the transcription activator to a target site. In the case of an activator domain, delivery by ZF-DBDs results in the upregulation of the target gene product.22 VP16 is a transactivation domain found in the Herpes simplex 1, virus which primes transcription of the virally encoded IE genes. VP16 contains a carboxy-terminal region with an acidic sequence at its terminus that is crucial to gene activation.23 This terminal motif can be attached to DNA-binding domains that recognize specific promotor sequences to activate transcription.24 Hence, linkage of a ZF-DBD to a VP16 domain achieves activation of specific target genes.25 5 Zinc Fingers (ZFs) are protein/DNA interaction domains capable of targeting specific sequences of DNA.