ALBERT

Description: Fetal hemoglobin (HbF) is a major genetic modifier of the phenotypic heterogeneity in patients with the major β-globin disorders sickle cell disease (SCD) and β-thalassemia. Although the normal level of HbF postnatally is approximately 1% of total hemoglobin, some individuals have a condition known as hereditary persistence of fetal hemoglobin (HPFH), characterized by elevated synthesis of γ-globin in adulthood. HPFH is caused by small or large deletions in the β-globin locus (deletional HPFH), or point mutations in the Aγ-globin or Gγ-globin gene promoters (non-deletional HPFH). Pharmacological agents such as butyrate, decitabine, and hydroxyurea are effective in inducing HbF in vitro and in vivo. To date, hydroxyurea is the only drug approved for clinical use in sickle cell patients, although the efficacy level is variable between patients and the long-term effects of this drug remain uncertain. Therefore, current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin gene expression in adult life. Many studies have demonstrated the role of stage-specific transcription factors in β-like globin gene switching, indicating their potential as therapeutic targets in the treatment of β-hemoglobinopathies. In order to better understand the molecular pathways involved in the regulating γ-globin gene expression, we used β-YAC transgenic mice, produced with a 213 Kb β-globin locus yeast artificial chromosome, containing a 187 Kb human chromosomal insert encompassing the entire 82 Kb β-globin locus from 5'HS5 of the LCR to 3'HS1, approximately 20 Kb downstream from the β-globin gene. Four different transgenic mouse lines were included in this study: 1) wild β-YAC mice, with the normal sequence of the human β-globin locus; 2) mutant β-YAC mice with the Aγ-globin -117 G〉A HPFH mutation 3) mutant β-YAC mice with the Aγ-globin -175 T〉C HPFH mutation, and 4) mutant β-YAC mice with the Aγ-globin -195 T〉C HPFH mutation. Adult -175 and -195 mutant β-YAC mice displayed an HPFH phenotype with an increased level of HbF. As measured by HPLC, -175 HPFH mice had the highest average level of γ-globin chains [16.4% γ/(γ+β)], followed by -195 HPFH mice (8.4%). Wild-type β-YAC control mice averaged 2.8% and -117 Greek HPFH β-YAC control mice displayed an average of 7.4%. Measurement of Aγ-globin mRNA by RNase protection analysis (RNAP) supported the HPLC data; γ/(γ+β) was 34%, 12.1%, 14.1% and less than 0.5% for -175 HPFH, -195 HPFH, -117 HPFH and wild-type β-YAC animals, respectively. Relative mRNA levels as determined by RT-qPCR were consistent with the RNAP results. Currently, we are examining our -175 and -195 HPFH mice for pancellular versus heterocellular distribution of HbF. To examine the molecular basis for the -175 and 195 HPFH phenotypes, fetal livers of these animals were collected on day E18 of gestation, after the fetal-to-adult β-like globin switch occurred, for chromatin immunoprecipitation (ChIP) analysis of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2 and LDB1. Previous unpublished DNA-protein array and ChIP data, comparing human primary erythroid cell cultures from normal donors and -195 HPFH individuals, showed a 6-fold enrichment of YY1 recruitment to the -195 region of the normal Aγ-globin promoter and a 5-fold enrichment of PAX1 recruitment to the HPFH mutant promoter, suggesting that YY1 may act as an A γ-globin gene repressor and PAX1 may be an activator when the -195 mutation is present. Preliminary ChIP experiments in β-YAC mice showed a similar pattern with YY1 enriched 2-fold in wild-type mice and PAX1 enriched 2-fold in -195 HPFH animals. Regarding -175 HPFH and wild-type β-YAC samples, we found occupancy enrichment of LMO2, TAL1 and LDB1 proteins (1.5-fold, 9-fold and 2.5-fold, respectively) in the -175 region of the Aγ-globin gene promoter in -175 HPFH β-YAC mice. Recently published studies in cell lines have shown that these three proteins form a complex with GATA-1 to mediate long-range interactions between the LCR and β-like globin genes. These mouse models provide additional tools for studying the regulation of γ-globin gene expression and may reveal new targets for selectively activating HbF. Disclosures No relevant conflicts of interest to declare.

Description: A 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) was modified by homologous recombination to delete 2.9 Kb of cross-species conserved sequence similarity encompassing the LCR 5′HS4 (Δ5′HS4 β-YAC). Three transgenic mouse lines were established; each contained two intact copies of the β-globin locus as determined by long range restriction enzyme mapping (LRRM) and Southern blot hybridization analyses. Human ε-, γ- and β-globin, and mouse α- and ζ-globin mRNAs were measured by RNAse protection in hematopoietic tissues derived from staged embryos, fetuses and adult mice. No difference in the temporal pattern of globin transgene expression was observed between Δ5′HS4 β-YAC mice and wild-type β-YAC mice. In addition, quantitative per-copy human β-like globin mRNA levels were similar between Δ5′HS4 and wild-type β-YAC transgenic lines, although γ-globin gene expression was slightly increased in the fetal liver, while β-globin gene expression was slightly decreased in Δ5′HS4 β-YAC mice. These data are in contrast to data obtained from β-YAC mice containing a deletion of the 280 bp 5′HS4 core. In these mice, γ- and β-globin gene expression was significantly decreased during fetal definitive erythropoiesis and β-globin gene expression was decreased during adult definitive erythropoiesis. However, these data are consistent with the observation that deletion of the 5′HS core elements is more deleterious than large deletions of the 5′HSs. Together, the compiled deletion data supports the hypothesis that the LCR exists as a holocomplex in which the 5′HS cores form an active site and the flanking 5′HS regions constrain the holocomplex conformation. In this model, 5′HS core mutations are dominant negative, whereas larger deletions allow the LCR to fold into alternate holocomplex structures that function normally, albeit less efficiently. To complete the study on the contribution of the individual 5′HSs to LCR function, a 0.8 Kb 5′HS1 fragment was deleted in the 213 Kb β-YAC by homologous recombination. Two ΔHS1 β-YAC transgenic lines have been established; four additional founders were recently identified. Of the two lines, one contains two intact copies of the globin locus; the other contains four deleted copies, one of which extends from the LCR through just 5′ to the β-globin gene. For both lines, ε-globin gene expression was markedly reduced, approximately 5–10 fold, during primitive erythropoiesis. Developmental expression profiles and levels of the γ- and β-globin genes (in the line that contains loci including the β-globin gene) were unaffected by deletion of 5′HS1. Breeding of the remaining four founders to obtain F1 and F2 progeny for similar structure/function studies is in progress. Decreased expression of the β-globin gene is the first phenotype ascribed to a 5′HS1 mutation, suggesting that this HS does indeed have a role in LCR function.

Description: A 2.9 Kb deletion of 5′HS3 (Δ5′HS3) or a 234 bp deletion of the 5′HS3 core (Δ5′HS3c) in a 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) abrogate ε-globin gene expression during primitive erythropoiesis in β-YAC transgenic mice, suggesting that HS sequences of the LCR are involved directly in ε-globin gene activation. The reduction of ε-globin gene transcription in Δ5′HS3 or Δ5′HS3c β-YAC transgenics can be explained by two hypotheses. The first is site-specificity. The interaction between the LCR and the ε-globin gene promoter involves specific sequences of 5′HS3 and specific sequences of the ε-globin gene promoter. When 5′HS3 or its core is deleted, these interactions do not take place and ε-globin gene transcription is diminished. The second hypothesis is change in conformation of the LCR. Normally, in the embryonic stage, the LCR achieves a three-dimensional conformation that favors interaction with the first gene in the complex, i.e., the ε-globin gene. When 5′HS3 is deleted, an alternate conformation is assumed that decreases the chance that there will be an interaction between the LCR and the ε-globin gene. However, the LCR interacts with the next gene in order, the γ-globin gene. In Δ5′HS3c β-YAC mice, γ-globin gene expression is normal during primitive erythropoiesis, but is extinguished in the fetal stage of definitive erythropoiesis. These data suggest that a conformational change occurs in the Δ5′HS3c LCR during the switch from embryonic to definitive erythropoiesis, from one that supports γ-globin gene expression to one that does not. Alternately, the embryonic trans-acting environment may allow the mutant LCR to interact with and activate the γ-globin genes, but the fetal trans-acting environment may not support this interaction in the absence of the 5′HS3 core. To distinguish between these possibilities, β-YAC lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (βm), coupled to either an intact LCR, a 2.9 Kb 5′HS3 deletion or a 234 bp 5′HS3 core deletion. Δ5′HS3c Δε::βm β-YAC mice expressed βm-globin throughout development beginning at day 10 in the yolk sac. γ-globin was expressed in the embryonic yolk sac, but not in the fetal liver. Some wild-type β-globin was expressed in addition to βm-globin in adult mice. The γ-globin phenotype is consistent with published data on Δ5′HS3c β-YAC mice. Although ε-globin was not expressed in Δ5′HS3c β-YAC mice, βm-globin was expressed in Δ5′HS3c Δε::βm β-YAC embryos, demonstrating that the 5′HS3 core was necessary for ε-globin expression during embryonic erythropoiesis, but not for βm-globin expression. These data support a site specificity model of LCR HS-globin gene interaction. In addition, nuclear ligation experiments provided evidence for a specific physical interaction between 5′HS3 and the γ-globin promoter during fetal definitive erythropoiesis, further supporting a site specificity model.

Description: Abstract 2019 Poster Board I-1041 Hereditary persistence of fetal hemoglobin (HPFH) is a condition associated with continued fetal hemoglobin (HbF) production in adults, where normally only very low levels of HbF are found. Sickle cell disease (SCD) patients are phenotypically normal if they carry a compensatory HPFH mutation due to the high levels of HbF. Understanding the molecular mechanisms leading to reactivation or derepression of γ-globin gene expression will lead to the development of new or better therapies to treat SCD patients. In our long-established and highly-characterized model system, transgenic mice carrying wild-type human β-globin locus yeast artificial chromosomes (β-YACs) express predominantly γ-globin and a lesser amount of γ-globin in the primitive erythroid cells of the yolk sac, mostly β-globin and some γ-globin in the definitive erythroid cells of the fetal liver and nearly exclusively β-globin in the adult definitive red blood cells, as measured both at the transcript and protein levels. We recently identified a novel Aγ-globin gene silencer motif located at -566 relative to the mRNA CAP site in a GATA motif. Repression is mediated by binding a GATA-1-FOG-1-Mi2 protein complex. Since our initial studies of this GATA-1 repressor complex were performed using β-YAC transgenic mice in which a second copy of the Aγ-globin gene was introduced between the locus control region (LCR) and the γ-globin gene, our first goal was to test if this mutation was functional at the normally-located Aγ-globin globin gene. β-YAC transgenic mice were produced with the T〉G HPFH point mutation at the -566 GATA site of this gene. These mice display a mild HPFH phenotype during adult definitive erythropoiesis; γ-globin gene expression levels were increased approximately 3% compared to wild-type β-YAC mice. Expression of γ-globin is also elevated relative to wild-type β-YAC controls during primitive erythropoiesis in the embryonic yolk sac and definitive erythropoiesis in the fetal liver. Chromatin immunoprecipitation (ChIP) experiments using day E12 to E18 post-conception fetal liver samples from wild type β-YAC transgenic mice demonstrate that GATA-1 is recruited to this GATA silencer first at day E16, followed by recruitment of FOG-1 and Mi2 at day E17. In addition, ChIP experiments performed with day E18 samples from the -566 HPFH mice demonstrate that this point mutation disrupts the recruitment of GATA-1 to this site at a developmental stage when it normally binds as a repressor in wild-type β-YAC transgenic samples. GATA-2 does not bind at the -566 GATA motif when γ-globin is actively transcribed. Thus, GATA-2/GATA-1 competition does not play a role in the function of this silencer or the mechanism of HPFH at this site. In addition, BCL11A does not appear to be a component of this GATA-1 repressor complex. Taken together our data indicate that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that the presence of the -566 Aγ-globin HPFH mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

Description: Hereditary persistence of fetal hemoglobin (HPFH) is a result of mutations that prevent the silencing of the g-globin genes during the adult stage of definitive erythropoiesis. Two types of HPFH are recognized, deletional HPFH and non-deletional HPFH. Mutations in the later class have been identified in the proximal promoters of the Ag- and Gg-globin genes. Individuals homozygous for sickle cell disease or certain b-thalassemia mutations, that have in addition a HPFH mutation, do not suffer the deleterious effects of these diseases. These subjects provide the natural evidence supporting the clinical effort to reactivate fetal hemoglobin as the major treatment for SCD and b-thalassemias. Thus, understanding the molecular mechanisms regulating the g-globin genes is essential for identification of points of therapeutic intervention. Although the number of point mutations causing HPFH has grown over the years, the biochemical mechanisms affected by these alterations remains elusive. In addition, it is unlikely that all potential mutations have been identified in humans. A complete catalog of all potential HPFH point mutations, coupled with knowledge of the transcriptional processes affected by them will be an invaluable step towards effectively treating these diseases. We recently identified a novel T〉A HPFH mutation in a GATA site at position -566 of the Ag-globin promoter, the most distal in the promoter to date, that affects binding of a GATA-1-FOG-1-Mi2 repressor complex. Since this study utilized mutated human b-globin locus yeast artificial chromosome (b-YAC) transgenic mice, where a second copy of the Ag-globin gene was introduced near the locus control region, we produced b-YAC transgenic mice containing the -566 mutation at the normally located Ag-globin gene. These mice display a mild HPFH phenotype, an approximately 3% increase in g-globin gene expression, compared to wild-type b-YAC mice. Chromatin immunoprecipitation (ChIP) studies demonstrated that this mutation prevents GATA-1 binding when g-globin is repressed in post-conception day 18 (E18) fetal liver, whereas recruitment was observed in wild-type b-YAC transgenic samples from the same developmental stage. These data are consistent with the presence of a GATA-1-mediated repressor complex at this GATA site when g-globin is not expressed. GATA-1-mediated repression may be a general mechanism of g-globin silencing. To begin testing this hypothesis, we utilized previously generated Ag-globin -117 G〉A Greek HPFH b-YAC transgenic mice, which show a 5–8% increase in g-globin synthesis in adult erythropoiesis. Published data suggested that this mutation affects nearby GATA-1 binding. Our ChIP data confirmed these results, however the GATA-1 multi-protein complex that is affected may differ from that recruited to the -566 GATA binding site. Finally, we have developed a cell-based selection that is being used to identify a comprehensive set of Ag-globin HPFH promoter mutations. Chemical inducer of dimerization (CID)-dependent Ag-globin promoter-eGFP b-YAC bone marrow cells were derived from transgenic mice and mutagenized with N-ethyl, N-nitrosourea (ENU). These cells are normally GFP−; treatment with g-globin-inducers or the presence of the -117 Greek HPFH mutation results in GFP+ cells. GFP+ cells were collected by FACS and individual cell clones expanded so that genomic DNA could be isolated. Promoter proximal regions were amplified by four PCR primer sets and subjected to heteroduplex analysis with the corresponding wild-type Ag-globin promoter PCR products as the control amplicons. Twenty three heteroduplexes have been detected among 158 mutant clones screened. Most are clustered in the proximal promoter. These data suggest that we have produced HPFH mutations, likely consisting of those known in human populations, as well as novel sites that affect repressor binding or enhance recruitment of transcriptional activators.

Description: Understanding the molecular mechanisms of erythropoiesis is critical for treating anemia and other hematopoietic diseases, which affect roughly 3 million Americans and 28% of the global population. The role of post-translational modification (PTM) of proteins in regulating developmental and differentiation processes is understudied, but recently we established that O-GlcNAcylation regulates erythropoiesis. O-GlcNAc regulates numerous cellular functions, including stress response, transcription, and cell cycle progression. O-GlcNAc is a single O-linked β-N-acetyl-D-glucosamine moiety added to serine/threonine amino acids of nuclear, cytoplasmic, and mitochondrial proteins. O-GlcNAc transferase (OGT), which adds the modification, and O-GlcNAcase (OGA), which removes the modification, are responsible for the dynamic processing of the PTM. In response to environmental cues, the variable cycling of O-GlcNAc on and off proteins has potential effects on transcriptional pathways essential for differentiation. Previously, we demonstrated that O-GlcNAc plays a role in regulating human γ-globin gene transcription during development in human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice and derivative immortalized bone marrow cells. O-GlcNAcylation modulates the formation of a GATA-1-FOG-1-NuRD repressor complex that binds the -566 GATA site of the Aγ-globin promoter when γ-globin gene expression is silent. OGT and OGA interact with GATA-1 and CHD4, a component of the NuRD complex. O-GlcNAcylation of CHD4 stimulates the formation of this repressor complex, blocking O-GlcNAcylation of CHD4 maintains Aγ-globin gene expression. Thus, O-GlcNAc cycling is a novel γ-globin regulatory mechanism, which might be modulated to increase fetal hemoglobin (HbF). Since O-GlcNAcylation involves input from multiple metabolic pathways, the modification acts as a general sensor of cellular homeostasis. Thus, in response to environmental cues, the addition and removal of O-GlcNAc from proteins may be variably altered with potential effects on biochemical and transcriptional pathways essential for erythropoiesis. To better understand how O-GlcNAcylation affects erythropoiesis in vivo, we developed several new, innovative mouse models. These include erythroid-specific OGT or OGA conditional knockout mice, and transgenic mice with erythroid-specific enforced expression of human OGT or OGA. OGT is an essential gene; erythroid-specific knockout results in fetal death due to severe anemia between day E12-14. OGA is not essential for erythropoiesis; no overt phenotype is observed. Based on previous our previous studies, we hypothesize that at the onset of erythroid lineage commitment, GATA-1 functions as an adaptor protein to deliver OGT and OGA to erythroid-specific cis-regulatory DNA elements, where they modify transcription complex or chromatin proteins responsible for directing transcriptional networks necessary for normal erythroid development and terminal differentiation. Currently, we are exploring how GATA-1-adaptor function mediates changes in the global O-GlcNAcylation pattern following the GATA-2 to GATA-1 switch that triggers erythroid differentiation. We are also examining the roles of OGT and OGA in the formation and function of the GATA-1-FOG-1-NuRD γ-globin repressor complex. Novel CRISPR/Cas9-based genome targeting tools were developed to probe these questions. We present phenotypic and molecular data related to the hematopoietic system, including anemia, blood cell histology and morphology, standard blood indices, and β-like globin gene expression during embryonic, fetal, and adult stages of erythropoiesis in our mouse models. In addition, we will show preliminary data using the enzymatically dead dCas9 tools we have synthesized, dCas9-OGT and dCas9-OGA protein fusions that are delivered to cis-regulatory elements controlling erythroid-specific genes involved in erythropoiesis and globin gene switching. The therapeutic outcome will be the identification of erythroid-specific protein targets whose activity can be modulated by altering their O-GlcNAcylation status. We emphasize that because the O-GlcNAc cycle has pleiotropic effects within the cell, it is not a good direct target for therapeutic intervention. However, many of the target proteins are likely to be suitable for treatment venues. Disclosures No relevant conflicts of interest to declare.

Description: Sickle cell disease (SCD) affects millions of people around the world and is the most common inherited disease in the United States, affecting 1 of 1800 births and 1 of 400 African-American births. Patients with SCD have an improved clinical course when fetal hemoglobin (HbF) levels are increased, and this is the basis for treatment with hydroxyurea as a disease modifying therapy. Hydroxyurea has been shown to decrease the frequency of pain episodes and decrease the incidence of stroke in patients who are at risk based on transcranial doppler velocities. Hydroxyurea is one of two FDA approved treatments for SCD patients, so there is need for new therapies, including the identification of druggable transcriptional activators that specifically up-regulate the gamma-globin genes (HbF). Understanding the mechanisms underlying control of globin gene expression, particularly those involved in activation of gamma-globin gene expression, is important for developing new treatments for SCD. Metal-responsive transcription factor-1 (MTF-1) is a key regulator of zinc metabolism in higher eukaryotes that controls the metal-inducible expression of metallothioneins and a number of other genes directly involved in the intracellular sequestration and efflux transport of zinc. Bao et al published that adults with SCD showed increased red blood cells (RBCs), hemoglobin (Hb) and hematocrit levels after 3 months of zinc supplementation compared to the placebo group. Furthermore, previous studies demonstrated that MTF-1 plays an essential role in liver development and that MTF-1-deficient mice display an anemic phenotype, suggesting a role for MTF-1 in hematopoiesis. In our study, we observed a 2.4-fold increase in gamma-globin expression in K562 cells at 4 hours. We also demonstrated increased gamma-globin expression in adult blood from MTF-1 overexpression, human beta-globin locus yeast artificial chromosome (beta-YAC) bi-transgenic (bigenic) mouse lines at the mRNA level by quantitative real-time RT-PCR (qPCR) and at the protein level by FACS analysis. Lastly, gamma-globin gene expression was induced 12-fold in bone marrow cells (BMCs) derived from these bigenic mice compared to BMCs derived from beta-YAC-only mice, and 3-fold after 6 hours of zinc treatment in beta-YAC-only BMCs. Co-immunoprecipitation (Co-IP) analysis showed that GATA-2 associated with MTF-1 in MTF-1 overexpression, beta-YAC BMCs, but not in beta-YAC-only BMCs, suggesting that reactivation of gamma-globin expression by MTF-1 might be mediated by a MTF-1-GATA-2 protein complex. Chromatin immunoprecipitation (ChIP) experiments indicated that MTF-1 and GATA-2 co-occupy the same sites in the gamma-globin promoter. Our data suggest that activation of gamma globin by MTF-1 is mediated by protein-protein interaction with GATA-2 and that this multi-protein complex is targeted to GATA sites located in the gamma-globin gene-promoters via binding of the GATA-2 protein. Based on our data, we hypothesize that zinc supplementation will increase plasma HbF by inducing MTF-1 thus activating gamma-globin gene expression. This protein represents a potential new target in strategies to reactivate gamma-globin in hemoglobinopathies where higher levels of HbF would have beneficial effects. More clinical and basic science studies are needed to further characterize the role of zinc in globin gene expression and hemoglobin F levels in the clinical setting. Disclosures No relevant conflicts of interest to declare.