Β-globin Locus Yeast Artificial Chromosome Research Articles

O-Glcnacylation Modulates Erythropoiesis: O-Glcnac Transferase (OGT) Is an Essential Gene

Post-translational modifications (PTMs) can regulate protein activity; one such modification is O-GlcNAcylation. O-GlcNAcylation is involved in the regulation of many cellular processes such as stress response, cell cycle progression, and transcription. The O-GlcNAc PTM is the attachment of a single N-acetyl-glucosamine moiety to either a serine or threonine residue on nuclear and cytoplasmic proteins. O-GlcNAc is added to proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) in response to changes in extracellular signals and nutrients. A balance in protein levels exists between these two enzymes; an increase or decrease of one results in a like compensatory change in the other. Thus, the rate of O-GlcNAc addition and removal is a dynamic cycling event that is exquisitely controlled for a given target molecule, which may be exploited therapeutically to alter gene expression. We previously 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. OGT and OGA interact with Mi2β, a component of the GATA-1-FOG-Mi2b repressor complex that binds the -566 GATA site of the Aγ-globin promoter when γ-globin gene expression is silent. O-GlcNAcylation of Mi2βstimulates formation of this repressor complex, whereas removal of this PTM results in activation of 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 a 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 the mechanism as to how O-GlcNAcylation regulates β-like globin gene switching and whether it plays a role in erythropoiesis, we developed several new, innovative mouse models. These include erythroid-specific OGT conditional knockout mice, and transgenic mice with erythroid-specific enforced expression of human OGT or OGA to examine the effect of OGT loss-of-function or OGT/OGA gain-of-function on erythroid development in vivo . We utilized erythropoietin receptor promoter-Cre (ErGFPcre) mice or produced new tamoxifen-inducible μLCR-β-globin promoter-Cre (μLCR-β promoter-ERT2CreERT2) mice that were bred with floxed OGT mice to produce early erythroid progenitor-specific or tamoxifen-inducible erythroid-specific knockouts of OGT, respectively. In all instances, the final mice also contained the human β-YAC reporter transgene. Knockout of OGT using ErGFPcre results in fetal death during embryogenesis. Although conceptuses may be recovered up through embryonic day E12, they completely lack red blood cells and some organ anomalies are observed. An intermediate, anemic phenotype is observed when the knockout is incomplete. Adult floxed OGT, μLCR-β promoter-ERT2CreERT2 β-YAC mice treated with tamoxifen develop an anemia that is dose-time-dependent and ultimately correlates with the level of OGT still present. 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 for both the LOF and GOF mice.Understanding the molecular and cellular components of erythropoiesis is critical for treating anemia and other hematopoietic diseases which affect roughly 3 million Americans and 28% of the population worldwide. In a more general sense, our work may provide insight into the role of O-GlcNAc in regulating developmental and differentiation processes. DisclosuresNo relevant conflicts of interest to declare.

O-Glcnacylation Is Essential for Erythropoiesis

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.

Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells.

As high fetal hemoglobin levels ameliorate the underlying pathophysiological defects in sickle cell anemia and beta (β)-thalassemia, understanding the mechanisms that enforce silencing of fetal hemoglobin postnatally offers the promise of effective molecular therapy. Depletion of the Nucleosome Remodeling and Deacetylase complex member MBD2 causes a 10-20-fold increase in γ-globin gene expression in adult β-globin locus yeast artificial chromosome transgenic mice. To determine the effect of MBD2 depletion in human erythroid cells, genome editing technology was utilized to knockout MBD2 in Human Umbilical cord Derived Erythroid Progenitor-2 cells resulting in γ/γ+β mRNA levels of approximately 50% and approximately 40% fetal hemoglobin by high performance liquid chromatography. In contrast, MBD3 knockout had no appreciable effect on γ-globin expression. Knockdown of MBD2 in primary adult erythroid cells consistently increased γ/γ+β mRNA ratios by approximately 10-fold resulting in approximately 30-40% γ/γ+β mRNA levels and a corresponding increase in γ-globin protein. MBD2 exerts its repressive effects through recruitment of the chromatin remodeler CHD4 via a coiled-coil domain, and the histone deacetylase core complex via an intrinsically disordered region. Enforced expression of wild-type MBD2 in MBD2 knockout cells caused a 5-fold decrease in γ-globin mRNA while neither the coiled-coil mutant nor the intrinsically disordered region mutant MBD2 proteins had an inhibitory effect. Co-immunoprecipitation assays showed that the coiled-coil and intrinsically disorder region mutations disrupt complex formation by dissociating the CHD4 and the histone deacetylase core complex components, respectively. These results establish the MBD2 Nucleosome Remodeling and Deacetylase complex as a major silencer of fetal hemoglobin in human erythroid cells and point to the coiled-coil and intrinsically disordered region of MBD2 as potential therapeutic targets.

O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2β Protein at the Aγ-Globin Promoter

One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2β repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in β-globin locus yeast artificial chromosome (β-YAC) bone marrow cells. When WT β-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2β at the (A)γ-globin promoter is increased. In addition, OGT and Mi2β recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human β-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2β is modified with O-GlcNAc, and both OGT and OGA interact with Mi2β, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2β repressor complex at the -566 GATA motif within the promoter.

Original Research: Generation of non-deletional hereditary persistence of fetal hemoglobin β-globin locus yeast artificial chromosome transgenic mouse models: -175 Black HPFH and -195 Brazilian HPFH.

Fetal hemoglobin is a major genetic modifier of the phenotypic heterogeneity in patients with sickle cell disease and certain β-thalassemias. Normal levels of fetal hemoglobin postnatally are approximately 1% of total hemoglobin. Patients who have hereditary persistence of fetal hemoglobin, characterized by elevated synthesis of γ-globin in adulthood, show reduced disease pathophysiology. Hereditary persistence of fetal hemoglobin is caused by β-globin locus deletions (deletional hereditary persistence of fetal hemoglobin) or γ-globin gene promoter point mutations (non-deletional hereditary persistence of fetal hemoglobin). Current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin in adult life. To better understand these pathways, we generated new β-globin locus yeast artificial chromosome transgenic mice bearing the (A)γ-globin -175 T > C or -195 C > G hereditary persistence of fetal hemoglobin mutations to model naturally occurring hereditary persistence of fetal hemoglobin. Adult -175 and -195 mutant β-YAC mice displayed a hereditary persistence of fetal hemoglobin phenotype, as measured at the mRNA and protein levels. The molecular basis for these phenotypes was examined by chromatin immunoprecipitation of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2, and LDB1. In -175 HPFH versus wild-type samples, the occupancy of LMO2, TAL1 and LDB1 proteins was enriched in HPFH mice (5.8-fold, 5.2-fold and 2.7-fold, respectively), a result that concurs with a recent study in cell lines showing that these proteins form a complex with GATA-1 to mediate long-range interactions between the locus control region and the (A)γ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of (A)γ-globin activation, in contrast to other hereditary persistence of fetal hemoglobin mutations that result in a loss of repression. The mice provide additional tools to study γ-globin gene expression and may reveal new targets for selectively activating fetal hemoglobin.

Induction of Fetal Hemoglobin and Reduction of Disease Pathology in Sickle Cell Mice By a Synthetic Zinc Finger Gamma-Globin Activator

A plethora of research has established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the only FDA-approved treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Thus, research has focused on identifying agents that specifically reactivate γ-globin gene expression during adult definitive erythropoiesis, with minimal off-target effects. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the -117 region of the A γ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in K562 cells, murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene, in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients, and in vivo, in gg1-VP64 β-YAC double transgenic (bigenic) mice. Transgenic mice with enforced expression of the gg1-VP64 fusion protein only in the erythroid-megakaryocytic compartment were crossed into the Townes sickle cell knock-in mouse (Jackson Laboratory) background. Compared with control sickle cell (HbSS) mice, gg1-VP64 ATF sickle cell (gg1-HbSS) mice had hematological values at levels found in wild-type homozygous or heterozygous adult hemoglobin (HbAA or HbAS, respectively) mice. For example, average RBC (106/mm3) was 11.7 for wild-type mice and 12.9 for gg1 HbSS, compared to 8.2 for HbSS mice. Average HGB (g/dl) was 15.1 for wild-type mice and gg1 HbSS mice, versus 10.0 for HbSS mice. Average HCT was 52.5% for wild-type mice, 53.7% for gg1 HbSS mice, but only 41.5% for HbSS mice. Finally, average WBC (103/mm3) was 9.4 for wild-type mice, 9.0 for gg1 HbSS mice and 91.0 for HbSS mice. HPLC and Western blot analysis to determine the effect of gg1-VP64 on HbF synthesis are underway. In addition, we are examining mice for numbers of HbF-positive cells, mature cells, and reticulocytes, as well as looking at organ damage. Our results demonstrate that the ATF class of reagent may be an effective gene therapy for treatment of SCD. DisclosuresMakala:Georgia Regents University: Employment.

Generation of Non-Deletional Hereditary Persistence of Fetal Hemoglobin (HPFH) Beta-Yac Transgenic Mouse Models: -175 Black HPFH and -195 Brazilian HPFH

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. DisclosuresNo relevant conflicts of interest to declare.

O-Glcnacylation Regulates γ-Globin Transcription

Patients with sickle cell disease (SCD), caused by mutation of the adult β-globin gene, are phenotypically normal if they carry compensatory mutations that result in continued expression of the fetal γ-globin genes, a condition termed hereditary persistence of fetal hemoglobin (HPFH). Thus, a logical clinical goal for treatment of SCD is to up-regulate γ-globin synthesis using compounds that are specific for increasing fetal hemoglobin (HbF) without pleiotropic effects on cellular homeostasis. Developmental regulation of the γ-globin genes is complex and normal silencing during the adult stage of erythropoiesis likely results from a combination of the loss of transcriptional activators and the gain of transcriptional repressor complexes. One mode of γ-globin silencing occurs at the GATA binding sites located at -566 or -567 relative to the Aγ-globin or Gγ-globin CAP sites respectively, and is mediated through the DNA binding moiety of GATA-1 and its recruitment of co-repressor partners, FOG-1 and Mi-2 (NuRD complex). Modifications of repressor complexes can regulate gene transcription; one such modification is O-GlcNAcylation. The O-GlcNAc post-translational modification is the attachment of a single N-acetyl-glucosamine moiety to either a serine or threonine residue on nuclear and cytoplasmic proteins. O-GlcNAc is added to proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) in response to changes in extracellular signals and nutrients. A dynamic balance in protein levels also exists between these two enzymes; an increase or decrease of one results in a like compensatory change in the other. Thus, the rate of O-GlcNAc addition and removal is a dynamic cycling event that is exquisitely controlled for a given target molecule, which may offer a point of intervention in the turning off or on of gene expression. O-GlcNAcylation is involved in the regulation of many cellular processes such as stress response, cell cycle progression, and transcription. Potentially, O-GlcNAc plays a pivotal role in regulating transcription of the human γ-globin genes. We induced human erythroleukemia cell line K562 with sodium butyrate to differentiate toward the erythroid lineage and observed the expected increase of γ-globin gene expression. A robust increase of γ-globin gene expression was measured after pharmacological inhibition of OGA using Thiamet-G (TMG). Using chromatin immunoprecipitation (ChIP), we demonstrated that OGT and OGA are recruited to the -566 region of the Aγ-globin promoter, the same region occupied by the GATA-1-FOG-1-Mi-2 (NuRD) repressor complex. However, OGT recruitment to this region was decreased when O-GlcNAc levels were artificially elevated by OGA inhibition with TMG. When γ-globin expression was not induced, Mi-2 was modified with O-GlcNAc and interacted with both OGT and OGA. After induction, O-GlcNAcylation of Mi-2 was reduced and Mi2 no longer interacted with OGT. Stable K562 cells were generated in which OGA was knocked down using shRNA. Following induction of these cells with sodium butyrate, γ-globin gene expression was higher compared to control cells. These data suggest that the dynamic cycling of O-GlcNAc on the Mi-2 (NuRD) moiety contributes towards regulation of γ-globin transcription. Concurrent ChIP experiments in human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice demonstrated that GATA-1, Mi2 and OGT were recruited to the -566 Aγ-globin GATA silencer site in day E18 fetal liver when γ-globin is repressed, but not in day E12 fetal liver when γ-globin is expressed. These data demonstrate that O-GlcNAc cycling is a novel mechanism regulating γ-globin gene expression and will provide new avenues to explore in how alterations in gene regulation lead to the onset, progression, and severity of hematological disease. Disclosures:No relevant conflicts of interest to declare.

A Cell-Based High-Throughput Screen For Novel Inducers Of Fetal Hemoglobin For Treatment Of Sickle Cell Disease, Cooley's Anemia and β-Thalassemias

Decades of research has established that the most effective treatment for sickle cell disease (SCD) and Cooley's anemia is increased fetal hemoglobin (HbF). Certain β-thalassemias may also benefit from fetal hemoglobin induction. Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the most widely available treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Identification of a drug specific for inducing or reactivating γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. An assay system developed in our lab to understand the mechanisms underlying the γ- to β-globin gene expression switch during development allowed us to generate a cell-based assay that was adapted for a HTS of 121,085 compounds from the libraries of the KU-HTS Laboratory (Prestwick, MicroSource, CMLD, Chembridge and ChemDiv compound libraries) and LCGC (OCL compound library). Transgenic mice were produced using a modified 213 Kb human β-globin locus yeast artificial chromosome (β-YAC). Two gene fusions were introduced into the β-YAC via homologous recombination in the host yeast, firefly luciferase was fused to the Aγ-globin promoter and Renilla luciferase was fused to the β-globin promoter. The resultant YAC was microinjected into fertilized mouse oocytes to produce transgenic mice. We used these mice to derive chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) containing the γ-luc/β-luc β-YAC, which were employed in the HTS. We identified 232 primary screen actives that induced γ-globin 2-fold or higher. A 4-assay, 10-point dose-response secondary screen using the same CID-dependent γ-luc/β-luc β-YAC BMCs reconfirmed that 211 of these active compounds induced γ-globin ≥2-fold with minimal or no β-globin induction, minimal cytotoxicity and did not directly inhibit purified luciferase enzyme. Additional secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the characteristics of seven of these 233 hits that were cherry-picked for further analysis. Four of the compounds were particularly promising, numbers 7, 42, 87 and 208. In CID-dependent wild-type β-YAC BMCs using the optimal dose for each compound, γ-globin mRNA induction ranged from 3- to 42-fold compared to 10-fold with sodium butyrate as measured by real-time qRT-PCR; F-cells ranged from 9.9-29.9% compared to 0.7% untreated and 15.9% treated with sodium butyrate as measured by flow cytometry. In human primary erythroid progenitor cells, the mRNA change was 1.6- to 3-fold compared to 1.75-fold with sodium butyrate and F-cells ranged from 9.1-29% compared to 5.7% untreated and 39.4% treated with sodium butyrate. Lead compounds will be tested in a pre-clinical β-YAC transgenic mouse model to determine their ability to induce HbF in vivo to aid development of these compounds for future clinical applications in hemoglobinopathies. Disclosures:No relevant conflicts of interest to declare.

LCR 5′ hypersensitive site specificity for globin gene activation within the active chromatin hub

The DNaseI hypersensitive sites (HSs) of the human β-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human β-globin locus yeast artificial chromosome (β-YAC) lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (βm), coupled to an intact LCR, a 5′HS3 complete deletion (5′ΔHS3) or a 5′HS3 core deletion (5′ΔHS3c). The 5′ΔHS3c mice expressed βm-globin throughout development; γ-globin was co-expressed in the embryonic yolk sac, but not in the fetal liver; and wild-type β-globin was co-expressed in adult mice. Although the 5′HS3 core was not required for βm-globin expression, previous work showed that the 5′HS3 core is necessary for ε-globin expression during embryonic erythropoiesis. A similar phenotype was observed in 5′HS complete deletion mice, except βm-globin expression was higher during primitive erythropoiesis and γ-globin expression continued into fetal definitive erythropoiesis. These data support a site specificity model of LCR HS-globin gene interaction.

Induction of Fetal Hemoglobin by Transcriptional Co-Activators MTF-1 and TSPYL1

Abstract 353Sickle cell disease (SCD) impacts one of 400 African-Americans born each year. Augmentation of fetal hemoglobin (HbF) levels is widely accepted as the most effective method for treating SCD, but hydroxyurea (HU) is currently the only approved drug that increases HbF. Thus, there is a need for the development of new therapies for this disease, including the identification of transcriptional activators that specifically up-regulate γ-globin (HbF). Developmental regulation of human β-like globin gene switching is controlled by several parameters, including cis- and trans-acting transcriptional determinants. Understanding the mechanisms underlying control of globin gene expression, particularly those involved in activation of γ-globin expression (HbF) 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. 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, when murine MTF-1 was expression was enforced, we observed a 5-fold increase in γ-globin expression in K562 cells. We also demonstrated increased γ-globin expression in adult blood from MTF-1 human β-globin locus yeast artificial chromosome (β-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, γ-globin gene expression was induced 12-fold in bone marrow cells (BMCs) derived from these bigenic mice compared to BMCs derived from β-YAC-only mice, and 3-fold after 6 hours of zinc treatment in β-YAC-only BMCs. Corroborative studies including zinc-deficient and zinc replete diets in β-YAC mice and erythroid-specific MTF-1 loss-of-function in loxP-flanked-MTF-1 LCR-β-globin promoter-Cre β-YAC mice further support a role for MTF-1 in g-globin gene expression. Chromatin immunoprecipitation (ChIP) analysis did not show recruitment of MTF-1 to any γ-globin gene-proximal metal response elements (MREs), the DNA motif that MTF-1 binds to control zinc metabolism genes. However, GATA-2 co-immunoprecipitated with MTF-1 in MTF-1 β-YAC BMCs, but not in β-YAC-only BMCs, suggesting that reactivation of γ-globin expression by MTF-1 might be mediated by a MTF-1-GATA-2 protein complex. ChIP experiments indicated that MTF-1 and GATA-2 co-occupy the same sites in the γ-globin promoter. Two of the stronger co-recruitment regions contain not only GATA sites, but also non-canonical MREs that vary by 1 or 2 bp from the canonical 7 bp MRE core. Interestingly, GATA-2 was induced 2-fold in adult blood of MTF-1 β-YAC mice, and also 3.5-fold in MTF-1 β-YAC BMCs treated with zinc for 6 hours. Our data suggest that activation of γ-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 γ-globin gene-promoters via binding of the GATA-2 protein.In a previous study we identified testis-specific protein Y-like 1 (TSPYL1) as a candidate gene involved in activation of γ-globin (de Andrade et al., 2006, Blood Cells, Mol. & Dis. 37:82). TSPYL1 mRNA level was increased 2–5 fold in deletional hereditary persistence of fetal hemoglobin (HPFH-2) subjects and decreased in a carrier of the Sicilian δβ-thalassemia trait. TSPYL1 is a transcription factor that is a member of the nucleosome assembly protein (NAP) family. TSPYL1 is not a DNA-binding protein; thus it exerts its effect through protein-protein interactions. When we enforced expression of human TSPYL1 in K562 cells an 11-fold induction of γ-globin expression was obtained. A reduction of γ-globin expression was observed following TSPYL1 knockdown in K562 cells. qPCR analysis of blood from TSPYL1 β-YAC bigenic mice showed that γ-globin expression was increased 4–12-fold. Taken together, our data strongly support the evidence that MTF-1 and TSPYL1 reactivate γ-globin expression in adult erythropoiesis. These two proteins represent potential new targets in strategies to reactivate γ-globin in hemoglobinopathies where higher levels of HbF would have beneficial effects. Disclosures:No relevant conflicts of interest to declare.

Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site

Autonomous silencing of gamma-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the (A)gamma-globin gene was identified between -730 and -378 relative to the mRNA start site. A marked copy of the (A)gamma-globin gene inserted between locus control region 5' DNase I-hypersensitive site 1 and the epsilon-globin gene was transcriptionally silenced in adult beta-globin locus yeast artificial chromosome (beta-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the -566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low gamma-globin levels, whereas only a weak signal was detected when gamma-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from beta-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the (A)gamma-globin -566 or (G)gamma-globin -567 GATA site when gamma-globin expression was low (day 18) but not when gamma-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, gamma-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the -566/-567 GATA sites of the proximal gamma-globin promoters.

Silencing of γ-Globin Gene Expression during Adult Definitive Erythropoiesis Is Mediated by a GATA-1 Repressor Complex.

Autonomous silencing of gene expression is one mechanism operative in the control of human β-like globin gene switching. Experiments using variously truncated Aγ-globin genes linked to LCR sequences suggested that a region of the Aγ-globin gene between -730 to -378 relative to the mRNA CAP site may function as an adult stage-specific silencer element. A marked copy of the Aγ-globin gene (Aγm-globin) was inserted between LCR 5′ HS1 and the ε-globin gene in a human β-globin locus yeast artificial chromosome (Aγm 5′ ε β-YAC). The Aγm-globin gene was autonomously silenced in Aγm 5′ ε β-YAC transgenic mice, even in the absence of an adult β-globin gene. A -730 to -378 deletion of the Aγm-globin gene was introduced into the Aγm 5′ ε β-YAC to produce a Δ1s Aγm 5′ ε β-YAC. Transgenic lines containing intact β-globin loci expressed the Δ1s Aγm-globin gene in embryonic yolk sac, fetal liver, and adult blood. To further delineate the function of the Δ1s fragment, transient transfection assays and protein-DNA interaction assays were performed. The Δ1s fragment was found to act as a repressor of a constitutively active SV40 promoter in K562 cells. DNaseI footprinting analysis and electromobility shift assays demonstrated GATA-1-binding at a site -570 bp upstream of the Aγ-globin CAP site. Recently generated β-YAC transgenic mice containing a T>G point mutation at the -570 GATA site of the normally-located Aγ-globin gene displayed a HPFH phenotype. Together, these data suggested that the -730 to -378 Aγ-globin gene region contains a silencer element at the -570 GATA site that binds a GATA-1 repressor complex during the adult stage of definitive erythropoiesis to silence expression of the Aγ-globin gene. Previous studies suggested that when GATA-1 functions as a repressor, it interacts with components of the MeCp1/NuRD complex. This complex may remodel chromatin into a repressed state, leading to silenced Aγ-globin gene expression during adult definitive erythropoiesis. The presence of components of the MeCP1/NuRD complex was assessed in uninduced (γ-globin repressor present) and induced (γ-globin repressor absent) erythroid cells (K562 and KU812) and non-erythroid cells (HFF) by Western blot analysis using an antibody to Mi2, which is a component of the NuRD complex. Mi2 protein was observed in erythroid cells when the levels of γ-globin were low (uninduced K562 or KU812 cells), whereas only a weak signal was detected when γ-globin expression was induced in these cells. The Mi2 signal in the HFF cells was even weaker. Chromatin immunoprecipitation (ChIP) using fetal liver samples from day E12 and E18 conceptuses of wild-type β-YAC transgenics showed that GATA-1, FOG-1 and Mi2 proteins co-localize to the -570 GATA site of the Aγ-globin gene in samples where γ-globin is silenced (E18 fetal liver), but not in samples where γ-globin is expressed (E12 fetal liver). Our data strongly suggest that the MeCP1/NuRD complex interacts with GATA-1 protein to form a repressor that may be involved in silencing Aγ-globin gene expression. In addition, we show that GATA-1, FOG-1 and Mi2 are recruited to the analogous -567 GATA site of Gγ-globin, in a pattern that parallels that of Aγ-globin. However the binding of these proteins to Gγ-globin is weaker than that observed for Aγ-globin. These data suggest that GATA-1-mediated repression is common to both γ-globin genes, but that other mechanisms function in the differential regulation of the two γ-globin genes.

Deletion of the human β-globin LCR 5′HS4 or 5′HS1 differentially affects β-like globin gene expression in β-YAC transgenic mice

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′ hypersensitive site (HS) 4 (Δ5′HS4 β-YAC). In three transgenic mouse lines, completion of the γ- to β-globin switch during definitive erythropoiesis was delayed relative to wild-type β-YAC mice. In addition, quantitative per-copy human β-like globin mRNA levels were similar to wild-type β-YAC transgenic lines, although β-globin gene expression was slightly decreased in the day 12 fetal liver of Δ5′HS4 β-YAC mice. A 0.8 kb 5′HS1 fragment was similarly deleted in the YAC. Three Δ5′HS1 β-YAC transgenic lines were established. ε-globin gene expression was markedly reduced, approximately 16 fold, during primitive erythropoiesis compared to wild-type β-YAC mice, but γ-globin expression levels were unaffected. However, during the fetal stage of definitive erythropoiesis, γ-globin gene expression was decreased approximately 4 fold at day 12 and approximately 5 fold at day 14. Temporal developmental expression profiles of the β-like globin genes were unaffected by deletion of 5′HS1. Decreased expression of the ε- and γ-globin genes is the first phenotype ascribed to a 5′HS1 mutation in the human β-globin locus, suggesting that this HS does indeed have a role in LCR function beyond simply a combined synergism with the other LCR HSs.

Human β-globin locus control region hypersensitive site specificity for globin gene activation during erythropoiesis

Although the human β-globin locus control region (LCR) functions as a holocomplex within an active chromatin hub, we provide evidence that within the aggregate hypersensitive site (HS) activation domain of the holocomplex, the individual HSs still mediate preferential activation of the globin genes during development. 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 e-globin gene expression during primitive erythropoiesis in β-YAC transgenic mice, suggesting that 5′HS3 sequences of the LCR are involved directly in e-globin gene activation. The reduction of e-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 e-globin gene promoter involves specific sequences of 5′HS3 and specific sequences of the e-globin gene promoter. When 5′HS3 or its core is deleted, these interactions do not take place and e-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, the e-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 e-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 e-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 Δe::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 e-globin was not expressed in Δ5′HS3c β-YAC mice, βm-globin was expressed in Δ5′HS3c Δe::βm β-YAC embryos, demonstrating that the 5′HS3 core was necessary for e-globin expression during embryonic erythropoiesis, but not for βm-globin expression. A similar phenotype was observed in Δ5′HS3 Δe::βm β-YAC mice, except βm-globin expression was higher in the day 10 yolk sac and γ-globin expression continued into the fetal liver stage of definitive erythropoiesis consistent with results published on Δ5′HS3 β-YAC mice. These data support a site specificity model of LCR HS-globin gene interaction.

Autonomous Silencing as Well as Competition Controls γ-Globin Gene Expression during Development

To investigate the control of the gamma-globin gene during development, we produced transgenic mice in which sequences of the beta-gene promoter were replaced by equivalent sequences of the gamma-gene promoter in the context of a human beta-globin locus yeast artificial chromosome (betaYAC) and analyzed the effects on globin gene expression during development. Replacement of 1,077 nucleotides (nt) of the beta-gene promoter by 1,359 nt of the gamma promoter resulted in striking inhibition of the gamma-promoter/beta-gene expression in the adult stage of development, providing direct evidence that the expression of the gamma gene in the adult is mainly controlled by autonomous silencing. Measurements of the expression of the gamma promoter/beta-globin gene as well as the wild gamma genes showed that gene competition is also involved in the control of gamma-gene expression in the fetal stage of development. We conclude that autonomous silencing is the main mechanism controlling gamma-gene expression in the adult, while autonomous silencing as well as competition between gamma and beta genes contributes to the control of gamma to beta switching during fetal development.

Human β-Globin Locus Control Region Hypersensitive Site Specificity for Globin Gene Activation during Erythropoiesis.

Although the human β-globin locus control region (LCR) functions as a holocomplex within an active chromatin hub, we provide evidence that within the aggregate hypersensitive site (HS) activation domain of the holocomplex, the individual HSs still mediate preferential activation of the globin genes during development. 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 5′HS3 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, 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. A similar phenotype was observed in Δ5′HS3 Δε::βm β-YAC mice, except βm-globin expression was higher in the day 10 yolk sac and γ-globin expression continued into the fetal liver stage of definitive erythropoiesis consistent with results published on Δ5′HS3 β-YAC mice. These data support a site specificity model of LCR HS-globin gene interaction.

γ-Globin Gene Expression in Chemical Inducer of Dimerization (CID)-dependent Multipotential Cells Established from Human β-Globin Locus Yeast Artificial Chromosome (β-YAC) Transgenic Mice

Identification of trans-acting factors or drugs capable of reactivating gamma-globin gene expression is complicated by the lack of suitable cell lines. Human K562 cells co-express epsilon- and gamma-globin but not beta-globin; transgenic mouse erythroleukemia 585 cells express predominantly human beta-globin but also gamma-globin; and transgenic murine GM979 cells co-express human gamma-and beta-globin. Human beta-globin locus yeast artificial chromosome transgenic mice display correct developmental regulation of beta-like globin gene expression. We rationalized that cells established from the adult bone marrow of these mice might express exclusively beta-globin and therefore could be employed to select or screen inducers of gamma-globin expression. A thrombopoietin receptor derivative that brings the proliferative status of primary mouse bone marrow cells under control of a chemical inducer of dimerization was employed to institute and maintain these cell populations. Human beta-globin was expressed, but gamma-globin was not; a similar expression pattern was observed in cells derived from fetal liver. gamma-Globin expression was induced upon exposure to 5-azacytidine, in cells derived from -117 Greek hereditary persistence of fetal hemoglobin human beta-globin locus yeast artificial chromosome (beta-YAC) mice, showing that the hereditary persistence of fetal hemoglobin (HPFH) phenotype was maintained in these cells or was reactivated by an artificial zinc finger-gamma-globin transcription factor and the previously identified fetal globin transactivators fetal Krüppel-like factor (FKLF) and fetal globin-increasing factor (FGIF). These cells may be useful for identifying transcription factors that reactivate gamma-globin synthesis or screening gamma-globin inducers for the treatment of sickle cell disease or beta-thalassemia.

Genome Architecture of the Human β-Globin Locus Affects Developmental Regulation of Gene Expression

To test the role of gene order in globin gene expression, mutant human beta-globin locus yeast artificial chromosome constructs were used, each having one additional globin gene encoding a "marked" transcript (epsilon(m), gamma(m), or beta(m)) integrated at different locations within the locus. When a beta(m)-globin gene was placed between the locus control region (LCR) and the epsilon-globin gene, beta(m)-globin expression dominated primitive and definitive erythropoiesis; only beta(m)-globin mRNA was detected during the fetal and adult definitive stages of erythropoiesis. When an (A)gamma(m)-globin gene was placed at the same location, (A)gamma(m)-globin was expressed during embryonic erythropoiesis and the fetal liver stage of definitive erythropoiesis but was silenced during the adult stage. The downstream wild-type gamma-globin genes were not expressed. When an epsilon(m)-globin gene was placed between the delta- and beta-globin genes, it remained silent during embryonic erythropoiesis; only the LCR-proximal wild-type epsilon-globin gene was expressed. Placement of a beta(m)-globin gene upstream of the (G)gamma-globin gene resulted in expression of beta(m)-globin in embryonic cells and in a significant decrease in expression of the downstream wild-type beta-globin gene. These results indicate that distance from the LCR, an inherent property of spatial gene order, is a major determinant of temporal gene expression during development.

LCR 5′HS3 Displays Specificity for ε-Globin Gene Activation during Primitive Erythropoiesis and γ-Globin Gene Activation during Fetal Definitive Erythropoiesis.

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.