Human Beta-globin Locus Research Articles

Mechanisms of Pol II Recruitment to the Human Beta-Globin Locus Control Region Super-Enhancer

The human ꞵ-globin gene locus consists of five genes that encode subunits of hemoglobin. The locus control region (LCR) regulates these genes, acting as a super-enhancer to promote high-level of β-globin expression in erythroid cells. The LCR, like other super-enhancers, is known to recruit transcription complexes and generate non-coding enhancer RNA (eRNA). Past research has suggested that super-enhancers form phase-separated transcription initiation domains that concentrate RNA polymerase II (Pol II) and transfer Pol II to target gene promoters during transient looping interactions. The aim of this project is to examine the role of TFIIH, involved transcription initiation, in enhancer RNA production and Pol II transfer in the globin gene locus. The LCR was immobilized on magnetic beads and incubated with erythroid nuclear protein extracts. Proteins not bound were washed away and bound proteins were subjected to western blotting experiments. The data show that TFIIH is bound at the LCR and thus may be involved in eRNA production. Cut & Run experiments were performed to examine the localization of TFIIH in the globin gene locus in intact cells. In the future, the laboratory plans to deplete TFIIH and examine production of eRNA and Pol II recruitment at the LCR.

Establishing a Link between Zinc Homeostasis and Globin Gene Expression

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.

Chromatin priming of genes in development: Concepts, mechanisms and consequences.

During ontogeny, cells progress through multiple alternate differentiation states by activating distinct gene regulatory networks. In this review, we highlight the important role of chromatin priming in facilitating gene activation during lineage specification and in maintaining an epigenetic memory of previous gene activation. We show that chromatin priming is part of a hugely diverse repertoire of regulatory mechanisms that genes use to ensure that they are expressed at the correct time, in the correct cell type, and at the correct level, but also that they react to signals. We also emphasize how increasing our knowledge of these principles could inform our understanding of developmental failure and disease.

A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells.

Mammalian chromosome replication starts from distinct sites; however, the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. Here we identify a replication-initiation determinant (RepID) protein that binds a subset of replication-initiation sites. A large fraction of RepID-binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from RepID-bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID-depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication-initiation events. These observations are consistent with a model, suggesting that RepID facilitates replication initiation at a distinct group of human replication origins.

576. DNA Ends Matter: The Impact of Using CRISPR/Cas9 Variants on DNA Repair Pathway Choices and Editing Profiles at the HBB Locus

Sickle Cell Anemia is an inherited recessive disorder caused by a single point mutation in the human beta globin (HBB) gene resulting in an abnormal type of hemoglobin. Here we report targeting the HBB locus using CRISPR/Cas9 technology for correction of Sickle Cell Disease, which affects nearly 1 million people worldwide. Cas9 and its variants can be used to introduce a variety of breaks including blunt double stranded break (DSB), single nicks, or dual nicks leaving either a 3’ or 5’ overhang. The type of cut and donor used can play a role in triggering different repair pathways, thus, resulting in various editing profiles. Using a single strand oligonucleotide (ssODN), we characterize different DNA repair outcomes including indel mutations resulting from Non Homologous End-Joining (NHEJ), Homology-Dependent Repair (HDR) using the donor as a template, and, finally, Gene Conversion (a kind of HDR event) using the closely related HBD gene as an endogenous template. Repair using homologous sequences from the HBD gene results in partial gene-conversion yielding a chimeric HBB-HBD gene that corrects the sickle cell point mutation. We observed that the Cas9 nickase/gRNA pair leaving a 5’ overhang displayed a significantly higher frequency of gene conversion and gene correction than other Cas9-induced DNA end structures. We also provide evidence that overexpression or down-regulation of critical factors in the repair pathways influence the repair pathway balance.In summary, we demonstrate that the frequency of various repair outcomes under different conditions offers insight into the mechanisms of repair of Cas9-induced DNA cleavage. The data support a therapeutic approach in which correction of the sickle-cell mutation is efficiently mediated through HDR using a donor template or by gene-conversion using the endogenous HBD gene.

560. Therapeutic Editing of the HBB Locus Using the Endogenous HBD Locus as a Donor Template

Sickle Cell Anemia is a recessive disorder caused by a single point mutation in the human beta globin (HBB) gene. Affecting nearly 1 million people worldwide, this disease is severely lacking in long-term treatment options and is a prime candidate for a gene editing therapeutic approach. Here we report the use of the CRISPR/Cas system to target the human HBB gene in the region of the sickle cell anemia-causing mutation.Utilizing two different Cas9 nickases as well as the wild type nuclease, we are able to introduce blunt double-strand breaks, single strand nicks, and dual-nicks in which the nicks are placed on opposite strands and leave either 3’ or 5’ overhangs of varying lengths. Using either single strand oligonucleotide (ssODN) or plasmid DNA donors, we characterize several different DNA repair outcomes including indel mutations resulting from non-homologous end-joining, homology-dependent repair (HDR) using the donor as a template, and finally HDR using the closely related HBD gene as an endogenous template. Repair using homologous sequences from the HBD gene results in partial gene-conversion yielding a chimeric HBB-HBD gene. The region of gene conversion includes the sequence most commonly mutated in sickle cell anemia. The frequency of this event depends on the nature of the break. The data support a therapeutic approach in which correction of the sickle-cell mutation is efficiently mediated through HDR using a donor template or by gene-conversion using the endogenous HBD gene.

Targeting KLF1 for the Treatment of Sickle Cell Disease Using Antisense Oligonucleotides

Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein in the fetus during the last months of embryonic development and the first few months of life after birth. HbF has a slightly greater oxygen binding affinity than adult hemoglobin (HbA, α2β2) and inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD.Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of KLF1, we can promote production of HbF through the upregulation of γ-globin expression.Since rodents don’t express γ-globin, we have employed both human and engineered mouse cell lines to demonstrate upregulation of γ-globin mRNA expression in vitro. We used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse KLF1, and treated with antisense oligonucleotides (ASOs) targeting mouse KLF1. After 7 days of free uptake with the ASOs, we observed a 6-fold increase of human γ-globin mRNA expression after achieving 65% mRNA reduction of mouse KLF1 compared to the untreated control. We were also able to demonstrate significant upregulation of human γ-globin protein expression in these cells by western blot. We have shown similar results in a human erythroleukemia cell line, K562, using ASOs targeting human KLF1. K562 cells were electroporated with the KLF1 ASOs and 4 days later, we observed a 5-fold increase of human γ-globin mRNA expression after achieving 40% mRNA reduction of human KLF1 compared to the untreated control. These data indicate that targeting mouse or human KLF1 with ASO treatment can cause an increase in human γ-globin expression in vitrothat is necessary for the upregulation of fetal hemoglobin.We have also shown that we are able to target the bone marrow in both mice and rats through subcutaneous administration of our KLF1 ASOs. In wild type mice, at a dose of 100 mpk/wk for 4 weeks, we observed KLF1 target reduction of 88% and a β-globin reduction of 58% compared to the saline control in whole bone marrow. In Sprague-Dawley rats, at a dose of 50 mpk/wk for 4 weeks, we observed KLF1 target reduction of 83% and a β-globin reduction of 77% compared to the saline control in whole bone marrow. Therefore, we are able to achieve significant β-globin mRNA reduction in the bone marrow in both mice and rats after subcutaneous administration of KLF ASOs. These data indicate that reducing KLF1 with antisense oligonucleotides is a viable option for the treatment of sickle cell anemia. DisclosuresPeralta:Isis Pharmaceuticals, Inc.: Employment. Low:Isis Pharmaceuticals, Inc.: Employment. Booten:Isis Pharmaceuticals, Inc.: Employment. Zhou:Univeristy of Alabama at Birmingham: Employment. Kim:Isis Pharmaceuticals, Inc.: Employment. Freier:Isis Pharmaceuticals, Inc.: Employment. Guo:Isis Pharmaceuticals, Inc.: Employment. Murray:Isis Pharmaceuticals, Inc.: Employment. Townes:University of Alabama at Birmingham: Employment. Hung:Isis Pharmaceuticals, Inc.: Employment.

Targeting BCL11A and KLF1 For The Treatment Of Sickle Cell Disease and β-Thalassemia In Vitro using Antisense Oligonucleotides

Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein with greater oxygen binding affinity in the fetus during the last months of embryonic development and the first few months of life after birth. HbF inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD as well as β-thalassemia. In β-thalassemia, there is reduced or absent synthesis of the β-globin gene, causing ineffective erythropoiesis.B-cell lymphoma/leukemia 11A (BCL11A) is a transcription factor in the zinc-finger protein family and is expressed in B cells and erythroid cells. BCL11A represses fetal hemoglobin expression by binding to the GGCCCGG motif in the β-globin promoter region. Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of BCL11A and KLF1, we can promote production of HbF through the upregulation of γ-globin expression.To demonstrate upregulation of γ-globin mRNA expression in vitro, we used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse BCL11A and KLF1 (Tim Townes, Univ. of Alabama at Birmingham). Antisense oligonucleotides (ASOs) targeting mouse BCL11A or mouse KLF1 were added to the cells in a dose-dependent manner. Seven days later, with free uptake of the ASOs into the cells, we observed dose-dependent reduction of mouse BCL11A mRNA (IC50 = 0.7 μM) and mouse KLF1 mRNA (IC50 = 3 μM). Consequently, we observed a 300 +/- 8% upregulation of human γ-globin mRNA expression after achieving ∼90% reduction in BCL11A mRNA expression after ASO treatment compared to the untreated control cells. Similarly, KLF1 ASO treatment caused a 500 +/- 58% up regulation of human γ-globin mRNA expression after achieving ∼80% mRNA reduction in KLF expression. These data indicate that targeting BCL11A and/or KLF1 with ASO treatment can cause an increase in γ-globin expression that is necessary for the upregulation of fetal hemoglobin and may be used for the treatment of sickle-cell anemia and β-thalassemia. Disclosures:Peralta: Isis Pharmaceuticals, Inc.: Employment. Low: Isis Pharmaceuticals, Inc.: Employment. Kim: Isis Pharmaceuticals, Inc.: Employment. Murray: Isis Pharmaceuticals, Inc.: Employment. Guo: Isis Pharmaceuticals, Inc.: Employment. Freier: Isis Pharmaceuticals, Inc.: Employment. Townes: University of Alabama at Birmingham: Employment. Hung: Isis Pharmaceuticals, Inc.: Employment.

Induction of Fetal Hemoglobin by Inactivation of HDAC1 or HDAC2 without Altering Cellular Proliferation

Abstract 354Histone deacetylase (HDAC) inhibitors are effective inducers of fetal hemoglobin, and prior studies have shown that selective inactivation of HDAC1 or HDAC2 is sufficient to induce fetal hemoglobin in vitro. In our current work, we demonstrate that HDAC1 and HDAC2 are attractive targets for clinical translation for two reasons: 1) Selective inhibition will decrease off-target effects that currently limit the use of hydroxyurea and pan-HDAC inhibitors, and 2) HDAC inhibitors induce fetal hemoglobin in various preclinical models, and they can be combined with hydroxyurea to achieve further fetal hemoglobin induction. To investigate off-target effects, we selectively inactivated HDAC1, HDAC2 or HDAC3 in human erythroid progenitor cells, and examined the effect of each knockdown on cellular cytotoxicity and cell cycle progression. Although knockdown of HDAC3 negatively influenced growth, selective knockdown of HDAC1 or HDAC2 had no effect on expansion of erythroid progenitors. In addition, knockdown of HDAC2 does not block cell cycle progression. These data support the possibility that an HDAC1- or HDAC2-specific inhibitor may offer a therapeutic advantage by reducing side effects, while maintaining robust HbF induction. Armed with this knockdown data, we are now investigating HDAC inhibitor compounds of various selectivity in in vitro and in vivo models. To perform optimal clinical trials, and ultimately to benefit the most sickle cell disease patients, it would be ideal to combine HDAC inhibitor treatment with hydroxyurea. A combination treatment approach may ameliorate some of the limitations of hydroxyurea use, such as the unpredictable effect on fetal hemoglobin levels, and the lack of benefit in beta thalassemia patients. First, we combined HDAC2 inactivation with hydroxyurea treatment in vitro. Human bone marrow-derived CD34+ cells were infected with lentiviruses containing an shRNA targeting either HDAC2 or a luciferase control gene. The cells were then treated on day 4 of erythroid differentiation with hydroxyurea (10–20 uM dose). Compared to the untreated luciferase control samples, we observed a 7–9-fold increase in gamma-globin expression in the untreated HDAC2-knockdown samples, a 2.5-fold increase in the hydroxyurea-treated luciferase control samples, and a trend toward an additive effect on gamma-globin induction in the cells where HDAC2 knockdown was combined with hydroxyurea treatment. To investigate the effects of HDAC inhibitors in vivo, we administered compounds to BCL11A conditional knockout transgenic mice (by erythroid-selective EpoR-GFP Cre) containing the human beta-globin locus. As reported previously, BCL11A inactivation powerfully de-repressed gamma-globin expression, and administration of an HDAC inhibitor, SAHA, led to a further elevation of gamma-globin mRNA. We now demonstrate that administration of another pan-HDAC inhibitor, panobinostat (LBH589), results in an additional 1.5- to 2.5-fold increase in gamma-globin mRNA relative to pre-treatment baseline. We are currently evaluating the combination of panobinostat and hydroxyurea in these mice to confirm that the compounds have an additive effect in vivo as well as in vitro. Taken together, these experiments indicate that inhibiting HDAC1 or HDAC2 is a promising therapeutic approach to increasing fetal hemoglobin levels in patients with beta-hemoglobinopathies, both alone and in combination with hydroxyurea. Disclosures:Bradner:Acetylon: .

“Definitive” Erythropoiesis Has Distinct Developmental Origins and Globin Expression Patterns in the Mammalian Embryo.

Abstract 2539Poster Board II-516A transient wave of primitive erythropoiesis begins at embryonic day 7.5 (E7.5) in the mouse as yolk sac-derived primitive erythroid progenitors (EryP-CFC) generate precursors that mature in the circulation and expand in numbers until E12.5. A second wave of erythroid progenitors (BFU-E) originates in the yolk sac beginning at E8.25 that generate definitive erythroid cells in vitro. These BFU-E colonize the newly forming liver beginning at E10.5, prior to the initial appearance there of adult-repopulating hematopoietic stem cells (HSCs) between E11.5-12.5. This wave of definitive erythroid yolk sac progenitors is proposed to be the source of new blood cells required by the growing embryo after the expansion of primitive erythroid cells has ceased and before HSC-derived hematopoiesis can fulfill the erythropoietic needs of the embryo. We utilized multispectral imaging flow cytometry both to distinguish erythroid lineages and to define specific stages of erythroid precursor maturation in the mouse embryo. Consistent with this model, we found that small numbers of definitive erythrocytes first enter the embryonic circulation beginning at E11.5. All maturational stages of erythroid precursors were observed in the E11.5 liver, consistent with these first definitive erythrocytes having rapidly completed their maturation in the liver. The expression of βH1 and εy-beta globin genes is thought to be limited to primitive erythroid cells. Surprisingly, examination of globin gene expression by in situ hybridization revealed high levels of βH1-, but not εy-globin, transcripts in the parenchyma of E11.5-12.5 livers. RT-PCR analysis of globin mRNAs confirmed the expression of βH1- and adult β1-, but not εy-globin, in E11.5 liver-derived definitive (ckit+, Ter119lo) proerythroblasts sorted by flow cytometry to remove contaminating primitive (ckit-, Ter119+) erythroid cells. A similar pattern of globin gene expression was found in individual definitive erythroid colonies derived from E9.5 yolk sac and from early fetal liver. In vitro differentiation of definitive erythroid progenitors from E9.5 yolk sac revealed a maturational “switch” from βH1- and β1-globins to predominantly β1-globin. βH1-globin transcripts were not observed in proerythroblasts from bone marrow or E16.5 liver or in erythroid colonies from later fetal liver. ChIP analysis revealed that hyperacetylated domains encompass all beta globin genes in primitive erythroid cells but only the adult β1- and β2-globin genes in E16.5 liver proerythroblasts. Consistent with their unique gene expression, E11.5 liver proerythroblasts have hyperacetylated domains encompassing the βh1-, β1- and β2-, but not εy-globin genes. We also examined human globin transgene expression in mice carrying a single copy of the human beta globin locus. Because of the overlapping presence and changing proportion of primitive and definitive erythroid cells during development, we analyzed sorted cell populations whose identities were confirmed by murine globin gene expression. We confirmed that primitive erythroid cells express higher levels of γ- than ε-globin and little β-globin. E11.5 proerythroblasts and cultured E9.5 progenitors express γ- and β-, but not ε-globin. E16.5 liver proerythroblasts express β- and low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. In summary, two forms of definitive erythropoiesis emerge in the murine embryo, each with distinct globin expression patterns and chromatin modifications of the β-globin locus. While both lineages predominantly express adult globins, the first, yolk sac-derived lineage uniquely expresses low levels of the embryonic βH1-globin gene as well as the human γ-globin transgene. The second definitive erythroid lineage, found in the later fetal liver and postnatal marrow, expresses only adult murine globins as well as low levels of the human γ-globin transgene only in the fetus. Our studies reveal a surprising complexity to the ontogeny of erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

A Zinc-Finger Transcriptional Activator Designed to Interact with the Gamma-Globin Gene Promoters Enhances Fetal Hemoglobin Production in Erythroid Cells Derived From Normal and Beta-Thalassemic CD34+ Cells.

Abstract 3567Poster Board III-504Fetal hemoglobin (α2γ2; HbF) is a potent genetic modifier of the severity of beta-thalassemia and sickle cell anemia. Clinical studies indicate that moderate elevation in production of HbF achieved through heritable persistence of HbF or administration of hydroxyurea, effectively reduce the severity of beta-chain defects. Accordingly, we are exploring strategies to maintain expression of the endogenous gamma-globin genes following lentiviral vector-mediated gene transfer. The artificial zinc-finger transcription factor (GG1-VP64) was designed to interact with sequences in the proximal gamma-globin gene promoters and has been shown to enhance gamma-globin expression in human erythroleukemia cells and mouse marrow cells which are transgenic for the human beta-globin locus. Here, we describe studies designed to evaluate the impact of expression of GG1-VP64 on gamma-globin expression by maturing adult erythroblasts derived from CD34+ cells of normal and thalassemic donors. We utilized an in vitro culture model of human erythropoiesis in which late stage erythroblasts are derived from human CD34+ hematopoietic cells. In this system, cytokine-mobilized peripheral blood or steady state bone marrow CD34+ cells from adults yielded erythroblasts containing 2% or less HbF. The lentiviral vector encodes for bicistronic expression of the GG1-VP64 transactivator and GFP under transcriptional control of the beta-spectrin or ankyrin-1 promoter which give low but progressive increase in expression during erythroid development. Three normal donor CD34+ cells were transduced 48 hours after initiation of culture by overnight exposure to the GG1-VP64 vector or GFP control vector. Approximately 50-60% of the cells were successfully transduced with the control and GG1-VP64 vectors as monitored by flow cytometry analysis for GFP expression. Control vector transduction had no effect on cell proliferation or differentiation monitored by consistent increases in cell numbers and the appearance of CD71 (transferrin receptor) and CD235 (glycophorin A) on most cells (>98% and >80%, respectively) whereas GG1-VP64 gene transfer reduced cell proliferation slightly without affecting erythroid differentiation. Erythroblasts derived from GFP transduced cells expressed low levels of HbF (1.7+/−0.6%) whereas those derived from cells transduced with GG1-VP64 demonstrated induction of HbF ranging from 12-21% with an average vector copy number of 0.8 to 1.0. When cells from a normal donor were sorted into GFP- and GFP+ populations, significant levels of HbF were present only in the GFP+ fraction. We next tested the GG1-VP64 transactivator in three independent studies using bone marrow CD34+ cells from two patients with beta-thalassemia major. Gene transfer was effective as reflected by 74+/−6% (control) and 47+/− 2% (GG1-VP64) GFP marking in bulk cultures. Again, GG1-VP64 gene transfer in beta-thalassemia CD34+ cells reduced cell growth somewhat but did not perturb erythroid differentiation as monitored by the appearance of transferrin receptor (>98%) and Glycophorin A (>80%) as well as cell morphology. Erythroblasts derived from GFP transduced cells expressed levels of HbF in the range of 26+/−5% whereas those derived from cells transduced with GG1-VP64 demonstrated a 2-fold induction of HbF to 52+/−9% with an average vector copy number of 0.5-0.9. Our data show that lentiviral-mediated, enforced expression of GG1-VP64 under the control of erythroid-specific promoters induced significant amounts of HbF in normal and thalassemic erythroblasts derived from adult CD34+ cells without altering their capacity for erythroid maturation following transduction. These observations demonstrate the potential for sequence specific enhancement of HbF in patients with beta-thalassemia or sickle cell anemia. Disclosures:No relevant conflicts of interest to declare.

ErythRED, a hESC line enabling identification of erythroid cells

A human embryonic stem cell (hESC) line that enabled globin-expressing cells to be easily recognized would facilitate optimization of erythroid differentiation in vitro and aid in the identification of hESC-derived erythroid cells in transplanted animals. We describe a genetically modified hESC line, ErythRED, in which expression of RFP, controlled by regulatory sequences from the human beta-globin locus control region, is restricted to maturing erythroid cells.

All of the human β‐type globin genes compete for LCR enhancer activity in embryonic erythroid cells of yeast artificial chromosome transgenic mice

In primitive erythroid cells of human beta-globin locus transgenic mice (TgM), the locus control region (LCR)-proximal epsilon- and gamma-globin genes are transcribed, whereas the distal delta- and beta-globin genes are silent. It is generally accepted that the beta-globin gene is competitively suppressed by gamma-globin gene expression at this developmental stage. Previously, however, we observed that epsilon-globin gene expression was severely attenuated when its distance from the LCR was extended, implying that beta-globin gene might also be silenced because of its great distance from the LCR. Here, to clarify the beta-globin gene silencing mechanism, we established TgM lines carrying either gamma- or epsilon- plus gamma-globin promoter deletions, without significantly altering the distance between the beta-globin gene and the LCR. Precocious expression of delta- and beta-globin genes was observed in primitive erythroid cells of mutant, but not wild-type TgM, which was most evident when both the epsilon and gamma promoters were deleted. Thus, we clearly demonstrated that the repression of the delta- and beta-globin genes in primitive erythroid cells is dominated by competitive silencing by the epsilon- and gamma-globin gene promoters, and that epsilon- and the other beta-like globin genes might be activated by two distinct mechanisms by the LCR.

Developmental and species-divergent globin switching are driven by BCL11A

The contribution of changes in cis-regulatory elements or trans-acting factors to interspecies differences in gene expression is not well understood. The mammalian beta-globin loci have served as a model for gene regulation during development. Transgenic mice containing the human beta-globin locus, consisting of the linked embryonic (epsilon), fetal (gamma) and adult (beta) genes, have been used as a system to investigate the temporal switch from fetal to adult haemoglobin, as occurs in humans. Here we show that the human gamma-globin (HBG) genes in these mice behave as murine embryonic globin genes, revealing a limitation of the model and demonstrating that critical differences in the trans-acting milieu have arisen during mammalian evolution. We show that the expression of BCL11A, a repressor of human gamma-globin expression identified by genome-wide association studies, differs between mouse and human. Developmental silencing of the mouse embryonic globin and human gamma-globin genes fails to occur in mice in the absence of BCL11A. Thus, BCL11A is a critical mediator of species-divergent globin switching. By comparing the ontogeny of beta-globin gene regulation in mice and humans, we have shown that alterations in the expression of a trans-acting factor constitute a critical driver of gene expression changes during evolution.

Differential requirement of a distal regulatory region for pre-initiation complex formation at globin gene promoters

Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human β-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the β-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual β-like globin genes. Moreover, in transgenic mice expressing the entire human β-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter β-like globin gene expression. Here, we show that abnormal expression of human β-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human ɛ- and γ-promoters, but not at the β-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human γ-promoters. We also find that HS2 is dispensable for high-level human β-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement.

Apolipoprotein E knock-out and knock-in mice: atherosclerosis, metabolic syndrome, and beyond

Given the multiple differences between mice and men, it was once thought that mice could not be used to model atherosclerosis, principally a human disease. Apolipoprotein E-deficient (apoEKO) mice have convincingly changed this view, and the ability to model human-like plaques in these mice has provided scientists a platform to study multiple facets of atherogenesis and to explore potential therapeutic interventions. In addition to its well-established role in lipoprotein metabolism, recent observations of reduced adiposity and improved glucose homeostasis in apoEKO mice suggest that apoE may also play a key role in energy metabolism in peripheral organs, including adipose tissue. Finally, along with apoEKO mice, knockin mice expressing human apoE isoforms in place of endogenous mouse apoE have provided insights into how quantitative and qualitative genetic alterations interact with the environment in the pathogenesis of complex human diseases.

PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA methylation in gene silencing.

Mammalian gene silencing is established through methylation of histones and DNA, although the order in which these modifications occur remains contentious. Using the human beta-globin locus as a model, we demonstrate that symmetric methylation of histone H4 arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for subsequent DNA methylation. H4R3me2s serves as a direct binding target for the DNA methyltransferase DNMT3A, which interacts through the ADD domain containing the PHD motif. Loss of the H4R3me2s mark through short hairpin RNA-mediated knockdown of PRMT5 leads to reduced DNMT3A binding, loss of DNA methylation and gene activation. In primary erythroid progenitors from adult bone marrow, H4R3me2s marks the inactive methylated globin genes coincident with localization of PRMT5. Our findings define DNMT3A as both a reader and a writer of repressive epigenetic marks, thereby directly linking histone and DNA methylation in gene silencing.

Expression of green fluorescent protein under the regulation of human locus control region elements HS2 and HS3 in transgenic mice

Human beta-globin locus control region (LCR) is composed of five DNase I hypersensitive sites (HSs). We previously demonstrated that when HS2 and HS3 were constructed together in one vector and integrated into one position in transgenic mice, the two cis-elements showed a marked synergy in regulating the spatial and temporal expression of beta-globin transgene. This study is to investigate whether these two elements still show a synergy or superposition when they are not located in one construct. HS2/GFP and HS3/GFP transgenic mice, with the transgene encoding green fluorescent protein (GFP) driven by beta-globin promoter and under the control of HS2 (HS2/GFP mice) and HS3 (HS3/GFP mice), were mated to generate double-heterozygotes (HS2/GFP + HS3/GFP mice). The expression and integration status of GFP genes in transgenic mice were analyzed. Our data showed that there was no difference in the percentages of GFP positive cells in peripheral blood between double-heterozygotes and their corresponding founders (HS2/GFP or HS3/GFP). Interestingly, it was observed that exogenous GFP genes were highly expressed in another transgenic mouse pedigree with HS2 element integrated twice into the same chromosome at two different loci. However, when one site was lost in some offspring because of recombination, the percentages of GFP positive cells in peripheral blood as well as in some erythroid tissues were decreased obviously. These results demonstrated that HS2 and HS3 elements would not show any synergy or superposition if they were located on different chromosomes in transgenic mice, and that HS2 element containing the core and flanking sequences might not be able to overcome the position effects and control position dependent expression of linked genes in transgenic mice.

A mechanism for Ikaros regulation of human globin gene switching

The human beta globin locus consists of an upstream LCR and functional genes arranged sequentially in the order of their expression during development: 5'-HBE1, HBG2, HBG1, HBD, HBB-3'. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Here we show that the transcription factor Ikaros plays a major role in the formation of the beta-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation, there is concomitant marked down-regulation of HBB, and up-regulation of HBG expression. We show for the first time Ikaros and its family member Eos, bind to critical cis elements implicated in haemoglobin switching and deletional hereditary persistence of fetal haemoglobin (HPFH). Chromatin conformation capture (3C) data demonstrated that Ikaros facilitates long-distance DNA looping between the LCR and a region upstream of HBD. This study provides new insights into the mechanism of stage-specific assembly of the beta-globin ACH, and HPFH.

Recruitment of Rad50 and MCM Complexes on the Human Beta Globin Locus Control Region (LCR): A Novel Role for LCR Hypersensitive Site 4 (HS4).

The human beta globin locus control region (LCR) aids in the establishment of an open chromatin configuration thereby facilitating the expression of linked downstream globin genes in a temporal and spatial manner along the developmental ontogeny. The LCR also functions as a classical enhancer of the beta like globin genes. The four DNAase-I hypersensitive regions (HS 1–4) on the LCR are believed to act as nucleation centers for the assembly of multi-protein transcriptional complexes that interact with the distant gene promoters. Present work is focused on characterizing the protein complex assemblies on the HS4 region and delineating their function in the context of goblin gene regulation. Our finding shows an assembly of a 2–5 MDa complex on the HS4 locus that is both erythroid restricted and sequence specific. Electrophoretic mobility shift assays (EMSA) were performed using K562 and HeLa nuclear extracts and ∼35mer double stranded oligonucleotides with 7 bp overlaps tiling the entire core HS4 sequence. We detected prominent K562 specific bands formed with an AT rich 39 bp HS4 sequence. Sequence specificity of DNA-protein complex formation on this AT rich sequence was determined by competitive EMSA assays. Gel supershift assays with a wide range of antibodies and subsequent partial purification of this multiprotein complex followed by the LC-MS/MS analysis reveal the presence of a mini chromosome maintenance (MCM) complex that is associated with the DNA replication, DNA repair associated Rad50-Mre-11-NBS-1 complex, histone acetyl transferase p300 and DNA binding transcription factors ILF2 (NF45) and ILF3 (NF90). The association of these proteins was determined by co-immunoprecipitation experiments. Chromatin immunoprecipitation (ChIP) experiments demonstrate the in vivo recruitment of these proteins on the HS4 region of the LCR. We are currently working on further characterization and functional studies of this complex and its significance on the role of the LCR in DNA replication and repair.