Renee Neades

Minor histocompatibility antigens on transfused leukoreduced units of red blood cells induce bone marrow transplant rejection in a mouse model

When successful, human leukocyte antigen (HLA)-matched bone marrow transplantation with reduced-intensity conditioning is a cure for several nonmalignant hematologic disorders that require chronic transfusion, such as sickle cell disease and aplastic anemia. However, there are unusually high bone marrow transplant (BMT) rejection rates in these patients. Rejection correlates with the number of transfusions before bone marrow transplantation, and it has been hypothesized that preimmunization to antigens on transfused blood may prime BMT rejection. Using a novel mouse model of red blood cell (RBC) transfusion and major histocompatibility complex-matched bone marrow transplantation, we report that transfusion of RBC products induced BMT rejection across minor histocompatibility antigen (mHA) barriers. It has been proposed that contaminating leukocytes are responsible for transfusion-induced BMT rejection; however, filter leukoreduction did not prevent rejection in the current studies. Moreover, we generated a novel transgenic mouse with RBC-specific expression of a model mHA and demonstrated that transfusion of RBCs induced a CD8(+) T-cell response. Together, these data suggest that mHAs on RBCs themselves are capable of inducing BMT rejection. Cellular immunization to mHAs is neither monitored nor managed by current transfusion medicine practice; however, the current data suggest that mHAs on RBCs may represent an unappreciated and significant consequence of RBC transfusion.

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

ABSTRACT Autonomous silencing of γ-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the Aγ-globin gene was identified between −730 and −378 relative to the mRNA start site. A marked copy of the Aγ-globin gene inserted between locus control region 5′ DNase I-hypersensitive site 1 and the ε-globin gene was transcriptionally silenced in adult β-globin locus yeast artificial chromosome (β-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 γ-globin levels, whereas only a weak signal was detected when γ-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from β-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the Aγ-globin −566 or Gγ-globin −567 GATA site when γ-globin expression was low (day 18) but not when γ-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, γ-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 γ-globin promoters.

Mi2β Is Required for γ-Globin Gene Silencing: Temporal Assembly of a GATA-1-FOG-1-Mi2 Repressor Complex in β-YAC Transgenic Mice

Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the −566 GATA motif of the Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the −566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1–mediated repressor complex was disrupted by the −566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting −566 Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.

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 γ-globin gene expression is complicated by the lack of suitable cell lines. Human K562 cells co-express ϵ- and γ-globin but not β-globin; transgenic mouse erythroleukemia 585 cells express predominantly human β-globin but also γ-globin; and transgenic murine GM979 cells co-express human γ-and β-globin. Human β-globin locus yeast artificial chromosome transgenic mice display correct developmental regulation of β-like globin gene expression. We rationalized that cells established from the adult bone marrow of these mice might express exclusively β-globin and therefore could be employed to select or screen inducers of γ-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 β-globin was expressed, but γ-globin was not; a similar expression pattern was observed in cells derived from fetal liver. γ-Globin expression was induced upon exposure to 5-azacytidine, in cells derived from –117 Greek hereditary persistence of fetal hemoglobin human β-globin locus yeast artificial chromosome (β-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-γ-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 γ-globin synthesis or screening γ-globin inducers for the treatment of sickle cell disease or β-thalassemia.

S-PHASE ARREST IN MOUSE KERATINOCYTES EXPOSED TO MULTIPLE DOSES OF ULTRAVIOLET B/A RADIATION

Exposure to solar ultraviolet (UV) radiation is believed to cause most human skin carcinomas. Despite the large body of evidence connecting UV exposure with skin cancer, the frequency and level of human exposure to repetitive doses of UV light will most likely continue for occupational and recreational reasons. By investigating the cellular response of keratinocytes to multiple, physiologically relevant doses of UV, we hope to better understand the processes involved in UV‐induced skin cancer. In this study, we used a UV exposure model to investigate the cell‐cycle response of keratinocytes exposed to multiple doses of UV‐B/A radiation in which the UV‐C component (wavelengths below 290 nm) had been filtered out. Our results indicated that exposure of asynchronous mouse keratinocytes to three doses of 200 J/m2 UV‐B/A radiation at 30 min intervals produced an inhibition of DNA synthesis and S‐phase arrest between 7 and 25 h after the last irradiation. The S‐phase arrest was not due to a reduction in the level of cyclin E and A proteins but was accompanied by inhibition of cyclin‐dependent kinase 2 (cdk2) activity. We observed a similar pattern of cdk2 inhibition induced by multiple UV‐B/A irradiations in mouse embryo fibroblasts from p21WAF null mice, indicating that the inhibition of cdk2 was independent of p21WAF in these cells. Mol. Carcinog. 23:159–167, 1998.

Induction of Fetal Hemoglobin In Vivo Mediated by a Synthetic γ-Globin Zinc Finger Activator

Sickle cell disease (SCD) and β-thalassemia patients are phenotypically normal if they carry compensatory hereditary persistence of fetal hemoglobin (HPFH) mutations that result in increased levels of fetal hemoglobin (HbF, γ-globin chains) in adulthood. Thus, research has focused on manipulating the reactivation of γ-globin gene expression during adult definitive erythropoiesis as the most promising therapy to treat these hemoglobinopathies. 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 murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene and in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients. Herein, we report that gg1-VP64 increased γ-globin gene expression in vivo, in peripheral blood samples from gg1-VP64 β-YAC double-transgenic (bigenic) mice. Our results demonstrate that ATFs function in an animal model to increase gene expression. Thus, this class of reagent may be an effective gene therapy for treatment of some inherited diseases.

A Cell-Based High-Throughput Screen for Novel Chemical Inducers of Fetal Hemoglobin for Treatment of Hemoglobinopathies

Decades of research have established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Identification of a drug specific for inducing 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. Assay systems developed in our labs to understand the mechanisms underlying the γ- to β-globin gene expression switch during development has allowed us to generate a cell-based assay that was adapted for a HTS of 121,035 compounds. Using chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) derived from human γ-globin promoter-firefly luciferase β-globin promoter-Renilla luciferase β-globin yeast artificial chromosome (γ-luc β-luc β-YAC) transgenic mice, we were able to identify 232 lead chemical compounds that induced γ-globin 2-fold or higher, with minimal or no β-globin induction, minimal cytotoxicity and that did not directly influence the luciferase enzyme. Secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the induction profiles of seven of the 232 hits that were cherry-picked for further analysis.

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 Aγ-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 Aγ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of Aγ-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.