Yeast Artificial Chromosome Transgenic Mice 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.

Ataxic Symptoms in Huntington's Disease Transgenic Mouse Model Are Alleviated by Chlorzoxazone.

Huntington’s disease (HD) is a hereditary neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein, Striatum atrophy in HD leads to a progressive disturbance of psychiatric, motor, and cognitive function. Recent studies of HD patients revealed that the degeneration of cerebellum is also observed independently from the striatal atrophy during early HD stage and may contribute to the motor impairment and ataxia observed in HD. Cerebellar Purkinje cells (PCs) are responsible for the proper cerebellar pathways functioning and motor control. Recent studies on mouse models of HD have shown that the abnormality of the biochemical functions of PCs are observed in HD, suggesting the contribution of PC dysfunction and death to the impaired movement coordination observed in HD. To investigate ataxic symptoms in HD we performed a series of experiments with the yeast artificial chromosome transgenic mouse model of HD (YAC128). Using extracellular single-unit recording method we found that the portion of the cerebellar PCs with bursting and irregular patterns of spontaneous activity drastically rises in aged YAC128 HD mice when compared with wild type littermates. Previous studies demonstrated that SK channels are responsible for the cerebellar PC pacemaker activity and that positive modulation of SK channel activity exerted beneficial effects in different ataxic mouse models. Here we studied effects of the SK channels modulator chlorzoxazone (CHZ) on the motor behavior of YAC128 HD mice and also on the electrophysiological activity and neuroanatomy of the cerebellar PCs from these mice. We determined that the long-term intraperitoneal injections of CHZ alleviated the progressive impairment in the firing pattern of YAC128 PCs. We also demonstrated that treatment with CHZ rescued age-dependent motor incoordination and improved the cerebellar morphology in YAC128 mice. We propose that abnormal changes in the PC firing patterns might be a one of the possible causes of ataxic symptoms in HD and in other polyglutamine disorders and that the pharmacological activation of SK channels may serve as a potential way to improve the activity of cerebellar PCs and relieve the ataxic phenotype in HD patients.

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.

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.

A novel GAA-repeat-expansion-based mouse model of Friedreich's ataxia.

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced levels of frataxin protein. We have previously reported the generation of human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing 90–190 GAA repeats, but the presence of multiple GAA repeats within these mice is considered suboptimal. We now describe the cellular, molecular and behavioural characterisation of a newly developed YAC transgenic FRDA mouse model, designated YG8sR, which we have shown by DNA sequencing to contain a single pure GAA repeat expansion. The founder YG8sR mouse contained 120 GAA repeats but, due to intergenerational expansion, we have now established a colony of YG8sR mice that contain ~200 GAA repeats. We show that YG8sR mice have a single copy of the FXN transgene, which is integrated at a single site as confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We have identified significant behavioural deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8sR FRDA mice compared with control Y47R and wild-type (WT) mice. We have also detected increased somatic GAA repeat instability in the brain and cerebellum of YG8sR mice, together with significantly reduced expression of FXN, FAST-1 and frataxin, and reduced aconitase activity, compared with Y47R mice. Furthermore, we have confirmed the presence of pathological vacuoles within neurons of the dorsal root ganglia (DRG) of YG8sR mice. These novel GAA-repeat-expansion-based YAC transgenic FRDA mice, which exhibit progressive FRDA-like pathology, represent an excellent model for the investigation of FRDA disease mechanisms and therapy.

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.

Cellular, molecular and functional characterisation of YAC transgenic mouse models of Friedreich ataxia.

BackgroundFriedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene. We have previously established and performed preliminary characterisation of several human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing GAA repeat expansions, Y47R (9 GAA repeats), YG8R (90 and 190 GAA repeats) and YG22R (190 GAA repeats).Methodology/Principal FindingsWe now report extended cellular, molecular and functional characterisation of these FXN YAC transgenic mouse models. FXN transgene copy number analysis of the FRDA mice demonstrated that the YG22R and Y47R lines each have a single copy of the FXN transgene while the YG8R line has two copies. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We identified significant functional deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8R and YG22R FRDA mice compared to Y47R and wild-type control mice. We also confirmed increased somatic GAA repeat instability in the cerebellum and brain of YG22R and YG8R mice, together with significantly reduced levels of FXN mRNA and protein in the brain and liver of YG8R and YG22R compared to Y47R.Conclusions/SignificanceTogether these studies provide a detailed characterisation of our GAA repeat expansion-based YAC transgenic FRDA mouse models that will help investigations of FRDA disease mechanisms and therapy.

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.

The Chicken HS4 Insulator Element Does Not Protect the H19 ICR from Differential DNA Methylation in Yeast Artificial Chromosome Transgenic Mouse

Mono-allelic expression at the mouse IGF2/H19 locus is controlled by differential allelic DNA methylation of the imprinting control region (ICR). Because a randomly integrated H19 ICR fragment, when incorporated into the genome of transgenic mice (TgM), was allele-specifically methylated in somatic, but not in germ cells, it was suggested that allele-discriminating epigenetic signature, set within or somewhere outside of the Tg H19 ICR fragment in germ cells, was later translated into a differential DNA methylation pattern. To test if the chicken β-globin HS4 (cHS4) chromatin insulator might interfere with methylation imprinting establishment at the H19 ICR, we inserted the H19 ICR fragment, flanked by a set of floxed cHS4 core sequences, into a human β-globin locus YAC and generated TgM (insulated ICR' TgM). As controls, the cHS4 sequences were removed from one side (5'HS4-deleted ICR') or both sides (pseudo-WT ICR') of the insulated ICR' by in vivo cre-loxP recombination. The data show that while maternally inherited transgenic H19 ICR was not methylated in insulated ICR' TgM, it was significantly methylated upon paternal transmission, though the level was lower than in the pseudo-WT ICR' control. Because this reduced level of methylation was also observed in the 5'HS4-deleted ICR' TgM, we speculate that the phenotype is due to VEZF1-dependent demethylation activity, rather than the insulator function, borne in cHS4. Collectively, although we cannot rule out the possibility that cHS4 is incapable of blocking an allele-discriminating signal from outside of the transgene, the epigenetic signature appears to be marked intrinsically within the H19 ICR.

Sox-Oct motifs contribute to maintenance of the unmethylated H19 ICR in YAC transgenic mice

Abnormal methylation at the maternally inherited H19 imprinted control region (H19 ICR) is one of the causative alterations leading to pathogenesis of Beckwith-Wiedemann syndrome (BWS). Recently, it was shown in human BWS patients, as well as mouse cell culture experiments, that Sox-Oct motifs (SOM) in the H19 ICR might play a role in protecting the maternal ICR from de novo DNA methylation. By grafting a mouse H19 ICR fragment into a human β-globin yeast artificial chromosome (YAC) followed by analysis in transgenic mice (TgM), we showed previously that the fragment carried sufficient information to establish and maintain differential methylation after fertilization. To examine possible functions of the SOM in the establishment and/or maintenance of differential methylation, two kinds of YAC-TgM were generated in this study. In the ΔSOM TgM, carrying the mouse H19 ICR bearing an SOM deletion, a maternally inherited transgenic ICR exhibited increased levels of methylation around the deletion site, in comparison to the wild-type control, after implantation. In the λ + CTCF + b (LCb) TgM, carrying a 2.3 kb λ DNA fragment supplemented with the fragment b including the SOM and four CTCF binding sites, maternally and some of the paternally inherited LCb fragments were significantly less methylated when compared with a control λ + CTCF fragment that was supplemented only with additional CTCF sites; the λ + CTCF was substantially methylated regardless of the parent of origin after implantation. These results demonstrated that the SOM in the maternal H19 ICR was required for maintaining surrounding sequences in the unmethylated state in vivo.

The H19 imprinting control region mediates preimplantation imprinted methylation of nearby sequences in yeast artificial chromosome transgenic mice.

In the mouse Igf2/H19 imprinted locus, differential methylation of the imprinting control region (H19 ICR) is established during spermatogenesis and is maintained in offspring throughout development. Previously, however, we observed that the paternal H19 ICR, when analyzed in yeast artificial chromosome transgenic mice (YAC-TgM), was preferentially methylated only after fertilization. To identify the DNA sequences that confer methylation imprinting, we divided the H19 ICR into two fragments (1.7 and 1.2 kb), ligated them to both ends of a λ DNA fragment into which CTCF binding sites had been inserted, and analyzed this in YAC-TgM. The maternally inherited λ sequence, normally methylated after implantation in the absence of H19 ICR sequences, became hypomethylated, demonstrating protective activity against methylation within the ICR. Meanwhile, the paternally inherited λ sequence was hypermethylated before implantation only when a 1.7-kb fragment was ligated. Consistently, when two subfragments of the H19 ICR were individually investigated for their activities in YAC-TgM, only the 1.7-kb fragment was capable of introducing paternal allele-specific DNA methylation. These results show that postfertilization methylation imprinting is conferred by a paternal allele-specific methylation activity present in a 1.7-kb DNA fragment of the H19 ICR, while maternal allele-specific activities protect the allele from de novo DNA methylation.

Dysregulation of Mitochondrial Calcium Signaling and Superoxide Flashes Cause Mitochondrial Genomic DNA Damage in Huntington Disease

Huntington disease (HD) is an inherited, fatal neurodegenerative disorder characterized by the progressive loss of striatal medium spiny neurons. Indications of oxidative stress are apparent in brain tissues from both HD patients and HD mouse models; however, the origin of this oxidant stress remains a mystery. Here, we used a yeast artificial chromosome transgenic mouse model of HD (YAC128) to investigate the potential connections between dysregulation of cytosolic Ca(2+) signaling and mitochondrial oxidative damage in HD cells. We found that YAC128 mouse embryonic fibroblasts exhibit a strikingly higher level of mitochondrial matrix Ca(2+) loading and elevated superoxide generation compared with WT cells, indicating that both mitochondrial Ca(2+) signaling and superoxide generation are dysregulated in HD cells. The excessive mitochondrial oxidant stress is critically dependent on mitochondrial Ca(2+) loading in HD cells, because blocking mitochondrial Ca(2+) uptake abolished elevated superoxide generation. Similar results were obtained using neurons from HD model mice and fibroblast cells from HD patients. More importantly, mitochondrial Ca(2+) loading in HD cells caused a 2-fold higher level of mitochondrial genomic DNA (mtDNA) damage due to the excessive oxidant generation. This study provides strong evidence to support a new causal link between dysregulated mitochondrial Ca(2+) signaling, elevated mitochondrial oxidant stress, and mtDNA damage in HD. Our results also indicate that reducing mitochondrial Ca(2+) uptake could be a therapeutic strategy for HD.

Sequences in the H19 ICR that are transcribed as small RNA in oocytes are dispensable for methylation imprinting in YAC transgenic mice

Allele-specific methylation of the endogenous H19 imprinting control region (ICR) is established in sperm. We previously showed that the paternal H19 ICR in yeast artificial chromosome (YAC) transgenic mice (TgM) was preferentially methylated in somatic cells, but not in germ cells, suggesting that differential methylation could be established after fertilization. In this report, we discovered small RNA molecules in growing oocytes, the nucleotide sequences of which mapped to the H19 ICR. To test if these small RNA sequences play a role in the establishment of differential methylation, we deleted the sequences from the H19 ICR DNA and generated YAC TgM. In somatic cells of these mice, methylation imprinting of the transgene was normally established. In addition, the mutant fragment was not methylated in sperm and eggs. These data demonstrate that sequences in the H19 ICR that correspond to the small RNA sequences are dispensable for methylation imprinting in YAC TgM.

P38 MAPK is involved in enhanced NMDA receptor-dependent excitotoxicity in YAC transgenic mouse model of Huntington disease

Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the protein huntingtin (htt). Previous studies have shown enhanced N-methyl-d-aspartate (NMDA)-induced excitotoxicity in neuronal models of HD, mediated in part by increased NMDA receptor (NMDAR) GluN2B subunit binding with the postsynaptic density protein-95 (PSD-95). In cultured hippocampal neurons, the NMDAR-activated p38 Mitogen-activated Protein Kinase (MAPK) death pathway is disrupted by a peptide (Tat-NR2B9c) that uncouples GluN2B from PSD-95, whereas NMDAR-mediated activation of c-Jun N-terminal Kinase (JNK) MAPK is PSD-95-independent. To investigate the mechanism by which Tat-NR2B9c protects striatal medium spiny neurons (MSNs) from mutant htt (mhtt)-enhanced NMDAR toxicity, we compared striatal tissue and cultured MSNs from presymptomatic yeast artificial chromosome (YAC) mice expressing htt with 128 polyQ (YAC128) to those from YAC18 and/or WT mice as controls. Similar to the previously published shift of GluN2B-containing NMDARs to extrasynaptic sites, we found increased PSD-95 localization as well as elevated PSD-95-GluN2B interactions in the striatal non-PSD (extrasynaptic) fraction from YAC128 mice. Notably, basal levels of both activated p38 and JNK MAPKs were elevated in the YAC128 striatum. NMDA stimulation of acute slices increased activation of p38 and JNK in WT and YAC128 striatum, but Tat-NR2B9c pretreatment reduced only the p38 activation in YAC128. In cultured MSNs, p38 MAPK inhibition reduced YAC128 NMDAR-mediated cell death to WT levels, and occluded the Tat-NR2B9c peptide protective effect; in contrast, inhibition of JNK had a similar protective effect in cultured MSNs from both WT and YAC128 mice. Our results suggest that altered activation of p38 MAPK contributes to mhtt enhancement of GluN2B/PSD-95 toxic signaling.

Dantrolene is neuroprotective in Huntington's disease transgenic mouse model

BackgroundHuntington's disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine expansion in the Huntingtin protein which results in the selective degeneration of striatal medium spiny neurons (MSNs). Our group has previously demonstrated that calcium (Ca2+) signaling is abnormal in MSNs from the yeast artificial chromosome transgenic mouse model of HD (YAC128). Moreover, we demonstrated that deranged intracellular Ca2+ signaling sensitizes YAC128 MSNs to glutamate-induced excitotoxicity when compared to wild type (WT) MSNs. In previous studies we also observed abnormal neuronal Ca2+ signaling in neurons from spinocerebellar ataxia 2 (SCA2) and spinocerebellar ataxia 3 (SCA3) mouse models and demonstrated that treatment with dantrolene, a ryanodine receptor antagonist and clinically relevant Ca2+ signaling stabilizer, was neuroprotective in experiments with these mouse models. The aim of the current study was to evaluate potential beneficial effects of dantrolene in experiments with YAC128 HD mouse model.ResultsThe application of caffeine and glutamate resulted in increased Ca2+ release from intracellular stores in YAC128 MSN cultures when compared to WT MSN cultures. Pre-treatment with dantrolene protected YAC128 MSNs from glutamate excitotoxicty, with an effective concentration of 100 nM and above. Feeding dantrolene (5 mg/kg) twice a week to YAC128 mice between 2 months and 11.5 months of age resulted in significantly improved performance in the beam-walking and gait-walking assays. Neuropathological analysis revealed that long-term dantrolene feeding to YAC128 mice significantly reduced the loss of NeuN-positive striatal neurons and reduced formation of Httexp nuclear aggregates.ConclusionsOur results support the hypothesis that deranged Ca2+ signaling plays an important role in HD pathology. Our data also implicate the RyanRs as a potential therapeutic target for the treatment of HD and demonstrate that RyanR inhibitors and Ca2+ signaling stabilizers such as dantrolene should be considered as potential therapeutics for the treatment of HD and other polyQ-expansion disorders.

Analysis of Fetal Hemoglobin Expression within Humanized Sickle Cell Disease Mice Overexpressing the TR2/4 Transgene.

AbstractAbstract 1619 Background:Sickle Cell Disease (SCD) is a chronic debilitating hematologic condition caused by a missense mutation within the adult beta globin gene leading to significant morbidity and mortality. Increased Fetal Hemoglobin production has been shown to significantly ameliorate SCD symptoms and improve survival. A novel specific DNA-binding factor DRED (direct repeat erythroid definitive) was recently identified that regulated epsilon and gamma globin gene expression (Tanimoto et al Genes Dev 2000). Purification of DRED revealed that it harbored the nuclear orphan hormone receptors, TR2/TR4, as its DNA binding core (Tanabe et al EMBO 2002). Overexpression of TR2/TR4 Transgene within Human Beta Globin Yeast Artificial Chromosome Transgenic Mice resulted in 4-fold induction of the gamma globin mRNA levels (Tanabe et al EMBO 2007). Therefore, we wanted to determine if the overexpression of TR2/TR4 within a humanized sickle cell disease model would result in fetal hemoglobin induction. Methods:Humanized Homozygous Knock-In UAB-Sickle Cell (UAB-Hbahα/hα Hbbhβs/hβs) Mice (Wu et al Blood 2006) was mated to TR2/TR4 Overexpressing Mice (TgTR2/TR4) to generate homozygous SS-TR2/TR4 compound heterozygotes (UAB-Hba hα/hα Hbb hβs/hβs TgTR2/TR4). We generated four 2–3 month old homozygous SS-TR2/TR4 transgenic mice and compared hemoglobin F levels, complete blood cell counts and % body weight (liver, spleen, kidney) to six 2–3 month old homozygous SS mice (Hbahα/hα Hbb hβs/hβs)without the overexpressing TgTR2/TR4. Tail PCR genotyping of all sickle cell mice (with and without TgTR2/TR4) and Hemoglobin F(Hgb F) and Sickle (HgbS) levels were confirmed by HPLC Hemoglobin electrophoresis. Results:The mean Hgb F: 7.8% (n=6, sd 1.63+/−) in the homozygous SS control mice vs. 16.5% (n=4, sd 2.64+/−)in the homozygous SS-TR2/4 Mice (2 Fold higher). Hematologic profile revealed a mean Hct: 25.2 (n=6, sd 5.50 +/−) mean MCV: 75.4 (n=6, sd 10+/−) and a mean WBC: 22.6 (n= 6, sd 13.9 +/−) in the homozygous SS control mice vs. a mean Hct: 31.25(n=4, sd 6.89+/−), mean MCV: 61(n=4, sd 3.5+/−) mean WBC: 16.3(n= 4, sd 5.99+/−) in the homozygous SS-TR2/TR4 mice. Lastly, initial organ (spleen, liver, kidney) pathology evaluation revealed decreased % body weight (bw) in homozygous SS TR2/TR4 Mice vs. homozygous SS controls: 1) Spleen %bw: 4.3% vs. 3.5% TgTR2/TR4), 2) Liver % bw: 8.8% vs. 7.7% TgTR2/TR4), and 3) Kidney %bw: 1.14% vs. 1.02% TgTR2/TR4). Conclusions:Our preliminary analysis revealed that TR2/TR4 overexpression within a humanized sickle cell disease mouse model resulted in a 2-fold induction of fetal hemoglobin based on HPLC hemoglobin electrophoresis. Further, increased TR2/TR4 overexpression improved anemia and organomegaly within sickle cell disease mice. TR2/TR4 may be an attractive target for fetal hemoglobin induction for the treatment of sickle cell disease. Ongoing studies will determine if TR2/TR4 decreases organ specific disease pathology. We will also determine the cellular distribution of fetal hemoglobin in future studies. Disclosures:No relevant conflicts of interest to declare.

Tetrabenazine is neuroprotective in Huntington's disease mice

BackgroundHuntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in Huntingtin protein (Htt). PolyQ expansion in Httexp causes selective degeneration of striatal medium spiny neurons (MSN) in HD patients. A number of previous studies suggested that dopamine signaling plays an important role in HD pathogenesis. A specific inhibitor of vesicular monoamine transporter (VMAT2) tetrabenazine (TBZ) has been recently approved by Food and Drug Administration for treatment of HD patients in the USA. TBZ acts by reducing dopaminergic input to the striatum.ResultsIn previous studies we demonstrated that long-term feeding with TBZ (combined with L-Dopa) alleviated the motor deficits and reduced the striatal neuronal loss in the yeast artificial chromosome transgenic mouse model of HD (YAC128 mice). To further investigate a potential beneficial effects of TBZ for HD treatment, we here repeated TBZ evaluation in YAC128 mice starting TBZ treatment at 2 months of age ("early" TBZ group) and at 6 months of age ("late" TBZ group). In agreement with our previous studies, we found that both "early" and "late" TBZ treatments alleviated motor deficits and reduced striatal cell loss in YAC128 mice. In addition, we have been able to recapitulate and quantify depression-like symptoms in TBZ-treated mice, reminiscent of common side effects observed in HD patients taking TBZ.ConclusionsOur results further support therapeutic value of TBZ for treatment of HD but also highlight the need to develop more specific dopamine antagonists which are less prone to side-effects.

Estrogen attenuates vascular remodeling in Lp(a) transgenic mice

ObjectiveAlthough it is well known that Lipoprotein(a) (Lp(a)) is an atherogenic lipoprotein and an independent risk factor for cardiovascular disease, there is no confirmed therapy to decrease Lp(a) or prevent atherosclerosis induced by Lp(a). Thus, it is mandatory to develop novel therapy to prevent atherosclerosis in high Lp(a) concentration. Here, we focused on the effect of estrogen on Lp(a) level and Lp(a)-induced vascular remodeling. MethodsWe employed Lp(a) transgenic mice (human apo(a) yeast artificial chromosome (YAC) and human apoB double transgenic mice). Vascular remodeling was induced by ligation of the common carotid artery and the effect of estrogen was evaluated in female mice after ovariectomy with or without estrogen replacement. ResultsEstrogen deficiency caused by ovariectomy increased serum Lp(a), and continuous replacement of 17β-estradiol (20μg/kg/day) reversed the change. In the vascular remodeling model induced by carotid artery occlusion, neointima formation was significantly increased in ovariectomized female Lp(a) transgenic mice, but few in male Lp(a) transgenic mice, as compared to wild FVB mice. Importantly, continuous replacement of estrogen in ovariectomized mice significantly attenuated it. In cultured endothelial cells and macrophages, addition of Lp(a) increased mRNA of ICAM-1, VCAM-1, E-selectin and MCP-1 in endothelial cells and TNF-α, IL-1β and MCP-1 in macrophages in a dose-dependent manner. Importantly, pre-treatment with estrogen attenuated these changes in a dose-dependent manner. ConclusionEstrogen negatively regulates both plasma Lp(a) level and Lp(a)-induced vascular remodeling, suggesting that estrogen might be a strong candidate to reduce serum Lp(a) concentration.

CTCF binding is not the epigenetic mark that establishes post-fertilization methylation imprinting in the transgenic H19 ICR

Imprinted expression of the mouse Igf2/H19 locus is controlled by parent-of-origin-specific methylation of the imprinting control region (ICR). We previously demonstrated that when placed in a heterologous genomic context, the H19 ICR fragment contains an intrinsic activity that allows it to acquire differential methylation in somatic cells but not in germ cells. In the present study, we investigated the requirements for the CTCF-binding sites of the ICR in the acquisition of post-fertilization methylation. To this end, two mutant ICR fragments were introduced into the human beta-globin locus in a yeast artificial chromosome transgenic mouse (TgM) model: 4xMut had mutations in all four ICR CTCF-binding sites that prevented CTCF binding but retained the methylation target CpG motifs, and -9CG harbored mutations in the CpG motifs within the CTCF-binding sites but each site retained constitutive CTCF-binding activity. In TgM germ cells and pre-implantation blastocysts, the absence of CTCF-binding sites (4xMut) did not lead to hypermethylation of the transgenic H19 ICR. However, after implantation, the mutations of CTCF sites (4xMut and -9CG) affected the maintenance of methylation. These results demonstrated that although the CTCF-binding sites are indispensable for maintenance of the unmethylated state of the maternal ICR in post-implantation embryos, they are not required to establish paternal-allele-specific methylation of the transgenic H19 ICR in pre-implantation embryos.