Yeast Artificial Chromosome Research Articles

PGC-1α agonism induces fetal hemoglobin and exerts antisickling effects in sickle cell disease.

Sickle cell disease is a growing health burden afflicting millions around the world. Clinical observation and laboratory studies have shown that the severity of sickle cell disease is ameliorated in individuals who have elevated levels of fetal hemoglobin. Additional pharmacologic agents to induce sufficient fetal hemoglobin to diminish clinical severity is an unmet medical need. We recently found that up-regulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) can induce fetal hemoglobin synthesis in human primary erythroblasts. Here, we report that a small molecule, SR-18292, increases PGC-1α leading to enhanced fetal hemoglobin expression in human erythroid cells, β-globin yeast artificial chromosome mice, and sickle cell disease mice. In SR-18292-treated sickle mice, sickled red blood cells are significantly reduced, and disease complications are alleviated. SR-18292, or agents in its class, could be a promising additional therapeutic for sickle cell disease.

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus.

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acidcycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.

High-level expression of leghemoglobin in Kluyveromyces marxianus by remodeling the heme metabolism pathway.

Soy leghemoglobin, when bound to heme, imparts a meat-like color and flavor and can serve as a substitute for animal-derived proteins. Enhancing cellular heme synthesis improves the recombinant expression of leghemoglobin in yeast. To achieve high-level expression of leghemoglobin A (LBA) in Kluyveromyces marxianus, a food-safe yeast, large-scale heme synthesis modules were transferred into K. marxianus using yeast artificial chromosomes (KmYACs). These modules contained up to 8 native and heterologous genes to promote the supply of heme precursors and downstream synthesis. Next, eight genes inhibiting heme or LBA synthesis were individually or combinatorially deleted, with the lsc1Δssn3Δ mutant yielding the best results. Subsequently, heme synthesis modules were combined with the lsc1Δssn3Δ mutant. In the resulting strains, the module genes were all actively expressed. Among these module genes, heterologous S. cerevisiae genes in the downstream heme synthesis pathway significantly enhanced the expression of their counterparts in K. marxianus, resulting in high heme content and LBA yield. After optimizing the medium recipe by adjusting the concentrations of glucose, glycine, and FeSO4·7H2O, a heme content of 66.32mg/L and an intracellular LBA titer of 7.27g/L were achieved in the engineered strain in a 5L fermentor. This represents the highest intracellular expression of leghemoglobin in microorganisms to date. The leghemoglobin produced by K. marxianus can be utilized as a safe ingredient for plant-based protein products.

Beyond Motor Deficits: Environmental Enrichment Mitigates Huntington's Disease Effects in YAC128 Mice.

Huntington's disease (HD) is a neurodegenerative genetic disorder characterized by motor, psychiatric, cognitive, and peripheral symptoms without effective therapy. Evidence suggests that lifestyle factors can modulate disease onset and progression, and environmental enrichment (EE) has emerged as a potential approach to mitigate the progression and severity of neurodegenerative processes. Wild-type (WT) and yeast artificial chromosome (YAC) 128 mice were exposed to different EE conditions. Animals from cohort 1 were exposed to EE between postnatal days 21 and 60, and animals from cohort 2 were exposed to EE between postnatal days 60 and 120. Motor and non-motor behavioral tests were employed to evaluate the effects of EE on HD progression. Monoamine levels, hippocampal cell proliferation, neuronal differentiation, and dendritic arborization were also assessed. Here we show that EE had an antidepressant-like effect and slowed the progression of motor deficits in HD mice. It also reduced monoamine levels, which correlated with better motor performance, particularly in the striatum. EE also modulated neuronal differentiation in the YAC128 hippocampus. These results confirm that EE can impact behavior, hippocampal neuroplasticity, and monoamine levels in YAC128 mice, suggesting this could be a therapeutic strategy to modulate neuroplasticity deficits in HD. However, further research is needed to fully understand EE's mechanisms and long-term effects as an adjuvant therapy for this debilitating condition.

Reverse genetic systems of SARS-CoV-2 for antiviral research

Reverse genetic systems are widely used to engineer recombinant viruses with desired mutations. In response to the COVID-19 pandemic, four types of reverse genetic systems have been developed for SARS-CoV-2: (i) a full-length infectious clone that can be used to prepare recombinant SARS-CoV-2 at biosafety level 3 (BSL3), (ii) a trans-complementation system that can be used to produce single-round infectious SARS-CoV-2 at BSL2, (iii) an attenuated SARS-CoV-2 vaccine candidate (with deletions of viral accessory genes) that may be developed for veterinary use as well as for antiviral screening at BSL2, and (iv) replicon systems with deletions of viral structural genes that can be used at BSL2. Each of these genetic systems has its advantages and disadvantages that can be used to address different questions for basic and translational research. Due to the long genomic size and bacteria-toxic sequences of SARS-CoV-2, several experimental approaches have been established to rescue recombinant viruses and replicons, including (i) in vitro DNA ligation, (ii) bacterial artificial chromosome (BAC) system, (iii) yeast artificial chromosome (YAC) system, and (iv) circular polymerase extension reaction (CPER). This review summarizes the current status of SARS-CoV-2 genetic systems and their applications for studying viral replication, pathogenesis, vaccines, and therapeutics.

Alternative processing of human HTT mRNA with implications for Huntington's disease therapeutics.

Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington's disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualize HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. Homogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to produce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington's disease. These approaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted antisense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood.

Novel reverse genetics of genotype I and III Japanese encephalitis viruses assembled through transformation associated recombination in yeast: The reporter viruses expressing a green fluorescent protein for the antiviral screening assay

Japanese encephalitis virus (JEV) belongs to a zoonotic flavivirus and is the main cause of viral encephalitis in humans throughout Asia. During the past two decades, both genotypes I (G) and III (GIII) JEVs co-existed in many Asian countries, and the prevalent strains have shifted from GIII to GI. Since all licensed JE vaccines are derived from GIII strains and no specific treatment is available, the development of novel vaccines and specific antiviral drugs against both genotypes is urgently required. To solve the unstable issue of the JEV infectious cDNA clone and establish a reliable antiviral screening assay, we established the yeast artificial chromosome (YAC)-based reverse genetics systems for GI and GIII JEV strains through transformation-associated recombination (TAR) technology in yeast. The YAC-based infectious clones of GI and GIII JEV exhibited high genetic stability both in yeast and E. coli. Using these reverse genetics systems, recombinant EGFP-reporter viruses were generated and remained stable for at least 10 passages in vitro. An image-based antiviral assay for JEV was developed with the EGFP-reporter viruses, and two drugs were identified to have a broad-spectrum inhibitory effect on GI and GIII JEV replication, which provide potential new therapeutic for the treatment of multiple genotypes JEV infection.

Exogenous artificial DNA forms chromatin structure with active transcription in yeast

Yeast artificial chromosomes (YACs) are important tools for sequencing, gene cloning, and transferring large quantities of genetic information. However, the structure and activity of YAC chromatin, as well as the unintended impacts of introducing foreign DNA sequences on DNA-associated biochemical events, have not been widely explored. Here, we showed that abundant genetic elements like TATA box and transcription factor-binding motifs occurred unintentionally in a previously reported data-carrying chromosome (dChr). In addition, we used state-of-the-art sequencing technologies to comprehensively profile the genetic, epigenetic, transcriptional, and proteomic characteristics of the exogenous dChr. We found that the data-carrying DNA formed active chromatin with high chromatin accessibility and H3K4 tri-methylation levels. The dChr also displayed highly pervasive transcriptional ability and transcribed hundreds of noncoding RNAs. The results demonstrated that exogenous artificial chromosomes formed chromatin structures and did not remain as naked or loose plasmids. A better understanding of the YAC chromatin nature will improve our ability to design better data-storage chromosomes.

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.

Cytogenetic and Molecular Delineation of a Region of Chromosome 7 Commonly Deleted in Malignant Myeloid Diseases

Loss of a whole chromosome 7 or a deletion of the long arm, del(7q), are recurring abnormalities in malignant myeloid diseases. To determine the location of genes on 7q that are likely to play a role in leukemogenesis, we examined the deleted chromosome 7 homologs in a series of 81 patients with therapy-related or de novo myelodysplastic syndrome or acute myeloid leukemia. Our analysis showed that the deletions were interstitial and that there were two distinct deleted segments of 7q. The majority of patients (65 of 81 [80%]) had proximal breakpoints in bands q11-22 and distal breakpoints in q31-36; the smallest overlapping deleted segment was within q22. The remaining 16 patients had deletions involving the distal q arm with a commonly deleted segment of q32-33. To define the proximal deleted segment at 7q22 at a molecular level, we used fluorescence in situ hybridization with a panel of mapped yeast artificial chromosome (YAC) clones from 7q to examine 15 patients with deletion breakpoints in 7q22. We determined that the smallest overlapping deleted segment is contained in a well-defined YAC contig that spans 2 to 3 Mb. These studies delineate the region of 7q that must be searched to isolate a putative myeloid leukemia suppressor gene, and provide the necessary cloned DNA for more detailed physical mapping and gene isolation.

Growth of Saccharomyces cerevisiae and Preparation of DNA.

This protocol describes methods for isolation of total DNA from a strain of Sacchromyces cerevisiae carrying a recombinant yeast artificial chromosome (YAC). This method is appropriate for preparing DNA that will be subjected to regular agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, polymerase chain reaction (PCR), or other methods that do not require intact high-molecular-weight DNA. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. Drop dialysis should be used to exchange buffers. The expected yield from a 10-mL culture is 2-4 µg of yeast DNA.

Neural stem cells derived from the developing forebrain of YAC128 mice exhibit pathological features of Huntington's disease.

ObjectivesHuntington's disease (HD) is a devastating neurodegenerative disease caused by polyglutamine (polyQ) expansion in the huntingtin (HTT) gene. Mutant huntingtin (mHTT) is the main cause of HD and is associated with impaired mitochondrial dynamics, ubiquitin‐proteasome system and autophagy, as well as tauopathy. In this study, we aimed to establish a new neural stem cell line for HD studies.Materials and methodsYAC128 mice are a yeast artificial chromosome (YAC)‐based transgenic mouse model of HD. These mice express a full‐length human mutant HTT gene with 128 CAG repeats and exhibit various pathophysiological features of HD. In this study, we isolated a new neural stem cell line from the forebrains of YAC128 mouse embryos (E12.5) and analysed its characteristics using cellular and biochemical methods.ResultsCompared to wild‐type (WT) NSCs, the YAC128 NSC line exhibited greater proliferation and migration capacity. In addition to mHTT expression, increased intracellular Ca2+ levels and dysfunctional mitochondrial membrane potential were observed in the YAC128 NSCs. YAC128 NSCs had defects in mitochondrial dynamics, including a deficit in mitochondrial axonal transport and unbalanced fusion and fission processes. YAC128 NSCs also displayed decreased voltage response variability and Na+ current amplitude. Additionally, the ubiquitin‐proteasome and autophagy systems were impaired in the YAC128 NSCs.ConclusionsWe have established a new neural stem line from YAC128 transgenic mice, which may serve as a useful resource for studying HD pathogenesis and drug screening.

Comparative studies of glial fibrillary acidic protein and brain-derived neurotrophic factor expression in two transgenic mouse models of Alzheimer's disease.

In Alzheimer's disease (AD) glial fibrillary acidic protein (GFAP) is expressed by reactive astrocytes surrounding β-amyloid (Aβ) plaques, whereas brain-derived neurotrophic factor (BDNF) levels are typically reduced. We compared the expression of GFAP, BDNF, and its precursor proBDNF in the dorsal hippocampus of two transgenic AD mouse models. APPSwe YAC mice expressing the APPSwe transgene on a yeast artificial chromosome (YAC) were assessed at age 4 and 21months, and APPSwe/PS1dE9 mice co-expressing mutant amyloid precursor protein (APPSwe) and presenilin-1 (PS1dE9) were assessed at age 4 and 9months. Significantly increased (1.4-fold) GFAP expression was observed in APPSwe YAC c.f. wild-type (Wt) mice aged 21months, when Aβ deposition was first evident in these mice. In APPSwe/PS1dE9 mice aged 4 and 9months, GFAP expression was significantly increased (1.6- and 3.1-fold, respectively) c.f. Wt mice, and was associated with robust Aβ deposition at 9months. BDNF expression was significantly lower in 4- and 21-month old APPSwe YAC mice (0.8- and 0.6-fold, respectively) c.f. age-matched Wt mice, whereas proBDNF expression was significantly higher (10-fold) in the APPSwe YAC c.f. Wt mice aged 21 months. In APPSwe/PS1dE9 mice aged 4 months, BDNF expression was significantly lower (0.4-fold) c.f. age-matched Wt mice and was equivalent to that in 9-month old mice of both genotypes; proBDNF expression mirrored that of BDNF in this strain. These findings support a role for reactive astrocytes and neuroinflammation, rather than BDNF, in the spatial memory deficits previously reported for APPSwe YAC and APPSwe/PS1dE9 mice.

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform.

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.

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.

Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10.

Large expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)n or a (ATCCT)n/(ATCCC)n interruption within the (ATTCT)n repeat is associated with the robust phenotype of ataxia and epilepsy while mostly pure (ATTCT)n may have reduced penetrance. Large repeat expansions of SCA10, and many other microsatellite expansions, can exceed 10,000 base pairs (bp) in size. Conventional next generation sequencing (NGS) technologies are ineffective in determining internal sequence contents or size of these expanded repeats. Using repeat primed PCR (RP-PCR) in conjunction with a high-sensitivity pulsed-field capillary electrophoresis fragment analyzer (FEMTO-Pulse, Agilent, Santa Clara, CA) (RP-FEMTO hereafter), we successfully determined sequence content of large expansion repeats in genomic DNA of SCA10 patients and transformed yeast artificial chromosomes containing SCA10 repeats. This RP-FEMTO is a simple and economical methodology which could complement emerging NGS for very long sequence reads such as Single Molecule, Real-Time (SMRT) and nanopore sequencing technologies.

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.

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.

Genetic Assays to Study Repeat Fragility in Saccharomyces cerevisiae.

Trinucleotide repeats are common in the human genome and can undergo changes in repeat number and cause length-dependent chromosome fragility. Expanded CAG repeats have been linked to over 14 human diseases and are considered hotspots for breakage and genomic rearrangement. Here we describe two Saccharomyces cerevisiae based assays that evaluate the rate of chromosome breakage that occurs within a repeat tract (fragility), with variations that allow the role of transcription to be evaluated. The first fragility assay utilizes end-loss and subsequent telomere addition as the main mode of repair of a yeast artificial chromosome (YAC). The second fragility assay relies on the fact that a chromosomal break stimulates recombination-mediated repair. A PCR-based assay can be used to evaluate instability of the repeat in the same conditions used to measure repeat fragility. These assays have contributed to understanding the genetic mechanisms that cause chromosome breaks and tract-length changes at unstable trinucleotide repeats.

Spiral ganglion cell degeneration-induced deafness as a consequence of reduced GATA factor activity.

Zinc-finger transcription factors GATA2 and GATA3 are both expressed in the developing inner ear, although their overlapping versus distinct activities in adult definitive inner ear are not well understood. We show here that GATA2 and GATA3 are co-expressed in cochlear spiral ganglion cells and redundantly function in the maintenance of spiral ganglion cells and auditory neural circuitry. Notably, Gata2 and Gata3 compound heterozygous mutant mice had a diminished number of spiral ganglion cells due to enhanced apoptosis, which resulted in progressive hearing loss. The decrease in spiral ganglion cellularity was associated with lowered expression of neurotrophin receptor TrkC that is an essential factor for spiral ganglion cell survival. We further show that Gata2 null mutants that additionally bear a Gata2 YAC (yeast artificial chromosome) that counteracts the lethal hematopoietic deficiency due to complete Gata2 loss nonetheless failed to complement the deficiency in neonatal spiral ganglion neurons. Furthermore, cochlea-specific Gata2 deletion mice also had fewer spiral ganglion cells and resultant hearing impairment. These results show that GATA2 and GATA3 redundantly function to maintain spiral ganglion cells and hearing. We propose possible mechanisms underlying hearing loss in human GATA2- or GATA3-related genetic disorders.