Mitochondria Profoundly Influence Apolipoprotein E Biology.

Background: Mitochondria can trigger Alzheimer's disease (AD)-associated molecular phenomena, but how mitochondria impact apolipoprotein E (APOE; apoE) is not well known.

Objective: Consider whether and how mitochondrial biology influences APOE and apoE biology.

Methods: We measured APOE expression in human SH-SY5Y neuronal cells with different forms of mitochondrial dysfunction including total, chronic mitochondrial DNA (mtDNA) depletion (ρ0 cells); acute, partial mtDNA depletion; and toxin-induced mitochondrial dysfunction.

O-GlcNAcylation and O-GlcNAc Cycling Regulate Gene Transcription: Emerging Roles in Cancer.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar post-translational modification (PTM) of intracellular proteins linking nutrient flux through the Hexosamine Biosynthetic Pathway (HBP) to the control of cis-regulatory elements in the genome. Aberrant O-GlcNAcylation is associated with the development, progression, and alterations in gene expression in cancer. O-GlcNAc cycling is defined as the addition and subsequent removal of the modification by O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) provides a novel method for cells to regulate various aspects of gene expression, including RNA polymerase function, epigenetic dynamics, and transcription factor activity.

NRF2 mediates γ-globin gene regulation through epigenetic modifications in a β-YAC transgenic mouse model.

Sickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model.

Episomal vectors based on S/MAR and the β-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient γ-globin activation in haematopoietic cells.

We report the development of episomal vectors for the specific γ-globin transcription activation in its native position by activator Zif-VP64, based on the Scaffold/Matrix Attachment Region (S/MAR) for episomal retention and the β-globin Replicator, the DNA replication-Initiation Region from the β-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV- Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine β-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34 cells, with transfection efficiencies of 46.3 ± 5.

An intergenic non-coding RNA promoter required for histone modifications in the human β-globin chromatin domain.

Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown.

Conjugate prodrug AN-233 induces fetal hemoglobin expression in sickle erythroid progenitors and β-YAC transgenic mice.

Pharmacologic induction of fetal hemoglobin (HbF) is an effective strategy for treating sickle cell disease (SCD) by ameliorating disease severity. Hydroxyurea is the only FDA-approved agent that induces HbF, but significant non-responders and requirement for frequent monitoring of blood counts for drug toxicity limit clinical usefulness. Therefore, we investigated a novel prodrug conjugate of butyric acid (BA) and δ-aminolevulinate (ALA) as a potential HbF inducing agent, using erythroid precursors and a preclinical β-YAC mouse model.

Mutational mimics of allosteric effectors: a genome editing design to validate allosteric drug targets.

Development of drugs that allosterically regulate enzyme functions to treat disease is a costly venture. Amino acid susbstitutions that mimic allosteric effectors in vitro will identify therapeutic regulatory targets enhancing the likelihood of developing a disease treatment at a reasonable cost. We demonstrate the potential of this approach utilizing human liver pyruvate kinase (hLPYK) as a model.

-GlcNAc homeostasis contributes to cell fate decisions during hematopoiesis.

The addition of a single β-d-GlcNAc sugar (-GlcNAc) by -GlcNAc-transferase (OGT) and -GlcNAc removal by -GlcNAcase (OGA) maintain homeostatic -GlcNAc levels on cellular proteins. Changes in protein -GlcNAcylation regulate cellular differentiation and cell fate decisions, but how these changes affect erythropoiesis, an essential process in blood cell formation, remains unclear. Here, we investigated the role of -GlcNAcylation in erythropoiesis by using G1E-ER4 cells, which carry the erythroid-specific transcription factor GATA-binding protein 1 (GATA-1) fused to the estrogen receptor (GATA-1-ER) and therefore undergo erythropoiesis after β-estradiol (E) addition.

A mutation affecting polycystin-1 mediated heterotrimeric G-protein signaling causes PKD.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of renal cysts that ultimately destroy kidney function. Mutations in the PKD1 and PKD2 genes cause ADPKD. Their protein products, polycystin-1 (PC1) and polycystin-2 (PC2) have been proposed to form a calcium-permeable receptor-channel complex; however the mechanisms by which they function are almost completely unknown.

Mouse Models of Erythropoiesis and Associated Diseases.

Animal models of erythropoiesis have been, and will continue to be, important tools for understanding molecular mechanisms underlying the development of this cell lineage and the pathophysiology associated with various human erythropoietic diseases. In this regard, the mouse is probably the most valuable animal model available to investigators. The physiology and short gestational period of mice make them ideal for studying developmental processes and modeling human diseases.

Sustained GlcNAcylation reprograms mitochondrial function to regulate energy metabolism.

Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked -acetylglucosamine (-GlcNAcylation) via overexpression of the GlcNAc-regulating enzymes GlcNAc transferase (OGT) or GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in GlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function.

Activation of Fetal γ-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains.

Reactivation of γ-globin expression has been shown to ameliorate disease phenotypes associated with mutations in the adult β-globin gene, including sickle cell disease. Specific mutations in the promoter of the γ-globin genes are known to prevent repression of the genes in the adult and thus lead to hereditary persistence of fetal hemoglobin. One such hereditary persistence of fetal hemoglobin is associated with a sequence located 567 bp upstream of the Gγ-globin gene which assembles a GATA-containing repressor complex.

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

One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2β repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in β-globin locus yeast artificial chromosome (β-YAC) bone marrow cells.

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.

O-GlcNAcase Expression is Sensitive to Changes in O-GlcNAc Homeostasis.

O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification involving an attachment of a single β-N-acetylglucosamine moiety to serine or threonine residues in nuclear and cytoplasmic proteins. Cellular O-GlcNAc levels are regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove the modification, respectively. The levels of O-GlcNAc can rapidly change in response to fluctuations in the extracellular environment; however, O-GlcNAcylation returns to a baseline level quickly after stimulus removal.

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.

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 (A)γ-globin gene.

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

The DNaseI hypersensitive sites (HSs) of the human β-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human β-globin locus yeast artificial chromosome (β-YAC) lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (β(m)), coupled to an intact LCR, a 5'HS3 complete deletion (5'ΔHS3) or a 5'HS3 core deletion (5'ΔHS3c).

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.

Transgenic expression of human LAMA5 suppresses murine Lama5 mRNA and laminin α5 protein deposition.

Laminin α5 is required for kidney glomerular basement membrane (GBM) assembly, and mice with targeted deletions of the Lama5 gene fail to form glomeruli. As a tool to begin to understand factors regulating the expression of the LAMA5 gene, we generated transgenic mice carrying the human LAMA5 locus in a bacterial artificial chromosome. These mice deposited human laminin α5 protein into basement membranes in heart, liver, spleen and kidney.

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach.

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system.

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo.

Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells.

Mutation in erythroid specific transcription factor KLF1 causes Hereditary Spherocytosis in the Nan hemolytic anemia mouse model.

KLF1 regulates definitive erythropoiesis of red blood cells by facilitating transcription through high affinity binding to CACCC elements within its erythroid specific target genes including those encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins in humans lead to the hemolytic anemia Hereditary Spherocytosis (HS) which includes a subpopulation with no known genetic defect. Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in the mouse model Nan.

Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production.

Through the shuffling of predefined modular zinc finger domains with predictable target site recognition in vitro, we have generated a large repertoire of artificial transcription factors with five zinc finger domains (TF(ZF)s). Here we report an effective strategy for the selection of ATF libraries by coupling expression of transcriptional activators of the promoter of interest to the enhanced production of retroviral vector particles transferring the TF(ZF) encoding gene. Using this strategy, we successfully selected specific TF(ZF)s that upregulate the expression of the gamma-globin promoter.