Ankyrin Gene Research Articles

Analysis of the Mobilities of Band 3 Populations Associated with Ankyrin Protein and Junctional Complexes in Intact Murine Erythrocytes

Current models of the erythrocyte membrane depict three populations of band 3: (i) a population tethered to spectrin via ankyrin, (ii) a fraction attached to the spectrin-actin junctional complex via adducin, and (iii) a freely diffusing population. Because many studies of band 3 diffusion also distinguish three populations of the polypeptide, it has been speculated that the three populations envisioned in membrane models correspond to the three fractions observed in diffusion analyses. To test this hypothesis, we characterized band 3 diffusion by single-particle tracking in wild-type and ankyrin- and adducin-deficient erythrocytes. We report that ∼40% of total band 3 in wild-type murine erythrocytes is attached to ankyrin, whereas ∼33% is immobilized by adducin, and ∼27% is not attached to any cytoskeletal anchor. More detailed analyses reveal that mobilities of individual ankyrin- and adducin-tethered band 3 molecules are heterogeneous, varying by nearly 2 orders of magnitude and that there is considerable overlap in diffusion coefficients for adducin and ankyrin-tethered populations. Taken together, the data suggest that although the ankyrin- and adducin-immobilized band 3 can be monitored separately, significant heterogeneity still exists within each population, suggesting that structural and compositional properties likely vary considerably within each band 3 complex.

Role of MicroRNA-486-5p Overexpression In CML CD34+ Cells In Modulating BCR-ABL Mediated Hematopoietic Stem/Progenitor Cell Transformation and Imatinib Sensitivity

Abstract 1667MicroRNAs are key regulators of gene expression that regulate normal differentiation and contribute to malignant transformation of hematopoietic cells. Using microRNA microarrays we identified increased expression of miR-486 in chronic myeloid leukemia (CML) compared to normal CD34+ cells. In both normal and CML cells, miR-486 expression level was significantly higher in MEP compared to HSC, GMP and CMP populations. Treatment with Imatinib resulted in reduced expression of miR-486-5p in CML CD34+ cells, suggesting that upregulation of miR-486-5p expression was at least in part BCR-ABL kinase dependent. Consistent with this ectopic expression of BCR-ABL in cord blood CD34+ cells using retroviral vectors resulted in 4.2 fold increase in miR-486-5p expression. miR-486-5p is located within the last intron of the Ankyrin-1 gene on chromosome 8 and is enriched in muscle cells. However, the role of miR-486-5p in normal and leukemic hematopoiesis has not been evaluated. To explore the role of miR-486-5p in growth and differentiation of hematopoietic progenitor cells (HSPC), we first overexpressed hsa-miR-486-5p pre-microRNA in normal CD34+ cells using lentiviral vectors. CB CD34+ cells overexpressing miRNA-486-5p generated modestly increased numbers of cells (1.22 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO for 6 days compared to cells expressing control vectors, with increased numbers of erythroid cells and reduced numbers of myeloid cells. We further investigated the role of miR-486-5p on growth and differentiation of normal and leukemic HSPC by inhibiting miR-486-5p expression using a modified pmiRZip lentivirus vector expressing an anti-miR-486-5p sequence and comparing to cells expressing a control scrambled anti-miRNA sequence. Expression of anti-miR-486-5p resulted in reduced proliferation of normal CD34+ cells (32±10% inhibition) and BCR-ABL transformed CD34+ cells (38±7 % inhibition) with significantly greater inhibition of erythroid compared to myeloid cells. Anti-miR486-5p expression resulted in significantly increased apoptosis of BCR-ABL-transformed CD34+ cells but not normal CD34+ cells (CML CD34+ cells: scramble 11.1±2.4%, anti-miR-486-5p 14.7±1.7 % p=0.02; Normal CD34+ cells: scramble 9.7±5.4%, anti-486-5p 13.4±7.9% p=0.15). Importantly, anti-miR-486-5p significantly enhanced the sensitivity of BCR-ABL transformed CD34+ cells to imatinib-mediated apoptosis [combination of scramble with IM: 17.7±8.1%; anti-miR-486-5p with IM: 26.4±13%]. A search for conserved miR-486-5p target genes in the TargetScan database identified the important hematopoietic negative regulatory factors Foxo1 and Pten amongst the highest ranking targets. Using pMIR-REPORT constructs containing miR-486-5p seed sites within the Foxo1 and Pten 3’-UTR we showed that Foxo1 and Pten are direct targets of miR-486-5p. Expression of anti-miR-486-5p increased Foxo1 and Pten protein expression and decreased active Akt in normal and CML CD34+ cells. Knockdown of Foxo1 using shRNA partly blocked the suppressive effects of anti-miR486-5p on the growth of CD34+ cells. In summary, we have shown that miR-486-5p expression is modulated during hematopoietic differentiation and plays an important role in regulating hematopoietic progenitor growth and differentiation towards the erythroid lineage. We further show that miR-486-5p expression is enhanced in CML CD34+ cells, related at least in part to BCR-ABL kinase activity, and contributes to enhanced progenitor growth and survival. Inhibition of miR-486-5p results in enhanced sensitivity of CML CD34+ cells to IM-induced apoptosis. miR-486-5p effects are mediated at least in part through inhibition of Foxo1 and Pten expression. We conclude that miR-486-5p represents a novel regulatory mechanism that promotes erythroid differentiation in normal hematopoiesis and modulates Bcr-Abl-mediated transformation and tyrosine kinase inhibitor sensitivity in CML progenitors. Disclosures:No relevant conflicts of interest to declare.

Multigenic Families in Ichnovirus: A Tissue and Host Specificity Study through Expression Analysis of Vankyrins from Hyposoter didymator Ichnovirus

The viral ankyrin (vankyrin) gene family is represented in all polydnavirus (PDVs) genomes and encodes proteins homologous to I-kappaBs, inhibitors of NF-kappaB transcription factors. The structural similarities led to the hypothesis that vankyrins mimic eukaryotic factors to subvert important physiological pathways in the infected host. Here, we identified nine vankyrin genes in the genome of the Hyposoter didymator Ichnovirus (HdIV). Time-course gene expression experiments indicate that all members are expressed throughout parasitism of Spodoptera frugiperda, as assessed using RNA extracted from whole larvae. To study tissue and/or species specificity transcriptions, the expression of HdIV vankyrin genes were compared between HdIV-injected larvae of S. frugiperda and S. littoralis. The transcriptional profiles were similar in the two species, including the largely predominant expression of Hd27-vank1 in all tissues examined. However, in various insect cell lines, the expression patterns of HdIV vankyrins differed according to species. No clear relationship between vankyrin expression patterns and abundance of vankyrin-bearing genomic segments were found in the lepidopteran cell lines. Moreover, in these cells, the amount of vankyrin-bearing genomic segments differed substantially between cytosol and nuclei of infected cells, implying the existence of an unexpected step regulating the copy number of HdIV segments in cell nuclei. Our in vitro results reveal a host-specific transcriptional profile of vankyrins that may be related to the success of parasitism in different hosts. In Spodoptera hosts, the predominant expression of Hd27-vank1 suggests that this protein might have pleiotropic functions during parasitism of these insect species.

Altered expression of endogenous transforming growth factor β1 and early calcification related genes in rat endplate

To explore the relationship between endogenous transforming growth factor (TGF)-β1 and calcification-related genes through an in vitro degeneration model by propagating rat endplate chondrocytes during a natural degeneration process. Endplate chondrocytes were extracted from rat lumbar vertebrae, isolated by enzyme digestion and P2 and P4 generations selected for a 6-day in vitro culture. The specimens were photographed microscopically to observe the cellular differences by alizarin red staining. Type II collagen marker gene, transcription factor SOX-9 gene and metabolism-related genes proteoglycan. matrix metalloproteinase (MMP)-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 were detected by RT-PCR to verify the degeneration model. Based on this model, the changes of growth factor TGF-β1 and calcification-related genes ankyrin (ANK), ectonucleotide pyrophosphatase (ENPP), tissue-nonspecific alkaline phosphatase (TNAP) were continuously tested. Compared with P2 cells, P4 cells tended to assume a spindle-shaped morphology. And alizarin red staining showed no change between them. The level of transcription factor SOX-9 of P4 cells (P4/P2 = 0.0690, P = 0.0489) was significantly lower than that of P2 cells. Type II collagen (P4/P2 = 0.0535, P = 0.009) and proteoglycan (P4/P2 = 0.2672, P = 0.0343) were also significantly lower than those of P2 cells. No significant changes were observed in other metabolism-related genes. TGF-β1 (P4/P2 = 0.5934, P = 0.0482) was significantly lower. The expressions of TNAP (P4/P2 = 0.0385, P = 0.0139) and ANK (P4/P2 = 0.2121, P = 0.0009) were significantly lower. But ENPP showed no significant change. P4 endplate chondrocytes undergo natural degeneration in vitro with the rising passage number. Type II collagen, SOX-9 and proteoglycan are significantly reduced. Endogenous TGF-β1 gene and calcification-related genes are down-regulated. The decrease of ANK gene may be caused by the down-regulation of endogenous TGF-β1. Modulating the expression of endogenous TGF-β1 gene in endplate chondrocytes may become a new therapeutic approach for the degeneration of intervertebral disc.

Regulation of DMD pathology by an ankyrin-encoded miRNA

BackgroundDuchenne muscular dystrophy (DMD) is an X-linked myopathy resulting from the production of a nonfunctional dystrophin protein. MicroRNA (miRNA) are small 21- to 24-nucleotide RNA that can regulate both individual genes and entire cell signaling pathways. Previously, we identified several mRNA, both muscle-enriched and inflammation-induced, that are dysregulated in the skeletal muscles of DMD patients. One particularly muscle-enriched miRNA, miR-486, is significantly downregulated in dystrophin-deficient mouse and human skeletal muscles. miR-486 is embedded within the ANKYRIN1(ANK1) gene locus, which is transcribed as either a long (erythroid-enriched) or a short (heart muscle- and skeletal muscle-enriched) isoform, depending on the cell and tissue types.ResultsInhibition of miR-486 in normal muscle myoblasts results in inhibited migration and failure to repair a wound in primary myoblast cell cultures. Conversely, overexpression of miR-486 in primary myoblast cell cultures results in increased proliferation with no changes in cellular apoptosis. Using bioinformatics and miRNA reporter assays, we have identified platelet-derived growth factor receptor β, along with several other downstream targets of the phosphatase and tensin homolog deleted on chromosome 10/AKT (PTEN/AKT) pathway, as being modulated by miR-486. The generation of muscle-specific transgenic mice that overexpress miR-486 revealed that miR-486 alters the cell cycle kinetics of regenerated myofibers in vivo, as these mice had impaired muscle regeneration.ConclusionsThese studies demonstrate a link for miR-486 as a regulator of the PTEN/AKT pathway in dystrophin-deficient muscle and an important factor in the regulation of DMD muscle pathology.

Association between promoter polymorphisms in a key cytoskeletal gene (Ankyrin 1) and intramuscular fat and water-holding capacity in porcine muscle

Ankyrin 1 (ANK1) is a positional and functional candidate gene for both bovine and porcine meat quality. The objectives of this study were: (1) to determine if the gene expression levels of ANK1 are associated with pig meat quality traits; (2) to examine polymorphisms in the promoter region of the porcine ANK1 gene for association with meat quality in diverse breeds; and (3) to search for putative transcription factor binding sites predicted to be altered by such polymorphisms. ANK1 gene expression was positively correlated with drip loss (%) in the Large White breed. Twelve novel SNPs were discovered in a 761bp region of the ANK1 promoter in three phenotypically diverse F1 cross populations. Five were subsequently selected for association analysis with meat quality traits and genotyped in three pure pig breeds (Pietrain n=98, Duroc n=99 and Large White n=98). Two of these five SNPs were associated with meat quality traits at the Bonferroni significance threshold: g.-606G>A with drip loss % in the Pietrain breed and g.-272G>A with intramuscular fat (IMF) in the LTL and SM. Following haplotype construction from SNP genotypes, Haplotype 3 was found to be associated with drip loss % at the Bonferroni level of significance in the Pietrain breed and Haplotype 5 was associated at the Bonferroni level with IMF in two muscles in the Large White breed. Further associations were observed at the nominal significance threshold. Our conclusion from this study is that SNPs in the ANK1 gene promoter could potentially contribute to genome-assisted selection SNP panels to improve IMF and water holding capacity on a breed basis.

Novel interactions of ankyrins-G at the costameres: The muscle-specific Obscurin/Titin-Binding-related Domain (OTBD) binds plectin and filamin C

Ankyrins, the adapters of the spectrin skeleton, are involved in local accumulation and stabilization of integral proteins to the appropriate membrane domains. In striated muscle, tissue-dependent alternative splicing generates unique Ank3 gene products (ankyrins-G); they share the Obscurin/Titin-Binding-related Domain (OTBD), a muscle-specific insert of the C-terminal domain which is highly conserved among ankyrin genes, and binds obscurin and titin to Ank1 gene products. We previously proposed that OTBD sequences constitute a novel domain of protein–protein interactions which confers ankyrins with specific cellular functions in muscle. Here we searched for muscle proteins binding to ankyrin-G OTBD by yeast two hybrid assay, and we found plectin and filamin C, two organizing elements of the cytoskeleton with essential roles in myogenesis, muscle cell cytoarchitecture, and muscle disease. The three proteins coimmunoprecipitate from skeletal muscle extracts and colocalize at costameres in adult muscle fibers. During in vitro myogenesis, muscle ankyrins-G are first expressed in postmitotic myocytes undergoing fusion to myotubes. In western blots of subcellular fractions from C2C12 cells, the majority of muscle ankyrins-G appear associated with membrane compartments. Occasional but not extensive co-localization at nascent costameres suggested that ankyrin-G interactions with plectin and filamin C are not involved in costamere assembly; they would rather reinforce stability and/or modulate molecular interactions in sarcolemma microdomains by establishing novel links between muscle-specific ankyrins-G and the two costameric dystrophin-associated glycoprotein and integrin-based protein complexes. These results report the first protein–protein interactions involving the ankyrin-G OTBD domain and support the hypothesis that OTBD sequences confer ankyrins with a gain of function in vertebrates, bringing further consolidation and resilience of the linkage between sarcomeres and sarcolemma.

Nodes of Ranvier Act as Barriers to Restrict Invasion of Flanking Paranodal Domains in Myelinated Axons

Accumulation of voltage-gated sodium (Na(v)) channels at nodes of Ranvier is paramount for action potential propagation along myelinated fibers, yet the mechanisms governing nodal development, organization, and stabilization remain unresolved. Here, we report that genetic ablation of the neuron-specific isoform of Neurofascin (Nfasc(NF¹⁸⁶)) in vivo results in nodal disorganization, including loss of Na(v) channel and ankyrin-G (AnkG) enrichment at nodes in the peripheral nervous system (PNS) and central nervous system (CNS). Interestingly, the presence of paranodal domains failed to rescue nodal organization in the PNS and the CNS. Most importantly, using ultrastructural analysis, we demonstrate that the paranodal domains invade the nodal space in Nfasc(NF¹⁸⁶) mutant axons and occlude node formation. Our results suggest that Nfasc(NF¹⁸⁶)-dependent assembly of the nodal complex acts as a molecular boundary to restrict the movement of flanking paranodal domains into the nodal area, thereby facilitating the stereotypic axonal domain organization and saltatory conduction along myelinated axons.

Mutation of a barrier insulator in the human ankyrin-1 gene is associated with hereditary spherocytosis

Defects of the ankyrin-1 gene are the most common cause in humans of hereditary spherocytosis, an inherited anemia that affects patients of all ethnic groups. In some kindreds, linked -108/-153 nucleotide substitutions have been found in the upstream region of the ankyrin gene promoter that is active in erythroid cells. In vivo, the ankyrin erythroid promoter and its upstream region direct position-independent, uniform expression, a property of barrier insulators. Using human erythroid cell lines and primary cells and transgenic mice, here we have demonstrated that a region upstream of the erythroid promoter is a barrier insulator in vivo in erythroid cells. The region exhibited both functional and structural characteristics of a barrier, including prevention of gene silencing in an in vivo functional assay, appropriate chromatin configuration, and occupancy by barrier-associated proteins. Fragments with the -108/-153 spherocytosis-associated mutations failed to function as barrier insulators in vivo and demonstrated perturbations in barrier-associated chromatin configuration. In transgenic mice, flanking a mutant -108/-153 ankyrin gene promoter with the well-characterized chicken HS4 barrier insulator restored position-independent, uniform expression at levels comparable to wild-type. These data indicate that an upstream region of the ankyrin-1 erythroid promoter acts as a barrier insulator and identify disruption of the barrier element as a potential pathogenetic mechanism of human disease.

An Active Chromatin Hub Involving the Three Alternate Promoter/First Exons of the Ankyrin-1 (ANK-1) Gene

AbstractAbstract 3862The Ankyrin-1 (ANK-1) gene is an example of the many mammalian genes with alternate promoters in which transcripts containing distinct first exons are fused to a common exon 2 in different cell types. The ANK-1E promoter/first exon is active in only erythroid cells and is located between a promoter/first exon located 40 kb upstream that is expressed exclusively in brain and muscle cells (ANK-1B) and a promoter/first exon located 16 kb downstream that is expressed in many cell types (ANK-1A). We have previously shown that the ANK-1E promoter/first exon is flanked by two DNase I hypersensitive sites (HS). The ANK-1E 5′HS is located in the promoter region, while two adjacent HS (3′HS 1&2) are located ∼6 kb downstream. We hypothesize that the erythroid-specific activation of the ANK-1E promoter/first exon involves both changes in transcription factor occupancy and in chromatin structure. Using whole-genome Chromatin Immune Precipitation (ChIPSeq) and whole-genome mRNA analysis (RNASeq), we have made a detailed study of Erythroid Krupple-like Factor (EKLF) occupancy and mRNA levels in sorted mouse erythroid progenitor cells and definitive erythroblasts. In erythroid progenitor cells, EKLF occupies the Ank-1E 5′HS, and the level of Ank-1E mRNA is relatively low. In erythroblasts, EKLF is not detected at either Ank-1E 5′HS or the Ank-1E 3′HS and the Ank-1 mRNA level is increased more than 2-fold, consistent with EKLF acting as a transcriptional repressor. We have also shown that the ANK-1E 5′HS and the 3′HS region are both occupied by the transcription factors GATA-1 and NF-E2, although EMSA assays have shown that NF-E2 does not bind to the 5′HS region, and that GATA-1 does not bind to the 3′HS region in vitro. Addition of the NF-E2 site in 3′HS1 to the ANK-1E promoter increases expression 4-fold indicating a potential interaction of these two regions. We have recently demonstrated that both the ANK-1E 5′HS and 3′HS2 act as barrier elements that prevent gene silencing in cultured erythroid cells and transgenic mice. Consistent with the functional observations, in erythroid cells both ANK-1E 5′HS and the 3′HS region are occupied by the barrier-associated proteins USF-1 and -2, PRMT-1 and -4, CTCF and SRC1, while the ANK-1A region is occupied by the barrier-associated proteins USF-2, PRMT-1 and -4, but not USF-1 and CTCF. We hypothesized that the HS flanking the ANK-1E promoter/first exon form a chromatin loop that brings both the transcription factors and barrier proteins into proximity. Using the chromatin conformation capture assay we demonstrated an interaction between the Ank-1E 5′HS and the 3′HS region. Analysis of the three dimensional structure of the 100 kb region upstream of the mouse Ank-1 exon 2 region was performed using the chromatin conformation capture carbon copy (5C) assay. We designed a set of 12 forward primers on the three Ank-1 promoter/first exons and the HS that flank them, and a set of 264 reverse primers to analyze all interactions in this region. In mouse fetal liver erythroid cells, the strongest interaction is between Ank-1E 5′HS and a region 15 kb 3′ to the Ank-1B region. In addition, the Ank-1B promoter/first exon region interacts with both the Ank-1A region and a region 30 kb downstream of the Ank-1B region forming a ‘rosette’ that includes Ank-1E. We propose a model in which the formation of an active chromatin hub in erythroid cells which is facilitated by erythroid transcription factors and stabilized by the barrier-associated proteins. Disclosures:No relevant conflicts of interest to declare.

MicroRNA-486-5p Targets Foxo1 and Regulates Human Hematopoietic Stem Cell Proliferation and Erythroid Differentiation

AbstractAbstract 3871Previous studies have supported a critical role for specific miRNA in regulating hematopoiesis. However the relative abundance and specificity for most miRNAs remains to be investigated, and the role of expressed miRNA in regulating cell fate and function remains poorly understood. Using microRNA microarrays we identified increased expression of miR-486 in chronic myeloid leukemia (CML) compared to normal CD34+ cells. miR-486 is located within the last intron of the Ankyrin-1 gene on chromosome 8 and is reported to be enriched in muscle cells. The expression pattern of miR-486 in hematopoietic cells and its roles in hematopoietic regulation are not known. In CB cells, miR-486 expression level was highest in MEP and was low in HSC. There was 16-fold increased expression of miR-486 during in vitro erythroid differentiation of CB Lin-CD34+CD38– cells, associated with 5-fold increase in Ankyrin-1 gene expression. To explore the role of miR-486 in growth and differentiation of hematopoietic stem and progenitor cells (HSPC), we first expressed hsa-miR-486-5p in CB CD34+ cells using lentiviral vectors. CB CD34+ cells transduced with this vector demonstrated 2–3 fold increased expression of miRNA-486-5p compared to cells transduced with a control vector (Ctrl). CB CD34+ cells expressing miR-486-5p generated modestly increased numbers of cells (1.22 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO for 6 days. Increased numbers of erythroid cells and reduced numbers of myeloid cells were generated in culture (GPA+ cells: Ctrl 58% and miR-486-5p 72.2%; CD33+ cells: Ctrl 30.7% and miR-486-5p 21.9%;, CD11b cells: Ctrl 33.5% and miR-486-5p 21.5%). To further investigate the effect of inhibition of miR-486-5p on growth and differentiation of HSPC, we inhibited miR-486 expression in CB CD34+ cells using a modified miRZip anti-miRNA lentivirus vectors (SBI) expressing anti-miR-486-5p and compared to cells expressing a control scrambled anti-miRNA sequence. Anti-miR-486-5p expressing CB CD34+ cells generated reduced number of cells in growth factor (GF) culture (67.5% inhibition) compared to controls. Greater inhibition of erythroid compared to myeloid cells was seen (GPA+ cells: 62.5% inhibition; CD33+ cells: 37.1% inhibition compared to controls at day 6). Anti-miR-486-5p expressing CB CD34+ cells also demonstrated reduced colony formation (BFU-E: 67% inhibition;, CFU-GM 16% inhibition), and reduced proliferation (43.88% inhibition of proliferation index) compared to controls. Similar results were observed with CB Lin-CD34+CD38- cells transduced with anti-486-5p virus (GPA+ cells: 67% inhibition; CD33+ cells: 30 % inhibition). The number of CD34+ cells was however maintained after culture (117% for miR-486-5p compared to scramble). These results indicate an important role for miR-486-5p in preservation, proliferation and erythroid differentiation of HSC. A search for evolutionarily conserved miR-486-5p targets using Targetscan 5.1 identified Foxo1, a member of the Foxo subfamily of forkhead transcription factors which play negative regulatory roles in hematopoiesis, as the highest ranking target. To demonstrate that Foxo1 is a direct target of miR-486-5p, we generated pMIR-REPORT™ constructs containing two miR-486-5p seed sites (182 and 658) within the Foxo1 3′-UTR. These constructs were cotransfected into HEK293T cells along with a miR-486-5p expression plasmid or empty control vector. Expression of miR-486-5p resulted in a 65% reduction in luciferase activity. Expression of anti-miR-486-5p resulted in increased Foxo1 protein expression in CB CD34+ cells. Expression of miR-486-5p also resulted in 50% decrease of Foxo1 protein expression. Using a Fas-L promoter-luciferase reporter we found that inhibition of miR486-5p increased Foxo1 transactivation activity in HEK293T cells. These results demonstrate that Foxo1 is a direct target of miR-486-5p. We conclude that miR-486-5p expression is modulated during normal hematopoietic differentiation and in leukemic hematopoiesis. Our results indicate a regulatory role for miR-486-5p in the growth hematopoietic stem cells and their erythroid differentiation. We show that miR-486-5p directly inhibits Foxo1 expression, which may potentially play an important role in its hematopoietic regulatory function. Disclosures:No relevant conflicts of interest to declare.

A Common Regulatory Signature Associated with Barrier Insulators in Human Primary Erythroid Cells

AbstractAbstract 3868Insulators are DNA sequences and associated binding proteins that establish and/or maintain boundaries between regions of active and silenced chromatin domains. In higher organisms, there are 2 types of insulators, enhancer-blocking insulators, which establish chromatin domains to separate enhancers and promoters, and barrier insulators, which create a barrier to protect against heterochromatin-mediated gene silencing. Despite their role as critical regulators of tissue-specific gene expression, barrier insulators are poorly understood in mammalian cells, with much of our knowledge from studies of the barrier insulator in the chicken β-globin locus, cHS4. The DNA region of cHS4 that functions as a barrier binds upstream stimulatory factor (USF) proteins that recruit histone methyltransferase (HMT) activity, and histone acetyltransferase (HAT) activity, supporting a model that recruitment of enzymes and other proteins associated with activating histone modifications block the mechanism(s) that lead to spreading of gene-silencing. Our goal is to identify a regulatory signature associated with barrier insulators in erythroid cells. We utilized chromatin immunoprecipitation coupled with ultrahigh throughput Solexa sequencing (ChIP-seq) to generate genome-wide maps of regulatory and chromatin modifying proteins in erythroid cells. To generate cells for ChIP, human CD34+ cells were cultured in serum free media with erythropoietin to yield a population of CD71+/GPA+ erythroid cells (R3/R4 population). First, a genome-wide map of USF1 and USF2 occupancy in primary erythroid cells was created. A total of 19213 USF1 and 20115 USF2 sites of occupancy were identified. USF1 and USF2 frequently heterodimerize; co-localization was present at 15882 sites (83% of USF1 and 79% of USF2 sites). USF heterodimers were commonly located near proximal promoters (within 1KB of TSS, 48% of sites) and enhancers (>1kb from RefSeq gene, 30% of sites). To analyze co-localizing barrier-associated arginine methyltransferases from erythroid cells, ChIP-seq was performed with PRMT1 and PRMT4/CARM1. A total of 7062 PRMT1 sites and 15900 PRMT4 sites were identified. PRMT1 and PRMT4 were commonly found at sites of USF occupancy, with 6120 sites demonstrating occupancy of all four factors, consistent with the hypothesis that the USF proteins frequently recruit HMT's in mammalian cells. Sites of PRMT/USF co-occupancy were more likely to be at proximal promoters (68%) than sites of USF occupancy alone. Genome-wide occupancy of four acetyltransferases commonly found in erythroid cells, CBP, p300, PCAF, and SRC1, was also studied using ChIP-seq. 6804, 46932, 25688, and 25833 sites of occupancy were found for CBP, p300, PCAF, and SRC1 respectively. Co-localization with the p300, PCAF, and SRC1 with the USF/PRMT binding sites was common, occurring in 3825 sites. These sites were most commonly located near proximal promoters (71%) and enhancers (17%). In contrast, CBP co-localized with the USF/PRMT/p300/PCAF/SRC regions in only 10 locations and sites of CBP occupancy were more commonly found at enhancers (64%) and introns (29%) than at promoters (0.4%). Detection of barrier insulators near gene promoters is not surprising. Recent studies have revealed many similarities between barriers and promoters, including binding of specific transcription factors, and have led to the suggestion that barrier insulators have evolved as specialized derivatives of gene promoters, each with specific, yet discrete function. The regulatory protein CTCF mediates enhancer-blocking insulator activity. ChIP-seq was utilized to create a genome-wide map of CTCF binding in erythroid cells. 38503 sites of CTCF occupancy were identified. These sites were located at enhancers (41%), introns, (28%) and proximal promoters (18%). 4459 CTCF sites (12%) co-localized with regions of USF/PRMT/p300/PCAF/SRC binding. These sites most commonly occurred at promoters (65%) and enhancers (19%). The role of CTCF in barrier insulator function is controversial; our data are consistent with recent data demonstrating its mark at chromatin boundaries. The signature composed of USF/PRMT/p300/PCAF/SRC/CTCF was found in the well characterized functional erythroid barrier located in the ankyrin-1 gene proximal promoter region. These data indicate that a common regulatory signature is likely associated with barrier elements in erythroid cells. Disclosures:No relevant conflicts of interest to declare.

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K(ATP) channels directly associate with ankyrin-B in heart via the K(ATP) channel alpha-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K(ATP) channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K(ATP) channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.

Functional Analysis of a Novelcis-Acting Regulatory Region within the Human Ankyrin Gene (ANK-1) Promoter

The characterization of atypical mutations in loci associated with diseases is a powerful tool to discover novel regulatory elements. We previously identified a dinucleotide deletion in the human ankyrin-1 gene (ANK-1) promoter that underlies ankyrin-deficient hereditary spherocytosis. The presence of the deletion was associated with a decrease in promoter function both in vitro and in vivo establishing it as a causative hereditary spherocytosis mutation. The dinucleotide deletion is located in the 5' untranslated region of the ANK-1 gene and disrupts the binding of TATA binding protein and TFIID, components of the preinitiation complex. We hypothesized that the nucleotides surrounding the mutation define an uncharacterized regulatory sequence. To test this hypothesis, we generated a library of more than 16,000 ANK-1 promoters with degenerate sequence around the mutation and cloned the functional promoter sequences after cell-free transcription. We identified the wild type and three additional sequences, from which we derived a consensus. The sequences were shown to be functional in cell-free transcription, transient-transfection, and transgenic mouse assays. One sequence increased ANK-1 promoter function 5-fold, while randomly chosen sequences decreased ANK-1 promoter function. Our results demonstrate a novel functional motif in the ANK-1 promoter.

A Modified ANK-1 Promoter Directs Uniform, Copy Number Dependent Gamma Globin Gene Expression at Therapeutic Levels of a in a Lentivirus Vector.

Abstract 3565Poster Board III-502Successful gene therapy for hemoglobin disorders beta-thalassemia and sickle cell disease (SCD) has been hindered by inefficient expression of beta-like globin genes from viral vectors. It is estimated that transduction of ∼25% of hematopoietic stem cells (HSC) to express a beta-like globin gene at ∼20% the level of endogenous alpha-globin would be therapeutic. The expression of the beta-like globin genes in their native locus is a precisely regulated process that relies on the coordinated activities of several cis-acting enhancer elements that make up the beta-globin locus control region (LCR). Expression of therapeutic levels of beta-like globin in mouse models and patient cells requires inclusion of LCR elements. These LCR elements are susceptible to silencing, reduce virus titer and can potentially activate oncogenes at the site of proviral insertion. To minimize these risks we have developed a novel approach using non-globin erythroid promoters to drive high levels of globin gene expression independent of enhancers. Previously, we showed that a minimal 276-bp promoter of the human ankyrin gene (ANK-1) does not contain an enhancer element, but does contain a barrier element that allows erythroid-specific, uniform, position independent and copy-number dependent expression of a linked gamma-globin gene. However, the level of gamma-globin expression from the ANK-1 promoter (4% of mouse alpha-globin per copy) was sub-therapeutic. TFIID, a component of the transcription initiation complex, binds a 9-bp sequence (TGCGGTGAG) within the wild type ANK-1 promoter. A dinucleotide deletion within this sequence that disrupts the binding of TFIID and results in ankyrin deficient Hereditary Spherocytosis (Gallagher et al. Hum Mol Genet.14: 2501-9. 2005). We hypothesized that sequence modifications of the ANK1 promoter in this region would increase the affinity for TFIID and increase expression. Mutational analysis of the wildtype TFIID site identified a number of sequence variants that increase expression. An ANK-1 promoter sequence variant that differs from the wildtype TFIID sequence in the first three nucleotides (GCGGGTGAG; GC-ANK-1), results in a 7-fold increase in transcriptional activity in K562 cells. We developed five independent transgenic mouse lines that carry this GC-ANK-1 promoter linked to a human gamma globin gene. Expression in all five lines is erythroid-specific, uniform, position independent and copy-number dependent. Moreover, the average level of gamma-globin mRNA is 34 ± 14 % of mouse alpha-globin mRNA per transgene copy, a 8.5-fold increase in gamma-globin expression over mice harboring the wildtype ANK-1 promoter. We developed a simplified lentiviral vector that contains the CG-ANK-1/gamma-globin gene. This vector was produced at high titer and transduced 57% (23/40) of mouse colony forming unit Spleen (CFU-S) cells. In CFU-S containing single copies of the provirus, the level of gamma-globin mRNA was 6.8 ± 2.9 % of mouse alpha-globin per copy. In CFU-S with two and three copies of the GC-ANK-1 gamma-globin provirus, expression of gamma-globin mRNA averaged 20% ± 1.6% and 30% of mouse alpha-globin per copy respectively, demonstrating copy-number dependent of expression. Overall, an analysis of 16 CFU-S foci demonstrated that the CG-ANK-1/gamma-globin vector expressed gamma globin mRNA at levels that average 8.5 ± 2.0% of mouse alpha-globin. Our data demonstrate that just two copies of the simplified CG-ANK-1/gamma-globin lentiviral vector could potentially produce the levels of gamma-globin necessary for effective treatment of the hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.

Ankyrin protein networks in membrane formation and stabilization

In eukaryotic cells, ankyrins serve as adaptor proteins that link membrane proteins to the underlying cytoskeleton. These adaptor proteins form protein complexes consisting of integral membrane proteins, signalling molecules and cytoskeletal components. With their modular architecture and ability to interact with many proteins, ankyrins organize and stabilize these protein networks, thereby establishing the infrastructure of membrane domains with specialized functions. To this end, ankyrin collaborates with a number of proteins including cytoskeletal proteins, cell adhesion molecules and large structural proteins. This review addresses the targeting and stabilization of protein networks related to ankyrin interactions with the cytoskeletal protein β-spectrin, L1-cell adhesion molecules and the large myofibrillar protein obscurin. The significance of these interactions for differential targeting of cardiac proteins and neuronal membrane formation is also presented. Finally, this review concludes with a discussion about ankyrin dysfunction in human diseases such as haemolytic anaemia, cardiac arrhythmia and neurological disorders.

Sequence and gene organization of 24 circles from the Cotesia plutellae bracovirus genome

Twenty-four genomic segments of Cotesia plutellae bracovirus (CpBV) were completely sequenced, and their genomic structures were analyzed. The aggregated genome size is 351,299 bp long and exhibits an average GC content of approximately 34.6%. Average coding density is about 32.3%, and 125 putative open reading frames (ORFs) are predicted. More than half (52.5%) of predicted genes are annotated as hypothetical, but they share sequence similarities with those of other bracoviral genomes. The annotated ORFs can be classified into the known bracoviral families, in which a family of protein tyrosine phosphatase is the largest, including 36 ORFs, suggesting a significant role during parasitization. In addition, 8 and 7 ORFs encode ankyrin-like and EP1-like genes, respectively. Some predicted genes are known only in Cotesia-associated bracoviral genomes. Phylogenetic analyses based on PTP, ankyrin and EP1-like gene groups revealed no correlation between bracoviruses.

Insulator Elements in Erythrocyte Membrane Genes

Insulators are DNA sequences and associated binding proteins that establish and/or maintain the boundaries between euchromatin and heterochromatin. One type of insulator establishes chromatin domains to separate enhancers and promoters and prevent their interaction (enhancer-blocking insulators) whereas another type creates a barrier to protect against heterochromatin-mediated gene silencing (barrier insulators). In the well-characterized chicken beta-globin LCR 5′HS4 insulator, binding of CTCF mediates enhancer-blocking functions and USF proteins mediate barrier activity. Because varying transcripts of erythrocyte membrane protein genes are expressed in both erythroid and nonerythroid cells, we hypothesized that that insulator elements that bind USF and CTCF participate in their regulation. Advances in technology have permitted rapid identification of DNA sequences bound by transcription factors and other DNA-associated proteins on a genome-wide scale. Coupling chromatin immunoprecipitation to microarrays that contain genomic regions (ChIP-chip) is a high resolution technique available for mapping protein-DNA interactions in vivo. We used ChIP-chip to identify CTCF and USF factor binding sites with potential insulator function that regulate membrane protein gene expression in erythroid cells. ChIP was performed with K562 and HeLa cells using antibodies against CTCF, USF1, and USF2. DNA obtained from these IPs was hybridized to a custom NimbleGen high-density human genomic DNA microarray. Chip probes were ~50bp in length, Tm ≥76°C, tiled ~65bp apart. Regions of repetitive DNA excluded. The chip included 15 erythrocyte membrane protein genes, most encoding complex loci with multiple tissue-, cell-, and developmental stage-specific transcripts, including alpha spectrin, beta spectrin, ankyrin, spectrin, band 3, ƒnalpha adducin, beta adducin, gamma adducin, ICAM4, erythroid associated membrane protein, protein 4.1R, protein 4.2, dematin, beta actin, tropomodulin, and tropomyosin. Each gene plus 50 to 100kb of flanking DNA were included on the chip. Binding sites on the custom DNA array were identified using the Tamalpais peak calling algorithm using L1–L3 level of stringency (Genom Res 16:595, 2006) or Tilescope using a signal cutoff of 0.9 (Genome Biol 8:R81, 2007). In K562 chromatin, 117 sites of CTCF occupancy were identified. There were 49 sites in 5′ flanking DNA, 3 in promoters, 9 in coding sequence, 20 in introns, 1 in 3′ untranslated region, and 35 in 3′ flanking DNA. Sites of USF1 and USF2 occupancy were identified in 7/15 and 9/15 genes, respectively. 15 USF1 sites were identified; 10 in 5′ flanking DNA, 1 in a promoter (<500bp from transcription start site), and 4 in 3′ flanking DNA. 22 sites of USF2 occupancy were identified; 9 in 5′ flanking DNA, 1 in a promoter, 4 in introns, and 8 in 3′ flanking DNA. USF1 and USF2 frequently heterodimerize and 10 sites bound both USF1 and USF2. To validate ChIP-chip results, a site binding USF2 near the ankyrin gene erythroid promoter was further analyzed. This site was chosen because the USF2-associated sequence directs uniform, copy-number dependent expression of a linked reporter gene in transgenic mice, the erythroid ankyrin promoter is positioned between 2 alternate, nonerythroid ankyrin promoters, and DNaseI mapping revealed that this region is contained within an erythroid-specific chromatin domain. Quantitative ChIP confirmed USF2 occupancy, as well as USF1 occupancy, in this region. For functional analysis, we used a gene silencing/position-effect variegation (PEV) assay to determine whether this region possess barrier element function. Using a HS2-beta globin promoter-GFP cassette as negative control and a cHS4-HS2-beta globin promoter-GFP-cHS4 cassette as positive control, the ankyrin promoter region fragment (p<0.001) functioned as barrier elements in this PEV assay. These data demonstrate that there are numerous binding sites for insulator-associated DNA binding proteins in erythrocyte membrane protein genes and indicate that these elements likely play a significant role in the regulation of tissue-specific expression of many genes expressed in erythroid cells.

Ankyrin-linked hereditary spherocytosis in an African-American kindred.

Mutations of ankyrin-1 are the most frequent cause of the inherited hemolytic anemia, hereditary spherocytosis (HS), in people of European ancestry. Ankyrin-1, which provides the primary linkage between the erythrocyte membrane skeleton and the plasma membrane, has numerous isoforms generated by alternative splicing, alternate polyadenylation, use of tissue-specific promoters, and alternate NH(2) or COOH-termini. Mutation detection in erythrocyte membrane protein genes, including ankyrin, has been a challenge, primarily due to the large size of these genes, and the apparent frequent occurrence of HS-associated null alleles. Using denaturing high-performance liquid chromatography (DHPLC), we screened the ankyrin gene of the proband of a large, three generation African-American kindred with ankyrin-deficient HS. DHPLC yielded an abnormal chromatogram for exon 1. Examination of the corresponding exon 1 sequence in genomic DNA from the proband revealed heterozygosity for a mutation of the initiator methionine (ATG to ATA Met 1 Ile). Coupled in vitrotranscription/translation studies with rabbit reticulocyte lysates demonstrated that the wild-type ankyrin erythroid cDNA initiates only from the known initiator methionine, indicating that the use of alternate initiator methionine is not a mechanism of isoform diversity in erythroid cells. The mutant ankyrin allele, unlike some initiator methionine mutations that utilize downstream codons for translation initiation, was associated with a null allele. This is the first report describing ankyrin-linked HS in an African-American kindred.

A Presynaptic Giant Ankyrin Stabilizes the NMJ through Regulation of Presynaptic Microtubules and Transsynaptic Cell Adhesion

In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.