KRAB zinc-finger proteins localise to novel KAP1-containing foci that are adjacent to PML nuclear bodies

The KRAB-zinc finger proteins (KRAB-ZFPs) represent a very large, but poorly understood, family of transcriptional regulators in mammals. They are thought to repress transcription via their interaction with KRAB-associated protein 1 (KAP1), which then assembles a complex of chromatin modifiers to lay down histone marks that are associated with inactive chromatin. Studies of KRAB-ZFP/KAP1-mediated gene silencing, using reporter constructs and ectopically expressed proteins, have shown colocalisation of both KAP1 and repressed reporter target genes to domains of constitutive heterochromatin in the nucleus. However, we show here that although KAP1 does indeed become recruited to pericentric heterochromatin during differentiation of mouse embryonic stem (ES) cells, endogenous KRAB-ZFPs do not. Rather, KRAB-ZFPs and KAP1 relocalise to novel nucleoplasmic foci that we have termed KRAB- and KAP1-associated (KAKA) foci. HP1s can also concentrate in these foci and there is a close spatial relationship between KAKA nuclear foci and PML nuclear bodies. Finally, we reveal differential requirements for the recruitment of KAP1 to pericentric heterochromatin and KAKA foci, and suggest that KAKA foci may contain sumoylated KAP1 - the form of the protein that is active in transcriptional repression.

A genome-wide analysis of 'Bounty' descendants implicates several novel variants in migraine susceptibility

Migraine is a common neurological disease with a complex genetic aetiology. The disease affects ~12% of the Caucasian population and females are three times more likely than males to be diagnosed. In an effort to identify loci involved in migraine susceptibility, we performed a pedigree-based genome-wide association study of the isolated population of Norfolk Island, which has a high prevalence of migraine. This unique population originates from a small number of British and Polynesian founders who are descendents of the Bounty mutiny and forms a very large multigenerational pedigree (Bellis et al.; Human Genetics, 124(5):543-5542, 2008). These population genetic features may facilitate disease gene mapping strategies (Peltonen et al.; Nat Rev Genet, 1(3):182-90, 2000. In this study, we identified a high heritability of migraine in the Norfolk Island population (h (2) = 0.53, P = 0.016). We performed a pedigree-based GWAS and utilised a statistical and pathological prioritisation approach to implicate a number of variants in migraine. An SNP located in the zinc finger protein 555 (ZNF555) gene (rs4807347) showed evidence of statistical association in our Norfolk Island pedigree (P = 9.6 × 10(-6)) as well as replication in a large independent and unrelated cohort with >500 migraineurs. In addition, we utilised a biological prioritisation to implicate four SNPs, in within the ADARB2 gene, two SNPs within the GRM7 gene and a single SNP in close proximity to a HTR7 gene. Association of SNPs within these neurotransmitter-related genes suggests a disrupted serotoninergic system that is perhaps specific to the Norfolk Island pedigree, but that might provide clues to understanding migraine more generally.

Affinity adsorption of plasmid DNA

The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 ± 0.0013, 0.0095 ± 0.0016, and 0.0080 ± 0.0006 mg, respectively, to 50 μL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 ± 0.0043, 0.0219 ± 0.0035, and 0.0190 ± 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.

Identity-by-Descent Mapping to Detect Rare Variants Conferring Susceptibility to Multiple Sclerosis

Genome-wide association studies (GWAS) have identified around 60 common variants associated with multiple sclerosis (MS), but these loci only explain a fraction of the heritability of MS. Some missing heritability may be caused by rare variants that have been suggested to play an important role in the aetiology of complex diseases such as MS. However current genetic and statistical methods for detecting rare variants are expensive and time consuming. 'Population-based linkage analysis' (PBLA) or so called identity-by-descent (IBD) mapping is a novel way to detect rare variants in extant GWAS datasets. We employed BEAGLE fastIBD to search for rare MS variants utilising IBD mapping in a large GWAS dataset of 3,543 cases and 5,898 controls. We identified a genome-wide significant linkage signal on chromosome 19 (LOD = 4.65; p = 1.9×10-6). Network analysis of cases and controls sharing haplotypes on chromosome 19 further strengthened the association as there are more large networks of cases sharing haplotypes than controls. This linkage region includes a cluster of zinc finger genes of unknown function. Analysis of genome wide transcriptome data suggests that genes in this zinc finger cluster may be involved in very early developmental regulation of the CNS. Our study also indicates that BEAGLE fastIBD allowed identification of rare variants in large unrelated population with moderate computational intensity. Even with the development of whole-genome sequencing, IBD mapping still may be a promising way to narrow down the region of interest for sequencing priority. © 2013 Lin et al.

The genetic associations of acute anterior uveitis and their overlap with the genetics of ankylosing spondylitis

Acute anterior uveitis (AAU) involves inflammation of the iris and ciliary body of the eye. It occurs both in isolation and as a complication of ankylosing spondylitis (AS). It is strongly associated with HLA-B*27, but previous studies have suggested that further genetic factors may confer additional risk. We sought to investigate this using the Illumina Exomechip microarray, to compare 1504 cases with AS and AAU, 1805 with AS but no AAU and 21 133 healthy controls. We also used a heterogeneity test to test the differences in effect size between AS with AAU and AS without AAU. In the analysis comparing AS+AAU+ cases versus controls, HLA-B*27 and HLA-A*02:01 were significantly associated with the presence of AAU (P<10−300 and P=6 × 10−8, respectively). Secondary independent association with PSORS1C3 (P=4.7 × 10−5) and TAP2 (P=1.1 × 10−5) were observed in the major histocompatibility complex. There was a new suggestive association with a low-frequency variant at zinc-finger protein 154 in the AS without AAU versus control analysis (zinc-finger protein 154 (ZNF154), P=2.2 × 10−6). Heterogeneity testing showed that rs30187 in ERAP1 has a larger effect on AAU compared with that in AS alone. These findings also suggest that variants in ERAP1 have a differential impact on the risk of AAU when compared with AS, and hence the genetic risk for AAU differs from AS.

Biochemical characterization of Aprataxin, the protein deficient in Ataxia with Oculomotor Apraxia type 1

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

Preparation, Analysis and Use of an Affinity Adsorbent for the Purification of GST Fusion Protein

Methods are presented for the preparation, ligand density analysis and use of an affinity adsorbent for the purification of a glutathione S-transferase (GST) fusion protein in packed and expanded bed chromatographic processes. The protein is composed of GST fused to a zinc finger transcription factor (ZnF). Glutathione, the affinity ligand for GST purification, is covalently immobilized to a solid-phase adsorbent (Streamline™). The GST–ZnF fusion protein displays a dissociation constant of 0.6 x10-6 M to glutathione immobilized to Streamline™. Ligand density optimization, fusion protein elution conditions (pH and glutathione concentration) and ligand orientation are briefly discussed.

Plasmid DNA purification via the use of a dual affinity protein

Methods are presented for the production, affinity purification and analysis of plasmid DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion protein, is used for the capture and purification of the pDNA. The protein is composed of GST, which displays affinity for glutathione immobilized to a solid-phase adsorbent, fused to a zinc finger transcription factor, which displays affinity for a target 9-base pair sequence contained within the target pDNA. A Picogreen™ fluorescence assay and/or anx ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.

Genetic polymorphisms in DPF3 associated with risk of breast cancer and lymph node metastases

Background Several studies have identified rare genetic variations responsible for many cases of familial breast cancer but their contribution to total breast cancer incidence is relatively small. More common genetic variations with low penetrance have been postulated to account for a higher proportion of the population risk of breast cancer. Methods and Results In an effort to identify genes that influence non-familial breast cancer risk, we tested over 25,000 single nucleotide polymorphisms (SNPs) located within approximately 14,000 genes in a large-scale case-control study in 254 German women with breast cancer and 268 age-matched women without malignant disease. We identified a marker on chromosome 14q24.3-q31.1 that was marginally associated with breast cancer status (OR = 1.5, P = 0.07). Genotypes for this SNP were also significantly associated with indicators of breast cancer severity, including presence of lymph node metastases ( P = 0.006) and earlier age of onset ( P = 0.01). The association with breast cancer status was replicated in two independent samples (OR = 1.35, P = 0.05). High-density association fine mapping showed that the association spanned about 80 kb of the zinc-finger gene DPF3 (also known as CERD4 ). One SNP in intron 1 was found to be more strongly associated with breast cancer status in all three sample collections (OR = 1.6, P = 0.003) as well as with increased lymph node metastases ( P = 0.01) and tumor size ( P = 0.01). Conclusion Polymorphisms in the 5' region of DPF3 were associated with increased risk of breast cancer development, lymph node metastases, age of onset, and tumor size in women of European ancestry. This large-scale association study suggests that genetic variation in DPF3 contributes to breast cancer susceptibility and severity.

Complete genomic sequence of a Rubus yellow net virus isolate and detection of genome-wide pararetrovirus-derived small RNAs

Rubus yellow net virus (RYNV) was cloned and sequenced from a red raspberry (Rubus idaeus L.) plant exhibiting symptoms of mosaic and mottling in the leaves. Its genomic sequence indicates that it is a distinct member of the genus Badnavirus, with 7932. bp and seven ORFs, the first three corresponding in size and location to the ORFs found in the type member Commelina yellow mottle virus. Bioinformatic analysis of the genomic sequence detected several features including nucleic acid binding motifs, multiple zinc finger-like sequences and domains associated with cellular signaling. Subsequent sequencing of the small RNAs (sRNAs) from RYNV-infected R. idaeus leaf tissue was used to determine any RYNV sequences targeted by RNA silencing and identified abundant virus-derived small RNAs (vsRNAs). The majority of the vsRNAs were 22-nt in length. We observed a highly uneven genome-wide distribution of vsRNAs with strong clustering to small defined regions distributed over both strands of the RYNV genome. Together, our data show that sequences of the aphid-transmitted pararetrovirus RYNV are targeted in red raspberry by the interfering RNA pathway, a predominant antiviral defense mechanism in plants. © 2013.

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)18-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

Zinc supplementation at conventional doses does not improve the disturbance of taste perception in hemodialysis patients

Objective: To determine the effect of zinc supplementation on taste perception in a group of hemodialysis patients. Design and Setting: Double-blind randomized placebo-controlled study in a teaching hospital dialysis unit. Patients: Fifteen stable hemodialysis patients randomized to placebo (6 male, 2 female; median age, 67; range, 30 to 72 years) or treatment (5 male, 2 female; median age, 60; range, 31 to 76 years). Intervention: Treatment group received zinc sulfate 220 mg per day for 6 weeks, and the placebo group received an apparently identical dummy pill. Main Outcome Measures: Taste scores by visual analogue scales, normalized protein catabolic rate and plasma, whole blood and red cell zinc levels. Results: At baseline, sweet and salt tastes were identified correctly by both groups. Sour was often confused with salt. Sour solutions of different concentrations were not distinguishable. Taste scores were not different after 6 weeks for either group. There was no significant increment in zinc levels or normalized protein catabolic rate for either group. Conclusion: We found a disturbance of taste perception in hemodialysis patients, particularly for the sour modality, which was not corrected by this regimen of zinc supplementation. These results cast doubts on the conclusions of earlier studies that indicated an improvement in taste after zinc supplementation.