Structural diversity of supercoiled DNA Irobalieva, Rossitza N; Fogg, Jonathan M; Catanese, Jr, Daniel J ...
Nature communications,
10/2015, Letnik:
6, Številka:
1
Journal Article
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By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) ...remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.
Genetic risk scores (GRSs) have been associated with coronary heart disease (CHD) in large studies. We asked whether expanding an established 27-variant GRS (GRS27) to a 50-variant GRS (GRS50) ...improved CHD prediction and whether GRSs are independent of self-reported family history of CHD.
The association between GRSs and incident CHD was assessed in Cox models adjusting for established risk factors in 23 595 participants of the Malmö Diet and Cancer study--a prospective, population-based study. During a median follow-up of 14.4 years, 2213 participants experienced a first CHD event. After adjustment for established risk factors, both GRS27 and GRS50 were associated with incident CHD hazard ratio (HR) = 1.70 for high (top quintile) vs. low (bottom quintile) of GRS27; 95% confidence interval (CI): 1.48-1.94; Ptrend = 1.6 × 10(-15) and HR = 1.92 for GRS50; 95% CI: 1.67-2.20; Ptrend = 6.2 × 10(-22). Adding 23 single nucleotide polymorphisms (SNPs) to GRS27 improved risk prediction (P = 3 × 10(-6)). Further adjustment for self-reported family history did not appreciably change the risk estimates of either GRS27 (HR = 1.65; 95% CI: 1.45-1.89) or GRS50 (HR = 1.87; 95% CI: 1.63-2.14). The addition of GRS50 to established risk factors, including self-reported family history, improved discrimination (P < 0.0001) and reclassification (continuous net reclassification improvement index = 0.17, P < 0.0001). In young participants (below median age), those with high GRS50 had 2.4-fold greater risk (95% CI: 1.85-3.12) than those with low GRS50.
The addition of 23 SNPs to an existing GRS27 improved CHD risk prediction and was independent of self-reported family history. Coronary heart disease risk assessment by GRS could be particularly useful in young individuals.
Psoriasis is a common inflammatory skin disease with complex genetics and different degrees of prevalence across ethnic populations. Here we present the largest trans-ethnic genome-wide meta-analysis ...(GWMA) of psoriasis in 15,369 cases and 19,517 controls of Caucasian and Chinese ancestries. We identify four novel associations at LOC144817, COG6, RUNX1 and TP63, as well as three novel secondary associations within IFIH1 and IL12B. Fine-mapping analysis of MHC region demonstrates an important role for all three HLA class I genes and a complex and heterogeneous pattern of HLA associations between Caucasian and Chinese populations. Further, trans-ethnic comparison suggests population-specific effect or allelic heterogeneity for 11 loci. These population-specific effects contribute significantly to the ethnic diversity of psoriasis prevalence. This study not only provides novel biological insights into the involvement of immune and keratinocyte development mechanism, but also demonstrates a complex and heterogeneous genetic architecture of psoriasis susceptibility across ethnic populations.
Supercoiled DNAs varying from 281 to 5302 bp were subjected to shear forces generated by aerosolization or sonication. DNA shearing strongly correlated with length. Typical sized plasmids (≥ 3000 bp) ...degraded rapidly. DNAs 2000-3000 bp persisted ~10 min. Even in the absence of condensing agents, supercoiled DNA <1200 bp survived nebulization, and increased forces of sonication were necessary to shear it. Circular vectors were considerably more resistant to shearing than linear vectors of the same length. DNA supercoiling afforded additional protection. These results show the potential of shear-resistant Minivector DNAs to overcome one of the major challenges associated with gene therapy delivery.
We performed a multitiered, case-control association study of psoriasis in three independent sample sets of white North American individuals (1,446 cases and 1,432 controls) with 25,215 genecentric ...single-nucleotide polymorphisms (SNPs) and found a highly significant association with an
IL12B 3′-untranslated-region SNP (
rs3212227), confirming the results of a small Japanese study. This SNP was significant in all three sample sets (odds ratio OR
common 0.64, combined
P P
comb=7.85×10
−10). A Monte Carlo simulation to address multiple testing suggests that this association is not a type I error. The coding regions of
IL12B were resequenced in 96 individuals with psoriasis, and 30 additional
IL12B-region SNPs were genotyped. Haplotypes were estimated, and genotype-conditioned analyses identified a second risk allele (
rs6887695) located ∼60 kb upstream of the
IL12B coding region that exhibited association with psoriasis after adjustment for
rs3212227. Together, these two SNPs mark a common
IL12B risk haplotype (OR
common 1.40,
P
comb=8.11×10
−9) and a less frequent protective haplotype (OR
common 0.58,
P
comb=5.65×10
−12), which were statistically significant in all three studies. Since
IL12B encodes the common IL-12p40 subunit of IL-12 and IL-23, we individually genotyped 17 SNPs in the genes encoding the other chains of these cytokines (
IL12A and
IL23A) and their receptors (
IL12RB1, IL12RB2, and
IL23R). Haplotype analyses identified two
IL23R missense SNPs that together mark a common psoriasis-associated haplotype in all three studies (OR
common 1.44,
P
comb=3.13×10
−6). Individuals homozygous for both the
IL12B and the
IL23R predisposing haplotypes have an increased risk of disease (OR
common 1.66,
P
comb=1.33×10
−8). These data, and the previous observation that administration of an antibody specific for the IL-12p40 subunit to patients with psoriasis is highly efficacious, suggest that these genes play a fundamental role in psoriasis pathogenesis.
Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM ...grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 to 100 nm in diameter. Exosomes, although isolated from unfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins.
With more than 240 million people infected, hepatitis B virus (HBV) is a major health concern. The inability to mimic the complexity of the liver using cell lines and regular primary human hepatocyte ...(PHH) cultures pose significant limitations for studying host/pathogen interactions. Here, we describe a 3D microfluidic PHH system permissive to HBV infection, which can be maintained for at least 40 days. This system enables the recapitulation of all steps of the HBV life cycle, including the replication of patient-derived HBV and the maintenance of HBV cccDNA. We show that innate immune and cytokine responses following infection with HBV mimic those observed in HBV-infected patients, thus allowing the dissection of pathways important for immune evasion and validation of biomarkers. Additionally, we demonstrate that the co-culture of PHH with other non-parenchymal cells enables the identification of the cellular origin of immune effectors, thus providing a valuable preclinical platform for HBV research.
Age- and sex-related alterations in gene transcription have been demonstrated, however the underlying mechanisms are unresolved. Neuroepigenetic pathways regulate gene transcription in the brain. ...Here, we measure in vivo expression of the epigenetic enzymes, histone deacetylases (HDACs), across healthy human aging and between sexes using
CMartinostat positron emission tomography (PET) neuroimaging (n = 41). Relative HDAC expression increases with age in cerebral white matter, and correlates with age-associated disruptions in white matter microstructure. A post mortem study confirmed that HDAC1 and HDAC2 paralogs are elevated in white matter tissue from elderly donors. There are also sex-specific in vivo HDAC expression differences in brain regions associated with emotion and memory, including the amygdala and hippocampus. Hippocampus and white matter HDAC expression negatively correlates with emotion regulation skills (n = 23). Age and sex are associated with HDAC expression in vivo, which could drive age- and sex-related transcriptional changes and impact human behavior.
Atrial fibrillation (AF) is prevalent and there is a clinical need for biomarkers to identify individuals at higher risk for AF. Fixed throughout a life course and assayable early in life, genetic ...biomarkers may meet this need. Here, we investigate whether multiple single nucleotide polymorphisms together as an AF genetic risk score (AF-GRS) can improve prediction of one's risk for AF.
In 27 471 participants of the Malmö Diet and Cancer Study, a prospective, community-based cohort, we used Cox models that adjusted for established AF risk factors to assess the association of AF-GRS with incident AF and ischemic stroke. Median follow-up was 14.4 years for incident AF and 14.5 years for ischemic stroke. The AF-GRS comprised 12 single nucleotide polymorphisms that had been previously shown to be associated with AF at genome-wide significance.
During follow-up, 2160 participants experienced a first AF event and 1495 had a first ischemic stroke event. Participants in the top AF-GRS quintile were at increased risk for incident AF (hazard ratio, 2.00; 95% confidence interval, 1.73-2.31; P=2.7×10(-21)) and ischemic stroke (hazard ratio, 1.23; 95% confidence interval, 1.04-1.46; P=0.02) when compared with the bottom quintile. Addition of the AF-GRS to established AF risk factors modestly improved both discrimination and reclassification (P<0.0001 for both).
An AF-GRS can identify 20% of individuals who are at ≈2-fold increased risk for incident AF and at 23% increased risk for ischemic stroke. Targeting diagnostic or therapeutic interventions to this subset may prove clinically useful.
Rheumatoid arthritis (RA) is the most common systemic autoimmune disease, affecting ∼1% of the adult population worldwide, with an estimated heritability of 60%. To identify genes involved in RA ...susceptibility, we investigated the association between putative functional single-nucleotide polymorphisms (SNPs) and RA among white individuals by use of a case-control study design; a second sample was tested for replication. Here we report the association of RA susceptibility with the minor allele of a missense SNP in
PTPN22 (discovery-study allelic
P=6.6×10
−4; replication-study allelic
P=5.6×10
−8), which encodes a hematopoietic-specific protein tyrosine phosphatase also known as “Lyp.” We show that the risk allele, which is present in ∼17% of white individuals from the general population and in ∼28% of white individuals with RA, disrupts the P1 proline-rich motif that is important for interaction with Csk, potentially altering these proteins' normal function as negative regulators of T-cell activation. The minor allele of this SNP recently was implicated in type 1 diabetes, suggesting that the variant phosphatase may increase overall reactivity of the immune system and may heighten an individual carrier’s risk for autoimmune disease.
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