Justin M. O’Sullivan

Cohesin mutations in myeloid malignancies: underlying mechanisms

Recently, whole genome sequencing approaches have pinpointed mutations in genes that were previously not associated with cancer. For Acute Myeloid Leukaemia (AML), and other myeloid disorders, these approaches revealed a high prevalence of mutations in genes encoding the chromosome cohesion complex, cohesin. Cohesin mutations represent a novel genetic pathway for AML, but how AML arises from these mutations is unknown. This review will explore the potential mechanisms by which cohesin mutations contribute to AML and other myeloid malignancies.

Biochar in Co-Contaminated Soil Manipulates Arsenic Solubility and Microbiological Community Structure, and Promotes Organochlorine Degradation

We examined the effect of biochar on the water-soluble arsenic (As) concentration and the extent of organochlorine degradation in a co-contaminated historic sheep-dip soil during a 180-d glasshouse incubation experiment. Soil microbial activity, bacterial community and structure diversity were also investigated. Biochar made from willow feedstock (Salix sp) was pyrolysed at 350 or 550°C and added to soil at rates of 10 g kg-1 and 20 g kg-1 (representing 30 t ha-1 and 60 t ha-1). The isomers of hexachlorocyclohexane (HCH) alpha-HCH and gamma-HCH (lindane), underwent 10-fold and 4-fold reductions in concentration as a function of biochar treatment. Biochar also resulted in a significant reduction in soil DDT levels (P < 0.01), and increased the DDE:DDT ratio. Soil microbial activity was significantly increased (P < 0.01) under all biochar treatments after 60 days of treatment compared to the control. 16S amplicon sequencing revealed that biochar-amended soil contained more members of the Chryseobacterium, Flavobacterium, Dyadobacter and Pseudomonadaceae which are known bioremediators of hydrocarbons. We hypothesise that a recorded short-term reduction in the soluble As concentration due to biochar amendment allowed native soil microbial communities to overcome As-related stress. We propose that increased microbiological activity (dehydrogenase activity) due to biochar amendment was responsible for enhanced degradation of organochlorines in the soil. Biochar therefore partially overcame the co-contaminant effect of As, allowing for enhanced natural attenuation of organochlorines in soil.

A diffusion model for the coordination of DNA replication in Schizosaccharomyces pombe

The locations of proteins and epigenetic marks on the chromosomal DNA sequence are believed to demarcate the eukaryotic genome into distinct structural and functional domains that contribute to gene regulation and genome organization. However, how these proteins and epigenetic marks are organized in three dimensions remains unknown. Recent advances in proximity-ligation methodologies and high resolution microscopy have begun to expand our understanding of these spatial relationships. Here we use polymer models to examine the spatial organization of epigenetic marks, euchromatin and heterochromatin, and origins of replication within the Schizosaccharomyces pombe genome. These models incorporate data from microscopy and proximity-ligation experiments that inform on the positions of certain elements and contacts within and between chromosomes. Our results show a striking degree of compartmentalization of epigenetic and genomic features and lead to the proposal of a diffusion based mechanism, centred on the spindle pole body, for the coordination of DNA replication in S. pombe.

Connecting SNPs in Diabetes: A Spatial Analysis of Meta-GWAS Loci

Meta-analyses of genome-wide association studies (GWAS) have improved our understanding of the genetic foundations of a number of diseases, including diabetes. However, single nucleotide polymorphisms (SNPs) that are identified by GWAS, especially those that fall outside of gene regions, do not always clearly link to the underlying biology. Despite this, these SNPs have often been validated through re-sequencing efforts as not just tag SNPs, but as causative SNPs, and so must play a role in disease development or progression. In this study, we show how the 3D genome (spatial connections) and trans-expression Quantitative Trait Loci connect diabetes loci from different GWAS meta-analyses, informing the backbone of regulatory networks. Our findings include a three-way functional–spatial connection between the TM6SF2, CTRB1–BCAR1, and CELSR2–PSRC1 loci (rs201189528, rs7202844, and rs7202844, respectively) connected through the KCNIP3 and BCAR1/BCAR3 loci, respectively. These spatial hubs serve as an example of how loci in genes with little biological connection to disease come together to contribute to the diabetes phenotype.

Linkages between changes in the 3D organization of the genome and transcription during myotube differentiation in vitro

BackgroundThe spatial organization of eukaryotic genomes facilitates and reflects the underlying nuclear processes that are occurring in the cell. As such, the spatial organization of a genome represents a window on the genome biology that enables analysis of the nuclear regulatory processes that contribute to mammalian development.MethodsIn this study, Hi-C and RNA-seq were used to capture the genome organization and transcriptome in mouse muscle progenitor cells (C2C12 myoblasts) before and after differentiation to myotubes, in the presence or absence of the cytidine analogue AraC.ResultsWe observed significant local and global developmental changes despite high levels of correlation between the myotubes and myoblast genomes. Notably, the genes that exhibited the greatest variation in transcript levels between the different developmental stages were predominately within the euchromatic compartment. There was significant re-structuring and changes in the expression of replication-dependent histone variants within the HIST1 locus. Finally, treating terminally differentiated myotubes with AraC resulted in additional changes to the transcriptome and 3D genome organization of sets of genes that were all involved in pyroptosis.ConclusionsCollectively, our results provide evidence for muscle cell-specific responses to developmental and environmental stimuli mediated through a chromatin structure mechanism.

The nucleolus: a raft adrift in the nuclear sea or the keystone in nuclear structure?

Abstract The nucleolus is a prominent nuclear structure that is the site of ribosomal RNA (rRNA) transcription, and hence ribosome biogenesis. Cellular demand for ribosomes, and hence rRNA, is tightly linked to cell growth and the rRNA makes up the majority of all the RNA within a cell. To fulfill the cellular demand for rRNA, the ribosomal RNA (rDNA) genes are amplified to high copy number and transcribed at very high rates. As such, understanding the rDNA has profound consequences for our comprehension of genome and transcriptional organization in cells. In this review, we address the question of whether the nucleolus is a raft adrift the sea of nuclear DNA, or actively contributes to genome organization. We present evidence supporting the idea that the nucleolus, and the rDNA contained therein, play more roles in the biology of the cell than simply ribosome biogenesis. We propose that the nucleolus and the rDNA are central factors in the spatial organization of the genome, and that rapid alterations in nucleolar structure in response to changing conditions manifest themselves in altered genomic structures that have functional consequences. Finally, we discuss some predictions that result from the nucleolus having a central role in nuclear organization.

Intergenic GWAS SNPs are Key Components of the Spatial and Regulatory Network for Human Growth

Meta-analysis of genome-wide association studies has resulted in the identification of hundreds of genetic variants associated with growth and stature. Determining how these genetic variants influence growth is important, but most are non-coding, and there is little understanding of how these variants contribute to adult height. To determine the mechanisms by which human variation contributes to growth, we combined spatial genomic connectivity (high-throughput conformation capture) with functional (gene expression, expression Quantitative Trait Loci) data to determine how non-genic loci associated with infant length, pubertal and adult height and contribute to gene regulatory networks. This approach identified intergenic single-nucleotide polymorphisms (SNPs) ∼85 kb upstream of FBXW11 that spatially connect with distant loci. These regulatory connections are reinforced by evidence of SNP-enhancer effects and altered expression in genes influencing the action of human growth hormone. Functional assays provided evidence for enhancer activity of the intergenic region near FBXW11 that harbors SNP rs12153391, which is associated with an expression Quantitative Trait Loci. Our results suggest that variants in this locus have genome-wide effects as key modifiers of growth (both overgrowth and short stature) acting through a regulatory network. We believe that the genes and pathways connected with this regulatory network are potential targets that could be investigated for diagnostic, prenatal and carrier testing for growth disorders. Finally, the regulatory networks we generated illustrate the power of using existing datasets to interrogate the contribution of intergenic SNPs to common syndromes/diseases.

GWAS on prolonged gestation (post-term birth): analysis of successive Finnish birth cohorts

Background Gestation is a crucial timepoint in human development. Deviation from a term gestational age correlates with both acute and long-term adverse health effects for the child. Both being born preterm and post-term, that is, having short and long gestational ages, are heritable and influenced by the prenatal and perinatal environment. Despite the obvious heritable component, specific genetic influences underlying differences in gestational age are poorly understood. Methods We investigated the genetic architecture of gestational age in 9141 individuals, including 1167 born post-term, across two Northern Finland cohorts born in 1966 or 1986. Results Here we identify one globally significant intronic genetic variant within the ADAMTS13 gene that is associated with prolonged gestation (p=4.85×10−8). Additional variants that reached suggestive levels of significance were identified within introns at the ARGHAP42 and TKT genes, and in the upstream (5’) intergenic regions of the B3GALT5 and SSBP2 genes. The variants near the ADAMTS13, B3GALT5, SSBP2 and TKT loci are linked to alterations in gene expression levels (cis-eQTLs). Luciferase assays confirmed the allele specific enhancer activity for the BGALT5 and TKT loci. Conclusions Our findings provide the first evidence of a specific genetic influence associated with prolonged gestation. This study forms a foundation for a better understanding of the genetic and long-term health risks faced by induced and post-term individuals. The long-term risks for induced individuals who have a previously overlooked post-term potential may be a major issue for current health providers.

A non-coding genetic variant maximally associated with serum urate levels is functionally linked to HNF4A-dependent PDZK1 expression

&NA; The precise molecular mechanisms by which urate‐associated genetic variants affect urate levels are unknown. Here, we tested for functional linkage of the maximally associated genetic variant rs1967017 at the PDZK1 locus to elevated PDZK1 expression. We performed expression quantitative trait loci (eQTL) and likelihood analyses and gene expression assays. Zebrafish were used to evaluate tissue‐specific gene expression. Luciferase assays in HEK293 and HepG2 cells measured the effect of rs1967017 on transcription amplitude. Probabilistic Annotation Integrator analysis revealed rs1967017 as most likely to be causal and rs1967017 was an eQTL for PDZK1 in the intestine. The region harboring rs1967017 was capable of directly driving green fluorescent protein expression in the kidney, liver and intestine of zebrafish embryos, consistent with a conserved ability to confer tissue‐specific expression. Small interfering RNA depletion of HNF4A reduced endogenous PDZK1 expression in HepG2 cells. Luciferase assays showed that the T allele of rs1967017 gains enhancer activity relative to the urate‐decreasing C allele, with T allele enhancer activity abrogated by HNF4A depletion. HNF4A physically binds the rs1967017 region, suggesting direct transcriptional regulation of PDZK1 by HNF4A. Computational prediction of increased motif strength, together with our functional assays, suggests that the urate‐increasing T allele of rs1967017 strengthens a binding site for the transcription factor HNF4A. Our and other data predict that the urate‐raising T allele of rs1967017 enhances HNF4A binding to the PDZK1 promoter, thereby increasing PDZK1 expression. As PDZK1 is a scaffold protein for many ion channel transporters, increased expression can be predicted to increase activity of urate transporters and alter excretion of urate.

Early-Life Nutrition, Epigenetics, and Altered Energy Balance Later in Life

Abstract Obesity and related metabolic disorders arise as a consequence of an imbalance between energy intake and energy expenditure. Observations in human populations and experimental evidence have provided evidence that alterations in the early-life nutritional environment can lead to perturbations in the systems regulating energy balance, thus predisposing to obesity and metabolic dysfunction in later life. This concept is preferentially termed the “developmental origins of health and disease” and needs to be viewed as a transgenerational phenomenon that can perpetuate a cycle of disease across generations. The mechanisms by which early-life nutritional insults have long-term effects on offspring health remain poorly defined. However, intervention studies have implicated altered wiring of the hypothalamic circuits, epigenetic effects, and programmed changes in adipogenesis in the observed long-term changes to the regulation of energy balance. Here we summarize the current evidence for early-life programming of disordered energy balance and its consequences for the health of offspring across the life course.