Does HPA-axis activity mediate the relationship between obstetric complications and externalizing behavior problems? The TRAILS study

To examine whether HPA-axis activity mediates the relationship between obstetric complications (OCs) and externalizing behavior problems, and to investigate whether this model is different for boys and girls. In a population-based cohort of 1,768 10- to 12-year-old early adolescents, we assessed the cortisol awakening response and evening cortisol levels. Externalizing behavior problems were assessed using the Child Behavior Checklist and the Youth Self-Report. OCs were retrospectively assessed in a parent interview. OCs significantly predicted externalizing behavior problems, but OCs did not predict HPA-axis activity. Thus, the mediation model was not supported. In addition to the relationship between HPA-axis activity and externalizing behavior problems, which is specific for girls, there is also a relationship between OCs and externalizing behavior problems. However, these two mechanisms are not related to each other indicating that HPA-axis activity is not a mediator in the relationship between OCs and externalizing behavior problems. Future research should focus on understanding the mechanism through which OCs cause externalizing behavior problems

The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1

Cyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cdk1 inhibitors, is also detrimental for proliferating cells, but has been associated with defects in mitotic progression, and the formation of aneuploid daughter cells. Here, we used a large-scale RNAi screen to identify the human genes that critically determine the cellular toxicity of Cdk1 inhibition. We show that Cdk1 inhibition leads to fatal sister chromatid alignment errors and mitotic arrest in the spindle checkpoint. These problems start early in mitosis and are alleviated by depletion of isoform 1 of PRC1 (PRC1-1), by gene ablation of its binding partner KIF4, or by abrogation of KIF4 motor activity. Our results show that, normally, Cdk1 activity must rise above the level required for mitotic entry. This prevents KIF4-dependent PRC1-1 translocation to astral microtubule tips and safeguards proper chromosome congression. We conclude that cell death in response to Cdk1 inhibitors directly relates to chromosome alignment defects generated by insufficient repression of PRC1-1 and KIF4 during prometaphase

HPA-axis activity and externalizing behavior problems in early adolescents from the general population:the role of comorbidity and gender The TRAILS study

Contradictory findings on the relationship between hypothalamus-pituitary-adrenal (HPA)-axis activity and externalizing behavior problems could be due to studies not accounting for issues of comorbidity and gender. In a population-based cohort of 1768 (10- to 12-year-old) early adolescents, we used a person-oriented approach and a variable-oriented approach to investigate whether comorbidity with internalizing behavior problems and gender moderate the relationship between HPA-axis activity (cortisol awakening response and evening cortisol levels) and externalizing behavior problems. We found that: (1) in early adolescents with pure externalizing behavior problems, there was a particularly strong effect of gender, in that girls showed significantly higher total cortisol levels after awakening (AUC(G) levels) and a significantly higher cortisol awakening response (AUC(I) levels) than boys. (2) Girls with pure externalizing behavior problems showed a significantly higher cortisol awakening response (AUC(I) levels) than girls without behavior problems or girls with comorbid internalizing behavior problems. This effect was absent in boys. (3) Externalizing behavior problems, in contrast to internalizing behavior problems, were associated with higher evening cortisol levels. This effect might, however, result from girls with externalizing behavior problems showing the highest evening cortisol levels. Overall, we were unable to find the expected relationships between comorbidity and HPA-axis activity, and found girls with pure externalizing behavior problems to form a distinct group with regard to their HPA-axis activity. There is need for prospective longitudinal studies of externalizing behavior problems in boys and girls in relation to their HPA-axis activity. It would be useful to consider how other risk factors such as life events and family and parenting factors as well as genetic risks affect the complex relationship between externalizing behavior problems and HPA-axis activity

Subanesthetic ketamine treatment promotes abnormal interactions between neural subsystems and alters the properties of functional brain networks

Acute treatment with subanesthetic ketamine, a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, is widely utilized as a translational model for schizophrenia. However, how acute NMDA receptor blockade impacts on brain functioning at a systems level, to elicit translationally relevant symptomatology and behavioral deficits, has not yet been determined. Here, for the first time, we apply established and recently validated topological measures from network science to brain imaging data gained from ketamine-treated mice to elucidate how acute NMDA receptor blockade impacts on the properties of functional brain networks. We show that the effects of acute ketamine treatment on the global properties of these networks are divergent from those widely reported in schizophrenia. Where acute NMDA receptor blockade promotes hyperconnectivity in functional brain networks, pronounced dysconnectivity is found in schizophrenia. We also show that acute ketamine treatment increases the connectivity and importance of prefrontal and thalamic brain regions in brain networks, a finding also divergent to alterations seen in schizophrenia. In addition, we characterize how ketamine impacts on bipartite functional interactions between neural subsystems. A key feature includes the enhancement of prefrontal cortex (PFC)-neuromodulatory subsystem connectivity in ketamine-treated animals, a finding consistent with the known effects of ketamine on PFC neurotransmitter levels. Overall, our data suggest that, at a systems level, acute ketamine-induced alterations in brain network connectivity do not parallel those seen in chronic schizophrenia. Hence, the mechanisms through which acute ketamine treatment induces translationally relevant symptomatology may differ from those in chronic schizophrenia. Future effort should therefore be dedicated to resolve the conflicting observations between this putative translational model and schizophrenia

A variant of the castor zinc finger 1 (CASZ1) gene is differentially associated with the clinical classification of chronic venous disease

Recent reports have suggested a reproducible association between the rs11121615 SNP, located within an intron of the castor zinc finger 1 (CASZ1) gene, and varicose veins. This study aimed to determine if this variant is also differentially associated with the various clinical classifications of chronic venous disease (CVD). The rs11121615 SNP was genotyped in two independent cohorts from New Zealand (n = 1876 controls /1606 CVD cases) and the Netherlands (n = 1626/2966). Participants were clinically assessed using well-established CVD criteria. The association between the rs11121615 C-allele and varicose veins was validated in both cohorts. This was strongest in those with higher clinical severity classes and was not significant in those with non-varicose vein CVD. Functional analysis of the rs11121615 variant demonstrated that the risk allele was associated with increased enhancer activity. This study demonstrates that the CASZ1 gene associated C-allele of rs11121615 has a significant, reproducible, association with CVD (CEAP C ≥ 2 meta-odds ratio 1.31, 95% CI 1.27-1.34, P = 1 × 10-98, PHet = 0.25), but not with non-varicose vein (CEAP C1, telangiectasia or reticular veins) forms of venous disease. The effect size of this association therefore appears to be susceptible to influence by phenotypic heterogeneity, particularly if a cohort includes a large number of cases with lower severity CVD

Transcriptional regulation of the Runx1 gene

Runx1 is crucial for blood cell development and is one of the genes most frequently translocated or mutated in leukaemia, thereby contributing to disease pathogenesis. Runx1 is transcribed from two promoters, P1 and P2, and the resulting transcripts give rise to different protein isoforms. Runx1-P2 is required for the generation of definitive haematopoietic progenitor and stem cells, whereas Runx1-P1 plays a role in controlling their population size. The multisubunit protein complex cohesin is best known for its role in sister chromatid cohesion from G2 phase to mitosis. Cohesin also cooperates with the insulator protein CCCTC-binding factor (CTCF) to regulate expression of genes. Cohesin and CTCF spatially connect gene regulatory elements with promoters, thereby structuring the genome in three-dimensional space. Cohesin was previously shown to be essential for runx1 expression in haematopoietic progenitor cells in zebrafish. CTCF however, was not required for expression of runx1 during zebrafish development, but instead restricted its expression in multipotent cells of the tail bud. A large intronic region that harbours gene regulatory elements resides between the two promoters of Runx1 in all vertebrates characterized to date. In mouse, this region includes a haematopoietic-specific enhancer located 24 kilobase pair (kb) from the mouse Runx1-P1 transcriptional start site (TSS). In zebrafish, four regulatory elements located +13, +14, +39 and +89 kb downstream of the P1 TSS, were identified based on the binding of cohesin, CTCF or on the presence of enhancer histone modifications. However, their functionality in regulating runx1 expression was unknown. In my thesis, I investigated the mechanism by which cohesin regulates Runx1 expression. I hypothesised it does so by controlling the formation of chromatin interactions between regulatory elements and the Runx1 promoters. I found that cohesin controls isoform-specific expression of runx1 during zebrafish development, and that the regulation of Runx1 expression by cohesin is conserved in mammalian cells. Three of the regulatory elements identified in zebrafish, termed +13, +14 and +89, were capable of functioning as insulators in a CTCF-dependent manner, suggesting they may be involved in insulating regulatory activity within certain genomic regions surrounding runx1. Determining the function of the +39 element requires further investigation. I used circular chromosome conformation capture (4C) to identify novel regulatory elements that interact with the Runx1 promoters and the +24 enhancer in a mouse haematopoietic progenitor cell line, HPC7. 4C interaction data showed that Runx1 is located within a topologically associated domain in which not many other genes are present, indicating that regulatory elements located in this region are likely involved in regulating transcription from Runx1. The active promoter in the HPC7 cell line, P1, interacted with two conserved non-coding regulatory elements, as well as with the +24 enhancer. Each regulatory element recruits multiple blood transcription factors in HPC7 cells, suggesting they function as haematopoietic-specific enhancers. The inactive P2 promoter did not interact with any conserved non-coding elements. Cohesin and CTCF did not bind to the putative enhancer elements, and do not seem to be directly involved in the formation of their interaction with Runx1-P1. Instead, cohesin and CTCF might organise the chromatin domain in which Runx1 resides, which in turn controls the availability of regulatory elements to interact with Runx1. Overall, the findings of my study suggest that spatial and temporal regulation of Runx1 gene expression could be orchestrated by cohesin, through its role in organising chromatin in three-dimensional space

A DNA Contact Map for the Mouse Runx1 Gene Identifies Novel Haematopoietic Enhancers

Abstract The transcription factor Runx1 is essential for definitive haematopoiesis, and the RUNX1 gene is frequently translocated or mutated in leukaemia. Runx1 is transcribed from two promoters, P1 and P2, to give rise to different protein isoforms. Although the expression of Runx1 must be tightly regulated for normal blood development, the mechanisms that regulate Runx1 isoform expression during haematopoiesis remain poorly understood. Gene regulatory elements located in non-coding DNA are likely to be important for Runx1 transcription. Here we use circular chromosome conformation capture sequencing to identify DNA interactions with the P1 and P2 promoters of Runx1, and the previously identified +24 enhancer, in the mouse multipotent haematopoietic progenitor cell line HPC-7. The active promoter, P1, interacts with nine non-coding regions that are occupied by transcription factors within a 1 Mb topologically associated domain. Eight of nine regions function as blood-specific enhancers in zebrafish, of which two were previously shown to harbour blood-specific enhancer activity in mice. Interestingly, the +24 enhancer interacted with multiple distant regions on chromosome 16, suggesting it may regulate the expression of additional genes. The Runx1 DNA contact map identifies connections with multiple novel and known haematopoietic enhancers that are likely to be involved in regulating Runx1 expression in haematopoietic progenitor cells

Transcriptional Regulation of RUNX1: An Informatics Analysis

The RUNX1/AML1 gene encodes a developmental transcription factor that is an important regulator of haematopoiesis in vertebrates. Genetic disruptions to the RUNX1 gene are frequently associated with acute myeloid leukaemia. Gene regulatory elements (REs), such as enhancers located in non-coding DNA, are likely to be important for Runx1 transcription. Non-coding elements that modulate Runx1 expression have been investigated over several decades, but how and when these REs function remains poorly understood. Here we used bioinformatic methods and functional data to characterise the regulatory landscape of vertebrate Runx1. We identified REs that are conserved between human and mouse, many of which produce enhancer RNAs in diverse tissues. Genome-wide association studies detected single nucleotide polymorphisms in REs, some of which correlate with gene expression quantitative trait loci in tissues in which the RE is active. Our analyses also suggest that REs can be variant in haematological malignancies. In summary, our analysis identifies features of the RUNX1 regulatory landscape that are likely to be important for the regulation of this gene in normal and malignant haematopoiesis

DNA methylation profiling identifies a high effect genetic variant for lipoprotein(a) levels

Changes in whole blood DNA methylation levels at several CpG sites have been associated with circulating blood lipids, specifically high-density lipoprotein and triglycerides. This study performs a discovery and validation epigenome-wide association study (EWAS) for circulating lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular diseases. Whole-blood DNA methylation profiles were assessed in a cohort of 1020 elderly individuals using the Illumina EPIC array and independent validation in 359 elderly males using the Illumina 450 k array. Plasma Lp(a) was measured using an apolipoprotein(a)-size-independent ELISA. Epigenome-wide rank regression analysis identified and validated a single CpG site, cg17028067 located in intron 1 of the LPA gene, that was significantly associated with plasma Lp(a) levels after correction for multiple testing. Genotyping of the site identified a relatively uncommon SNP (rs76735376, MAF <0.02) at the CpG site that largely explained the observed methylation effect. Rs76735376 is an expression quantitative trait loci for the LPA gene and could affect expression by altering enhancer activity. This EWAS for plasma Lp(a) identified a single CpG site within LPA. This association is due to an uncommon, but highly effective genetic variant, which was not in significant linkage disequilibrium with other variants known to influence Lp(a) levels or apo(a) isoform size. This study highlights the utility of CpG site methylation to identify potentially important genetic associations that would not be readily apparent in a comparable size genetic association study