With an increasing number of vertebrate genomes being sequenced in draft or finished form, unique opportunities for decoding the language of DNA sequence through comparative genome alignments have ...arisen. However, novel tools and strategies are required to accommodate this large volume of genomic information and to facilitate the transfer of predictions generated by comparative sequence alignment to researchers focused on experimental annotation of genome function. Here, we present the ECR Browser, a tool that provides easy and dynamic access to whole genome alignments of human, mouse, rat and fish sequences. This web-based tool (http://ecrbrowser.dcode.org) provides the starting point for discovery of novel genes, identification of distant gene regulatory elements and prediction of transcription factor binding sites. The genome alignment portal of the ECR Browser also permits fast and automated alignments of any user-submitted sequence to the genome of choice. The interconnection of the ECR Browser with other DNA sequence analysis tools creates a unique portal for studying and exploring vertebrate genomes.
Ischemic preconditioning (IPC) of one or two limbs improves performance of exercise that recruits the same limb(s). However, it is unclear whether IPC application to another limb than that in ...exercise is also effective and which mechanisms are involved. We investigated the effect of remote IPC (RIPC) on muscle fatigue, time to task failure, forearm hemodynamics, and deoxygenation during handgrip exercise. Thirteen men underwent RIPC in the lower limbs or a control intervention (CON), in random order, and then performed a constant load rhythmic handgrip protocol until task failure. Rates of contraction and relaxation (ΔForce/ΔTime) were used as indices of fatigue. Brachial artery blood flow and conductance, besides forearm microvascular deoxygenation, were assessed during exercise. RIPC attenuated the slowing of contraction and relaxation throughout exercise (P < 0.05 vs CON) and increased time to task failure by 11.2% (95% confidence interval: 0.7–21.7%, P <0.05 vs CON). There was no significant difference in blood flow, conductance, and deoxygenation between conditions throughout exercise (P > 0.05). In conclusion, RIPC applied to the lower limbs delayed the development of fatigue during handgrip exercise, prolonged time to task failure, but was not accompanied by changes in forearm hemodynamics and deoxygenation.
Clustering of multiple transcription factor binding sites (TFBSs) for the same transcription factor (TF) is a common feature of cis-regulatory modules in invertebrate animals, but the occurrence of ...such homotypic clusters of TFBSs (HCTs) in the human genome has remained largely unknown. To explore whether HCTs are also common in human and other vertebrates, we used known binding motifs for vertebrate TFs and a hidden Markov model-based approach to detect HCTs in the human, mouse, chicken, and fugu genomes, and examined their association with cis-regulatory modules. We found that evolutionarily conserved HCTs occupy nearly 2% of the human genome, with experimental evidence for individual TFs supporting their binding to predicted HCTs. More than half of the promoters of human genes contain HCTs, with a distribution around the transcription start site in agreement with the experimental data from the ENCODE project. In addition, almost half of the 487 experimentally validated developmental enhancers contain them as well--a number more than 25-fold larger than expected by chance. We also found evidence of negative selection acting on TFBSs within HCTs, as the conservation of TFBSs is stronger than the conservation of sequences separating them. The important role of HCTs as components of developmental enhancers is additionally supported by a strong correlation between HCTs and the binding of the enhancer-associated coactivator protein Ep300 (also known as p300). Experimental validation of HCT-containing elements in both zebrafish and mouse suggest that HCTs could be used to predict both the presence of enhancers and their tissue specificity, and are thus a feature that can be effectively used in deciphering the gene regulatory code. In conclusion, our results indicate that HCTs are a pervasive feature of human cis-regulatory modules and suggest that they play an important role in gene regulation in the human and other vertebrate genomes.
Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking ...these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.
Dietary factors influence male reproductive function in both experimental and epidemiological studies. However, there are currently no specific dietary guidelines for male preconception health. Here, ...we use the Nutritional Geometry framework to examine the effects of dietary macronutrient balance on reproductive traits in C57BL/6 J male mice. Dietary effects are observed in a range of morphological, testicular and spermatozoa traits, although the relative influence of protein, fat, carbohydrate, and their interactions differ depending on the trait being examined. Interestingly, dietary fat has a positive influence on sperm motility and antioxidant capacity, differing to typical high fat diet studies where calorie content is not controlled for. Moreover, body adiposity is not significantly correlated with any of the reproductive traits measured in this study. These results demonstrate the importance of macronutrient balance and calorie intake on reproductive function and support the need to develop specific, targeted, preconception dietary guidelines for males.
Multi-isotope calibration (MICal) is a novel approach to calibration for inductively coupled plasma mass spectrometry (ICP-MS). In MICal, only two calibration solutions are required: solution A, ...composed of 50% v v
−1
of sample and 50% v v
−1
of a standard solution containing the analytes, and solution B, composed of 50% v v
−1
of sample and 50% v v
−1
of a blank solution. MICal is based on monitoring the signal intensities of several isotopes of the same analyte in solutions A and B. By plotting the analytical signals from solution A in the
x
-axis, and from solution B in the
y
-axis, the analyte concentration in the sample is calculated using the slope of that graph and the concentration of the reference standard added to solution A. As both solutions contain the same amount of sample, matrix-matching is easily achieved. In this proof-of-concept study, MICal was applied to the determination of Ba, Cd, Se, Sn, and Zn in seven certified reference materials with different matrices (e.g., plant materials, flours, and water). In most cases, MICal results presented no statistical difference from the certified values at a 95% confidence level. The new strategy was also compared with traditional calibration methods such as external calibration, internal standardization and standard additions, and recoveries were generally better for MICal. This is a simple, accurate, and fast alternative method for matrix-matching calibration in ICP-MS.
Graphical abstract
Multi-isotope calibration: fast and innovative matrix-matching calibration for ICP-MS.
Transcriptional mediators of cell stress pathways, including HIF1α, ATF4, and p53, are key to normal development and play critical roles in disease, including ischemia and cancer. Despite their ...importance, mechanisms by which pathways mediated by these transcription factors interact with one another are not fully understood. In addressing the controversial role of HIF1α in cardiomyocytes (CMs) during heart development, we discovered a mid-gestational requirement for HIF1α for proliferation of hypoxic CMs, involving metabolic switching and a complex interplay among HIF1α, ATF4, and p53. Loss of HIF1α resulted in activation of ATF4 and p53, the latter inhibiting CM proliferation. Bioinformatic and biochemical analyses revealed unexpected mechanisms by which HIF1α intersects with ATF4 and p53 pathways. Our results highlight previously undescribed roles of HIF1α and interactions among major cell stress pathways that could be targeted to enhance proliferation of CMs in ischemia and may have relevance to other diseases, including cancer.
•HIF1α is required for proliferation of a subset of cardiomyocytes at mid-gestation•HIF1α targets genes regulating both energy metabolism and the cell cycle•HIF1α promotes Mif expression to prevent p53 activation•HIF1α represses ATF4 signaling
Guimarães-Camboa et al. demonstrate that before completion of cardiac angiogenesis, subsets of highly proliferative fetal cardiomyocytes display nuclear accumulation of HIF1α. Cardiac ablation of HIF1α and subsequent mechanistic analyses suggest that this transcription factor promotes proliferation of hypoxic fetal cardiomyocytes by regulating multiple cellular functions, including ATF4 and p53 signaling.
Inherited cardiomyopathy associates with a range of phenotypes, mediated by genetic and nongenetic factors. Noninherited cardiomyopathy also displays varying progression and outcomes. Expression of ...cardiomyopathy genes is under the regulatory control of promoters and enhancers, and human genetic variation in promoters and enhancers may contribute to this variability.
We superimposed epigenomic profiling from hearts and cardiomyocytes, including promoter-capture chromatin conformation information, to identify enhancers for 2 cardiomyopathy genes,
and
. Enhancer function was validated in human cardiomyocytes derived from induced pluripotent stem cells. We also conducted a genome-wide search to ascertain genomic variation in enhancers positioned to alter cardiac expression and correlated one of these variants to cardiomyopathy progression using biobank data.
Multiple enhancers were identified and validated for
and
, including a key enhancer that regulates the switch from
expression to
expression. Deletion of this enhancer resulted in a dose-dependent increase in
and faster contractile rate in engineered heart tissues. We searched for genomic variation in enhancer sequences across the genome, with a focus on nucleotide changes that create or interrupt transcription factor binding sites. The sequence variant, rs875908, disrupts a T-Box Transcription Factor 5 binding motif and maps to an enhancer region 2 kilobases from the transcriptional start site of
Gene editing to remove the enhancer that harbors this variant markedly reduced
expression in human cardiomyocytes. Using biobank-derived data, rs875908 associated with longitudinal echocardiographic features of cardiomyopathy.
Enhancers regulate cardiomyopathy gene expression, and genomic variation within these enhancer regions associates with cardiomyopathic progression over time. This integrated approach identified noncoding modifiers of cardiomyopathy and is applicable to other cardiac genes.
In 2015, high rates of microcephaly were reported in Northeast Brazil following the first South American Zika virus (ZIKV) outbreak. Reported microcephaly rates in other Zika-affected areas were ...significantly lower, suggesting alternate causes or the involvement of arboviral cofactors in exacerbating microcephaly rates.
We merged data from multiple national reporting databases in Brazil to estimate exposure to 9 known or hypothesized causes of microcephaly for every pregnancy nationwide since the beginning of the ZIKV outbreak; this generated between 3.6 and 5.4 million cases (depending on analysis) over the time period 1 January 2015-23 May 2017. The association between ZIKV and microcephaly was statistically tested against models with alternative causes or with effect modifiers. We found no evidence for alternative non-ZIKV causes of the 2015-2017 microcephaly outbreak, nor that concurrent exposure to arbovirus infection or vaccination modified risk. We estimate an absolute risk of microcephaly of 40.8 (95% CI 34.2-49.3) per 10,000 births and a relative risk of 16.8 (95% CI 3.2-369.1) given ZIKV infection in the first or second trimester of pregnancy; however, because ZIKV infection rates were highly variable, most pregnant women in Brazil during the ZIKV outbreak will have been subject to lower risk levels. Statistically significant associations of ZIKV with other birth defects were also detected, but at lower relative risks than that of microcephaly (relative risk < 1.5). Our analysis was limited by missing data prior to the establishment of nationwide ZIKV surveillance, and its findings may be affected by unmeasured confounding causes of microcephaly not available in routinely collected surveillance data.
This study strengthens the evidence that congenital ZIKV infection, particularly in the first 2 trimesters of pregnancy, is associated with microcephaly and less frequently with other birth defects. The finding of no alternative causes for geographic differences in microcephaly rate leads us to hypothesize that the Northeast region was disproportionately affected by this Zika outbreak, with 94% of an estimated 8.5 million total cases occurring in this region, suggesting a need for seroprevalence surveys to determine the underlying reason.
Annotators of the human genome are increasingly exploiting comparisons with genomes at both the distal and proximal evolutionary edges of the vertebrate tree. Despite the sequence similarity between ...primates, comparisons among members of this clade are beginning to identify primate- as well as human-specific functional elements. At the distal evolutionary extreme, comparing the human genome to that of non-mammal vertebrates such as fish has proved to be a powerful filter to prioritize sequences that most probably have significant functional activity in all vertebrates.
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