Transcriptome profiling reveals expression signatures of cranial neural crest cells arising from different axial levels

Background: Cranial neural crest cells (NCCs) are a unique embryonic cell type which give rise to a diverse array of derivatives extending from neurons and glia through to bone and cartilage. Depending on their point of origin along the antero-posterior axis cranial NCCs are rapidly sorted into distinct migratory streams that give rise to axial specific structures. These migratory streams mirror the underlying segmentation of the brain with NCCs exiting the diencephalon and midbrain following distinct paths compared to those exiting the hindbrain rhombomeres (r). The genetic landscape of cranial NCCs arising at different axial levels remains unknown. Results: Here we have used RNA sequencing to uncover the transcriptional profiles of mouse cranial NCCs arising at different axial levels. Whole transcriptome analysis identified over 120 transcripts differentially expressed between NCCs arising anterior to r3 (referred to as r1-r2 migratory stream for simplicity) and the r4 migratory stream. Eight of the genes differentially expressed between these populations were validated by RT-PCR with 2 being further validated by in situ hybridisation. We also explored the expression of the Neuropilins (Nrp1 and Nrp2) and their co-receptors and show that the A-type Plexins are differentially expressed in different cranial NCC streams. Conclusions: Our analyses identify a large number of genes differentially regulated between cranial NCCs arising at different axial levels. This data provides a comprehensive description of the genetic landscape driving diversity of distinct cranial NCC streams and provides novel insight into the regulatory networks controlling the formation of specific skeletal elements and the mechanisms promoting migration along different paths.Rachael Lumb, Sam Buckberry, Genevieve Secker, David Lawrence and Quenten Schwar

The relationship between Vitamin D deficiency and leprosy in two English medieval populations

YesIn palaeopathology, a well-established approach to malnutrition and ill-health is the study of metabolic conditions. Leprosy is a mycobacterial disease that is manifested on the bones, and is commonly studied in archaeological contexts. Vitamin D is essential for maintaining a normal immune system, and thus a metabolic insufficiency could have a major effect in the resistance of an individual to invading pathogens. It has been indicated by clinical studies that there is an increase in the risk of contracting tuberculosis for individuals with Vitamin D deficiency, and like TB, leprosy is a disease of the poor, and it is more severe in individuals with low resistance to the pathogen. The project investigated the immunological aspect of leprosy by investigating the comorbidity of Vitamin D deficiency and the disease. During the study, the prevalence rates of Vitamin D deficiency (residual rickets and osteomalacia) were compared for adults in two medieval populations: adults with skeletal evidence of lepromatous leprosy from the leprosarium of St James and Mary Magdalene in Chichester (n=62) and adults from the non-leprous population found in Box Lane, Pontefract (n=52), both in England. Macroscopic analysis identified only one probable case of residual rickets and two possible cases of osteomalacia, providing no statistical significance in the relationship between the conditions. The present article focuses on these results, aiming to underline the reasons behind negative results in research, caused either by failed methodology or the insufficient collection of samples

Age estimation [editorial].

yesAssessing and interpreting dental and skeletal age-related changes in both the living and the dead is of interest to a wide range of disciplines (e.g. see Bittles and Collins 1986) including human biology, paediatrics, public health, palaeodemography, archaeology, palaeontology, human evolution, forensic anthropology and legal medicine. ... This special issue of Annals of Human Biology arises from the 55th annual symposium of the Society for the Study of Human Biology in association with the British Association for Biological Anthropological and Osteoarchaeology held in Oxford, UK, from 9–11 December 2014. Only a selection of the presentations are included here which encompass some of the major recent advances in age estimation from the dentition and skeleton

Quantitative allele-specific expression and DNA methylation analysis of H19, IGF2 and IGF2R in the human placenta across gestation reveals H19 imprinting plasticity

Extent: 11p.Imprinted genes play important roles in placental differentiation, growth and function, with profound effects on fetal development. In humans, H19 and IGF2 are imprinted, but imprinting of IGF2R remains controversial. The H19 non-coding RNA is a negative regulator of placental growth and altered placental imprinting of H19-IGF2 has been associated with pregnancy complications such as preeclampsia, which have been attributed to abnormal first trimester placentation. This suggests that changes in imprinting during the first trimester may precede aberrant placental morphogenesis. To better understand imprinting in the human placenta during early gestation, we quantified allele-specific expression for H19, IGF2 and IGF2R in first trimester (6–12 weeks gestation) and term placentae (37–42 weeks gestation) using pyrosequencing. Expression of IGF2R was biallelic, with a mean expression ratio of 49:51 (SD = 0.07), making transient imprinting unlikely. Expression from the repressed H19 alleles ranged from 1–25% and was higher (P<0.001) in first trimester (13.5±8.2%) compared to term (3.4±2.1%) placentae. Surprisingly, despite the known co-regulation of H19 and IGF2, little variation in expression of the repressed IGF2 alleles was observed (2.7±2.0%). To identify regulatory regions that may be responsible for variation in H19 allelic expression, we quantified DNA methylation in the H19-IGF2 imprinting control region and H19 transcription start site (TSS). Unexpectedly, we found positive correlations (P<0.01) between DNA methylation levels and expression of the repressed H19 allele at 5 CpG’s 2000 bp upstream of the H19 TSS. Additionally, DNA methylation was significantly higher (P<0.05) in first trimester compared with term placentae at 5 CpG’s 39–523 bp upstream of the TSS, but was not correlated with H19 repressed allele expression. Our data suggest that variation in H19 imprinting may contribute to early programming of placental phenotype and illustrate the need for quantitative and robust methodologies to further elucidate the role of imprinted genes in normal and pathological placental development.Sam Buckberry, Tina Bianco-Miotto, Stefan Hiendleder and Claire T. Robert

Large scale gene expression meta-analysis reveals tissue-specific, sex-biased gene expression in humans

The severity and prevalence of many diseases are known to differ between the sexes. Organ specific sex-biased gene expression may underpin these and other sexually dimorphic traits. To further our understanding of sex differences in transcriptional regulation, we performed meta-analyses of sex biased gene expression in multiple human tissues. We analyzed 22 publicly available human gene expression microarray data sets including over 2500 samples from 15 different tissues and 9 different organs. Briefly, by using an inverse-variance method we determined the effect size difference of gene expression between males and females. We found the greatest sex differences in gene expression in the brain, specifically in the anterior cingulate cortex, (1818 genes), followed by the heart (375 genes), kidney (224 genes), colon (218 genes), and thyroid (163 genes). More interestingly, we found different parts of the brain with varying numbers and identity of sex-biased genes, indicating that specific cortical regions may influence sexually dimorphic traits. The majority of sex-biased genes in other tissues such as the bladder, liver, lungs, and pancreas were on the sex chromosomes or involved in sex hormone production. On average in each tissue, 32% of autosomal genes that were expressed in a sex-biased fashion contained androgen or estrogen hormone response elements. Interestingly, across all tissues, we found approximately two-thirds of autosomal genes that were sex-biased were not under direct influence of sex hormones. To our knowledge this is the largest analysis of sex-biased gene expression in human tissues to date. We identified many sex-biased genes that were not under the direct influence of sex chromosome genes or sex hormones. These may provide targets for future development of sex-specific treatments for diseases.Benjamin T. Mayne, Tina Bianco-Miotto, Sam Buckberry, James Breen, Vicki Clifton, Cheryl Shoubridge and Claire T. Robert

MassiR: A method for predicting the sex of samples in gene expression microarray datasets

UNLABELLED: High-throughput gene expression microarrays are currently the most efficient method for transcriptome-wide expression analyses. Consequently, gene expression data available through public repositories have largely been obtained from microarray experiments. However, the metadata associated with many publicly available expression microarray datasets often lacks sample sex information, therefore limiting the reuse of these data in new analyses or larger meta-analyses where the effect of sex is to be considered. Here, we present the massiR package, which provides a method for researchers to predict the sex of samples in microarray datasets. Using information from microarray probes representing Y chromosome genes, this package implements unsupervised clustering methods to classify samples into male and female groups, providing an efficient way to identify or confirm the sex of samples in mammalian microarray datasets. AVAILABILITY AND IMPLEMENTATION: massiR is implemented as a Bioconductor package in R. The package and the vignette can be downloaded at bioconductor.org and are provided under a GPL-2 license.Sam Buckberry, Stephen J. Bent, Tina Bianco-Miotto and Claire T. Robert

Large-scale manipulation of promoter DNA methylation reveals context-specific transcriptional responses and stability

BACKGROUND: Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed. RESULTS: Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. CONCLUSIONS: These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-022-02728-5

Placental transcriptome co-expression analysis reveals conserved regulatory programs across gestation

Background: Mammalian development in utero is absolutely dependent on proper placental development, which is ultimately regulated by the placental genome. The regulation of the placental genome can be directly studied by exploring the underlying organisation of the placental transcriptome through a systematic analysis of gene-wise co-expression relationships. Results: In this study, we performed a comprehensive analysis of human placental co-expression using RNA sequencing and intergrated multiple transcriptome datasets spanning human gestation. We identified modules of co-expressed genes that are preserved across human gestation, and also identifed modules conserved in the mouse indicating conserved molecular networks involved in placental development and gene expression patterns more specific to late gestation. Analysis of co-expressed gene flanking sequences indicated that conserved co-expression modules in the placenta are regulated by a core set of transcription factors, including ZNF423 and EBF1. Additionally, we identified a gene co-expression module enriched for genes implicated in the pregnancy pathology preeclampsia. By using an independnet transcriptome dataset, we show that these co-expressed genes are differentially expressed in preeclampsia. Conclusions: This study represents a comprehensive characterisation of placental co-expression and provides insight into potential transcriptional regulators that govern conserved molecular programs fundamental to placental development.Sam Buckberry, Tina Bianco-Miotto, Stephen J. Bent, Vicki Clifton, Cheryl Shoubridge, Kartik Shankar and Claire T. Robert

Recurrent acquisition of cytosine methyltransferases into eukaryotic retrotransposons

Transposable elements are in a constant arms race with the silencing mechanisms of their host genomes. One silencing mechanism commonly used by many eukaryotes is dependent on cytosine methylation, a covalent modification of DNA deposited by C5 cytosine methyltransferases (DNMTs). Here, we report how two distantly related eukaryotic lineages, dinoflagellates and charophytes, have independently incorporated DNMTs into the coding regions of distinct retrotransposon classes. Concomitantly, we show that dinoflagellates of the genus Symbiodinium have evolved cytosine methylation patterns unlike any other eukaryote, with most of the genome methylated at CG dinucleotides. Finally, we demonstrate the ability of retrotransposon DNMTs to methylate CGs de novo, suggesting that retrotransposons could self-methylate retrotranscribed DNA. Together, this is an example of how retrotransposons incorporate host-derived genes involved in DNA methylation. In some cases, this event could have implications for the composition and regulation of the host epigenomic environment

The effect of Vdr gene ablation on global gene expression in the mouse placenta

Abstract not availableSam Buckberry, Fleur Spronk, Rebecca L. Wilson, Jessica A. Laurence, Tina Bianco-Miotto, Shalem Leemaqz, Sean O'Leary, Paul H. Anderson, Claire T. Robert