On the discovery of population-specific state transitions from single-cell RNA sequencing data
ABSTRACT: Single-cell RNA-sequencing (scRNA-seq) has quickly become an empowering technology to profile the transcriptomes of individual cells on a large scale. Many early analyses of differential expression have aimed at identifying differences between cell types (or clusters), and thus are focused on finding markers for cell populations either in a single sample or across multiple samples. More generally, such methods can compare expression levels in multiple sets of cells, thus leading to cross-condition analyses. However, given the emergence of replicated multi-condition scRNA-seq datasets, an area of increasing focus is making sample-level inferences, termed here as differential state analysis. For example, one could investigate the condition-specific responses of specific immune cell subsets across cells measured from patients within each condition, however, it is not clear which statistical framework best handles this situation. In this work, we surveyed the methods available to perform cross-condition differential state analyses, including cell-level mixed models and methods based on aggregated ``pseudobulk'' data. We developed a flexible simulation platform that mimics both single and multi-sample scRNA-seq data and provide robust tools for multi-condition analysis within the R package.
Project description:Nucleosomes are barriers to transcription in vitro, however, their effects on RNA polymerase in vivo are unknown. Here we describe a simple and general strategy to comprehensively map the positions of elongating and arrested RNA Polymerase II (RNAPII) at nucleotide resolution. Our results suggest that nucleosomes present significant, context-specific barriers to RNAPII in vivo that can be tuned by the incorporation of H2A.Z. 8 sets of two replicates each of paired-end and 3 sets of two replicates each of single-end samples were sequenced and analyzed.
Project description:Genetic analyses of speciation have focused nearly exclusively on retrospective analyses of reproductive isolation between highly divergent species. Yet, a full understanding of the speciation process must encompass analysis of the consequences of genomic divergence in young lineages still capable of exchanging genes under natural conditions. The accumulation of conditionally neutral genetic variation may lead to the evolution of divergent gene networks. In a hybrid background, such mutations may no longer compensate one another, resulting in the appearance of selectively disadvantageous traits, including disruption of gene expression regulation. Here, we documented genome-wide patterns of gene expression divergence between young lineages of normal and dwarf lake whitefish and their backcross hybrids for which strong, yet incomplete post-zygotic isolation barriers exist. A significant proportion (33%) of backcross hybrids showed developmental abnormalities not seen in parental forms and eventually leading to death. While the transcriptome of parental forms was nearly identical during embryonic development, suggesting a role for stabilizing selection, all hybrids displayed strongly divergent patterns of gene expression. By comparing healthy, surviving hybrids against moribund ones, we observed that over 2000 genes were misregulated in these abnormal embryos. In particular, misregulation was significantly biased towards essential developmental genes which were strongly underexpressed. Furthermore, genes previously documented to be highly transgressive (exaggerated inter-individual variance) were almost invariably underexpressed in hybrids. Our results thus clearly showed a transcriptome-wide signature of hybrid breakdown in young, incipient species and demonstrated a persuasive link between misexpression of essential developmental genes and post zygotic isolation. Samples of dwarf, normal, backcross-healthy and backcross-moribund were hybridized in a loop design, involving eight biological replicates for the backcross-healthy and backcross-moribund comparison and six for the others. Dye swap was performed between each replicate. As a result, we obtained a final set of 32 microarray slides.
Project description:The interplay between mitogenic and proinflammatory signaling pathways play key roles in determining the phenotypes and clinical outcomes of breast cancers. We have used global nuclear run-on coupled with deep sequencing to characterize the immediate transcriptional responses of MCF-7 breast cancer cells treated with estradiol, TNFα, or both. In addition, we have integrated these data with chromatin immunoprecipitation coupled with deep sequencing for estrogen receptor alpha (ERα), the pioneer factor FoxA1 and the p65 subunit of the NF-κB transcription factor. Our results indicate extensive transcriptional interplay between these two signaling pathways, which is observed for a number of classical mitogenic and proinflammatory protein-coding genes. In addition, GRO-seq has allowed us to capture the transcriptional crosstalk at the genomic locations encoding for long non-coding RNAs, a poorly characterized class of RNAs which have been shown to play important roles in cancer outcomes. The synergistic and antagonistic interplay between estrogen and TNFα signaling at the gene level is also evident in the patterns of ERα and NF-κB binding, which relocalize to new binding sites that are not occupied by either treatment alone. Interestingly, the chromatin accessibility of classical ERα binding sites is predetermined prior to estrogen treatment, whereas ERα binding sites gained upon co-treatment with TNFα require NF-κB and FoxA1 to promote chromatin accessibility de novo. Our data suggest that TNFα signaling recruits FoxA1 and NF-κB to latent ERα enhancer locations and directly impact ERα enhancer accessibility. Binding of ERα to latent enhancers upon co-treatment, results in increased enhancer transcription, target gene expression and altered cellular response. This provides a mechanistic framework for understanding the molecular basis for integration of mitogenic and proinflammatory signaling in breast cancer. Using GRO-seq and ChIP-seq (ER, FoxA1 and p65) to assay the molecular crosstalk of MCF-7 cells treated with E2, TNFα or both E2+TNFα.
Project description:Non-lymphoid tissues (NLTs) harbour a pool of adaptive immune cells distinct from their counterparts in lymphoid tissues, and their development and phenotype remains largely unexplored. We used scRNA-seq to survey CD4+ T regulatory (Treg) and memory T (Tmem) cells in spleen, lymph nodes, skin and colon in an unbiased way, in mouse. This cross-tissues comparison allows us to obtain marker genes for immune populations in specific locations, as well as examine each population's heterogeneity. Additionally, a continuous phenotype of Treg migration can be modelled from the mouse data, unravelling the transcriptional stages through which these cells transition between tissues.
Project description:Neisseria meningitidis (meningococcus) is an invasive bacterial pathogen that colonizes human vessels, causing thrombotic lesions and meningitis. Establishment of tight interactions with endothelial cells is crucial for meningococci to resist hemodynamic forces. Two endothelial receptors, CD147 and the β2-adrenergic receptor (β2AR), are sequentially engaged by meningococci to adhere and promote signaling events leading to vascular colonization, but their spatiotemporal coordination is unknown. Here we report that CD147 and β2AR form constitutive hetero-oligomeric complexes. The scaffolding protein alpha-actinin-4 directly binds to the cytosolic tail of CD147 and governs the assembly of CD147/β2AR complexes in highly-ordered clusters at bacterial adhesion sites. This multi-molecular assembly process increases the binding strength of meningococci to endothelial cells under shear stress, and creates molecular platforms for the elongation of membrane protrusions surrounding adherent bacteria. Thus, the specific organization of cellular receptors has major impacts on host-pathogen interaction.
Project description:Single-cell RNA sequencing (scRNA-seq) was used to study the various transcriptional states of individual CD4+ T cells during blood-stage Plasmodium chabaudi infection in mice. This is an experimental model of malaria in which CD4+ T cells are essential for controlling parasite numbers, and which is characterized by concurrent development of Th1 and Tfh cells. We have used Plasmodium-specific TCR transgenic CD4+ T cells to minimise the effects of TCR diversity on Th fate decisions. Activated antigen-specific cells were studied at days 0, 2, 3, 4 and 7. In addition, dendritic cells and monocytes were studied at days 0 and 3. Cell lysis, RT and cDNA preamplification was performed using Fluidigm C1 system.
Project description:The genetic mechanisms underlying hybridization are poorly understood despite their potentially important roles in speciation processes, adaptative evolution, and agronomical innovation. In this study, transcription profiles were compared among three populations of brook charr and their hybrids using microarrays to assess the influence of hybrid origin on modes of transcription regulation inheritance and on the mechanisms underlying growth. We found that twice as many transcripts were differently expressed between the domestic strain and the two wild populations (Rupert and Laval) than between wild ones, despite their deeper genetic distance. This could reflect the consequence of artificial selection during domestication. We detected that hybrids exhibited strikingly different patterns of mode of transcription regulation, being mostly additive (94%) for domestic × Rupert, and non-additive for Laval × domestic (45.7%) and Rupert × Laval hybrids (37.5%). Both heterosis and outbreeding depression for growth were observed among the crosses. Our results indicated that prevalence of dominance in transcription regulation seems related to growth heterosis, while prevalence of transgressive transcription regulation may be more related to outbreeding depression. Our study clearly shows, for the first time in vertebrates, that the consequences of hybridization on both the transcriptome level and the phenotype are highly dependent on the specific genetic architectures of crossed populations and therefore hardly predictable. Comparison between six crosses using a loop design: direct comparison of hybrids vs parental populations fishes for 8 families. Each individual was technically replicated on two bi-coloured distinct microarrays and dye-swapped.
Project description:Parental effects represent an important source of variation in offspring phenotypes. Depending on the specific mechanisms involved, parental effects may be caused to different degrees by either the maternal or paternal parent, and these effects may in turn act at different stages of development. In order to detect parental effects acting on gene transcription regulation and length phenotype during ontogeny, the transcriptomic profiles of two reciprocal hybrids from Laval נRupert and Laval נdomestic populations of brook charr were compared at hatching, yolk sac resorption, and 15 weeks after exogenous feeding. Using a salmonid cDNA microarray, our results show that parental effects modulated gene expression among reciprocal hybrids only at the yolk sac resorption stage. In addition, Laval נdomestic and Laval נRupert reciprocal hybrids differed in the magnitude of theses parental effects, with 199 and 630 differentially expressed transcripts, respectively. This corresponds to a maximum of 18.5% of the analyzed transcripts. These transcripts are functionally related to cell cycle, nucleic acid metabolism and intracellular protein traffic, which is consistent with observed differences associated with embryonic development and growth differences in other fish species. Our results thus illustrate how parental effects on patterns of gene transcription seem dependent on the genetic architecture of the parents. In addition, in absence of transcriptomic differences, the non-transcriptomic deposits could also act to highlight the offspring differences in length before the yolk sac resorption. Pair-wise direct comparisons between 8 reciprocal hybrids (LavalRupert vs RupertLaval and LavalDomestic vs DomesticLaval) were performed at each developmental stage. For each developmental stage, cDNA of 8 samples from each cross were hybridized for a total of 48 microarrays analyzed. The distribution of dyes is equilibrated among each cross (4 samples with cy3 and 4 samples with Al647).
Project description:Coronary artery disease (CAD) is the leading cause of mortality and morbidity driven by both genetic and environmental risk factors. Meta-analyses of genome-wide association studies (GWAS) have identified multiple single nucleotide polymorphisms (SNPs) associated with CAD and myocardial infarction (MI) susceptibility in multi-ethnic populations. The majority of these variants reside in non-coding regulatory regions and are co-inherited with hundreds of candidate regulatory SNPs. Herein, we use integrative genomic, epigenomic, and transcriptomic fine-mapping in human coronary artery smooth muscle cells (HCASMC) and tissues to identify causal regulatory variation and mechanisms responsible for CAD associations. Using these genome-wide maps we prioritize 65 candidate variants and perform allele-specific binding and expression analyses on 7 top candidates. We validate our findings in two independent cohorts of diseased human arterial expression quantitative trait loci (eQTL), which together demonstrate fundamental links between CAD associations and regulatory function in the appropriate disease context. We performed ATAC-seq, ChIP-seq, and RNA-seq on human coronary artery smooth muscle cells grown in SmGM-2 Smooth Muscle Growth Medium-2 including hEGF, insulin, hFGF-B and FBS, but without antibiotics (Lonza, #CC-3182). For ATAC-seq and RNA-seq we performed stimulations with growth factors (TGF-B1, PDGF-BB, PDGF-DD) versus serum-free control. We conducted two biological replicates for each condition using independent donors. For ATAC-seq experiments, sequencing was completed on an Illumina Hiseq 2500, paired-end 50bp reads. For ChIP-seq we performed immunoprecipitations using H3K27ac (Abcam ab4729). We conducted two biological replicates using HCASMC from independent donors, and also did an IgG control for these studies. For RNA-seq we also conducted two replicates using HCASMC from independent donors. For both ChIP-seq and RNA-seq experiments, sequencing was completed on an Illumina HiSeq 2500, paired-end 100bp reads. We also performed ex-vivo ATAC-seq on frozen tissues (isolated media) from normal and atherosclerotic human coronary arteries, using three independent donors for each. Sequencing was also completed on an Illumina HiSeq 2500, paired end 50bp reads.