Comparative Genomics Reveals Key Gain-of-Function Events in Foxp3 during Regulatory T Cell Evolution

The immune system has the ability to suppress undesirable responses, such as those against commensal bacteria, food, and paternal antigens in placenta pregnancy. The lineage-specific transcription factor Foxp3 orchestrates the development and function of regulatory T cells underlying this immunological tolerance. Despite the crucial role of Foxp3 in supporting immune homeostasis, little is known about its origin, evolution, and species conservation. We explore these questions using comparative genomics, structural modeling, and functional analyses. Our data reveal that key gain-of-function events occurred during the evolution of Foxp3 in higher vertebrates. We identify key conserved residues in its forkhead domain and show a detailed analysis of the N-terminal region of Foxp3, which is only conserved in mammals. These components are under purifying selection, and our mutational analyses demonstrate that they are essential for Foxp3 function. Our study points to critical functional adaptations in immune tolerance among higher vertebrates, and suggests that Foxp3-mediated transcriptional mechanisms emerged during mammalian evolution as a stepwise gain of functional domains that enabled Foxp3 to interact with a multitude of interaction partners

An atlas of mouse CD4(+) T cell transcriptomes.

BACKGROUND: CD4(+) T cells are key regulators of the adaptive immune system and can be divided into T helper (Th) cells and regulatory T (Treg) cells. During an immune response Th cells mature from a naive state into one of several effector subtypes that exhibit distinct functions. The transcriptional mechanisms that underlie the specific functional identity of CD4(+) T cells are not fully understood. RESULTS: To assist investigations into the transcriptional identity and regulatory processes of these cells we performed mRNA-sequencing on three murine T helper subtypes (Th1, Th2 and Th17) as well as on splenic Treg cells and induced Treg (iTreg) cells. Our integrated analysis of this dataset revealed the gene expression changes associated with these related but distinct cellular identities. Each cell subtype differentially expresses a wealth of 'subtype upregulated' genes, some of which are well known whilst others promise new insights into signalling processes and transcriptional regulation. We show that hundreds of genes are regulated purely by alternative splicing to extend our knowledge of the role of post-transcriptional regulation in cell differentiation. CONCLUSIONS: This CD4(+) transcriptome atlas provides a valuable resource for the study of CD4(+) T cell populations. To facilitate its use by others, we have made the data available in an easily accessible online resource at www.th-express.org

The Regulatory T Cell Lineage Factor Foxp3 Regulates Gene Expression through Several Distinct Mechanisms Mostly Independent of Direct DNA Binding.

The lineage factor Foxp3 is essential for the development and maintenance of regulatory T cells, but little is known about the mechanisms involved. Here, we demonstrate that an N-terminal proline-rich interaction region is crucial for Foxp3's function. Subdomains within this key region link Foxp3 to several independent mechanisms of transcriptional regulation. Our study suggests that Foxp3, even in the absence of its DNA-binding forkhead domain, acts as a bridge between DNA-binding interaction partners and proteins with effector function permitting it to regulate a large number of genes. We show that, in one such mechanism, Foxp3 recruits class I histone deacetylases to the promoters of target genes, counteracting activation-induced histone acetylation and thereby suppressing their expression

Vulnerability of the North Water ecosystem to climate change

High Arctic ecosystems and Indigenous livelihoods are tightly linked and exposed to climate change, yet assessing their sensitivity requires a long-term perspective. Here, we assess the vulnerability of the North Water polynya, a unique seaice ecosystem that sustains the world’s northernmost Inuit communities and several keystone Arctic species. We reconstruct mid-to-late Holocene changes in sea ice, marine primary production, and little auk colony dynamics through multi-proxy analysis of marine and lake sediment cores. Our results suggest a productive ecosystem by 4400–4200 cal yrs b2k coincident with the arrival of the first humans in Greenland. Climate forcing during the late Holocene, leading to periods of polynya instability and marine productivity decline, is strikingly coeval with the human abandonment of Greenland from c. 2200–1200 cal yrs b2k. Our long-term perspective highlights the future decline of the North Water ecosystem, due to climate warming and changing sea-ice conditions, as an important climate change risk

The Regulatory T Cell Lineage Factor Foxp3 Regulates Gene Expression through Several Distinct Mechanisms Mostly Independent of Direct DNA Binding.

The lineage factor Foxp3 is essential for the development and maintenance of regulatory T cells, but little is known about the mechanisms involved. Here, we demonstrate that an N-terminal proline-rich interaction region is crucial for Foxp3's function. Subdomains within this key region link Foxp3 to several independent mechanisms of transcriptional regulation. Our study suggests that Foxp3, even in the absence of its DNA-binding forkhead domain, acts as a bridge between DNA-binding interaction partners and proteins with effector function permitting it to regulate a large number of genes. We show that, in one such mechanism, Foxp3 recruits class I histone deacetylases to the promoters of target genes, counteracting activation-induced histone acetylation and thereby suppressing their expression

Novel Clade 2.3.4.4b Highly Pathogenic Avian Influenza A H5N8 and H5N5 Viruses in Denmark, 2020

Since late 2020, outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4.4b have emerged in Europe. To investigate the evolutionary history of these viruses, we performed genetic characterization on the first HPAI viruses found in Denmark during the autumn of 2020. H5N8 viruses from 14 wild birds and poultry, as well as one H5N5 virus from a wild bird, were characterized by whole genome sequencing and phylogenetic analysis. The Danish H5N8 viruses were found to be genetically similar to each other and to contemporary European clade 2.3.4.4b H5N8 viruses, while the Danish H5N5 virus was shown to be a unique genotype from the H5N5 viruses that circulated at the same time in Russia, Germany, and Belgium. Genetic analyses of one of the H5N8 viruses revealed the presence of a substitution (PB2-M64T) that is highly conserved in human seasonal influenza A viruses. Our analyses showed that the late 2020 clade 2.3.4.4b HPAI H5N8 viruses were most likely new incursions introduced by migrating birds to overwintering sites in Europe, rather than the result of continued circulation of H5N8 viruses from previous introductions to Europe in 2016/2017 and early 2020

Separation of FKH-mediated DNA binding from the scaffolding function of Foxp3.

<p>(A) Model of complex formation and DNA binding of wild type, FKH deletion mutant with an SV40 NLS sequence (Foxp3^ΔFKHnls) and proline-rich region (exon 1 to exon 3) deletion mutant (Foxp3^ΔProR) of Foxp3. Spheres represent putative Foxp3 interaction partners with (purple) or without (blue) their own DNA binding capability. (B) Quantification of the similarity in the gene expression profiles of naïve T_H cells transduced with expression vectors for wild type (Foxp3), GFP (control) or mutant Foxp3 genes: Foxp3^ΔFKHnls, Foxp3^ΔFKH, Foxp3^ΔProR. Transcriptomes from two independent experiments are shown. Euclidean distances were calculated from regularized log-transformed read counts for Foxp3-regulated genes. (C) Principal-component analysis of the transcriptomes calculated from gene expression data using regularized log-transformed read counts. Color spheres represent individual experiments. PC1, PC2 and PC3 account for 55.4%, 21.1% and 7.22% of the variance, respectively. PC values were calculated using all samples used in this study, but only the samples relevant to this figure are shown here. (D) Venn diagrams showing sets of Foxp3-regulated genes which are differentially expressed (adjusted <i>P</i>< 0.05) in a comparison between T_H::Foxp3 cells and those transduced with other constructs. The set of all Foxp3-regulated genes is represented by a grey ellipse, while colored ellipses represent sets of differentially expressed genes. (E) Venn diagrams showing sets of Foxp3-regulated genes that retain their Foxp3-like control in cells transduced with the indicated constructs. The set of all Foxp3-regulated genes is represented by a grey ellipse, while colored ellipses represent sets of genes retaining Foxp3-like control. (F) Comparison of Foxp3-regulated gene expression in T_H::control cells and those transduced with other constructs. Each dot represents an individual Foxp3-regulated gene. Genes are separated into those shown to be upregulated by Foxp3 expression and those that are downregulated. Differential gene expression was calculated from the two independent experiments using Wald tests on moderated log2 fold changes as implemented by the DESeq2 R package with <i>P</i> values adjusted for multiple testing using the procedure of Benjamini and Hochberg [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005251#pgen.1005251.ref058" target="_blank">58</a>]. Numeric annotations on the graphs indicate the number of Foxp3-upregulated and Foxp3-downregulated genes that are dysregulated by each mutant.</p

Mutational dissection of ProR of Foxp3 reveals distinct mechanisms of gene regulation.

<p>(A) Conservation plot (top) showing the average similarity score at individual amino acid positions from multiple sequence alignments of placental mammalian Foxp3 (red; seven species), non-placental mammalian Foxp3 (black; three species) and non-mammalian vertebrate Foxp3L (green; seven species). The ‘placental’ and ‘mammalian’ halves of the ProR were defined semi-arbitrarily based on the homology across different species. The plots were generated using EMBOSS plotcon with a window size of 20. The REL test as implemented in Datamonkey was used to perform a sliding window analysis (4 codons) of placental Foxp3s with calculated Bayes factors plotted in red (bottom). The average pairwise identity (averaged over 4 amino acids) between Foxp3s is plotted in green (dotted grey lines: exon boundaries). The positions of proline residues are denoted by purple bars. For orientation, a schematic representation of Foxp3 is shown at the bottom. m1-m5 denote regions of interest for which deletion mutants were generated. (B) Flow cytometry analysis of Foxp3 target gene expression in T_H cells transduced with wild type Foxp3 (blue), control vector (black) and Foxp3 mutants (red). Foxp3^Δe1: deletion of exon1; Foxp3^Δe1-2: deletion of exon 1–2, Foxp3^ΔProR: deletion of exon 1–3; Foxp3^Δm1: deletion of region m1; Foxp3^Δm2, Foxp3^Δm3, Foxp3^Δm4, Foxp3^Δm5: regions m1, m2, m3, m4, m5 were replaced by short stretches of alanines. Foxp3^Δm4.1 and Foxp3^Δm4.2 are outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005251#pgen.1005251.s002" target="_blank">S2A Fig</a>. Expression was measured in transduced cells rested 48 h post-transduction (plots were gated on rCD8a⁺ transduced cells and are representative of three independently transduced cell samples in parallel). For IL-2, resting cells were re-stimulated for 12 h with cell stimulation cocktail (PMA and ionomycin with protein transport inhibitor) before staining. (C) Summaries of normalized mean fluorescence intensity (MFI) in (B), shown as ratios of MFI (mean ± SD) of rCD8a⁺ cells/ rCD8a^- cells in each sample. Significant differences between T_H::Foxp3 and cells transduced with the other constructs were determined by one way analysis of variance (ANOVA) followed by Tukey’s post-hoc test(* indicates <i>P</i>≤0.05; ** indicates <i>P</i>≤0.01; *** indicates <i>P</i>≤0.001).</p