Progesterone receptor mediates ovulatory transcription through RUNX transcription factor interactions and chromatin remodelling.

Progesterone receptor (PGR) plays diverse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, rapid acute induction of PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for this specialized PGR function in ovulation is poorly understood. We have assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrate that stimulating ovulation rapidly reprograms chromatin accessibility in two-thirds of sites, correlating with altered gene expression. An ovary-specific PGR action involving interaction with RUNX transcription factors was observed with 70% of PGR-bound regions also bound by RUNX1. These transcriptional complexes direct PGR binding to proximal promoter regions. Additionally, direct PGR binding to the canonical NR3C motif enable chromatin accessibility. Together these PGR actions mediate induction of essential ovulatory genes. Our findings highlight a novel PGR transcriptional mechanism specific to ovulation, providing new targets for infertility treatments or new contraceptives that block ovulation.

A disrupted FOXP3 transcriptional signature underpins systemic regulatory T cell insufficiency in early pregnancy failure.

Regulatory T (Treg) cell defects are implicated in disorders of embryo implantation and placental development, but the origins of Treg cell dysfunction are unknown. Here, we comprehensively analyzed the phenotypes and transcriptional profile of peripheral blood Treg cells in individuals with early pregnancy failure (EPF). Compared to fertile subjects, EPF subjects had 32% fewer total Treg cells and 54% fewer CD45RA+CCR7+ naive Treg cells among CD4+ T cells, an altered Treg cell phenotype with reduced transcription factor FOXP3 and suppressive marker CTLA4 expression, and lower Treg:Th1 and Treg:Th17 ratios. RNA sequencing demonstrated an aberrant gene expression profile, with upregulation of pro-inflammatory genes including CSF2, IL4, IL17A, IL21, and IFNG in EPF Treg cells. In silico analysis revealed 25% of the Treg cell dysregulated genes are targets of FOXP3. We conclude that EPF is associated with systemic Treg cell defects arising due to disrupted FOXP3 transcriptional control and loss of lineage fidelity.

Parallel recovery of chromatin accessibility and gene expression dynamics from frozen human regulatory T cells.

Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories.

3DFAACTS-SNP: using regulatory T cell-specific epigenomics data to uncover candidate mechanisms of type 1 diabetes (T1D) risk.

BACKGROUND: Genome-wide association studies (GWAS) have enabled the discovery of single nucleotide polymorphisms (SNPs) that are significantly associated with many autoimmune diseases including type 1 diabetes (T1D). However, many of the identified variants lie in non-coding regions, limiting the identification of mechanisms that contribute to autoimmune disease progression. To address this problem, we developed a variant filtering workflow called 3DFAACTS-SNP to link genetic variants to target genes in a cell-specific manner. Here, we use 3DFAACTS-SNP to identify candidate SNPs and target genes associated with the loss of immune tolerance in regulatory T cells (Treg) in T1D. RESULTS: Using 3DFAACTS-SNP, we identified from a list of 1228 previously fine-mapped variants, 36 SNPs with plausible Treg-specific mechanisms of action. The integration of cell type-specific chromosome conformation capture data in 3DFAACTS-SNP identified 266 regulatory regions and 47 candidate target genes that interact with these variant-containing regions in Treg cells. We further demonstrated the utility of the workflow by applying it to three other SNP autoimmune datasets, identifying 16 Treg-centric candidate variants and 60 interacting genes. Finally, we demonstrate the broad utility of 3DFAACTS-SNP for functional annotation of all known common (> 10% allele frequency) variants from the Genome Aggregation Database (gnomAD). We identified 9376 candidate variants and 4968 candidate target genes, generating a list of potential sites for future T1D or other autoimmune disease research. CONCLUSIONS: We demonstrate that it is possible to further prioritise variants that contribute to T1D based on regulatory function, and illustrate the power of using cell type-specific multi-omics datasets to determine disease mechanisms. Our workflow can be customised to any cell type for which the individual datasets for functional annotation have been generated, giving broad applicability and utility.

Molecular Insights Into Regulatory T-Cell Adaptation to Self, Environment, and Host Tissues: Plasticity or Loss of Function in Autoimmune Disease.

There has been much interest in the ability of regulatory T cells (Treg) to switch function in vivo, either as a result of genetic risk of disease or in response to environmental and metabolic cues. The relationship between levels of FOXP3 and functional fitness plays a significant part in this plasticity. There is an emerging role for Treg in tissue repair that may be less dependent on FOXP3, and the molecular mechanisms underpinning this are not fully understood. As a result of detailed, high-resolution functional genomics, the gene regulatory networks and key functional mediators of Treg phenotype downstream of FOXP3 have been mapped, enabling a mechanistic insight into Treg function. This transcription factor-driven programming of T-cell function to generate Treg requires the switching on and off of key genes that form part of the Treg gene regulatory network and raises the possibility that this is reversible. It is plausible that subtle shifts in expression levels of specific genes, including transcription factors and non-coding RNAs, change the regulation of the Treg gene network. The subtle skewing of gene expression initiates changes in function, with the potential to promote chronic disease and/or to license appropriate inflammatory responses. In the case of autoimmunity, there is an underlying genetic risk, and the interplay of genetic and environmental cues is complex and impacts gene regulation networks frequently involving promoters and enhancers, the regulatory elements that control gene expression levels and responsiveness. These promoter-enhancer interactions can operate over long distances and are highly cell type specific. In autoimmunity, the genetic risk can result in changes in these enhancer/promoter interactions, and this mainly impacts genes which are expressed in T cells and hence impacts Treg/conventional T-cell (Tconv) function. Genetic risk may cause the subtle alterations to the responsiveness of gene regulatory networks which are controlled by or control FOXP3 and its target genes, and the application of assays of the 3D organization of chromatin, enabling the connection of non-coding regulatory regions to the genes they control, is revealing the direct impact of environmental/metabolic/genetic risk on T-cell function and is providing mechanistic insight into susceptibility to inflammatory and autoimmune conditions.

Importance of a N-terminal aspartate in the internalization of the neuropeptide Y Y2 receptor.

With human neuropeptide Y Y2 receptor expressed in the Chinese hamster ovary (CHO) cells, the Asp35Ala mutation, and especially the change of Pro34Asp35 to Ala34Ala35, decrease the compartmentalization and strongly accelerate internalization of the receptor. These changes are not associated with alterations in agonist affinity, G-protein interaction, dimerization, or level of expression of the mutated receptors relative to the wildtype receptor. The proline-flanked aspartate in the N-terminal extracellular segment of the neuropeptide Y Y2 receptor thus apparently has a large role in anchoring and compartmentalization of the receptor. However, the Pro34Ala mutation does not significantly affect the embedding and cycling of the receptor.