OCT4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human

Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs.

Single nucleus multi-omics regulatory landscape of the murine pituitary

To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation. We also identify transcriptional and chromatin accessibility programs distinguishing each major cell type. Regulons, which are co-regulated gene sets sharing binding sites for a common transcription factor driver, recapitulate cell type clustering. We identify both cell type-specific and sex-specific regulons that are highly correlated with promoter accessibility, but not with methylation state, supporting the centrality of chromatin accessibility in shaping cell-defining transcriptional programs. The sn multi-omics atlas is accessible at snpituitaryatlas.princeton.edu.

THU0047 THE SYNOVIUM REWIRES AN IMMUNOLOGICAL RHEOSTAT THAT DEFINES TWO FUNCTIONALLY DISPARATE PATHOGENIC CD4+HLA-DR+ SUBSETS IN HUMAN ARTHRITIS

Background:Despite advances in understanding how the adaptive T cell landscape is affected in human arthritis, specific T cell subset knowledge has yet to be utilised in clinical settings. We have previously discovered within active arthritic patients, a circulating pathogenic-like lymphocyte (CPLs; CD4+HLA-DR+) within the T-effector compartment, that is phenotypically similar to their synovial counterparts. CPLs are inflammatory, correlate with disease activity and overlap in synovial TCR repertoire. A similar inflammation-associated T-regulatory (iaTreg; CD4+HLA-DR+) subset, that is activated, poised to migrate to inflamed site and sharing synovial TCR overlap, suggest a common disease ontogeny that may exist between CPLs and iaTregs.Objectives:Here we seek to determine whether and how the synovial microenvironment plays a role in modulating these two functionally divergent (Teff/Treg compartments) yet pathogenically homologous subsets. This modulation, akin to an immunological rheostat, may be a feature of the disease process.Methods:We examined CD45+ immune cells from synovial and PBMCs (active JIA, inactive JIA, paediatric healthy) through mass cytometry (CyToF). CD4 T cells were sorted into CPLs, iaTregs, Teff and Treg through FACS Aria II, from active JIA PBMCs, paired JIA SFMCs and healthy paediatric PBMCs and examined through ngRNASEQ.Results:Mass cytometric analysis reveal a significant enrichment of synovium signatures in both circulatory CPLs and iaTregs subsets from active arthritic PBMCs, as compared with the conventional pool of Teff/Tregs. This immunological relationship between CPLs/iaTregs is reaffirmed by comparative differential gene expression (DEG) and phylogenetic tree analysis, which indicated transcriptomic convergence between circulatory pathogenic CPLs/iaTreg subsets and divergence from their respective conventional Teff/Treg pools. Circulatory CPLs/iaTregs exhibit (a) common pathway dysregulation in T cell signalling, (b) restriction in TCR oligoclonality and (c) common transcription factor drivers within the gene regulatory network, suggesting a common pathogenic mechanism acting on these two disparate compartments.To understand how the microenvironment plays a role in modulating these two subsets, we compared the transcriptome of CPLs/iaTreg and conventional Teff/Treg subsets from (a) healthy PBMCs, (b) JIA PBMCs and (c) paired JIA SFMCs. The convergence between CPLs/iaTreg increases across the spatial/disease continuum, culminating in 7 key common dysregulated pathways within synovium CPLs/iaTregs. Importantly we detected higher clonotypic sharing of TCRs in CPLs/iaTregs across the spatial and disease continuum, suggesting a common precursor driven by antigenic selection.Conclusion:Our data suggest that CPLs/iaTregs are dichotomic components of a systemic immune rheostat, shape through the synovium environment, modulating autoimmunity in human arthritis. As iaTreg and CPL most likely have the capacity to morph into each other, the molecular crossroads which control this plasticity represent novel therapeutic targets.Disclosure of Interests:None declared

Transcriptional regulator PAX4 links Receptor Tyrosine Kinases (RTKs) and cytoskeleton stability in Alzheimer’s disease and type 2 diabetes

Alzheimer’s Disease (AD) and Type 2 Diabetes (T2D) share a common hallmark of insulin resistance. Besides Insulin Receptor (IR), two non-canonical RTKs, ALK and RYK, exhibit significant and consistent functional downregulation in post-mortem AD and T2D tissues. Incidentally, both have Grb2 as a common downstream adapter and NOX4 as a common ROS producing factor. Here we show that Grb2 and NOX4 play critical roles in reducing the severity of both the diseases. The study demonstrates that the abundance of Grb2 in degenerative conditions, in conjunction with NOX4, reverse cytoskeletal degradation by counterbalancing the network of small GTPases. PAX4, a transcription factor for both Grb2 and NOX4, emerges as the key link between the common pathways of AD and T2D. Both ALK and RYK downregulation elevate the PAX4 level by reducing its suppressor ARX via Wnt/β-Catenin signaling pathway. For the first time, this study brings together RTKs other than Insulin Receptor (IR), their common transcription factor PAX4 and both AD and T2D pathologies on a common regulatory platform.

miRCoop: Identifying Cooperating miRNAs via Kernel Based Interaction Tests

Although miRNAs can cause widespread changes in expression programs, single miRNAs typically induce mild repression on their targets. Cooperativity is reported as one strategy to overcome this constraint. Expanding the catalog of synergistic miRNAs is critical for understanding gene regulation and for developing miRNA-based therapeutics. In this study, we develop miRCoop to identify synergistic miRNA pairs that have weak or no repression on the target mRNA, but when bound together, induce strong repression. miRCoop uses kernel-based interaction tests together with miRNA and mRNA target information. We apply our approach to kidney tumor patient data and identify 66 putative triplets. For 64 of these triplets, there is at least one common transcription factor that potentially regulates all participating RNAs of the triplet, supporting a functional association among them. Furthermore, we find that triplets are enriched for certain biological processes that are relevant to kidney cancer. Some of the synergistic miRNAs are very closely encoded in the genome, hinting a functional association among them. We believe miRCoop can aid our understanding of the complex regulatory interactions in different health and disease states of the cell and can help in designing miRNA-based therapies. Matlab code for the methodology is provided in https://github.com/guldenolgun/miRCoop.

Mouse prenatal platelet-forming lineages share a core transcriptional program but divergent dependence on MPL

Key Points Prenatal platelet-forming lineages are subject to common transcription factor controls despite distinct spatial and ancestral origins. Platelet-forming lineage production is MPL-independent on emergence, but MPL is required in the late fetus for efficient thrombopoiesis.