Biology subjects

Blelloch, R.

Publications and source records attributed to Blelloch, R..

12 recordsLinked to original sources

bioRxiv2026

KMT2C and KMT2D amplify GRHL2-driven enhancer activation

The activation of cis-regulatory enhancers is essential for cell fate specification by driving cell type-specific gene expression. Differentiation models are widely used to study enhancer biology but the asynchronous and interdependent nature of gene regulatory changes during cell state transitions can complicate mechanistic studies. To overcome these limitations, here we develop a tamoxifen-gated system for acute enhancer activation in embryonic stem cells (ESCs) based on GRHL2, a pioneer transcription factor which naturally becomes expressed as naive ESCs differentiate into the formative ESC state. Using this system, we investigate the functional relationship between GRHL2 and the histone mono-methyltransferases KMT2C and KMT2D (KMT2C/D). GRHL2 readily binds its target sites independent of KMT2C/D. However, in the absence of KMT2C/D, there are dramatic reductions in H3K4me1/2, P300 recruitment, and H3K27ac deposition at these sites as well as diminished transcriptional activation. Still, strikingly, a basal level of active enhancer mark acquisition and transcriptional activation occurs. Consistent with these findings, during the naive to formative ESC differentiation, GRHL2 enhancer remodeling and target expression is also strongly but incompletely dependent on KMT2C/D. Together, these results define a functional co-activator relationship in which KMT2C/D act as important amplifiers of GRHL2-driven enhancer activation in ESCs and establish a rapid inducible system for dissecting the kinetics and enzymatic dependencies of pioneer transcription factor mediated enhancer remodeling.

molecular biology↗

bioRxiv2024

Synergy between cis-regulatory elements can render cohesin dispensable for distal enhancer function

Enhancers are critical genetic elements controlling transcription from promoters, but the mechanisms by which they convey regulatory information across large genomic distances remain elusive. Here, we engineered pluripotent stem cells in which cohesin loop extrusion can be inducibly disrupted without causing confounding cell cycle defects. While evident, transcriptional dysregulation was cell-type specific, and not all loci with distal enhancers depend equally on cohesin extrusion. Using comparative genome editing, we demonstrate that enhancer-promoter communication across as little as 20 kilobases can rely on cohesin. However, promoter-proximal regulatory elements can support long-range, cohesin-independent enhancer action - either upon disabling extrusion or across strong CTCF insulators. Finally, transcriptional dynamics and the emergence of new embryonic cell types in response to differentiation cues remained largely robust to disrupting cohesin extrusion. Beyond establishing novel experimental strategies to study cohesin functions in enhancer biology, our work provides mechanistic insight accounting for both cell type- and genomic context-specificity.

molecular biology↗

bioRxiv2024

Coordinate post-transcriptional regulation by microRNAs and RNA binding proteins is critical for early embryonic cell fate decisions

Abstract/SummaryPost-transcriptional control by RNA binding proteins (RBPs) and microRNAs play central roles in mRNA stability and translation (1). However, little is known about how RBPs and microRNAs coordinate in developmental time to regulate cell fate. Here, we show that coordinate RBP and microRNA control of a single transcript, Profilin 2 (Pfn2), is essential for differentiation of embryonic stem cells (ESCs) into the primary germ layer lineages. The Pfn2 3untranslated region has a binding site for Iron Regulatory Proteins and a nearby binding site for ESC enriched microRNAs (2,3). Deletion of this microRNA site leads to increased PFN2 and reduced FGF signaling during pluripotency transition prior to germ layer formation (4). In contrast, deletion of the iron response element leads to decreased PFN2 and a Wnt signaling defect, reduced nuclear beta-catenin, and a subsequent block in mesendodermal lineages during early germ layer formation. The choreographed microRNA-IRE axis of control on the Pfn2 transcript is essential for two key signal transduction steps during ESC differentiation.

developmental biology↗

bioRxiv2024

The miR-290 and miR-302 clusters are essential for reprogramming of fibroblasts to induced pluripotent stem cells

The miR-290 and miR-302 clusters of microRNAs are highly expressed in naive and primed pluripotent stem cells, respectively. Ectopic expression of the embryonic stem cell-specific cell cycle regulating (ESCC) family of microRNAs arising from these two clusters dramatically enhances the reprogramming of both mouse and human somatic cells to induced pluripotency. Here, we used genetic knockouts to dissect the requirement for the miR-290 and miR-302 clusters during the reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs) with retrovirally introduced Oct4, Sox2, and Klf4. Knockout of either cluster alone did not negatively impact the efficiency of reprogramming. Resulting cells appeared identical to their embryonic stem cell microRNA cluster knockout counterparts. In contrast, the combined loss of both clusters blocked the formation of iPSCs. While rare double knockout clones could be isolated, they showed a dramatically reduced proliferation rate, a persistent inability to fully silence the exogenously introduced pluripotency factors, and a transcriptome distinct from individual miR-290 or miR-302 mutant ESC and iPSCs. Taken together, our data show that miR-290 and miR-302 are essential yet interchangeable in reprogramming to the induced pluripotent state. Impact StatementThe process by which somatic cell reprogramming yields induced pluripotent stem cells (iPSCs) is incompletely understood. MicroRNAs from the miR-290 and miR-302 clusters have been shown to greatly increase reprogramming efficiency, but their requirement in the process has not been studied. Here, we examine this requirement by genetically removing the miRNA clusters in somatic cells. We discover that somatic cells lacking either, but not both, of these miRNA clusters can form iPSC cells. This work thus provides new important insight into mechanisms underlying reprogramming to pluripotency.

developmental biology↗

bioRxiv2024

PVRL2 Suppresses Anti-tumor Immunity Through PVRIG- and TIGIT-Independent Pathways

PVRL2 is believed to act as an immune checkpoint protein in cancer; however, most insight into PVRL2s role is inferred from studies on its known receptor PVRIG. Here, we directly study PVRL2. PVRL2 levels are high in tumor cells and tumor-derived exosomes. Deletion of PVRL2 in multiple syngeneic mouse models of cancer shows a dramatic reduction in tumor growth that is immune dependent. This effect can be even greater than seen with deletion of PD-L1. PVRL2 functions by suppressing CD8 T and NK cells in the tumor microenvironment. Unexpectedly, the effect of PVRL2 loss on tumor growth remains in the absence of PVRIG. In contrast, PVRIG loss shows no additive effect in the absence of PVRL2. TIGIT blockade combined with PVRL2 deletion results in the greatest reduction in tumor growth. This effect is not recapitulated by the combined deletion of PVRL2 with its paralog PVR, the ligand for TIGIT. These data uncover PVRL2 as a distinct inhibitor of the anti-tumor immune response with functions beyond that of its known receptor PVRIG. Importantly, the data provide a strong rationale for combinatorial targeting of PVRL2 and TIGIT for cancer immunotherapy.

immunology↗

bioRxiv2024

A Multiomics, Spatiotemporal, and Single Cell Atlas for Mapping Cell-Type-Specific Dysregulation at the Maternal-Fetal Interface

The placenta, the first organ to functionally mature, undergoes disordered development in many pregnancy complications. Molecular investigations have been hampered by the extreme cellular heterogeneity of the placenta, and this complexity is further exaggerated at the maternal-fetal interface where maternal and fetal cells co-mingle. We generated the paired single nucleus epigenomes and transcriptome for each of [~]200,000 cells at the human maternal-fetal interface from early pregnancy to term. These data identified cell-type-specific transcriptional regulatory programs and uncovered key transcription factors driving the lineage differentiation of placental cytotrophoblasts. Integrating spatial single cell proteomics profiling, we localized the observed cell types in situ, and characterized the dynamic stages and distinct features of endothelial cells of maternal spiral arteries remodeled by extravillous cytotrophoblasts. Integrative analyses of the single cell data across gestation enabled fine-mapping of the developmental trajectories of cytotrophoblasts and decidual stromal cells, and defining the signature molecular profiles of known and novel cell (sub)types. To demonstrate clinical value, we integrated the reference single cell data with large-scale population genomes from pregnancy complications and identified the most vulnerable maternal and fetal cell types in preeclampsia, preterm birth, and miscarriage. This study presents the most comprehensive placental and decidual single cell resource across gestation to date, reveals new insights into the drivers of normal human placentation, and uncovers the cellular basis of dysfunction associated with common pregnancy complications.

genomics↗

bioRxiv2023

MLL3/MLL4 enzymatic activity shapes DNA replication timing

Mammalian genomes are replicated in a precise order during S phase, which is cell-type-specific1-3 and correlates with local transcriptional activity2,4-8, chromatin modifications9 and chromatin architecture1,10,11,12. However, the causal relationships between these features and the key regulators of DNA replication timing (RT) are largely unknown. Here, machine learning was applied to quantify chromatin features, including epigenetic marks, histone variants and chromatin architectural factors, best predicting local RT under steady-state and RT changes during early embryonic stem (ES) cell differentiation. About one-third of genome exhibited RT changes during the differentiation. Combined, chromatin features predicted steady-state RT and RT changes with high accuracy. Of these features, histone H3 lysine 4 monomethylation (H3K4me1) catalyzed by MLL3/4 (also known as KMT2C/D) emerged as a top predictor. Loss of Mll3/4 (but not Mll3 alone) or their enzymatic activity resulted in erasure of genome-wide RT dynamics during ES cell differentiation. Sites that normally gain H3K4me1 in a MLL3/4-dependent fashion during the transition failed to transition towards earlier RT, often with transcriptional activation unaffected. Further analysis revealed a requirement for MLL3/4 in promoting DNA replication initiation zones through MCM2 recruitment, providing a direct link for its role in regulating RT. Our results uncover MLL3/4-dependent H3K4me1 as a functional regulator of RT and highlight a causal relationship between the epigenome and RT that is largely uncoupled from transcription. These findings uncover a previously unknown role for MLL3/4-dependent chromatin functions which is likely relevant to the numerous diseases associated with MLL3/4 mutations.

genomics↗

bioRxiv2022

Loss of MLL3/4 decouples enhancer H3K4 monomethylation, H3K27 acetylation, and gene activation during ESC differentiation

Enhancers are essential in defining cell fates through the control of cell type specific gene expression. Enhancer activation is a multi-step process involving chromatin remodelers and histone modifiers including the monomethylation of H3K4 (H3K4me1) by MLL3 (KMT2C) and MLL4 (KMT2D). MLL3/4 are thought to be critical for enhancer activation and cognate gene expression including through the recruitment of acetyltransferases for H3K27. Here we test this model by evaluating the impact of MLL3/4 loss on chromatin and transcription during early embryonic stem cell differentiation. We find that MLL3/4 activity is required at most if not all sites that gain or lose H3K4me1 but is largely dispensable at sites that remain stably methylated during this transition. This requirement extends to H3K27 acetylation (H3K27ac) at most transitional sites. However, many sites gain H3K27ac independent of MLL3/4 or H3K4me1 including enhancers regulating key factors in early differentiation. Furthermore, despite the failure to gain active histone marks at thousands of enhancers, transcriptional activation of nearby genes is largely unaffected, thus uncoupling the regulation of these chromatin events from transcriptional changes during this transition. These data challenge current models of enhancer activation and imply distinct mechanisms between stable and dynamically changing enhancers. Collectively, our study highlights gaps in knowledge about the steps and epistatic relationships of enzymes necessary for enhancer activation and cognate gene transcription.

genomics↗

bioRxiv2022

Regionally distinct trophoblast regulate barrier function and invasion in the human placenta

The human placenta contains two specialized regions: the villous chorion where gases and nutrients are exchanged between maternal and fetal blood, and the smooth chorion which surrounds more than 70% of the developing fetus but whose cellular composition and function is poorly understood. Here, we use single cell RNA sequencing to compare the cell types and molecular programs between these two regions in the second trimester human placenta. Each region consists of progenitor cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs) with similar gene expression programs. While CTBs in the villous chorion differentiate into syncytiotrophoblasts, they take an alternative trajectory in the smooth chorion producing a previously unknown CTB population which we term smooth-chorion-specific CTBs (SC-CTBs). Marked by expression of region-specific cytokeratins, the SC-CTBs form a stratified epithelium above a basal layer of progenitor CTBs. They express epidermal and metabolic transcriptional programs consistent with a primary role in defense against physical stress and pathogens. Additionally, we show that SC-CTBs closely associate with EVTs and secrete factors that inhibit the migration of the EVTs. This restriction of EVT migration is in striking contrast to the villous region where EVTs migrate away from the chorion and invade deeply into the decidua. Together, these findings greatly expand our understanding of CTB differentiation in these distinct regions of the human placenta. This knowledge has broad implications for studies of the development, functions, and diseases of the human placenta. Impact StatementSingle cell RNA-sequencing of distinct regions of the human placenta identifies a smooth chorion-specific cytotrophoblast population responsible for unique functions of the smooth chorion, including acting as a barrier and restricting invasion.

developmental biology↗

bioRxiv2021

Cell-surface tethered promiscuous biotinylators enable small-scale surface proteomics of human exosomes

Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e7 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived exosomes, leading to the robust quantification of 1,020 cell and exosome surface proteins. We identified a newly-recognized subset of exosome-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer exosomes. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types.

biochemistry↗

bioRxiv2020

MicroRNA-dependent inhibition of PFN2 orchestrates ERK activation and pluripotent state transitions by regulating endocytosis

Profilin2 (PFN2) is a target of the embryonic stem cell (ESC) enriched miR-290 family of miRNAs and an actin/dynamin binding protein implicated in endocytosis. Here, we show that the miR-290-PFN2 pathway regulates many aspects of ESC biology. In the absence of microRNAs, PFN2 is upregulated in ESCs, with a resulting decrease in endocytosis. Reintroduction of miR-290, knockout of PFN2, or disruption of the PFN2 dynamin interacting domain in miRNA deficient cells reverses the endocytosis defect. The loss of miRNA suppression of PFN2 and associated reduction in endocytosis impairs ERK signaling, which in turn inhibits ESC cell cycle progression and differentiation from a naive to formative state. Mutagenesis of the single canonical conserved 3UTR miR-290 binding site of PFN2 in otherwise wild-type cells recapitulates these phenotypes. Together, these findings define an axis of post-transcriptional control, endocytosis, and signal transduction that is essential for ESC self-renewal and differentiation.

developmental biology↗

bioRxiv2018

Decoupling the impact of microRNAs on translational repression versus RNA degradation in embryonic stem cells

Translation and mRNA degradation are intimately connected, yet the mechanisms that regulate them are not fully understood. Here we studied the link between translation and mRNA stability in embryonic stem cells (ESCs). Transcripts showed a wide range of stabilities, which correlated with their translation levels. The protein DHH1 links translation to mRNA stability in yeast; however loss of the mammalian homolog, DDX6, in ESCs did not disrupt the correlation across transcripts. Instead, the loss of DDX6 led to upregulated translation of microRNA targets, without concurrent changes in mRNA stability. The Ddx6 knockout cells were phenotypically and molecularly similar to cells lacking all microRNAs (Dgcr8 knockout ESCs). These data show that the loss of DDX6 can separate the two canonical functions of microRNAs: translational repression and transcript destabilization. Furthermore, these data uncover a central role for translational repression independent of transcript destabilization in defining the downstream consequences of microRNA loss.

genetics↗