Regulate Cell Fate Decisions Research Articles

Engineering synthetic agonists for targeted activation of Notch signaling.

Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although there are several classes of Notch inhibitors, the mechanical force requirement for Notch receptor activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonist (SNAG) proteins by tethering affinity-matured Notch ligands to antibodies or cytokines that internalize their targets. This bispecific format enables SNAGs to "pull" on mechanosensitive Notch receptors, triggering their activation in the presence of a desired biomarker. We successfully developed SNAGs targeting six independent surface markers, including the tumor antigens PDL1, CD19, and HER2, and the immunostimulatory receptor CD40. SNAGs targeting CD40 increase expansion of central memory γδ T cells from peripheral blood, highlighting their potential to improve the phenotype and yield of low-abundance T cell subsets. These insights have broad implications for the pharmacological activation of mechanoreceptors and will expand our ability to modulate Notch signaling in biotechnology.

Cpt1a Drives primed-to-naïve pluripotency transition through lipid remodeling

Metabolism has been implicated in cell fate determination, particularly through epigenetic modifications. Similarly, lipid remodeling also plays a role in regulating cell fate. Here, we present comprehensive lipidomics analysis during BMP4-driven primed to naive pluripotency transition or BiPNT and demonstrate that lipid remodeling plays an essential role. We further identify Cpt1a as a rate-limiting factor in BiPNT, driving lipid remodeling and metabolic reprogramming while simultaneously increasing intracellular acetyl-CoA levels and enhancing H3K27ac at chromatin open sites. Perturbation of BiPNT by histone acetylation inhibitors suppresses lipid remodeling and pluripotency transition. Together, our study suggests that lipid remodeling promotes pluripotency transitions and further regulates cell fate decisions, implicating Cpt1a as a critical regulator between primed-naive cell fate control.

Abstract C059: Regulatory Elements of Pancreas Development License the Initiation of Pancreatic Ductal Adenocarcinoma

Abstract Mutations in DNA are a known cause of cancer. However, they become oncogenic only under specific cellular conditions and within certain tissue contexts. Tissue injury often leads to the aberrant activation of developmental pathways, eroding markers of cellular differentiation and giving rise to transient cellular states. These states resemble developmental progenitors, characterized by higher transcriptional plasticity. Oncogenes, particularly KRAS, co-opt cellular plasticity by acting on tissue-specific enhancers, preventing the resolution of acinar plasticity and maintaining the pancreas in a pro-inflammatory phase that progresses to cancer1,2. However, the specific role of tissue-specific enhancers in pancreas regeneration and cancer initiation remains unexplored, and strategies to target these regenerative processes are yet to be developed. Our laboratory investigates non-mutational mechanisms of cancer, with a special interest in the intersection of developmental biology and cancer, aiming to elucidate the processes of cellular plasticity and cancer initiation. Recent studies have shown that acinar cells possess remarkable cellular plasticity, giving rise to a diverse population of cellular states in pancreas regeneration and cancer contexts3-5. We have discovered that the transcription factor Sox4 is necessary for the specification of tuft cells and gastric metaplasia found in chronic pancreatitis and in pancreatic intraepithelial lesions (PanINs). Moreover, we have shown that cellular plasticity has a non-cell autonomous protective effect; losing Sox4 and cellular heterogeneity in PanINs results in a fibrotic and immunosuppressive tumor microenvironment and disease progression. Our results demonstrated that targeting cellular plasticity accelerates or prevents cancer initiation through autonomous and non-autonomous cellular processes6. In this study, we leverage a comprehensive resource of ncRNAs transcribed from enhancer elements of pancreas development, together with their inferred target genes and correlations with clinical outcomes. We focused on an ncRNA associated with the risk of pancreatic cancer. To study its function, we developed two novel mouse strains for tissue-specific deletion, enabling precise manipulation and study of its role in the pancreas and other tissues. Our phenotypic analysis demonstrates that the ncRNA is activated following tissue injury and is essential for developing PanINs. Moreover, we show that it regulates cell-cell communication after tissue injury. This mechanism operates independently of cellular plasticity but significantly creates a pro-inflammatory environment that favors cancer development. Our results demonstrate that enhancer elements are actionable targets to regulate cell fate decisions in regeneration and cancer. Additionally, KRAS-induced cancer initiation depends on developmental regulatory elements, opening the window to develop prophylactic interventions to delay tumor onset. Citation Format: Luis Arnes. Regulatory Elements of Pancreas Development License the Initiation of Pancreatic Ductal Adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C059.

BHLH transcription factors cooperate with chromatin remodelers to regulate cell fate decisions during Arabidopsis stomatal development.

The development of multicellular organisms requires coordinated changes in gene expression that are often mediated by the interaction between transcription factors (TFs) and their corresponding cis-regulatory elements (CREs). During development and differentiation, the accessibility of CREs is dynamically modulated by the epigenome. How the epigenome, CREs, and TFs together exert control over cell fate commitment remains to be fully understood. In the Arabidopsis leaf epidermis, meristemoids undergo a series of stereotyped cell divisions, then switch fate to commit to stomatal differentiation. Newly created or reanalyzed scRNA-seq and ChIP-seq data confirm that stomatal development involves distinctive phases of transcriptional regulation and that differentially regulated genes are bound by the stomatal basic helix-loop-helix (bHLH) TFs. Targets of the bHLHs often reside in repressive chromatin before activation. MNase-seq evidence further suggests that the repressive state can be overcome and remodeled upon activation by specific stomatal bHLHs. We propose that chromatin remodeling is mediated through the recruitment of a set of physical interactors that we identified through proximity labeling-the ATPase-dependent chromatin remodeling SWI/SNF complex and the histone acetyltransferase HAC1. The bHLHs and chromatin remodelers localize to overlapping genomic regions in a hierarchical order. Furthermore, plants with stage-specific knockdown of the SWI/SNF components or HAC1 fail to activate specific bHLH targets and display stomatal development defects. Together, these data converge on a model for how stomatal TFs and epigenetic machinery cooperatively regulate transcription and chromatin remodeling during progressive fate specification.

Abstract We046: Building Mass or Strength: Stage-specific Effects of Insulin and Wnt on Cell Fate Decisions in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background: Reactivation of the so-called fetal gene programs and pathways have been reported in failing hearts, however, it remains unclear whether this phenomenon is beneficial or detrimental to the remaining functional myocardium. During cardiac development, the orchestration of these embryonic growth pathways, facilitate temporal and stage-specific cell fate decisions such as required for specification and growth of the various myocardial compartments. Previously, we demonstrated that molecular activation of the Wnt pathway resulted in massive expansion of immature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Yet, it is unknown what exact cues guide the cell fate decisions of immature hiPSC-CMs to either proliferate or terminally differentiate into more mature cardiomyocytes. Purpose: This work uncovers the embryonic growth pathways that control the cues for proliferation versus terminal differentiation in functional immature hiPSC-CMs. Methods: We utilised spontaneously beating hiPSC-CMs at day 11 of differentiation and performed a combinatory screen for various dosages of CHIR99021/Wnt and Insulin/Akt pathway. Proliferation was determined by quantification of cell number and the proportion of Ki67 positive hiPSC-CMs. Terminal differentiation was evaluated by presence of more mature sarcomere markers and cell cycle exit. Results: Our combinatory screen for Insulin/Akt and CHIR99021/Wnt demonstrates synergistic effects on proliferation of immature hiPSC-CMs, whereas the absence of these pathway activators leads to rapid cell cycle exit and terminal differentiation of hiPSC-CMs. Detailed characterization reveals that CHIR99021/Wnt also importantly regulates sarcomere homeostasis and architecture in hiPSC-CMs. Moreover, we identify a novel temporal interplay between CHIR99021/Wnt via TCF and Insulin/Akt via FOXO as regulators of cell-fate decision in immature hiPSC-CMs. Conclusions: Molecular targeting of the embryonic growth pathways in acute and chronic forms of heart failure requires a detailed understanding of the specific effects on cardiomyocyte function and sarcomere architecture. This work provides a starting point to develop future strategies to target acute and chronic forms of heart failure via modulation of embryonic growth pathways.

Mapping the chromatin accessibility landscape of zebrafish embryogenesis at single-cell resolution by SPATAC-seq.

Currently, the dynamic accessible elements that determine regulatory programs responsible for the unique identity and function of each cell type during zebrafish embryogenesis lack detailed study. Here we present SPATAC-seq: a split-pool ligation-based assay for transposase-accessible chromatin using sequencing. Using SPATAC-seq, we profiled chromatin accessibility in more than 800,000 individual nuclei across 20 developmental stages spanning the sphere stage to the early larval protruding mouth stage. Using this chromatin accessibility map, we identified 604 cell states and inferred their developmental relationships. We also identified 959,040 candidate cis-regulatory elements (cCREs) and delineated development-specific cCREs, as well as transcription factors defining diverse cell identities. Importantly, enhancer reporter assays confirmed that the majority of tested cCREs exhibited robust enhanced green fluorescent protein expression in restricted cell types or tissues. Finally, we explored gene regulatory programs that drive pigment and notochord cell differentiation. Our work provides a valuable open resource for exploring driver regulators of cell fate decisions in zebrafish embryogenesis.

Stage specific effects of insulin and Wnt on cell fate decisions in human induced pluripotent stem cell-derived cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation Background Reactivation of the so-called fetal gene programs and pathways have been reported in failing hearts, however, it remains unclear whether this phenomenon is beneficial or detrimental to the remaining functional myocardium. During cardiac development, the orchestration of these embryonic growth pathways, facilitate temporal and stage-specific cell fate decisions such as required for specification and growth of the various myocardial compartments. Previously, we demonstrated that molecular activation of the Wnt pathway resulted in massive expansion of immature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Yet, it is unknown what exact cues guide the cell fate decisions of immature hiPSC-CMs to either proliferate or terminally differentiate into more mature cardiomyocytes. Purpose This work uncovers the embryonic growth pathways that control the cues for proliferation versus terminal differentiation in functional immature hiPSC-CMs. Methods We utilised spontaneously beating hiPSC-CMs at day 11 of differentiation and performed a combinatory screen for various dosages of CHIR99021/Wnt and Insulin/Akt pathway. Proliferation was determined by quantification of cell number and the proportion of Ki67 positive hiPSC-CMs. Terminal differentiation was evaluated by presence of more mature sarcomere markers and cell cycle exit. Results Our combinatory screen for Insulin/Akt and CHIR99021/Wnt demonstrates synergistic effects on proliferation of immature hiPSC-CMs, whereas the absence of these pathway activators leads to rapid cell cycle exit and terminal differentiation of hiPSC-CMs. Detailed characterization reveals that CHIR99021/Wnt also importantly regulates sarcomere homeostasis and architecture in hiPSC-CMs. Moreover, we identify a novel temporal interplay between CHIR99021/Wnt via TCF and Insulin/Akt via FOXO as regulators of cell-fate decision in immature hiPSC-CMs. Conclusions Molecular targeting of the embryonic growth pathways in acute and chronic forms of heart failure requires a detailed understanding of the specific effects on cardiomyocyte function and sarcomere architecture. This work provides a starting point to develop future strategies to target acute and chronic forms of heart failure via modulation of embryonic growth pathways.Graphical Summary

Abstract 2077: The Epigenetic Regulator PRDM16 Controls Smooth Muscle Phenotypic Modulation And Atherosclerosis Risk

Despite decades of progress, coronary artery disease (CAD) remains the top cause of death worldwide. Additionally, trends in outcomes have worsened recently, highlighting the critical need for additional treatments. Human genetics has identified over 300 loci associated with CAD, but understanding the molecular mechanisms leading to disease remains a huge barrier to developing new therapies. These CAD-associated loci are enriched in smooth muscle cells (SMC) of the vascular wall, but no current therapies target these cells. Since insulin resistance is an important risk factor for CAD, understanding the molecular functions of genes associated with both insulin resistance and CAD will prioritize therapeutic candidates, especially if expressed in SMCs. One promising candidate is the gene PRDM16 (PR domain containing 16), which is highly expressed in vascular tissue. PRDM16 regulates cell fate decisions and insulin resistance in adipose tissue, but its role in atherosclerosis and SMC function is unknown. Recent advances in lineage tracing and conditional SMC knockout mouse models in conjunction with single cell technologies have demonstrated the cellular trajectories of SMC into several cellular states, including fibromyocytes (FMC) and chondromyocytes (CMC). Here, we employ this innovative approach to understand the role of Prdm16 on SMC phenotypic modulation in vivo and its role in the development of FMC and CMC. We also demonstrate the impact of Prdm16 in atherosclerosis and other important lesion characteristics relating to disease risk. We validate these effects in vitro and employ epigenetic analysis to identify the gene regulatory mechanisms whereby PRDM16 mediates its effects on SMC phenotypic modulation. Collectively these data demonstrate that PRDM16 is a causal factor that promotes risk of CAD.

Diverse functions of cytochrome c in cell death and disease.

The redox-active protein cytochrome c is a highly positively charged hemoglobin that regulates cell fate decisions of life and death. Under normal physiological conditions, cytochrome c is localized in the mitochondrial intermembrane space, and its distribution can extend to the cytosol, nucleus, and extracellular space under specific pathological or stress-induced conditions. In the mitochondria, cytochrome c acts as an electron carrier in the electron transport chain, facilitating adenosine triphosphate synthesis, regulating cardiolipin peroxidation, and influencing reactive oxygen species dynamics. Upon cellular stress, it can be released into the cytosol, where it interacts with apoptotic peptidase activator 1 (APAF1) to form the apoptosome, initiating caspase-dependent apoptotic cell death. Additionally, following exposure to pro-apoptotic compounds, cytochrome c contributes to the survival of drug-tolerant persister cells. When translocated to the nucleus, it can induce chromatin condensation and disrupt nucleosome assembly. Upon its release into the extracellular space, cytochrome c may act as an immune mediator during cell death processes, highlighting its multifaceted role in cellular biology. In this review, we explore the diverse structural and functional aspects of cytochrome c in physiological and pathological responses. We summarize how posttranslational modifications of cytochrome c (e.g., phosphorylation, acetylation, tyrosine nitration, and oxidation), binding proteins (e.g., HIGD1A, CHCHD2, ITPR1, and nucleophosmin), and mutations (e.g., G41S, Y48H, and A51V) affect its function. Furthermore, we provide an overview of the latest advanced technologies utilized for detecting cytochrome c, along with potential therapeutic approaches related to this protein. These strategies hold tremendous promise in personalized health care, presenting opportunities for targeted interventions in a wide range of conditions, including neurodegenerative disorders, cardiovascular diseases, and cancer.

The chromatin accessibility dynamics during cell fate specifications in zebrafish early embryogenesis.

Chromatin accessibility plays a critical role in the regulation of cell fate decisions. Although gene expression changes have been extensively profiled at the single-cell level during early embryogenesis, the dynamics of chromatin accessibility at cis-regulatory elements remain poorly studied. Here, we used a plate-based single-cell ATAC-seq method to profile the chromatin accessibility dynamics of over 10000 nuclei from zebrafish embryos. We investigated several important time points immediately after zygotic genome activation (ZGA), covering key developmental stages up to dome. The results revealed key chromatin signatures in the first cell fate specifications when cells start to differentiate into enveloping layer (EVL) and yolk syncytial layer (YSL) cells. Finally, we uncovered many potential cell-type specific enhancers and transcription factor motifs that are important for the cell fate specifications.

Chimeric PRMT6 protein produced by an endogenous retrovirus promoter regulates cell fate decision in mouse preimplantation embryos†.

Murine endogenous retrovirus with leucine tRNA primer, also known as MERVL, is expressed during zygotic genome activation in mammalian embryos. Here we show that protein arginine N-methyltransferase 6 (Prmt6) forms a chimeric transcript with MT2B2, one of the long terminal repeat sequences of murine endogenous retrovirus with leucine tRNA primer, and is translated into an elongated chimeric protein (PRMT6MT2B2) whose function differs from that of the canonical PRMT6 protein (PRMT6CAN) in mouse preimplantation embryos. Overexpression of PRMT6CAN in fibroblast cells increased asymmetric dimethylation of the third arginine residue of both histone H2A (H2AR3me2a) and histone H4 (H4R3me2a), while overexpression of PRMT6MT2B2 increased only H2AR3me2a. In addition, overexpression of PRMT6MT2B2 in one blastomere of mouse two-cell embryos promoted cell proliferation and differentiation of the blastomere into epiblast cells at the blastocyst stage, while overexpression of PRMT6CAN repressed cell proliferation. This is the first report of the translation of a chimeric protein (PRMT6MT2B2) in mouse preimplantation embryos. Our results suggest that analyzing chimeric transcripts with murine endogenous retrovirus with leucine tRNA primer will provide insight into the relationship between zygotic genome activation and subsequent intra- and extra-cellular lineage determination.

RNA-binding proteins in bone pathophysiology.

Bone remodelling is a highly regulated process that maintains mineral homeostasis and preserves bone integrity. During this process, intricate communication among all bone cells is required. Indeed, adapt to changing functional situations in the bone, the resorption activity of osteoclasts is tightly balanced with the bone formation activity of osteoblasts. Recent studies have reported that RNA Binding Proteins (RBPs) are involved in bone cell activity regulation. RBPs are critical effectors of gene expression and essential regulators of cell fate decision, due to their ability to bind and regulate the activity of cellular RNAs. Thus, a better understanding of these regulation mechanisms at molecular and cellular levels could generate new knowledge on the pathophysiologic conditions of bone. In this Review, we provide an overview of the basic properties and functions of selected RBPs, focusing on their physiological and pathological roles in the bone.

Deciphering driver regulators of cell fate decisions from single-cell transcriptomics data with CEFCON

Single-cell technologies enable the dynamic analyses of cell fate mapping. However, capturing the gene regulatory relationships and identifying the driver factors that control cell fate decisions are still challenging. We present CEFCON, a network-based framework that first uses a graph neural network with attention mechanism to infer a cell-lineage-specific gene regulatory network (GRN) from single-cell RNA-sequencing data, and then models cell fate dynamics through network control theory to identify driver regulators and the associated gene modules, revealing their critical biological processes related to cell states. Extensive benchmarking tests consistently demonstrated the superiority of CEFCON in GRN construction, driver regulator identification, and gene module identification over baseline methods. When applied to the mouse hematopoietic stem cell differentiation data, CEFCON successfully identified driver regulators for three developmental lineages, which offered useful insights into their differentiation from a network control perspective. Overall, CEFCON provides a valuable tool for studying the underlying mechanisms of cell fate decisions from single-cell RNA-seq data.

Elevated Id2 expression causes defective meiosis and spermatogenesis in mice.

Inhibitors of DNA binding (ID) proteins mainly inhibit gene expression and regulate cell fate decisions by interacting with E-proteins. All four ID proteins (ID1-4) are present in the testis, and ID4 has a particularly important role in spermatogonial stem cell fate determination. Several lines of evidence indicate that ID proteins are involved in meiosis; however, functional experiments have not been conducted to validate this observation. In this study, we report that ID2 is enriched in spermatocytes and that forced ID2 expression in germ cells causes defects in spermatogenesis. A detailed analysis demonstrated that Id2 overexpression (Id2 OE) decreased the total number of spermatogonia and changed the dynamics of meiosis progression. Specifically, spermatocytes were enriched in the zygotene stage, and the proportion of pachytene spermatocytes was significantly decreased, indicating defects in the zygotene-pachytene transition. The number of MLH1-positive foci per cell was decreased in pachytene spermatocytes from Id2 OE testes, suggesting abnormalities in recombination. Transcriptome analysis revealed that forced Id2 expression changed the expression of a list of genes mainly associated with meiosis and spermatid development. ID2 protein is expressed in spermatocytes, and its genetic ablation in the germline does not affect spermatogenesis, likely due to genetic compensation of its family members. However, forced Id2 expression changes meiosis progression and causes defects in spermiogenesis. These data provide important evidence that ID proteins play pivotal roles in male meiosis and spermatid development.

Pre-Existing Chromatin States Regulate KLF4 Binding to Eliminate Leukemia Stem Cells

Transcription factors collaborate with chromatin's epigenetic states to regulate cell fate decisions. While recent in vitro work using cryo-EM (Sinha et al., Nature, 2023) suggested that histone modifications could regulate the activity of pioneer transcription factors, it was not clear whether these findings applied in vivo, or if chromatin states could influence transcription factor binding to affect cell fate. Our previous work (Liu et al., Leukemia,2014; Wang et al., Nat Commun,2019) showed that reprogramming factors (Oct4, Sox2, Klf4, and cMyc, collectively known as OSKM) selectively eliminated leukemia cells in vivo, with minimal impact on normal Hematopoietic Stem and Progenitor Cells (HSPCs). This suggested that cell fates induced by OSKM could be influenced by pre-existing chromatin states. Building on this hypothesis, we profiled the pre-existing chromatin states of leukemia cells and HSPCs using MNase-ChIP-seq, ATAC-seq, and WGBS-seq. In our Tet-on-induced models, Klf4 activation resulted in the targeting of distinct gene sets and exhibited similar dynamic changes in chromatin accessibility, despite these regions sharing the same active chromatin state. Using machine learning algorithms, we identified H3K18ac –a feature of the active chromatin states preferred by Klf4– as a dominant factor influencing Klf4's genomic binding. We then altered H3K18ac through an enzymatically deficient SIRT7, which is an H3K18ac deacetylase, and examined where Klf4 binds. We observed an increase in Klf4 binding at sites where H3K18ac levels were elevated. This result suggested that Klf4 binding was influenced by changes in H3K18ac. We exposed human CD34+ cells, several leukemia cell lines, and samples from leukemia patients to ATPO-253. This small molecule, currently in phase 1 clinical trials, has been found to enhance the levels of the KLF4 protein. This treatment led to an increase in KLF4 protein levels and we observed activation of apoptosis genes in the tested leukemia cell lines that possessed high pre-existing H3K18ac levels. In addition, we observed a decline across all tested leukemia samples, while the population of CD34+ cells essentially remained stable. In conclusion, our work demonstrated that pre-existing chromatin states, such as H3K18ac, regulate Klf4's genomic binding, thereby influencing distinct cell fates in leukemia cells and HSPCs. This opens potential avenues for clinical applications in targeted leukemia cell clearance using KLF4 or ATPO-253.

Adaptation of RiPCA for the Live-Cell Detection of mRNA-Protein Interactions.

RNA-binding proteins (RBPs) act as essential regulators of cell fate decisions, through their ability to bind and regulate the activity of cellular RNAs. For protein-coding mRNAs, RBPs control the localization, stability, degradation, and ultimately translation of mRNAs to impact gene expression. Disruption of the vast network of mRNA-protein interactions has been implicated in many human diseases, and accordingly, targeting these interactions has surfaced as a new frontier in RNA-targeted drug discovery. To catalyze this new field, methods are needed to enable the detection and subsequent screening of mRNA-RBP interactions, particularly in live cells. Using our laboratory's RNA-interaction with Protein-mediated Complementation Assay (RiPCA) technology, herein we describe its application to mRNA-protein interactions and present a guide for the development of future RiPCA assays for structurally diverse classes of mRNA-protein interactions.

Hdac1 and Hdac2 positively regulate Notch1 gain-of-function pathogenic signaling in committed osteoblasts of male mice.

Skeletal development requires precise extrinsic and intrinsic signals to regulate processes that form and maintain bone and cartilage. Notch1 is a highly conserved signaling receptor that regulates cell fate decisions by controlling the duration of transcriptional bursts. Epigenetic molecular events reversibly modify DNA and histone tails by influencing the spatial organization of chromatin and can fine-tune the outcome of a Notch1 transcriptional response. Histone deacetylase 1 and 2 (HDAC1 and HDAC2) are chromatin modifying enzymes that mediate osteoblast differentiation. While an HDAC1-Notch interaction has been studied in vitro and in Drosophila, its role in mammalian skeletal development and disorders is unclear. Osteosclerosis is a bone disorder with an abnormal increase in the number of osteoblasts and excessive bone formation. Here, we tested whether Hdac1/2 contribute to the pathogenesis of osteosclerosis in a murine model of the disease owing to conditionally cre-activated expression of the Notch1 intracellular domain in immature osteoblasts. Importantly, selective homozygous deletions of Hdac1/2 in osteoblasts partially alleviate osteosclerotic phenotypes (Col2.3kb-Cre; TGRosaN1ICD/+ ; Hdac1flox/flox ; Hdac2flox/flox ) with a 40% decrease in bone volume and a 22% decrease in trabecular thickness in 4 weeks old when compared to male mice with heterozygous deletions of Hdac1/2 (Col2.3 kb-Cre; TGRosaN1ICD/+ ; Hdac1flox/+ ; Hdac2flox/+ ). Osteoblast-specific deletion of Hdac1/2 in male and female mice results in no overt bone phenotype in the absence of the Notch1 gain-of-function (GOF) allele. These results provide evidence that Hdac1/2 contribute to Notch1 pathogenic signaling in the mammalian skeleton. Our study on epigenetic regulation of Notch1 GOF-induced osteosclerosis may facilitate further mechanistic studies of skeletal birth defects caused by Notch-related GOF mutations in human patients, such as Adams-Oliver disease, congenital heart disease, and lateral meningocele syndrome.

Chromatin-modifying enzymes as modulators of nuclear size during lineage differentiation

The mechanism of nuclear size determination and alteration during normal lineage development and cancer pathologies which is not fully understood. As recently reported, chromatin modification can change nuclear morphology. Therefore, we screened a range of pharmacological chemical compounds that impact the activity of chromatin-modifying enzymes, in order to get a clue of the specific types of chromatin-modifying enzymes that remarkably effect nuclear size and shape. We found that interrupted activity of chromatin-modifying enzymes is associated with nuclear shape abnormalities. Furthermore, the activity of chromatin-modifying enzymes perturbs cell fate determination in cellular maintenance and lineage commitment. Our results indicated that chromatin-modifying enzyme regulates cell fate decision during lineage differentiation and is associate with nuclear size alteration.

Haspin balances the ratio of asymmetric cell division through Wnt5a and regulates cell fate decisions in mouse embryonic stem cells

Many different types of stem cells utilize asymmetric cell division (ACD) to produce two daughter cells with distinct fates. Haspin-catalyzed phosphorylation of histone H3 at Thr3 (H3T3ph) plays important roles during mitosis, including ACD in stem cells. However, whether and how Haspin functions in ACD regulation remains unclear. Here, we report that Haspin knockout (Haspin-KO) mouse embryonic stem cells (mESCs) had increased ratio of ACD, which cumulatively regulates cell fate decisions. Furthermore, Wnt5a is significantly downregulated due to decreased Pax2 in Haspin-KO mESCs. Wnt5a knockdown mESCs phenocopied Haspin-KO cells while overexpression of Wnt5a in Haspin-KO cells rescued disproportionated ACD. Collectively, Haspin is indispensable for mESCs to maintain a balanced ratio of ACD, which is essential for normal development and homeostasis.

FACT regulates pluripotency through proximal and distal regulation of gene expression in murine embryonic stem cells

BackgroundThe FACT complex is a conserved histone chaperone with critical roles in transcription and histone deposition. FACT is essential in pluripotent and cancer cells, but otherwise dispensable for most mammalian cell types. FACT deletion or inhibition can block induction of pluripotent stem cells, yet the mechanism through which FACT regulates cell fate decisions remains unclear.ResultsTo explore the mechanism for FACT function, we generated AID-tagged murine embryonic cell lines for FACT subunit SPT16 and paired depletion with nascent transcription and chromatin accessibility analyses. We also analyzed SPT16 occupancy using CUT&RUN and found that SPT16 localizes to both promoter and enhancer elements, with a strong overlap in binding with OCT4, SOX2, and NANOG. Over a timecourse of SPT16 depletion, nucleosomes invade new loci, including promoters, regions bound by SPT16, OCT4, SOX2, and NANOG, and TSS-distal DNaseI hypersensitive sites. Simultaneously, transcription of Pou5f1 (encoding OCT4), Sox2, Nanog, and enhancer RNAs produced from these genes’ associated enhancers are downregulated.ConclusionsWe propose that FACT maintains cellular pluripotency through a precise nucleosome-based regulatory mechanism for appropriate expression of both coding and non-coding transcripts associated with pluripotency.