Expression Of Lineage Genes Research Articles

Myelination potential and injury susceptibility of grey versus white matter human oligodendrocytes.

Increasing evidence indicates heterogeneity in functional and molecular properties of oligodendrocyte lineage cells both during development and under pathologic conditions. In multiple sclerosis, remyelination of grey matter lesions exceeds that in white matter. Here we used cells derived from grey matter versus white matter regions of surgically resected human brain tissue samples, to compare the capacities of human A2B5-positive progenitor cells and mature oligodendrocytes to ensheath synthetic nanofibers, and relate differences to the molecular profiles of these cells. For both cell types, the percentage of ensheathing cells was greater for grey matter versus white matter cells. For both grey matter and white matter samples, the percentage of cells ensheathing nanofibers was greater for A2B5-positive cells versus mature oligodendrocytes. Grey matter A2B5-positive cells were more susceptible than white matter A2B5-positive cells to injury induced by metabolic insults. Bulk RNA sequencing indicated that separation by cell type (A2B5-positive vs mature oligodendrocytes) is more significant than by region but segregation for each cell type by region is apparent. Molecular features of grey matter versus white matter derived A2B5-positive and mature oligodendrocytes were lower expression of mature oligodendrocyte genes and increased expression of early oligodendrocyte lineage genes. Genes and pathways with increased expression in grey matter derived cells with relevance for myelination included those related to responses to external environment, cell-cell communication, cell migration, and cell adhesion. Immune and cell death related genes were up-regulated in grey matter derived cells. We observed a significant number of up-regulated genes shared between the stress/injury and myelination processes, providing a basis for these features. In contrast to oligodendrocyte lineage cells, no functional or molecular heterogeneity was detected in microglia maintained in vitro, likely reflecting the plasticity of these cells ex vivo. The combined functional and molecular data indicate that grey matter human oligodendrocytes have increased intrinsic capacity to myelinate but also increased injury susceptibility, in part reflecting their being at a stage earlier in the oligodendrocyte lineage.

Distinct dynamics of parental 5-hydroxymethylcytosine during human preimplantation development regulate early lineage gene expression

The conversion of DNA 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by TET enzymes represents a significant epigenetic modification, yet its role in early human embryos remains largely unknown. Here we showed that the early human embryo inherited a significant amount of 5hmCs from an oocyte, which unexpectedly underwent de novo hydroxymethylation during its growth. Furthermore, the generation of 5hmC in the paternal genome after fertilization roughly followed the maternal pattern, which was linked to DNA methylation dynamics and regions of sustained methylation. The 5hmCs persisted until the eight-cell stage and exhibited high enrichment at OTX2 binding sites, whereas knockdown of OTX2 in human embryos compromised the expression of early lineage genes. Specifically, the depletion of 5hmC affected the activation of embryonic genes, which was further evaluated by ectopically expressing mouse Tet3 in human early embryos. These findings revealed distinct dynamics of 5hmC and unravelled its multifaceted functions in early human embryonic development.

A Retinoic Acid:YAP1 signaling axis controls atrial lineage commitment.

Vitamin A/Retinoic Acid (Vit A/RA) signaling is essential for heart development. In cardiac progenitor cells (CPCs), RA signaling induces the expression of atrial lineage genes while repressing ventricular genes, thereby promoting the acquisition of an atrial cardiomyocyte cell fate. To achieve this, RA coordinates a complex regulatory network of downstream effectors that is not fully identified. To address this gap, we applied a functional genomics approach (i.e scRNAseq and snATACseq) to untreated and RA-treated human embryonic stem cells (hESCs)-derived CPCs. Unbiased analysis revealed that the Hippo effectors YAP1 and TEAD4 are integrated with the atrial transcription factor enhancer network, and that YAP1 is necessary for activation of RA-enhancers in CPCs. Furthermore, in vivo analysis of control and conditionally YAP1 KO mouse embryos (Sox2-cre) revealed that the expression of atrial lineage genes, such as NR2F2, is compromised by YAP1 deletion in the CPCs of the second heart field. Accordingly, we found that YAP1 is required for the formation of an atrial chamber but is dispensable for the formation of a ventricle, in hESC-derived patterned cardiac organoids. Overall, our findings revealed that YAP1 is a non-canonical effector of RA signaling essential for the acquisition of atrial lineages during cardiogenesis.

Epidermal growth factor receptor regulates lineage plasticity driving transformation to small cell lung cancer

Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) is clinically and genetically heterogeneous, with concurrent RB1/TP53 mutations, indicating an increased risk of transformation into small cell lung cancer (SCLC). When tumor cells convert into a different histological subtype, they lose their dependence on the original oncogenic driver, resulting in therapeutic resistance. However, the molecular details associated with this transformation remain unclear. It has been difficult to define molecular mechanisms of neuroendocrine (NE) transformation in lung cancer due to a lack of pre- and post-transformation clinical samples. In this study, we established a NSCLC cell line with concurrent RB1/TP53 mutations and built corresponding patient-derived xenograft (PDX) models to investigate the mechanisms underlying transformation to SCLC. Studying these PDX models, we demonstrate that EGFR loss facilitates lineage plasticity of lung adenocarcinoma initiated by biallelic mutations of TP53 and RB1. Gene expression analysis of these EGFR knockout tumors revealed altered expression of neuroendocrine synapse-associated lineage genes. There is an increased expression of epigenetic reprogramming factors like Sox2 and gene associated with neural development like NTRK in these EGFR knockout tumors. These findings uncovered the role of EGFR in the acquisition of plasticity, which is the ability of a cell to substantially modify its identity and take on a new phenotype, and defined a novel landscape of potential drivers of NE transformation in lung cancer.

Engineered neurogenesis in naïve adult rat cortex by Ngn2-mediated neuronal reprogramming of resident oligodendrocyte progenitor cells.

Adult tissue stem cells contribute to tissue homeostasis and repair but the long-lived neurons in the human adult cerebral cortex are not replaced, despite evidence for a limited regenerative response. However, the adult cortex contains a population of proliferating oligodendrocyte progenitor cells (OPCs). We examined the capacity of rat cortical OPCs to be re-specified to a neuronal lineage both in vitro and in vivo. Expressing the developmental transcription factor Neurogenin2 (Ngn2) in OPCs isolated from adult rat cortex resulted in their expression of early neuronal lineage markers and genes while downregulating expression of OPC markers and genes. Ngn2 induced progression through a neuronal lineage to express mature neuronal markers and functional activity as glutamatergic neurons. In vivo retroviral gene delivery of Ngn2 to naive adult rat cortex ensured restricted targeting to proliferating OPCs. Ngn2 expression in OPCs resulted in their lineage re-specification and transition through an immature neuronal morphology into mature pyramidal cortical neurons with spiny dendrites, axons, synaptic contacts, and subtype specification matching local cytoarchitecture. Lineage re-specification of rat cortical OPCs occurred without prior injury, demonstrating these glial progenitor cells need not be put into a reactive state to achieve lineage reprogramming. These results show it may be feasible to precisely engineer additional neurons directly in adult cerebral cortex for experimental study or potentially for therapeutic use to modify dysfunctional or damaged circuitry.

Comparison Between Cancellous Trabecular and Cortical Specimens from Human Lumbar Spine Samples as an Alternative Source of Mesenchymal Stromal Cells.

Due to their immunosuppressive potential and ability to differentiate into multiple musculoskeletal cell lineages, mesenchymal stromal cells (MSCs) became popular in clinical trials for the treatment of musculoskeletal disorders. The aim of this study was to isolate and characterize native populations of MSCs from human cortical and cancellous bone from the posterior elements of the lumbar spine and determine what source of MSCs yields better quality and quantity of cells to be potentially used for spinal fusion repair. We were able to show that MSCs from trabecular and cortical spine had the typical MSC morphology and expression markers; the ability to differentiate in adipocyte, chondrocyte, or osteoblast but they did not have a consistent pattern in the expression of the specific differentiation lineage genes. Moreover, MSCs from both sites demonstrated an immune suppression profile suggesting that these cells may have a more promising success in applications related to immunomodulation more than exploring their ability to drive osteogenesis to prevent nonunion in spine fusion procedures.

Generation and integration of hair follicle-primed spheroids in bioengineered skin constructs

Skin is a complex organ made up of different cell layers, appendages, connective tissues, and immune repertoires. These different components interact extensively to maintain the overall functions of the integumentary system. In particular, appendages such as hair follicles critically contribute to the skin’s function in thermoregulation, sensory perception, and homeostatic regeneration. Despite a strong need for better skin regenerative therapeutics, efforts to bio-engineer highly functional appendage-containing human reconstituted skin in vitro have not yielded much success. Here, we report methods in generating and incorporating hair follicle-primed heterotypic spheroids into epidermal-dermal skin constructs that induced invaginating outgrowths with follicle-like organization and lineage gene expression. By co-culturing epithelial keratinocytes (KCs) with dermal papilla (DP) cells in low attachment plates, we established the media and culture conditions that best supported the viability, signalling and remodelling of the cell aggregates to form 3D KC-DP spheroids with the expression of both DP inductiveness and hair follicle lineage genes. We show that long-term growth and maturation of KC cells in these spheroids was supported by incorporation into epidermal-dermal constructs but not in scaffold-less media. When cultured, the bio-fabricated constructs developed invaginations from the integrated spheroids with follicle-forming potential. The generation of these constructs is a step towards the development of functional hair-bearing skin mimetics.

PTPRU, a quiescence-induced receptor tyrosine phosphatase negatively regulates osteogenic differentiation of human mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are heterogeneous osteo-progenitors that are mainly responsible for bone regeneration and homeostasis. In vivo, a subpopulation of bone marrow MSCs persists in a quiescent state, providing a source of new cells for repair. Previously, we reported that induction of quiescence in hMSCs in vitro skews their differentiation potential in favour of osteogenesis while suppressing adipogenesis. Herein, we uncover a new role for a protein tyrosine phosphatase, receptor type U (PTPRU) in repressing osteogenesis during quiescence. A 75 kD PTPRU protein isoform was found to be specifically induced during quiescence and down-regulated during cell cycle reactivation. Using siRNA-mediated knockdown, we report that in proliferating hMSC, PTPRU preserves self-renewal, while in quiescent hMSC, PTPRU not only maintains reversibility of cell cycle arrest but also suppresses expression of osteogenic lineage genes. Knockdown of PTPRU in proliferating or quiescent hMSC de-represses osteogenic markers, and enhances induced osteogenic differentiation. We also show that PTPRU positively regulates a β-catenin-TCF transcriptional reporter. Taken together, our study suggests a role for a quiescence-induced 75kD PTPRU isoform in modulating bone differentiation in hMSC, potentially involving the Wnt pathway.

DNA promoter hypermethylation of melanocyte lineage genes determines melanoma phenotype.

Cellular stress contributes to the capacity of melanoma cells to undergo phenotype switching into highly migratory and drug-tolerant dedifferentiated states. Such dedifferentiated melanoma cell states are marked by loss of melanocyte-specific gene expression and increase of mesenchymal markers. Two crucial transcription factors, microphthalmia-associated transcription factor (MITF) and SRY-box transcription factor 10 (SOX10), important in melanoma development and progression, have been implicated in this process. In this study we describe that loss of MITF is associated with a distinct transcriptional program, MITF promoter hypermethylation, and poor patient survival in metastatic melanoma. From a comprehensive collection of melanoma cell lines, we observed that MITF-methylated cultures were subdivided in 2 distinct subtypes. Examining mRNA levels of neural crest-associated genes, we found that 1 subtype had lost the expression of several lineage genes, including SOX10. Intriguingly, SOX10 loss was associated with SOX10 gene promoter hypermethylation and distinct phenotypic and metastatic properties. Depletion of SOX10 in MITF-methylated melanoma cells using CRISPR/Cas9 supported these findings. In conclusion, this study describes the significance of melanoma state and the underlying functional properties explaining the aggressiveness of such states.

The human amniotic epithelium confers a bias to differentiate toward the neuroectoderm lineage in human embryonic stem cells.

Human embryonic stem cells (hESCs) derive from the epiblast and have pluripotent potential. To maintain the conventional conditions of the pluripotent potential in an undifferentiated state, inactivated mouse embryonic fibroblast (iMEF) is used as a feeder layer. However, it has been suggested that hESC under this conventional condition (hESC-iMEF) is an artifact that does not correspond to the in vitro counterpart of the human epiblast. Our previous studies demonstrated the use of an alternative feeder layer of human amniotic epithelial cells (hAECs) to derive and maintain hESC. We wondered if the hESC-hAEC culture could represent a different pluripotent stage than that of naïve or primed conventional conditions, simulating the stage in which the amniotic epithelium derives from the epiblast during peri-implantation. Like the conventional primed hESC-iMEF, hESC-hAEC has the same levels of expression as the 'pluripotency core' and does not express markers of naïve pluripotency. However, it presents a downregulation of HOX genes and genes associated with the endoderm and mesoderm, and it exhibits an increase in the expression of ectoderm lineage genes, specifically in the anterior neuroectoderm. Transcriptome analysis showed in hESC-hAEC an upregulated signature of genes coding for transcription factors involved in neural induction and forebrain development, and the ability to differentiate into a neural lineage was superior in comparison with conventional hESC-iMEF. We propose that the interaction of hESC with hAEC confers hESC a biased potential that resembles the anteriorized epiblast, which is predisposed to form the neural ectoderm.

Nuclear GSK-3β and Oncogenic KRas Lead to the Retention of Pancreatic Ductal Progenitor Cells Phenotypically Similar to Those Seen in IPMN.

Glycogen synthase kinase-3β (GSK-3β) is a downstream target of oncogenic KRas and can accumulate in the nucleus in pancreatic ductal adenocarcinoma (PDA). To determine the interplay between oncogenic KRas and nuclear GSK-3β in PDA development, we generated Lox-STOP-Lox (LSL) nuclear-targeted GSK-3β animals and crossed them with LSL-KRasG12D mice under the control of the Pdx1-cre transgene—referred to as KNGC. Interestingly, 4-week-old KNGC animals show a profound loss of acinar cells, the expansion of ductal cells, and the rapid development of cystic-like lesions reminiscent of intraductal papillary mucinous neoplasm (IPMN). RNA-sequencing identified the expression of several ductal cell lineage genes including AQP5. Significantly, the Aqp5+ ductal cell pool was proliferative, phenotypically distinct from quiescent pancreatic ductal cells, and deletion of AQP5 limited expansion of the ductal pool. Aqp5 is also highly expressed in human IPMN along with GSK-3β highlighting the putative role of Aqp5+ ductal cells in human preneoplastic lesion development. Altogether, these data identify nGSK-3β and KRasG12D as an important signaling node promoting the retention of pancreatic ductal progenitor cells, which could be used to further characterize pancreatic ductal development as well as lineage biomarkers related to IPMN and PDA.

PEA-OXA ameliorates allodynia, neuropsychiatric and adipose tissue remodeling induced by social isolation

Chronic social isolation generates a persistent state of stress associated with obesity along with some neuro-endocrine disorders and central behavioral sequelae (eg anxiety, depression, aggression, and allodynia). In this study, we evaluated the effect of social isolation on body weight, depressive- and anxious-aggressive-like behavior, as well as on phenotypic changes of adipocytes from visceral adipose tissue of control (group-housed) or socially isolated (single-housed) male mice. The effect of treatment with pentadecyl-2-oxazoline (PEA-OXA), a natural alpha2 antagonist and histamine H3 protean partial agonist, on these alterations was also evaluated. Single or group-housed mice treated with vehicle or PEA-OXA underwent body weight, mechanical allodynia, anxious-, depressive- and aggressive-like behavior measurements. Proliferation rate, apoptosis, senescence, expression of fat lineage genes, lipid droplets and proinflammatory cytokines were measured on white adipose tissue adipocytes from group- or single-housed mice. Single housed mice developed weight gain, mechanical allodynia at the von Frey test, aggressiveness in the resident intruder test, depression- and anxiety-like behavior in the tail suspension and hole drop tests, respectively. Single housed mice receiving PEA-OXA showed a general resolution of both, physical-metabolic and behavioral alterations associated with social isolation. Furthermore, adipocytes from the adipose tissue of socially isolated mice showed an evident inflamed phenotype (i.e. a reduced rate of proliferation, apoptosis, senescence, and ROS hyper-production together with an increased expression of IL-1β, IL-10, IL-17, and TNF-α and a decrease of IL-6). The treatment with PEA-OXA on adipocytes from single housed mice produced a protective/anti-inflammatory phenotype with an increased expression of brown adipose tissue biomarker. This study confirms that persistent stress caused by social isolation predisposes to obesity and neuropsychiatric disorders. PEA-OXA, through its multi-target activity on alpha2 adrenoceptor and histamine H3 receptors, which have recently aroused great interest in the neuropsychiatric field, reduces weight gain, systemic pro-inflammatory state, allodynia, and affective disorders associated with social isolation.

HIRA stabilizes skeletal muscle lineage identity

The epigenetic mechanisms coordinating the maintenance of adult cellular lineages and the inhibition of alternative cell fates remain poorly understood. Here we show that targeted ablation of the histone chaperone HIRA in myogenic cells leads to extensive transcriptional modifications, consistent with a role in maintaining skeletal muscle cellular identity. We demonstrate that conditional ablation of HIRA in muscle stem cells of adult mice compromises their capacity to regenerate and self-renew, leading to tissue repair failure. Chromatin analysis of Hira-deficient cells show a significant reduction of histone variant H3.3 deposition and H3K27ac modification at regulatory regions of muscle genes. Additionally, we find that genes from alternative lineages are ectopically expressed in Hira-mutant cells via MLL1/MLL2-mediated increase of H3K4me3 mark at silent promoter regions. Therefore, we conclude that HIRA sustains the chromatin landscape governing muscle cell lineage identity via incorporation of H3.3 at muscle gene regulatory regions, while preventing the expression of alternative lineage genes.

Tumor cell heterogeneity and its transcriptional bases in pancreatic cancer: a tale of two cell types and their many variants.

Pancreatic ductal adenocarcinoma (PDAC), one of the most highly lethal tumors, is characterized by complex histology, with a massive fibrotic stroma in which both pseudo-glandular structures and compact nests of abnormally differentiated tumor cells are embedded, in different proportions and with different mutual relationships in space. This complexity and the heterogeneity of the tumor component have hindered the development of a broadly accepted, clinically actionable classification of PDACs, either on a morphological or a molecular basis. Here, we discuss evidence suggesting that such heterogeneity can to a large extent, albeit not exclusively, be traced back to two main classes of PDAC cells that commonly coexist in the same tumor: cells that maintained their ability to differentiate toward endodermal, mucin-producing epithelia and epithelial cells unable to form glandular structures and instead characterized by various levels of squamous differentiation and the expression of mesenchymal lineage genes. The underlying gene regulatory networks and how they are controlled by distinct transcription factors, as well as the practical implications of these two different populations of tumor cells, are discussed.

Single Cell Transcriptome Mapping of Human Thymopoiesis Reveals a Continuum of Cell States during T-Lineage Specification and Commitment

The initial stages of thymopoiesis are characterized by the induction of T-lineage genes (specification) and the repression of alternative lineage genes (commitment). Multilineage (Thy1 and Thy2) and committed (Thy3) populations representing successive differentiation stages have been defined within CD34+ progenitor cells in the human thymus by the expression of CD7 and CD1a. However, due to intra-population heterogeneity and unstudied transitional states between populations, our understanding of the transcriptional programs that launch human thymopoiesis remains incomplete. Also, the interpretation of species specific gene expression profiles seen in these populations is confounded by species related differences in progenitor immunophenotypes. To our knowledge, whole transcriptome profiles of human thymic CD34+ progenitors at single cell resolution have not been reported. To resolve the transcriptional landscape of the initial stages of thymopoiesis, we performed single cell RNA-Seq (sRNA-Seq) of FACS sorted unfractionated human thymic CD34+CD4-CD8- cells from 3 donors using 10X (1 thymus, 5000 cells), Indrop (1 thymus, 2000 cells), or BD genomics (1 thymus, 192 index sorted cells) sequencing Averaged sRNA-Seq expression profiles recapitulated previous bulk RNASeq data validating our sRNA-Seq pipeline. Transcriptomes of index sorted cells mapped appropriately to those predicted to be Thy1,Thy2, or Thy3 cells from CD34, CD7, and CD1a RNA levels in the 10x and indrops data indicating the validity of identifying Thy1-3 cells by RNA levels. Seurat clustering showed that CD34+ thymocytes consist of a continuum of cell states rather than discrete populations. Nine cell states marked by gradual changes in CD2, CD44, CD7, CD1A, and CD34 RNA levels were seen: state 1 (Thy1), states 2-6 (Thy2), and states 7-9 (Thy3). Thy2 and Thy3 cells spanned several previously undescribed states with distinct expression profiles. Single cells co-expressing stem and T-lineage genes were seen in state 1 indicating an early onset of T-lineage priming that occurs prior to downregulation of stem cell genes. Within Thy2 cells, high expression of stem and alternative lineage genes was mostly restricted to states 2 and 3, which represent novel Thy2 subpopulations. State 2 cells co-expressed T-cell and high levels of innate immune genes, particularly IRF8. State 3 cells showed low CD2 expression and co-expressed T-cell and stem, myeloid, B-cell, and/ or NK genes. These results indicate concomitant priming of multiple lineage transcriptional programs rather than just the presence of unilineage gene program expressing subpopulations in the earliest thymic progenitor cells. In preliminary experiments, FACS sorted CD2 low Thy2 cells generated T-cells as well as a higher output of alternative lineage cells than CD2 high Thy2 cells. States 7-9 showed committed profiles with upregulated T-cell genes and silenced stem and alternative lineage genes. While state 9 (late Thy3) showed high RAG1 expression and cell cycle arrest, most Thy3 cells (states 7 and 8) were cycling indicating that commitment, which precedes T-cell receptor rearrangement, is not associated with proliferation arrest. Monocle pseudotime analysis finely resolved expression profiles along the differentiation trajectory within Thy1-3 populations: e.g. genes repressed earlier (e.g. MEIS1) vs later (e.g. DTX1). BCL11B, a key T-cell commitment gene was one of the earliest transcription factors (TF) to be induced during human thymopoiesis, and upregulation of TCF7 and BCL11B occurred concurrently. In contrast, in mice induction of Bcl11b occurs later after Tcf7 is fully upregulated. Thus, the initial stages of thymopoiesis are characterized by a continuum of multilineage transcriptional priming followed by a gradual transition to a T-lineage restricted gene program, a process similar to that recently described for lineage commitment of bone marrow hematopoietic progenitors but not previously reported in the thymus. SRNA-Seq yielded expression profiles along numerous data-points on the differentiation trajectory within heterogenous populations, providing high transcriptional resolution not possible with bulk data. We found differences in key TF expression profiles that could imply species specific regulatory mechanisms. Our data are a resource for elucidating transcriptional networks in T-cell development and T-cell leukemias. Disclosures No relevant conflicts of interest to declare.

Rho-mediated signaling promotes BRAF inhibitor resistance in de-differentiated melanoma cells.

Over half of cutaneous melanoma tumors have BRAFV600E/K mutations. Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses. In this study we demonstrate that approximately 50–60% of melanoma cell lines with vemurafenib resistance acquired in vitro show activation of RhoA family GTPases. In BRAFi-resistant melanoma cell lines and tumors, activation of RhoA is correlated with decreased expression of melanocyte lineage genes. Using a machine learning approach, we built gene expression-based models to predict drug sensitivity for 265 common anti-cancer compounds. We then projected these signatures onto the collection of TCGA cutaneous melanoma and found that poorly differentiated tumors were predicted to have increased sensitivity to multiple Rho kinase (ROCK) inhibitors. Two transcriptional effectors downstream of Rho, MRTF and YAP1, are activated in the RhoHigh BRAFi-resistant cell lines, and resistant cells are more sensitive to inhibition of these transcriptional mechanisms. Taken together, these results support the concept of targeting Rho-regulated gene transcription pathways as a promising therapeutic approach to restore sensitivity to BRAFi-resistant tumors or as a combination therapy to prevent the onset of drug resistance.

The Effect of the Repression of Oxidative Stress on Tenocyte Differentiation: A Preliminary Study of a Rat Cell Model Using a Novel Differential Tensile Strain Bioreactor.

Because of limitations in the current understanding of the exact pathogenesis of tendinopathy, and the lack of an optimal experimental model, effective therapy for the disease is currently unavailable. This study aims to prove that repression of oxidative stress modulates the differentiation of tendon-derived cells (TDCs) sustaining excessive tensile strains, and proposes a novel bioreactor capable of applying differential tensile strains to cultured cells simultaneously. TDCs, including tendon-derived stem cells, tenoblasts, tenocytes, and fibroblasts, were isolated from the patellar tendons of Sprague‒Dawley rats. Cyclic uniaxial stretching with 4% or 8% strain at 0.5 Hz for 8 h was applied to TDCs. TDCs subjected to 8% strain were treated with epigallocatechin gallate (EGCG), piracetam, or no medication. Genes representing non-tenocyte lineage (Pparg, Sox9, and Runx2) and type I and type III collagen were analyzed by quantitative polymerase chain reaction. The 8% strain group showed increased expression of non-tenocyte lineage genes and type III/type I collagen ratios compared with the control and 4% strain groups, and the increased expression was ameliorated with addition of EGCG and piracetam. The model developed in this work could be applied to future research on the pathophysiology of tendinopathy and development of treatment options for the disease. Repression of oxidative stress diminishes the expression of genes indicating aberrant differentiation in a rat cell model, which indicates potential therapeutic intervention of tendinopathy, the often relentlessly degenerate condition.

Abstract 5294: The PanNET-related histone H3.3 chaperone Daxx regulates lineage specification and tissue homeostasis in the pancreas

Abstract Progressive age-dependent replacement of canonical H3.1/2 with H3.3 in post-mitotic cells is a crucial mechanism for maintaining genome integrity at heterochromatic sites. The histone chaperone DAXX cooperates with the chromatin remodeller ATRX for the deposition of the non-canonical histone H3.3 at heterochromatin, while it prevents the expression of endogenous retroviral elements in another complex involving the SETDB1 methyltransferases. Loss of function mutations affecting ATRX/DAXX and MEN-1 have been recently identified as a subgroup of pancreatic neuroendocrine tumours (PanNETs) leading to poor prognosis and more aggressive phenotype compared to the ATRX/DAXX and MEN-1 wild-type counterparts. We developed a mouse model for conditional Daxx inactivation in mouse islets to understand the impact of DAXX loss on pancreas homeostasis and PanNETs development. We found that loss of Daxx in the endocrine pancreas, using the Insulin-1Cre driver, leads to aberrant glucose metabolism with the development of hyperglycemia and consequently insulin resistance in mice. We have also shown that loss of DAXX is linked to increased expression of α-cell lineage genes upon glucose stimulation of isolated islets in vitro, resembling the recently reported phenotype of PanNETs harboring ATRX/DAXX or MEN1 mutations. Nonetheless, loss of Daxx was not sufficient to lead to PanNET development. We are currently investigating the chromatin and transcriptional perturbations caused by Daxx loss and how these could be linked to the aberration of cell lineage specification and tissue dysfunction in the pancreas. More generally, this work will provide new insight into how epigenetic alterations contribute to misregulation of developmental programmes and cancer development in the pancreas. Citation Format: Teresa Sposito, Marie-Sophie Nguyen Tu, Guy A. Rutter, Marco Novelli, Chrissie Thirlwell, Paolo Salomoni. The PanNET-related histone H3.3 chaperone Daxx regulates lineage specification and tissue homeostasis in the pancreas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5294.

Gene duplication led to divergence of expression patterns, protein-protein interaction patterns and floral development functions of AGL6-like genes in the basal angiosperm Magnolia wufengensis (Magnoliaceae).

The MADS-box family genes play critical roles in the regulation of growth and development of flowering plants. AGAMOUS-LIKE 6 (AGL6)-like genes are one of the most enigmatic subfamilies of the MADS-box family because of highly variable expression patterns and ambiguous functions, which have long puzzled researchers. A lot of AGL6 homologs have been identified from gymnosperms and angiosperms. However, only a few have been characterized, especially for basal angiosperm taxa. Magnolia wufengensis is a woody basal angiosperm from the family Magnoliaceae. In the current study, the phylogenesis, expression and protein-protein interaction (PPI) patterns, and functions of two AGL6 homologs from M. wufengensis, MawuAGL6-1 and MawuAGL6-2, were analyzed. Phylogenetic analysis indicated that the two AGL6 duplicates may have arisen by gene duplication before the divergence of Magnoliaceae and Lauraceae, with the diversification of their expression and PPI patterns after gene duplication. Functional analysis revealed that, in addition to common functions in accelerating flowering, MawuAGL6-1 might be responsible for flower meristem determinacy, while MawuAGL6-2 is preferentially recruited to regulate tepal morphogenesis. These findings further advance our understanding of the evolution of phylogenesis, expression, interaction and functions of AGL6 lineage genes from basal angiosperms, as well as the entire AGL6 lineage genes, and the significance of AGL6 lineage genes in the evolution and biological diversity.

The Transcription Factor ERG Regulates Super-Enhancers Associated With an Endothelial-Specific Gene Expression Program.

The ETS (E-26 transformation-specific) transcription factor ERG (ETS-related gene) is essential for endothelial homeostasis, driving expression of lineage genes and repressing proinflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG's homeostatic function is lineage-specific, because aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. Chromatin immunoprecipitation with high-throughput sequencing in human umbilical vein endothelial cells showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4 (Delta-like protein 4), CLDN5 (claudin-5), VWF (von Willebrand factor), and CDH5 (VE-cadherin). Comparison between human umbilical vein endothelial cell and prostate cancer TMPRSS2 (transmembrane protease, serine-2):ERG fusion-positive human prostate epithelial cancer cell line (VCaP) cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and MED (Mediator complex subunit)-1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 (GATA-binding protein 2) and AP-1 (activator protein 1) is significantly lower compared with super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in endothelial cells and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms from genome-wide association studies lie within noncoding regions and perturb transcription factor recognition sequences in relevant cell types. Analysis of genome-wide association studies data shows significant enrichment of risk variants for cardiovascular disease and other diseases, at ERG endothelial enhancers and super-enhancers. The transcription factor ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of cardiovascular disease-associated single nucleotide polymorphisms at ERG super-enhancers suggests that ERG-dependent transcription modulates disease risk.