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Fig. S1. Supplementary data for large scale genomic aberrations illustrated in Figure 2, including: A) the distribution of mapped read density of WGS; B) PCR confirmation of translocation and fusion junctions of a superamplicon; and C) PCR and RT-PCR amplification of MGAM, ZFAND3, and the ZFAND3-MGAM fusion gene. Fig. S2. The distribution of mapped read density of WES, supplementary data for Figure 3. Fig. S3. Differentially expressed genes and functions among complex, simple carcinomas and normal mammary glands, supplementary data for Figure 3. Fig. S4. IHC images of histone modifications in canine normal mammary glands at 400X magnification, supplementary data for Figure 4. Fig. S5. IHC images of histone modifications in simple carcinomas, complex carcinomas and normal mammary glands at 200X magnification, supplementary data for Figure 5. Fig. S6. Supplementary data for homology between canine MCs and human breast cancers described in Figure 6. These include differentially expressed genes between ER+ and ER- carcinomas at FDR ≤ 0.1 and an example of PAM50 clustering indicating canine and human tumor IDs.

Replication timing organizes epigenome The temporal order of DNA replication is conserved from yeast to humans, but its biological significance remains unclear. Klein et al. eliminated the protein RIF1, a master regulator of replication timing, in several human cell lines. RIF1 loss during the G1 phase of the cell cycle resulted in a heterogeneous, nearly random replication timing program from the first S phase that persisted even in stable RIF1-null clones. Altered replication timing was followed by replication-dependent redistribution of active and repressive histone modifications and alterations in genome architecture. These results support a model in which replication timing orchestrates the epigenetic state of newly replicated chromatin. Science , this issue p. 371

Background: Alopecia Areata is a form of relapsing nonscarring hair loss, which is a poorly understood autoimmune phenomenon characterized by T-cell inflammation as a result of environmental and ge...

Physical interactions between distal regulatory elements have a key role in regulating gene expression, but the extent to which these interactions vary between cell types and contribute to cell-type-specific gene expression remains unclear. Here, to address these questions as part of phase III of the Encyclopedia of DNA Elements (ENCODE), we mapped cohesin-mediated chromatin loops, using chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), and analysed gene expression in 24 diverse human cell types, including core ENCODE cell lines. Twenty-eight per cent of all chromatin loops vary across cell types; these variations modestly correlate with changes in gene expression and are effective at grouping cell types according to their tissue of origin. The connectivity of genes corresponds to different functional classes, with housekeeping genes having few contacts, and dosage-sensitive genes being more connected to enhancer elements. This atlas of chromatin loops complements the diverse maps of regulatory architecture that comprise the ENCODE Encyclopedia, and will help to support emerging analyses of genome structure and function., A map of cohesin-mediated chromatin loops in 24 types of human cells identifies loops that show cell-type-specific variation, indicating that chromatin loops may help to specify cell-specific gene expression programs and functions.

Human beige adipocytes (BAs) have potential utility for the development of therapeutics to treat diabetes and obesity-associated diseases. Although several reports have described the generation of beige adipocytes in vitro, their potential utility in cell therapy and drug discovery has not been reported. Here, we describe the generation of BAs from human adipose-derived stem/stromal cells (ADSCs) in serum-free medium with efficiencies >90%. Molecular profiling of beige adipocytes shows them to be similar to primary BAs isolated from human tissue. In vitro, beige adipocytes exhibit uncoupled mitochondrial respiration and cAMP-induced lipolytic activity. Following transplantation, BAs increase whole-body energy expenditure and oxygen consumption, while reducing body-weight in recipient mice. Finally, we show the therapeutic utility of BAs in a platform for high-throughput drug screening (HTS). These findings demonstrate the potential utility of BAs as a cell therapeutic and as a tool for the identification of drugs to treat metabolic diseases., Methods to generate beige adipocytes from a human cell source are inefficient. Here, the authors present a protocol that efficiently generates beige adipocytes from human adipose-derived stem cells (ADSCs), which have potential utility in therapeutic development relating to metabolic diseases such as type 2 diabetes.

Aims Surgical Site Infection (SSI) is common after colorectal surgery where rates are reported as high as 30%. The aim of this project was to assess whether a collaborative approach to implement a 4-point care bundle could reduce SSI rates across a group of hospitals. Methods PreciSSIon is a Quality Improvement Project (QIP) involving 7 NHS hospitals, with a design based on the Institute for Healthcare Improvement’s Breakthrough Series collaborative model. The Public Health England SSI surveillance questionnaire was used to measure patient-reported SSI at 30 days post-op. An evidence based bundle of care was introduced including: 2% chlorhexidine skin preparation; dual ring wound protectors; triclosan-coated sutures for wound closure; and a second dose of antibiotics after 4 hours. Results All hospitals had implemented the care bundle by February 2020. By December 2020 6/7 hospitals had reduced SSI. Mean patient questionnaire response rate = 84.8%. Mean compliance with each of the above bundle components are as follows: 91%; 72%; 85%; and 92% respectively. Conclusions Implementing an evidence based care bundle, using a collaborative approach to QIP, was effective in reducing the rates of patient-reported SSI following colorectal surgery. The success of this project in the West of England suggests that similar methodology could be used to improve patient outcomes in other regions.

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a single α3-linked sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms that account for neural phenotypes associated with GM3SD are not known. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in iPSCs and NCCs from both variants, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated, GSLs compared to WT. Shifts in ceramide profiles associated with iPSC and NCC GSLs were also detected in GM3SD variants. Altered GSL profiles in the GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotnib, an inhibitor of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase increased protein O-GlcNAcylation and significantly rescued baseline and erlotnib-induced apoptosis. Collectively, these findings indicate broad effects on cell signaling during differentiation of GM3SD patient-derived iPSCs to NCCs. Thus, human GM3SD cells provide a novel platform to investigate structure/function relationships that connect GSL diversity to cell signaling, cell survival, and neural differentiation.

Brown adipocytes (BA) are a specialized fat cell which possesses a high capacity for fuel oxidation combined with heat production. The maintenance of high metabolic activity in BA requires elevated oxidation of fuel through the tricarboxylic acid cycle. Pyruvate carboxylase (PC) was previously proposed to be essential for coordination between fuel oxidation and thermogenesis. By differentiating human pluripotent stem cells to mature BA in vitro, we showed that ablation of PC gene by CRISPR Cas9 genome engineering did not impair the ability of stem cells to generate mature BA. However, brown adipocytes deficient for PC expression displayed a 35% reduction in ATP-linked respiration, but not thermogenesis under both basal and isoproterenol-stimulated conditions. This relatively mild impairment of ATP-link respiration in PC knockout BA was protected by increased spare mitochondrial respiratory capacity. Taken together, this study highlights the role of PC in supporting fuel oxidation rather than thermogenesis in human BA.

International audience; SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423*), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423*) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.

Summary The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties., Graphical Abstract, Highlights • Stable transcriptional reprogramming toward endoderm within one cell cycle • Replication timing can change in the first S phase of a cell fate change • Early discordance between Replication timing and Hi-C compartments • Alignment of replication timing to compartments increases in later cell cycles, The temporal order of DNA replication is regulated during development, and is highly correlated with gene expression, chromatin structure, and 3D-genome architecture. Gilbert and colleagues find that stable transcriptional changes of human embryonic stem cells to endoderm can occur within a single cell cycle, accompanied by discordance in replication timing and chromatin compartment that align in later differentiation stages.

Beige adipocytes (also known as brite adipocytes) have significant utility for numerous applications, such as drug screening, cell therapy, and disease modelling. However, a high-efficiency protocol from human adult adipose-derived stem/stromal stem cells (ADSC) has not been described. The protocol described here achieves beige adipocyte purities of >92% in a fully-defined, serum-free media cocktail, which enables these downstream applications. This method provides a significant leap forward over previously described, serum-based protocols that were inconsistent and inefficient.

Summary Vascularization is critical for organ homeostasis and function, but cell-based technologies that promote vascular regeneration are limited. This protocol describes steps to generate human pluripotent stem cell (hPSC)-derived vascular progenitors of the mesothelium lineage. This technology has several advantages for the generation of vascular cells. First and foremost, MesoT cells are multipotent progenitors that can generate smooth muscle cells and endothelial cells. MesoT cells therefore have potential utility in tissue repair, tissue engineering, and in vascularization of laboratory grown organs. For complete details on the use and execution of this protocol, please refer to Colunga et al. (2019)., Graphical Abstract, Highlights • Generation of vascular progenitors (MesoT) from human pluripotent cells (hPSCs) • MesoT cells are equivalent to in vivo vascular progenitor cells in the embryonic heart • hPSC-derived MesoT cells are multipotent and derived from the mesothelium lineage • Human MesoT cells have potential utility in regenerative medicine, Vascularization is critical for organ homeostasis and function, but cell-based technologies that promote vascular regeneration are limited. This protocol describes steps to generate human pluripotent stem cell (hPSC)-derived vascular progenitors of the mesothelium lineage. This technology has several advantages for the generation of vascular cells. First and foremost, MesoT cells are multipotent progenitors that can generate smooth muscle cells and endothelial cells. MesoT cells therefore have potential utility in tissue repair, tissue engineering, and in vascularization of laboratory grown organs.

Over the last 15 years connections between cell cycle control, maintenance of pluripotency, and control of cell fate decisions have been firmly established. With the emergence of powerful tools, such as highly-specific small molecule inhibitors for cyclin-dependent protein kinase (CDK) activity and single-cell imaging technologies, the mechanistic links between cyclins, CDKs and regulation in PSCs in mechanistic detail has been made possible. In this review, we discuss new developments that mechanistically link the CDK regulatory network to control of cell fate decisions, including maintenance of the pluripotent state. Overall, these findings have potential to impact the translational applications of stem cells in regenerative medicine, drug discovery and cancer treatment.

Madin-Darby canine kidney II (MDCKII) cells are used widely to study epithelial morphogenesis. To better understand this process, we performed time-course RNA-seq analysis of MDCKII 3D cystogenesis, along with polarized 2D cells for comparison. Our study reveals a biphasic change in the transcriptome after the 1st cell cycle. This change appears to be linked to translocation of β-catenin, supported by analyses with AVL9- or DENND5A-knockdown clones, and HNF1B mitotic bookmarking, supported by ATAC-seq study. Specifically, β-catenin is depleted from the nucleus and enriched at the cell-cell junctions following the 1st cell cycle, downregulating the MYC network and decreasing cell proliferation. Meanwhile, HNF1B is retained in the nucleus, upregulating its targets and contributing to the cell polarity establishment. Our study supports a qualitative change model for transcriptome remodeling during epithelial morphogenesis and that this qualitative change results from transcription factor redistribution during the first cell cycle.

DNA is replicated in a defined temporal order termed the replication timing (RT) program. RT is spatially segregated in the nucleus with early/late replication corresponding to Hi-C A/B chromatin compartments, respectively. Early replication is also associated with active histone modifications and transcriptional permissiveness. However, the mechanistic interplay between RT, chromatin state, and genome compartmentalization is largely unknown. Here we report that RT is central to epigenome maintenance and compartmentalization in both human embryonic stem cells (hESCs) and cancer cell line HCT116. Knockout (KO) of the conserved RT control factor RIF1, rather than causing discrete RT switches as previously suspected, lead to dramatically increased cell to cell heterogeneity of RT genome wide, despite RIF1’s enrichment in late replicating chromatin. RIF1 KO hESCs have a nearly random RT program, unlike all prior RIF1 KO cells, including HCT116, which show localized alterations. Regions that retain RT, which are prevalent in HCT116 but rare in hESCs, consist of large H3K9me3 domains revealing two independent mechanisms of RT regulation that are used to different extents in different cell types. RIF1 KO results in a striking genome wide downregulation of H3K27ac peaks and enrichment of H3K9me3 at large domains that remain late replicating, while H3K27me3 and H3K4me3 are re-distributed genome wide in a cell type specific manner. These histone modification changes coincided with global reorganization of genome compartments, transcription changes and a genome wide strengthening of TAD structures. Inducible degradation of RIF1 revealed that disruption of RT is upstream of genome compartmentalization changes. Our findings demonstrate that disruption of RT leads to widespread epigenetic mis-regulation, supporting previously speculative models in which the timing of chromatin assembly at the replication fork plays a key role in maintaining the global epigenetic state, which in turn drives genome architecture.

Vascular progenitor cells have been identified from perivascular cell fractions and peripheral blood and bone marrow mononuclear fractions. These vascular progenitors share the ability to generate some of the vascular lineages, including endothelial cells, smooth muscle cells, and pericytes. The potential therapeutic uses for vascular progenitor cells are broad and relate to stroke, ischemic disease, and in the engineering of whole organs and tissues that require a vascular component. This review summarizes the best characterized sources of vascular progenitor cells and discusses advances in 3D printing and electrospinning using blended polymers for the creation of biomimetic vascular grafts. These advances are pushing the field of regenerative medicine closer to the creation of small diameter vascular grafts with long-term clinical utility.

Extreme intestinal polyposis in pet dogs has not yet been reported in literature. We identified a dog patient who developed numerous intestinal polyps, with the severity resembling human classic familial adenomatous polyposis (FAP), except the jejunum-ileum junction being the most polyp-dense. We investigated this dog, in comparison with 22 other dogs with spontaneous intestinal tumors but no severe polyposis, and with numerous published human cancers. We found, not APC mutation, but three other alteration pathways as likely reasons of this canine extreme polyposis. First, somatic truncation mutation W411X of FBXW7, a component of an E3 ubiquitin ligase, over-activates MYC and cell cycle-promoting network, accelerating crypt cell proliferation. Second, genes of protein trafficking and localization are downregulated, likely associated with germline mutation G406D of STAMBPL1, a K63-deubiquitinase, and MYC network activation. This inhibits epithelial apical-basolateral polarity establishment, preventing crypt cell differentiation. Third, Bacteroides uniformis, a commensal gut anaerobe, thrives and expresses abundantly thioredoxin and nitroreductase. These bacterial products could reduce oxidative stress linked to host germline mutation R51X of CYB5RL, a cytochrome b5 reductase homologue, decreasing cell death. Our work emphasizes the close collaboration of alterations across the genome, transcriptome and microbiome in promoting tumorigenesis.

Brown adipocytes (BAs) are a potential cell source for the treatment of metabolic disease, including type 2 diabetes. In this report, human pluripotent stem cells (hPSCs) are subject to directed differentiation through a paraxial mesoderm progenitor state that generates BAs at high efficiency. Molecular analysis identifies potential regulatory networks for BA development, giving insight into development along this lineage. hPSC-derived BAs undergo elevated rates of glycolysis, uncoupled respiration, and lipolysis that are responsive to changes in cyclic AMP (cAMP)-dependent signaling, consistent with metabolic activity in BA tissue depots. Transplanted human BAs engraft into the inter-scapular region of recipient mice and exhibit thermogenic activity. Recipient animals have elevated metabolic activity, respiratory exchange ratios, and whole-body energy expenditure. Finally, transplanted BAs reduce circulating glucose levels in hyperglycemic animals. These data provide a roadmap for brown adipocyte development and indicate that BAs generated from hPSCs have potential as a tool for therapeutic development.

Summary Background Closure of an abdominal stoma, a common elective operation, is associated with frequent complications; one of the commonest and impactful is incisional hernia formation. We aimed to investigate whether biological mesh (collagen tissue matrix) can safely reduce the incidence of incisional hernias at the stoma closure site. Methods In this randomised controlled trial (ROCSS) done in 37 hospitals across three European countries (35 UK, one Denmark, one Netherlands), patients aged 18 years or older undergoing elective ileostomy or colostomy closure were randomly assigned using a computer-based algorithm in a 1:1 ratio to either biological mesh reinforcement or closure with sutures alone (control). Training in the novel technique was standardised across hospitals. Patients and outcome assessors were masked to treatment allocation. The primary outcome measure was occurrence of clinically detectable hernia 2 years after randomisation (intention to treat). A sample size of 790 patients was required to identify a 40% reduction (25% to 15%), with 90% power (15% drop-out rate). This study is registered with ClinicalTrials.gov, NCT02238964. Findings Between Nov 28, 2012, and Nov 11, 2015, of 1286 screened patients, 790 were randomly assigned. 394 (50%) patients were randomly assigned to mesh closure and 396 (50%) to standard closure. In the mesh group, 373 (95%) of 394 patients successfully received mesh and in the control group, three patients received mesh. The clinically detectable hernia rate, the primary outcome, at 2 years was 12% (39 of 323) in the mesh group and 20% (64 of 327) in the control group (adjusted relative risk [RR] 0·62, 95% CI 0·43–0·90; p=0·012). In 455 patients for whom 1 year postoperative CT scans were available, there was a lower radiologically defined hernia rate in mesh versus control groups (20 [9%] of 229 vs 47 [21%] of 226, adjusted RR 0·42, 95% CI 0·26–0·69; p

Summary: In this report we describe a human pluripotent stem cell-derived vascular progenitor (MesoT) cell of the mesothelium lineage. MesoT cells are multipotent and generate smooth muscle cells, endothelial cells, and pericytes and self-assemble into vessel-like networks in vitro. MesoT cells transplanted into mechanically damaged neonatal mouse heart migrate into the injured tissue and contribute to nascent coronary vessels in the repair zone. When seeded onto decellularized vascular scaffolds, MesoT cells differentiate into the major vascular lineages and self-assemble into vasculature capable of supporting peripheral blood flow following transplantation. These findings demonstrate in vivo functionality and the potential utility of MesoT cells in vascular engineering applications. : Colunga et al. describe a multipotent vascular progenitor cell that contributes to neovascularization of damaged tissue and that efficiently populates vascular scaffolds and self-assembles into functional vessels. These findings open up new opportunities for the vascularization of diseased and damaged tissue and for surgical vessel replacement. Keywords: stem cells, mesothelium, vascular progenitor, tissue engineering, regenerative medicine

Metabolism has been shown to alter cell fate in human pluripotent stem cells (hPSC). However, current understanding is almost exclusively based on work performed at 20% oxygen (air), with very few studies reporting on hPSC at physiological oxygen (5%). In this study, we integrated metabolic, transcriptomic, and epigenetic data to elucidate the impact of oxygen on hPSC. Using 13C-glucose labeling, we show that 5% oxygen increased the intracellular levels of glycolytic intermediates, glycogen, and the antioxidant response in hPSC. In contrast, 20% oxygen increased metabolite flux through the TCA cycle, activity of mitochondria, and ATP production. Acetylation of H3K9 and H3K27 was elevated at 5% oxygen while H3K27 trimethylation was decreased, conforming to a more open chromatin structure. RNA-seq analysis of 5% oxygen hPSC also indicated increases in glycolysis, lysine demethylases, and glucose-derived carbon metabolism, while increased methyltransferase and cell cycle activity was indicated at 20% oxygen. Our findings show that oxygen drives metabolite flux and specifies carbon fate in hPSC and, although the mechanism remains to be elucidated, oxygen was shown to alter methyltransferase and demethylase activity and the global epigenetic landscape.

Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/β-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/β-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity., The Myc transcription factor is a major regulator of stem cell (SC) self-renewal and pluripotency but how this integrates signals from other pathways is unclear. Here, the authors show that Myc activation triggers epigenetic memory in self renewing embryonic SCs via PRC2-mediated potentiation of the Wnt pathway.

For many years, stem cell metabolism was viewed as a byproduct of cell fate status rather than an active regulatory mechanism; however, there is now a growing appreciation that metabolic pathways influence epigenetic changes associated with lineage commitment, specification, and self-renewal. Here we review how metabolites generated during glycolytic and oxidative processes are utilized in enzymatic reactions leading to epigenetic modifications and transcriptional regulation. We discuss how "metabolic reprogramming" contributes to global epigenetic changes in the context of naive and primed pluripotent states, somatic reprogramming, and hematopoietic and skeletal muscle tissue stem cells, and we discuss the implications for regenerative medicine.

Summary Madin-Darby canine kidney II (MDCKII) cells are widely used to study epithelial morphogenesis. To better understand this process, we performed time course RNA-seq analysis of MDCKII 3D cystogenesis, along with polarized 2D cells for comparison. Our study reveals a biphasic change in the transcriptome that occurs after the first cell cycle and coincides with lumen establishment. This change appears to be linked to translocation of β-catenin, supported by analyses with AVL9- and DENND5A-knockdown clones, and regulation by HNF1B, supported by ATAC-seq study. These findings indicate a qualitative change model for transcriptome remodeling during epithelial morphogenesis, leading to cell proliferation decrease and cell polarity establishment. Furthermore, our study reveals that active mitochondria are retained and chromatin accessibility decreases in 3D cysts but not in 2D polarized cells. This indicates that 3D culture is a better model than 2D culture for studying epithelial morphogenesis., Graphical Abstract, Highlights • The transcriptome switches after the first cell cycle and during MDCKII lumenogenesis • The transcriptome switch is linked to β-catenin translocation and HNF1B activation • Chromatin accessibility decreases during MDCKII cystogenesis • Active mitochondria are maintained in 3D, but not 2D, epithelial morphogenesis, Developmental Biology; Embryology; Transcriptomics

Pluripotent stem cells retain the ability to differentiate into a wide-range of cell types while undergoing self-renewal. They also exhibit an unusual mode of cell cycle regulation, reflected by a cell cycle structure where G1- and G2-phases are truncated. When individual pluripotent stem cells are exposed to specification cues, they activate developmental programs and remodel the cell cycle so that the length of G1 and overall cell division times increase. The response of individual stem cells to pro-differentiation signals is strikingly heterogeneous, resulting in asynchronous differentiation. Recent evidence indicates that this phenomenon is due to cell cycle-dependent mechanisms that restrict the initial activation of developmental genes to the G1-phase. This suggests a broad biological mechanism where multipotent cells are ‘primed’ to initiate cell fate decisions during their transition through G1. Mechanisms underpinning commitment towards the differentiated state and its relationship to the cell cycle are discussed.

Summary Here we show that bivalent domains and chromosome architecture for bivalent genes are dynamically regulated during the cell cycle in human pluripotent cells. Central to this is the transient increase in H3K4-trimethylation at developmental genes during G1, thereby creating a “window of opportunity” for cell-fate specification. This mechanism is controlled by CDK2-dependent phosphorylation of the MLL2 (KMT2B) histone methyl-transferase, which facilitates its recruitment to developmental genes in G1. MLL2 binding is required for changes in chromosome architecture around developmental genes and establishes promoter-enhancer looping interactions in a cell-cycle-dependent manner. These cell-cycle-regulated loops are shown to be essential for activation of bivalent genes and pluripotency exit. These findings demonstrate that bivalent domains are established to control the cell-cycle-dependent activation of developmental genes so that differentiation initiates from the G1 phase., Highlights • Bivalent domains are unstable, dynamic, and cell-cycle regulated • CDK2 phosphorylates MLL2 and establishes bivalent domains in G1 • Chromosome remodeling in G1 is required for the “poised” pluripotent state, In this report, Dalton and colleagues show that developmental genes are primed for activation in G1 phase of the cell cycle by a mechanism requiring convergence of the cell-cycle machinery with cell signaling pathways. This priming mechanism involves the establishment of bivalent epigenetic domains and dynamic changes in chromosome architecture around developmental genes.

AVL9, DENND5A and NUPL1 are among the cancer driver candidate genes previously identified via dog-human comparison, and may function in epithelial cell polarity as indicated by bioinformatics analysis. To better understand their cellular functions and roles in cancer, we knocked down each gene in MDCKII cells through shRNA and performed three-dimensional culture. Compared to the control, the knockdown clones developed significantly more abnormal cysts, e.g., cysts with the lumen harboring dead and/or live cells, or cysts having multiple lumens. Further analysis revealed that abnormalities initiated at the first cell division and persisted throughout the entire cystogenesis process. For NUPL1-knockdown cells, abnormal cytogenesis largely arose from faulty cell divisions, notably monopolar spindles or spindles with poorly separated poles. For AVL9- or DENND5A-knockdown cells, abnormalities originated from both aberrant intracellular trafficking and defective mitosis. Moreover, while all knockdown clones displayed an accelerated rate of both cell proliferation and death, only AVL9- and DENND5A-knockdowns, but not NUPL1-knockdown, promoted cell migration. These observations indicate that NUPL1 contributes to bipolar spindle formation, whereas AVL9 and DENND5A participate in both intracellular trafficking and cell cycle progression. Our study shed lights on these genes' normal cellular functions and on how their alteration contributes to carcinogenesis.

As human pluripotent stem cells (hPSCs) exit pluripotency, they are thought to switch from a glycolytic mode of energy generation to one more dependent on oxidative phosphorylation. Here we show that, although metabolic switching occurs during early mesoderm and endoderm differentiation, high glycolytic flux is maintained and, in fact, essential during early ectoderm specification. The elevated glycolysis observed in hPSCs requires elevated MYC/MYCN activity. Metabolic switching during endodermal and mesodermal differentiation coincides with a reduction in MYC/MYCN and can be reversed by ectopically restoring MYC activity. During early ectodermal differentiation, sustained MYCN activity maintains the transcription of "switch" genes that are rate-limiting for metabolic activity and lineage commitment. Our work, therefore, shows that metabolic switching is lineage-specific and not a required step for exit of pluripotency in hPSCs and identifies MYC and MYCN as developmental regulators that couple metabolism to pluripotency and cell fate determination.

Cell fate decisions are closely linked to changes in metabolic activity. Over recent years this connection has been implicated in mechanisms underpinning embryonic development, reprogramming and disease pathogenesis. In addition to being important for supporting the energy demands of different cell types, metabolic switching from aerobic glycolysis to oxidative phosphorylation plays a critical role in controlling biosynthetic processes, intracellular redox state, epigenetic status and reactive oxygen species levels. These processes extend beyond ATP synthesis by impacting cell proliferation, differentiation, enzymatic activity, ageing and genomic integrity. This review will focus on how metabolic switching impacts decisions made by multipotent cells and discusses mechanisms by which this occurs.

Spontaneously occurring canine mammary cancer represents an excellent model of human breast cancer, but is greatly understudied. To better use this valuable resource, we performed whole-genome sequencing, whole-exome sequencing, RNA-seq, and/or high-density arrays on twelve canine mammary cancer cases, including seven simple carcinomas and four complex carcinomas. Canine simple carcinomas, which histologically match human breast carcinomas, harbor extensive genomic aberrations, many of which faithfully recapitulate key features of human breast cancer. Canine complex carcinomas, which are characterized by proliferation of both luminal and myoepithelial cells and are rare in human breast cancer, seem to lack genomic abnormalities. Instead, these tumors have about 35 chromatin-modification genes downregulated and are abnormally enriched with active histone modification H4-acetylation, whereas aberrantly depleted with repressive histone modification H3K9me3. Our findings indicate the likelihood that canine simple carcinomas arise from genomic aberrations, whereas complex carcinomas originate from epigenomic alterations, reinforcing their unique value. Canine complex carcinomas offer an ideal system to study myoepithelial cells, the second major cell lineage of the mammary gland. Canine simple carcinomas, which faithfully represent human breast carcinomas at the molecular level, provide indispensable models for basic and translational breast cancer research. Cancer Res; 74(18); 5045–56. ©2014 AACR.