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The oncogene yes-associated protein (YAP) controls liver tumor initiation and progression via cell extrinsic functions by creating a tumor-supporting environment in conjunction with cell autonomous mechanisms. However, how YAP controls organization of the microenvironment and in particular the vascular niche, which contributes to liver disease and hepatocarcinogenesis, is poorly understood. To investigate heterotypic cell communication, we dissected murine and human liver endothelial cell (EC) populations into liver sinusoidal endothelial cells (LSEC) and continuous endothelial cells (CEC) through histomorphological and molecular characterization. In YAPS127A-induced tumorigenesis, a gradual replacement of LSECs by CECs was associated with dynamic changes in the expression of genes involved in paracrine communication. The formation of new communication hubs connecting CECs and LSECs included the hepatocyte growth factor (Hgf)/c-Met signaling pathway. In hepatocytes and tumor cells, YAP/TEA domain transcription factor 4 (TEAD4)–dependent transcriptional induction of osteopontin (Opn) stimulated c-Met expression in EC with CEC phenotype, which sensitized these cells to the promigratory effects of LSEC-derived Hgf. In human hepatocellular carcinoma, the presence of a migration-associated tip-cell signature correlated with poor clinical outcome and the loss of LSEC marker gene expression. The occurrence of c-MET–expressing CECs in human liver cancer samples was confirmed at the single-cell level. In summary, YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell–derived factors modify the cross-talk between LSECs and CECs via the HGF/c-MET axis. Significance: YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between EC subpopulations.

European Research Council (ERC) Starting Grant 205905 (to O.O.) and Synergy Grant 856506—LIFEPLAN (to O.O. and T.R.); Spanish Ramón y Cajal Grant RYC-2014-16263 (to M.M.D.) (...), Delgado, M.D.M., Roslin, T., Tikhonov, G., Meyke, E., Lo, C., Gurarie, E., Abadonova, M., Abduraimov, O., Adrianova, O., Akimova, T., Akkiev, M., Ananin, A., Andreeva, E., Andriychuk, N., Antipin, M., Arzamascev, K., Babina, S., Babushkin, M., Bakin, O., Barabancova, A., Basilskaja, I., Belova, N., Belyaeva, N., Bespalova, T., Bisikalova, E., Bobretsovz, A., Bobrov, V., Bobrovskyi, V., Bochkareva, E., Bogdanov, G., Bolshakov, V., Bondarchuk, S., Bukharova, E., Butunina, A., Buyvolov, Y., Buyvolova, A., Bykov, Y., Chakhireva, E., Chashchina, O., Cherenkova, N., Chistjakov, S., Chuhontseva, S., Davydov, E.A., Demchenko, V., Diadicheva, E., Dobrolyubov, A., Dostoyevskaya, L., Drovnina, S., Drozdova, Z., Dubanaev, A., Dubrovsky, Y., Elsukov, S., Epova, L., Ermakova, O.S., Ermakova, O., Esengeldenova, A., Evstigneev, O., Fedchenko, I., Fedotova, V., Filatova, T., Gashev, S., Gavrilov, A., Gaydys, I., Golovcov, D., Goncharova, N., Gorbunova, E., Gordeeva, T., Grishchenko, V., Gromyko, L., Hohryakov, V., Hritankov, A., Ignatenko, E., Igosheva, S., Ivanova, U., Ivanova, N., Kalinkin, Y., Kaygorodova, E., Kazansky, F., Kiseleva, D., Knorrem, A., Kolpashikov, L., Korobov, E., Korolyova, H., Korotkikh, N., Kosenkov, G., Kossenko, S., Kotlugalyamova, E., Kozlovsky, E., Kozsheechkin, V., Kozurak, A., Kozyr, I., Krasnopevtseva, A., Krugliko, S., Kuberskaya, O., Kudryavtsev, A., Kulebyakina, E., Kulsha, Y., Kupriyanova, M., Kurbanbagamaev, M., Kutenkov, A., Kutenkova, N., Kuyantseva, N., Kuznetsov, A., Larin, E., Lebedev, P., Litvinov, K., Luzhkova, N., Mahmudov, A., Makovkina, L., Mamontov, V., Mayorova, S., Megalinskaja, I., Meydus, A., Minin, A., Mitrofanov, O., Motruk, M., Myslenkov, A., Nasonova, N., Nemtseva, N., Nesterova, I., Nezdoliy, T., Niroda, T., Novikova, T., Panicheva, D., Pavlov, A., Pavlova, K., Van, P., Podolski, S., Polikarpova, N., Tatiana Polyanskaya81, Pospelov, I., Pospelova, E., Prokhorov, I., Prokosheva, I., Puchnina, L., Putrashyk, I., Raiskaya, J., Rozhkov, Y., Rozhkova, O., Rudenko, M., Rybnikova, I., Rykova, S., Sahnevich, M., Samoylov, A., Sank, V., Sapelnikova, I., Sazonov, S., Selyunina, Z., Shalaeva, K., Shashkov, M., Shcherbakov, A., Shevchyk, V., Shubin, S., Shujskaja, E., Sibgatullin, R., Sikkilah, N., Sitnikova, E., Sivkov, A., Skok, N., Skorokhodova, S., Smirnova, E., Sokolova, G., Sopin, V., Spasovski, Y., Stepanov, S., Stratiy, V., Strekalovskaya, V., Sukhov, A., Suleymanova, G., Sultangareeva, L., Teleganova, V., Teplov, V., Teplova, V., Tertitsa, T., Timoshkin, V., Tirski, D., Tolmachev, A., Tomilin, A., Tselishcheva, L., Turgunov, M., Tyukh, Y., Van, V., Ershkova, E., Vasin, A., Vasina, A., Vekliuk, A., Vetchinnikova, L., Vinogradov, V., Volodchenko, N., Voloshina, I., Xoliqov, T., Yablonovska-Grishchenko, E., Yakovlev, V., Yakovleva, M., Yantser, O., Yarema, Y., Zahvatov, A., Zakharov, V., Zelenetskiy, N., Zheltukhin, A., Zubina, T., Kurhinen, J., Ovaskainen, O.

Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as a renewable resource for energy and bio-based products. Transcriptional regulators of the lignin biosynthetic pathway represent promising targets for tailoring lignin content and composition in plant secondary cell walls. However, knowledge about the transcriptional regulation of lignin biosynthesis in lignocellulosic feedstocks, such as Miscanthus, is limited. In Miscanthus leaves, MsSCM1 and MsMYB103 are expressed at growth stages associated with lignification. The ectopic expression of MsSCM1 and MsMYB103 in N. benthamiana leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin compositions. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 and MsMYB103 overexpression showed an extensive overlap with the response to the NAC master transcription factor MsSND1, but were distinct from each other, underscoring the inherent complexity of secondary cell wall formation. Furthermore, conserved and previously described promoter elements as well as novel and specific motifs could be identified from the target genes of the three transcription factors. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of lignin content and composition in Miscanthus towards a tailored biomass.

Stem cells are one of the foundational evolutionary novelties that allowed the independent emergence of multicellularity in the plant and animal lineages. In plants, the homeodomain (HD) transcription factor WUSCHEL (WUS) is essential for the maintenance of stem cells in the shoot apical meristem. WUS has been reported to bind to diverse DNA motifs and to act as transcriptional activator and repressor. However, the mechanisms underlying this remarkable behavior have remained unclear. Here, we quantitatively delineate WUS binding to three divergent DNA motifs and resolve the relevant structural underpinnings. We show that WUS exhibits a strong binding preference for TGAA repeat sequences, while retaining the ability to weakly bind to TAAT elements. This behavior is attributable to the formation of dimers through interactions of specific residues in the HD that stabilize WUS DNA interaction. Our results provide a mechanistic basis for dissecting WUS dependent regulatory networks in plant stem cell control., WUSCHEL is a homeodomain transcription factor that is essential for stem cell maintenance in the plant shoot apical meristem. Here, via structural and biochemical approaches, Sloan et al. show that strong WUSCHEL binding to preferential target motifs can be attributed to dimer formation that stabilizes DNA binding.

Next to cell autonomous mechanisms, the oncogene yes-associated protein (YAP) controls liver tumor initiation and progression via cell extrinsic functions creating a tumor-supporting environment. However, how YAP affects the microenvironment and in particular the vascular niche, which contributes to liver disease and hepatocarcinogenesis, is poorly understood.In this study, histo-morphological and molecular characterization of murine liver endothelial cells (ECs) populations and human single cell data revealed the presence of liver sinusoidal endothelial cells (LSECs) and capillary endothelial cells (CECs) in healthy liver tissue. In YAPS127A-induced tumorigenesis, a gradual replacement of LSECs by CECs was associated with dynamic changes in the expression of genes involved in EC subtype-specific paracrine communication. The formation of new potential communication hubs connecting CECs and LSECs included the hepatocyte growth factor (Hgf)/c-Met signaling pathway. In hepatocytes and tumor cells, YAP/TEA domain transcription factor 4 (TEAD4)-dependent transcriptional induction of osteopontin (Opn) stimulated c-Met expression in ECs with CEC phenotype, which sensitized these cells to the pro-migratory effects of LSEC-derived Hgf. In human HCCs, the presence of a migration-associated tip-cell signature correlated with poor clinical outcome and the loss of LSEC marker gene expression. In addition, the replacement of LSECs by CECs with exclusive c-MET expression in a CEC subpopulation was confirmed at the single cell level.In summary, YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs (e.g. the HGF/c-Met axis), in which tumor cell-derived factors modify the crosstalk between LSECs and CECs.

Acquisition of detailed structural and molecular information from intact biological samples, while preserving cellular three-dimensional structures, still represents a challenge for biological studies aiming to unravel system functions. Here we describe a novel X-ray-based methodology for analysis of gene expression pattern in intact murine brain ex vivo by microCT. The method relays on detection of bromine molecules in the products of enzymatic reaction generated by theβ-galactosidase (lacZ) gene reporter. To demonstrate the feasibility of the method, the analysis of the expression pattern of tRNA endonuclease 54 (Tsen54)-lacZ reporter gene in the whole-mount murine brain in semi-quantitative manner is performed. Mutations in Tsen54 gene causes pontocerebellar hypoplasia (PCH), severe neurodegenerative disorder with both mental and motor deficits. Comparing relative levels of Tsen54 gene expression, we have demonstrated that highest Tsen54 expression observed in anatomical brain substructures important for the normal motor and memory functions in mice. In the forebrain strong expression in perirhinal, retrosplenial and secondary motor areas was observed. In olfactory area Tsen54 is highly expressed in the nucleus of the lateral olfactory tract, anterior olfactory and bed nuclei, while in hypothalamus in lateral mammillary nucleus and preoptic area. In hindbrain Tsen54 is expressed in the reticular, cuneate and trigeminal nuclei of medulla, and in pontine gray of pons and in cerebellum, in the molecular and Purkinje cell layers. Delineating anatomical brain regions in which Tsen54 is strongly expressed will allow functionally address the role Tsen54 gene in normal physiology and in PCH disease.Significance StatementCharacterization of gene expression pattern in the brain of model organisms is critical for unravelling the gene function in normal physiology and disease. It is performed by optical imaging of the two-dimensional brain sections which then assembled in volume images. Here we applied microCT platform, which allows three-dimensional imaging of non transparent samples, for analysis of gene expression. This method based on detection by X-ray the bromine molecules presented in the products generated by enzymatic activity of b-galactosidase reporter gene. With this method we identify anatomical brain substructures in which Tsen54 gene, mutated in pontocerebellar hypoplasia disease, is expressed.

Enrichment of leukemic blasts with a stem cell phenotype correlates with poor survival in acute myeloid leukemia (AML). In this context, measurement of the stem cell marker aldehyde-dehydrogenase (ALDH) activity can distinguish poor prognosis cases with increased fractions of ALDH-positive cells (ALDH-numerous AML) and favorable outcome cases with low percentages (ALDH-rare AML). It has been shown that ALDH-numerous AML favor leukemic engraftment in xenotransplantation assays which suggests increased leukemic stem cell (LSC) potential. To test if this reflects an immature cell of origin, comparative gene-expression studies of CD34+ leukemic blasts were performed. This analysis revealed increased expression of LSC and HSC signatures in ALDH-numerous AML, whereas ALDH-rare AML were enriched for a progenitor signature. The enrichment of stemness-associated transcriptional programs suggests that ALDH-numerous AML derive from immature hematopoietic progenitors and offers an explanation for the poor prognosis and therapy resistance of this subgroup which is likely caused by inherited stem cell properties.

In this work, we applied three-dimensional microCT imaging to study murine embryogenesis in the range from immediate post-implantation period (embryonic day 5.5) to mid-gestation (embryonic day 12.5) with the resolution up to 1.4 µm/voxel. Also, we introduce an imaging procedure for non-invasive volumetric estimation of an entire litter of embryos within the maternal uterine structures. This method allows for an accurate, detailed and systematic morphometric analysis of both embryonic and extra-embryonic components during embryogenesis. Three-dimensional imaging of unperturbed embryos was performed to visualize the egg cylinder, primitive streak, gastrulation and early organogenesis stages of murine development in the C57Bl6/N mouse reference strain. Further, we applied our microCT imaging protocol to determine the earliest point when embryonic development is arrested in a mouse line with knockout for tRNA splicing endonuclease subunit Tsen54 gene. Our analysis determined that the embryonic development in Tsen54 null embryos does not proceed beyond implantation. We demonstrated that application of microCT imaging to entire litter of non-perturbed embryos greatly facilitate studies to unravel gene function during early embryogenesis and to determine the precise point at which embryonic development is arrested in mutant animals. The described method is inexpensive, does not require lengthy embryos dissection and can be applicable for detailed analysis of mutant mice at laboratory scale as well as for high-throughput projects. Electronic supplementary material The online version of this article (doi:10.1007/s00335-017-9723-6) contains supplementary material, which is available to authorized users.

During development cells become restricted in their differentiation potential by repressing alternative cell fates, and the Polycomb complex plays a crucial role in this process. However, how alternative fate genes are lineage-specifically silenced is unclear. We studied Ultrabithorax (Ubx), a multi-lineage transcription factor of the Hox class, in two tissue lineages using sorted nuclei and interfered with Ubx in mesodermal cells. We find that depletion of Ubx leads to the de-repression of genes normally expressed in other lineages. Ubx silences expression of alternative fate genes by retaining the Polycomb Group protein Pleiohomeotic at Ubx targeted genomic regions, thereby stabilizing repressive chromatin marks in a lineage-dependent manner. Our study demonstrates that Ubx stabilizes lineage choice by suppressing the multipotency encoded in the genome via its interaction with Pho. This mechanism may explain why the Hox code is maintained throughout the lifecycle, since it could set a block to transdifferentiation in adult cells.

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness., Spatial control of auxin signaling maintains a balance between stem-cell self-renewal and differentiation at the plant shoot apex. Here Ma et al. show that rheostatic control of auxin response by the WUSCHEL transcription factor maintains stem cells by conferring resistance to auxin mediated differentiation.

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.

During development cells become gradually restricted in their differentiation potential by the repression of alternative cell fates. While we know that the Polycomb complex plays a crucial role in this process, it still remains unclear how alternative fate genes are specifically targeted for silencing in different cell lineages. We address this question by studying Ultrabithorax (Ubx), a multi-lineage transcription factor (TF) of the Hox class, in the mesodermal and neuronal lineages using sorted nuclei ofDrosophilaembryos and by interfering with Ubx in mesodermal cells that have already initiated differentiation. We find that Ubx is a key regulator of lineage development, as its mesoderm-specific depletion leads to the de-repression of many genes normally expressed in other lineages. Ubx silences expression of alternative fate genes by interacting with and retaining the Polycomb Group (PcG) protein Pleiohomeotic (Pho) at Ubx targeted genomic regions, thereby setting repressive chromatin marks in a lineage-dependent manner. In sum, our study demonstrates that Ubx stabilizes lineage choice by suppressing the multi-potency encoded in the genome in a lineage-specific manner via its interaction with Pho. This mechanism may explain why the Hox code is maintained throughout the lifecycle, since it seems to set a block to transdifferentiation in many adult cells.

In the past decade, the importance of the niche to provide regulatory inputs to balance stem cell self-renewal and differentiation has become clear. However, the regulatory interplay between stem cells and their niche at the whole genome level is still poorly understood. To elucidate the mechanisms controlling stem cells and their progenies as they progress through their developmental program at the transcriptional level, we recorded the regulatory program of two independent cell lineages in the Drosophila testis stem cell model. To this end, we identified genes active in the soma or germline as well as genome-wide binding profiles of two essential transcription factors, Zfh-1 and Abd-A, expressed in somatic support cells and crucial for fate acquisition of both cell lineages. Our data identified key roles for TOR signalling, signal processing V-ATPase proton pumps and the nuclear transport engaged nucleoporins and we demonstrate their importance in controlling germline maintenance, proliferation and differentiation from the support side. To make our dataset publicly available and support quick and intuitive data mining, we generated an interactive online analysis tool. Applying our tool for comparative analysis, we uncovered conserved core gene sets of adult stem cells across species boundaries. We have tested the functional relevance of these genes in the Drosophila testis and intestine and find a striking over-representation of stem cell defects when the corresponding genes were depleted. In summary, our dataset and interactive platform represents a versatile tool for identifying novel gene networks active in diverse stem cell types and provides a valuable resource for elucidating the multifaceted regulatory inputs required to guide proper stem cell behaviour.

In the past decade, the importance of the niche to provide regulatory inputs to balance stem cell self-renewal and differentiation has become clear. However, the regulatory interplay between stem cells and their niche at the whole genome level is still poorly understood. To elucidate the mechanisms controlling stem cells and their progenies as they progress through their developmental program at the transcriptional level, we recorded the regulatory program of two independent cell lineages in theDrosophilatestis stem cell model. To this end, we identified genes active in the soma or germline as well as genome-wide binding profiles of two essential transcription factors, Zfh-1 and Abd-A, expressed in somatic support cells and crucial for fate acquisition of both cell lineages. Our data identified key roles for TOR signalling, signal processing V-ATPase proton pumps and the nuclear transport engaged nucleoporins and we demonstrate their importance in controlling germline maintenance, proliferation and differentiation from the support side. To make our dataset publicly available and support quick and intuitive data mining, we generated an interactive online analysis tool. Applying our tool for comparative analysis, we uncovered conserved core gene sets of adult stem cells across species boundaries. We have tested the functional relevance of these genes in theDrosophilatestis and intestine and find a striking overrepresentation of stem cell defects when the corresponding genes were depleted. In summary, our dataset and interactive platform represents a versatile tool for identifying novel gene networks active in diverse stem cell types and provides a valuable resource for elucidating the multifaceted regulatory inputs required to guide proper stem cell behaviour.

Formation of a precise vascular network within the central nervous system is of critical importance to assure delivery of oxygen and nutrients and for accurate functionality of neuronal networks. Vascularization of the spinal cord is a highly stereotypical process. However, the guidance cues controlling blood vessel patterning in this organ remain largely unknown. Here we describe a new neuro-vascular communication mechanism that controls vessel guidance in the developing spinal cord. We show that motor neuron columns remain avascular during a developmental time window, despite expressing high levels of the pro-angiogenic vascular endothelial growth factor (VEGF). We describe that motor neurons express the VEGF trapping receptor sFlt1 via a Neuropilin-1-dependent mechanism. Using a VEGF gain-of-function approach in mice and a motor neuron-specific sFlt1 loss-of-function approach in chicken, we show that motor neurons control blood vessel patterning by an autocrine mechanism that titrates motor neuron-derived VEGF via their own expression of sFlt1., The guidance cues regulating blood vessel patterning in the central nervous system remain unclear. Here, the authors show in mice and chicken developing spinal cord that motor neurons control blood vessel patterning by an autocrine mechanism titrating VEGF via the expression of its trapping receptor sFlt1.

We report the first experimental evidence for the mitogenic action of cerium(IV) oxide and cerium(III) fluoride nanoparticles (CONs and CFNs) on the regeneration of a whole organism – freshwater flatworms Schmidtea mediterranea (planarian). Both types of cerium-containing nanoparticles are shown to be a highly potent mitogen for planaria. Both CONs and CFNs, in micro- and nanomolar concentrations, markedly accelerate planarian blastema growth, due to the enhancement of cellular proliferation, causing an increase in the mitotic index and in the quantity of blastema cells in regenerating planaria. CONs provided maximum activity at concentrations which were two orders of magnitude lower than those for CeF3. The valence state of cerium in cerium-containing nanoparticles plays a significant role in the planarian regeneration mechanism: CeO2 nanoparticles containing predominantly Ce4+ species presumably scavenge wound induced reactive oxygen species and moderately activate gene expression processes, while the regenerative action of CeF3 nanoparticles containing only Ce3+ species is manifested in the pronounced expression of the genes involved in cell division, differentiation and migration. This is the first report on the effect of cerium-containing nanoparticles on tissue regeneration in vivo, further revealing the mechanisms of their biological action, which enhances the possibility of their use in cellular technologies.

Background C/EBPa and C/EBPb are transcription factors with tissue specific expression regulating several important cellular processes. They work by recruiting protein complexes to a common DNA recognition motif and both are able to compensate each other's absence in many cell types, thus showing functional redundancy. They also play distinct roles in specific cellular pathways and their abnormal functioning gives raise to different human pathologies. Methods To investigate the molecular basis of C/EBPa and C/EBPb specificity and redundancy we characterized their in vivo protein-protein interaction networks by Tandem Affinity Purification (TAP) and Mass Spectrometry (MS). To unravel the functional features of C/EBPa and C/EBPb proteomes we studied the statistical enrichment of binding partners related to Gene Ontology (GO) terms and KEGG pathways. Results Our data confirmed that the C/EBPa and C/EBPb regulate biological processes like cell proliferation, apoptosis and transformation. We found that both C/EBPa and C/EBPb are involved in other cellular pathways such as RNA maturation, RNA splicing and DNA repair. Specific interactions of C/EBPa with MRE11, RUVBL1 and RUVBL2 components of DNA repair system were confirmed by co-immunoprecipitation assays. Conclusions Our comparative analysis of the C/EBPa and C/EBPb proteomes provides an insight for understanding both their redundant and specific roles in cells indicating their involvement in new pathways. Such novel predicted functions are relevant to normal cellular processes and disease phenotypes controlled by these transcription factors. General significance Functional characterization of C/EBPa and C/EBPb proteomes suggests they can regulate novel pathways and indicate potential molecular targets for therapeutic intervention.

The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.

Copy number variation (CNV) has been associated increasingly with altered susceptibility to human disease. Large CNVs are likely to incur disease risk or resilience via predictable changes in gene dosage that are relatively straightforward to model using chromosomal engineering in mice. The classical class I major histocompatibility locus (MHC-I) contains a dense set of genes essential for innate immune system function in vertebrates. MHC-I genes are highly polymorphic and genetic variation in the region is associated with altered susceptibility to a wide variety of common diseases. Here we investigated the role of gene dosage within MHC-I on susceptibility to disease by engineering a mouse line carrying a 1.9-Mb duplication of this region [called Dp(MHC-I)]. Extensive phenotypic analysis of heterozygous (3N) Dp(MHC-I) animals did not reveal altered blood and stem cell parameters, susceptibility to high-fat diet, death by cancer, or contact dermatitis. However, several measures of disease severity in a model of atherosclerosis were improved, suggesting dosage-sensitive modulators of cardiovascular disease. Homozygous Dp(MHC-I)/Dp(MHC-I) mice demonstrated embryonic lethality. These mice serve as a model for studying the consequences of targeted gene dosage alteration in MHC-I with functional and evolutionary implications.

The identification of susceptibility genes for human disease is a major goal of current biomedical research. Both sequence and structural variation have emerged as major genetic sources of phenotypic variability and growing evidence points to copy number variation as a particularly important source of susceptibility for disease. Here we propose and validate a strategy to identify genes in which changes in dosage alter susceptibility to disease-relevant phenotypes in the mouse. Our approach relies on sensitized phenotypic screening of megabase-sized chromosomal deletion and deficiency lines carrying altered copy numbers of similar to 30 linked genes. This approach offers several advantages as a method to systematically identify genes involved in disease susceptibility. To examine the feasibility of such a screen, we performed sensitized phenotyping in five therapeutic areas (metabolic syndrome, immune dysfunction, atherosclerosis, cancer and behaviour) of a 0.8 Mb reciprocal chromosomal duplication and deficiency on chromosome 11 containing 27 genes. Gene dosage in the region significantly affected risk for high-fat diet-induced metabolic syndrome, antigen-induced immune hypersensitivity, ApoE-induced atherosclerosis, and home cage activity. Follow up studies on individual gene knockouts for two candidates in the region showed that copy number variation in Stat5 was responsible for the phenotypic variation in antigen-induced immune hypersensitivity and metabolic syndrome. These data demonstrate the power of sensitized phenotypic screening of segmental aneuploidy lines to identify disease susceptibility genes.

Udgivelsesdato: 2007-Feb-21 The C/EBPalpha transcription factor regulates hepatic nitrogen, glucose, lipid and iron metabolism. However, how it is able to independently control these processes is not known. Here, we use mouse knock-in mutagenesis to identify C/EBPalpha domains that specifically regulate hepatic gluconeogenesis and lipogenesis. In vivo deletion of a proline-histidine rich domain (PHR), dephosphorylated at S193 by insulin signaling, dysregulated genes involved in the generation of acetyl-CoA and NADPH for triglyceride synthesis and led to increased hepatic lipogenesis. These promoters bound SREBP-1 as well as C/EBPalpha, and the PHR was required for C/EBPalpha-SREBP transcriptional synergy. In contrast, the highly conserved C/EBPalpha CR4 domain was found to undergo liver-specific dephosphorylation of residues T222 and T226 upon fasting, and alanine mutation of these residues upregulated the hepatic expression of the gluconeogenic G6Pase and PEPCK mRNAs, but not PGC-1alpha, leading to glucose intolerance. Our results show that pathway-specific metabolic regulation can be achieved through a single transcription factor containing context-sensitive regulatory domains, and indicate C/EBPalpha phosphorylation as a PGC-1alpha-independent mechanism for regulating hepatic gluconeogenesis.

In this paper, we describe the unexpected outgrowth of B lineage cells from PU.1−/− fetal liver cultures. The cells express all early B cell genes tested, including the putative PU.1 target genes IL-7R and EBF but not B220, and can produce immunoglobulin M. However, we observed a delay in the PU.1−/− B cell outgrowth and reduced precursor frequencies, indicating that although PU.1 is not strictly required for B cell commitment, it facilitates B cell development. We also ablated PU.1 in CD19-expressing B lineage cells in vivo, using a Cre-lox approach that allows them to be tracked. PU.1 excision resulted in a shift from B-2 cells to B-1–like cells, which dramatically increased with the age of the mice. Our data indicate that this shift is predominantly caused by a B-2 to B-1 cell reprogramming. Furthermore, we found that B-2 cells express substantially more PU.1 than B-1 cells, which is consistent with the idea that maintenance of the B-2 cell phenotype requires relatively high levels of PU.1, but B-1 cells require little.

Newcastle disease virus (NDV) infects domesticated and wild birds throughout the world and has the possibility to cause outbreaks in chicken flocks in future. To assess the evolutionary characteristics of 10 NDV strains isolated from chickens in Kazakhstan during 1998 we investigated the phylogenetic relationships among these viruses and viruses described previously. For genotyping, fusion (F) gene phylogenetic analysis (nucleotide number 47-421) was performed using sequences of Kazakhstanian isolates as compared to sequences of selected NDV strains from GenBank. Phylogenetic analysis showed that all newly characterized strains belonged to the genetic group designated as VIIb. All strains possessed a virulent fusion cleavage site (RRQRR/F) belonging to velogenic or mesogenic pathotypes with intracerebral pathogenicity indexes (ICPI) varying from 1.05 to 1.87.

In Saccharomyces cerevisiae, the initiation of DNA replication is sequence specific (27; for a review, see reference 23). In mammalian cells, however, the mechanism underlying this process is still being defined. Although specific origins of replication have been mapped, initiation regions as large as 50 kb have also been described (for reviews, see references 20 and 22), making the potential role of sequence specificity less clear. In mammalian cells, the origins of DNA replication are activated in different intervals of S phase and the timing of the activation of these origins is precisely regulated. The regulation of the replication timing of a specific gene appears to reflect the transcriptional activity of surrounding sequences (for a review, see reference 58). Evidence that shows that the localization of some genes or even of an entire chromosome within the nucleus is nonrandom and is linked both to chromatin structure and replication timing has been presented (9, 16, 41; for a review, see reference 14). Thus, early- and late-replication foci tend to occupy different subnuclear domains in interphase cells (for a review, see reference 28). Recent observations suggest that nuclear structure might play an important role in defining when and where DNA replication initiates (for a review, see reference 21). The immunoglobulin heavy-chain (Igh) locus (consisting of, in the 5′-to-3′ direction, 15 heavy-chain variable [Vh] gene families, 12 heavy-chain diversity [Dh] segments, 4 heavy-chain joining [Jh] segments, an intronic enhancer, 8 constant-region [Ch] genes, and the 3′ regulatory region) is an important model system for the study of mammalian DNA replication. The Igh locus shows successive changes in transcription and DNA rearrangements during B-cell development (Fig. ​(Fig.1).1). V(D)J joining occurs at the pro-B-cell stage, preceded by transcriptional activation of Vh genes. Class switching occurs later in B-cell development. These processes result in unique combinations of V, D, J, and C segments encoding and transcribing individual heavy-chain (and light-chain) genes in individual B cells (for a review, see reference 45). B-cell lines representing different stages of B-cell development are available for the study of the replication and nuclear localization of the Igh locus. The cell lines used in the present study are listed in Table ​Table1.1. The mouse Igh locus is very large (approximately 3 Mb) but has been physically mapped and cloned in yeast artificial chromosome and bacterial artificial chromosome (BAC) arrays (13) and is being sequenced. The information derived from these arrays greatly facilitated the present study. FIG. 1. Rearrangements of the Igh locus during B-cell development.The germ line configuration of the Igh locus on murine chromosome 12 is shown (44, 57). In the 63-12 RAG2−/− pro-B-cell line, the entire locus is unrearranged and in germ line configuration, ... TABLE 1. Characteristics of B-lineage cell lines used in this study Previous studies using bromodeoxyuridine (BrdU) S-phase fractionation have shown that the replication timing of the Igh locus in non-B-cell lines, such as the murine erythroleukemia (MEL) cell line, is different from that in pre-B-cell lines (11, 34, 35). In MEL cells, an ∼400-kb sequence forms a transition region between an early-replication domain downstream of the 3′ regulatory region and a late-replication domain containing Vh genes (triphasic-replication pattern) (Fig. ​(Fig.2A).2A). Sequences within this transition region replicate 3′ to 5′ progressively later during S. A previous study using neutral-alkaline two-dimensional (N/A 2D) gel electrophoresis has shown that the transition region is formed by a single replicon through which replication forks progress in the 3′-to-5′ direction (25). In contrast, the entire Igh locus is replicated within the first hour of S in two pre-B-cell lines. Previous studies also demonstrated that part of the Igh-C region replicates early in S in plasma cell lines (34). FIG. 2. Replication timing of the murine Igh locus in B-lineage cell lines. The time in S phase when a particular segment is replicated is expressed in terms of the C value (y axis) and is plotted versus its distance in kilobases from the Cα gene (x axis), ... One explanation for the dramatically different replication programs detected in non-B cells and pre-B cells is that the DNA replication program is linked to the transcriptional status of the Igh locus. In MEL cells, the Igh locus is transcriptionally silent, while in pre-B-cell lines, this locus is actively transcribed. Since the pattern of transcription along the locus changes during B-cell development, we decided to examine the replication of the Igh locus in B-cell lines at different stages of B-cell development. The experiments presented here reveal two distinct programs of replication for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines, including those derived from RAG2−/− mice, the entire Igh locus is replicated early in S phase. The temporal pattern of Igh replication in other cell lines representing late stages of B-cell development (an immature-B-cell line and plasma cell lines) resembles that in non-B cells. The temporal-transition regions in the different cell lines are similar in length but encompass different sequences, reflecting individual DNA rearrangement events associated with VDJ and class switch recombination. The replication timing of the Igh locus in ex vivo-expanded pro-B cells and lipopolysaccharide (LPS)-stimulated spleen B cells is consistent with the data obtained from the cell lines. In this study, we address the potential mechanism for this dramatic difference in replication programs. N/A 2D gel electrophoresis demonstrates that replication forks progress in both directions through the examined Igh-C genes in cell lines in which the entire locus replicates early in S. Our results suggest that the early replication of the Igh locus is achieved through the activation of one or more replication origins. In contrast, in B cells in which the Igh locus replicates through a triphasic pattern, replication forks progress in a single 3′-to-5′ direction (opposite to the transcriptional orientation) through all the constant-region genes we examined. Our observations that the directions of replication fork progression can differ in B-lineage cell lines raise the possibility that changes in Igh replication are involved in processes of Igh rearrangement and expression. We have found that, associated with the changes in replication, the entire Igh locus is located away from the nuclear periphery when it is replicated early in S phase but is located near the nuclear periphery when it is replicated in a triphasic pattern.

We have previously shown that replication forks stall at a family of repeated sequences (FR) within the Epstein-Barr virus latent origin of replication oriP, both in a small plasmid and in the intact Epstein-Barr virus genome. Each of the 20 repeated sequences within the FR contains a binding site for Epstein-Barr nuclear antigen 1 (EBNA-1), the only viral protein required for latent replication. We showed that the EBNA-1 protein enhances the accumulation of paused replication forks at the FR. In this study, we have investigated a series of truncated EBNA-1 proteins to determine the portion of the EBNA-1 protein that is responsible for pausing of forks at the FR. Two-dimensional agarose gel electrophoresis was performed on the products of in vitro replication reactions in the presence of full-length EBNA-1 or proteins with various deletions to assess the extent of fork pausing at the FR. We conclude that a portion of the DNA binding domain is important for fork pausing. We also present evidence indicating that phosphorylation of the EBNA-1 protein or EBNA-1-truncated derivatives is not essential for pausing. To investigate the mechanism of EBNA-1-mediated pausing of replication forks, we asked whether EBNA-1 could inhibit the DNA unwinding activity of replicative helicases. We found that EBNA-1, when bound to the FR, inhibits DNA unwinding in vitro by SV40 T antigen and Escherichia coli dnaB helicases in an orientation-independent manner.

We have previously observed that replication and nuclear location of the murine Igh locus are developmentally regulated during B cell differentiation. In non-B, B, and plasma cells, sequences near the 3′ end of the Igh locus replicate early in S while upstream Vh sequences replicate late in S, and the Igh locus is located near the nuclear periphery. In fact, in MEL non-B cells, replication of a 500-kb segment containing Igh-C and flanking sequences occurs progressively later throughout S by 3′ to 5′ unidirectional fork movement. In contrast, in pro- and pre-B cells, the entire 3-Mb Igh locus is located away from the nuclear periphery and replicates early in S by forks progressing in both directions. In this study, using an 18-81 (pre-B) × BW5147 (T) cell fusion system in which Igh expression is extinguished, we found that in all Igh alleles, Vh sequences replicated later in S than 3′ Igh sequences (similar to that detected in BW5147), but the Igh locus was situated away from the nuclear periphery (similar to that observed in 18-81). Thus, pre-B cell-derived Igh genes had changes in replication timing, but not in nuclear location, whereas T cell-derived Igh genes changed their nuclear location but not their replication timing. These data are consistent with the silencing of a pre-B cell-specific replication program in the fusion hybrid cells and independent regulation of the nuclear location of Igh loci.

We previously described a mouse line that contains green myelomonocytic cells due to the knock-in of enhanced green fluorescence protein (EGFP) into the lysozyme M gene.1 We have now created a transgenic line with fluorescent erythroid cells using a β-globin locus control region driving the enhanced cyan fluorescence protein (ECFP) gene. These mice exhibit cyan fluorescent cells specifically in the erythroid compartment and in megakaryocyte-erythroid progenitors. Crossing the animals with lysozyme EGFP mice yielded a line in which live erythroid and myeloid cells can readily be distinguished by fluorescence microscopy and by fluorescence-activated cell-sorter scanner. This cross allowed unambiguous identification of unstained mixed erythroid-myeloid colonies for the first time. The new mouse lines should become useful tools to dissect the branching between erythroid and myelomonocytic cells during in vitro differentiation of definitive multipotent progenitors.

International audience; In non–B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, Ca, replicates early in S; other heavy chain con- stant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from Ca. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for ~400 kb, beginning downstream of the IgH3' regulatory region and continuing to the Dregion, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be acheived by a single replication fork.

DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.