Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In ...this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.
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► PRC1 interacts with Runx1 and CBFβ ► PRC1 and Runx1/CBFβ colocalize on chromatin and coregulate a subset of genes ► PRC1 and Runx1/CBFβ deficiencies generate partial phenocopies of one another in vivo ► Runx1 directly recruits PRC1-independent of PRC2
In zebrafish, hematopoietic stem cells (HSCs) are born in the developing aorta during embryogenesis. From the definitive wave of hematopoiesis onward, blood homeostasis relies on self-renewal and ...differentiation of progeny of existing HSCs, or clones, rather than de novo generation. Here, we describe an approach to quantify the number and size of HSC clones at various times throughout the lifespan of the animal using a fluorescent, multicolor labeling strategy. The system is based on combining the multicolor Zebrabow system with an inducible, early lateral plate mesoderm and hematopoietic lineage specific cre driver (draculin (drl)). The cre driver can be temporally controlled and activated in early hematopoiesis to introduce a color barcoding unique to each HSC and subsequently inherited by their daughter cells. Clonal diversity and dominance can be investigated in normal development and blood disease progression, such as blood cancers. This adoptable method allows researchers to obtain quantitative insight into clonality-defining events and their contribution to adult hematopoiesis.
The zebrafish (Danio rerio) is among the most widely used model animals in scientific research. Historically, these fish have been reared in the laboratory using simple methods developed by home ...aquarists. For laboratories with high demand for breeding and generation turn-over, however, there has been a shift away from this approach towards one that leverages techniques, tools, and feeds from commercial aquaculture to help accelerate growth rates and decrease generation times. While these advances have improved efficiency, the effects of feeding zebrafish diets that are designed to grow disparately related cold-water fish species to market size quickly are not well-understood. To explore the impacts that intensive feeding protocols may have on this species, groups of zebrafish larvae from two different wild-type lines were stocked into treatment tanks at a standard density of 10 fish per liter and were administered either a "high" or "low" food diet for a maximum of 63 days. During their growth phase, the "high" food diet group received at least 8x more rotifers and at least 2x more Artemia than the "low" food diet group. Growth, survival, and reproductive performance (fecundity and viability) were measured in these fish and in their offspring. We found that fish that were fed more grew more rapidly and were able to reproduce earlier than fish that were fed less, but they were also more likely to produce higher proportions of non-viable embryos.
Melanocytes in development and cancer Uong, Audrey; Zon, Leonard I.
Journal of cellular physiology,
January 2010, Letnik:
222, Številka:
1
Journal Article
The restrictive nature of the blood–brain barrier (BBB) creates a major challenge for brain drug delivery with current nanomedicines lacking the ability to cross the BBB. Extracellular vesicles (EVs) ...have been shown to contribute to the progression of a variety of brain diseases including metastatic brain cancer and have been suggested as promising therapeutics and drug delivery vehicles. However, the ability of native tumor-derived EVs to breach the BBB and the mechanism(s) involved in this process remain unknown. Here, we demonstrate that tumor-derived EVs can breach the intact BBB in vivo, and by using state-of-the-art in vitro and in vivo models of the BBB, we have identified transcytosis as the mechanism underlying this process. Moreover, high spatiotemporal resolution microscopy demonstrated that the endothelial recycling endocytic pathway is involved in this transcellular transport. We further identify and characterize the mechanism by which tumor-derived EVs circumvent the low physiologic rate of transcytosis in the BBB by decreasing the brain endothelial expression of rab7 and increasing the efficiency of their transport. These findings identify previously unknown mechanisms by which tumor-derived EVs breach an intact BBB during the course of brain metastasis and can be leveraged to guide and inform the development of drug delivery approaches to deliver therapeutic cargoes across the BBB for treatment of a variety of brain diseases including, but not limited to, brain malignancies.
Enhancers are the primary determinants of cell identity, but the regulatory components controlling enhancer turnover during lineage commitment remain largely unknown. Here we compare the enhancer ...landscape, transcriptional factor occupancy, and transcriptomic changes in human fetal and adult hematopoietic stem/progenitor cells and committed erythroid progenitors. We find that enhancers are modulated pervasively and direct lineage- and stage-specific transcription. GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. Examination of lineage-specific enhancers identifies transcription factors and their combinatorial patterns in enhancer turnover. Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription. Thus, genome-wide enhancer profiling coupled with in situ enhancer editing provide critical insights into the functional complexity of enhancers during development.
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•Pervasive changes in enhancer landscapes occur during blood stem cell specification•Analysis of developmentally regulated enhancers uncover driver TFs and combinations•Genomic editing reveals functional hierarchy of super-enhancer constituents•Functionally divergent GATA switch enhancers cooperate within enhancer clusters
Enhancers are crucial determinants of cell identity. Huang et al. compare enhancer landscapes in human hematopoietic stem/progenitor cells and erythroid progenitors. They uncover driver transcription factors and their combinatorial patterns in enhancer turnover. Genomic editing of constituent enhancers reveals functional hierarchy and diversity of enhancer clusters during hematopoiesis.
Transcriptome analysis of adult hematopoietic stem cells (HSCs) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is ...lacking. Here, we acquired the transcriptomes of developing HSCs purified from >2,500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network-biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knockdown in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSCs, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification.
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► Gene expression profiling of hematopoietic stem cells during embryonic development ► Expression profiles used to construct gene regulatory networks ► Multiple regulators of HSC development implicated ► ESC-derived HSCs resemble definitive HSCs but lack a Notch-signaling signature
Network-based analysis of HSC development using transcriptome profiling reveals stage-specific developmental regulators and differences relative to HSCs derived from ESCs.
The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry ...oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.
The most common mutation in human melanoma, BRAF(V600E), activates the serine/threonine kinase BRAF and causes excessive activity in the mitogen-activated protein kinase pathway. BRAF(V600E) ...mutations are also present in benign melanocytic naevi, highlighting the importance of additional genetic alterations in the genesis of malignant tumours. Such changes include recurrent copy number variations that result in the amplification of oncogenes. For certain amplifications, the large number of genes in the interval has precluded an understanding of the cooperating oncogenic events. Here we have used a zebrafish melanoma model to test genes in a recurrently amplified region of chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to accelerate melanoma formation significantly in zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing and gene expression analyses uncovered genes, including HOX genes, that are transcriptionally dysregulated in response to increased levels of SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.
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