Role of RNA modifications in Blood development and Regeneration.

Blood development and regeneration require rapid turnover of cells, and RNA modifications play a key role in it via regulating stemness and cell fate regulation. RNA modifications effect gene activity via post-transcriptional and translation-mediated mechanisms. Diverse molecular players involved in RNA modification processes are abundantly expressed by hematopoietic stem cells and lineages. Close to 150 RNA chemical modifications have been reported, but only m6A, I, Ψ, and m1A, a handful have been studied in cell fate regulation. The role of RNA modification in blood diseases and disorders is an emerging field and offers potential for therapeutic interventions. Knowledge of RNA modification and enzymatic activities could be used to design therapies in the future. Here, we summarize the recent advances in RNA modification and the epitranscriptome field and discuss their regulation of blood development and regeneration.

BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells.

Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.

A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma.

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein (TIE:EGFP). TIE:EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single-cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of SATB2, a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages localize to TIE:EGFP+ regions and preferentially phagocytose TIE:EGFP+ melanoma cells compared to TIE:EGFP- melanoma cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.

Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

Specific oncogene activation of the cell of origin in mucosal melanoma.

Mucosal melanoma (MM) is a deadly cancer derived from mucosal melanocytes. To test the consequences of MM genetics, we developed a zebrafish model in which all melanocytes experienced CCND1 expression and loss of PTEN and TP53. Surprisingly, melanoma only developed from melanocytes lining internal organs, analogous to the location of patient MM. We found that zebrafish MMs had a unique chromatin landscape from cutaneous melanoma. Internal melanocytes could be labeled using a MM-specific transcriptional enhancer. Normal zebrafish internal melanocytes shared a gene expression signature with MMs. Patient and zebrafish MMs have increased migratory neural crest gene and decreased antigen presentation gene expression, consistent with the increased metastatic behavior and decreased immunotherapy sensitivity of MM. Our work suggests the cell state of the originating melanocyte influences the behavior of derived melanomas. Our animal model phenotypically and transcriptionally mimics patient tumors, allowing this model to be used for MM therapeutic discovery.

Colorimetric Barcoding to Track, Isolate, and Analyze Hematopoietic Stem Cell Clones.

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.

G-CSF-induced hematopoietic stem cell mobilization from the embryonic hematopoietic niche does not require neutrophils and macrophages.

Hematopoietic stem cell transplantation requires the collection of hematopoietic cells from patients or stem cell donors. Granulocyte colony-stimulating factor (G-CSF) is widely used in the clinic to mobilize hematopoietic stem and progenitor cells (HSPCs) from the adult bone marrow niche into circulation, allowing a collection of HSPCs from the blood. The mechanism by which G-CSF acts to mobilize HSPCs is unclear, with some studies showing a direct stimulation of stem cells and others suggesting that myeloid cells are required. In this study, we developed a heat-inducible G-CSF transgenic zebrafish line to study HSPC mobilization in vivo. Live imaging of HSPCs after G-CSF induction revealed an increase in circulating HSPCs, demonstrating a successful HSPC mobilization. These mobilized HSPCs went on to prematurely colonize the kidney marrow, the adult zebrafish hematopoietic niche. We eliminated neutrophils or macrophages using a nitroreductase-based cell ablation system and found that G-CSF still mobilizes HSPCs from the niche. Our findings indicate that neutrophils and macrophages are not required for G-CSF-induced HSPC mobilization from the embryonic hematopoietic niche.

MITF regulates IDH1 and NNT and drives a transcriptional program protecting cutaneous melanoma from reactive oxygen species.

Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo . Some of the MITF target genes involved, such as IDH1 and NNT , are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state. One Sentence Summary: MITF promote melanoma survival via increasing ROS tolerance.

Structural basis for inactivation of PRC2 by G-quadruplex RNA.

Polycomb repressive complex 2 (PRC2) silences genes through trimethylation of histone H3K27. PRC2 associates with numerous precursor messenger RNAs (pre-mRNAs) and long noncoding RNAs (lncRNAs) with a binding preference for G-quadruplex RNA. In this work, we present a 3.3-Å-resolution cryo-electron microscopy structure of PRC2 bound to a G-quadruplex RNA. Notably, RNA mediates the dimerization of PRC2 by binding both protomers and inducing a protein interface composed of two copies of the catalytic subunit EZH2, thereby blocking nucleosome DNA interaction and histone H3 tail accessibility. Furthermore, an RNA-binding loop of EZH2 facilitates the handoff between RNA and DNA, another activity implicated in PRC2 regulation by RNA. We identified a gain-of-function mutation in this loop that activates PRC2 in zebrafish. Our results reveal mechanisms for RNA-mediated regulation of a chromatin-modifying enzyme.

Transcription factors interact with RNA to regulate genes.

Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA binding contributes to TF function by promoting the dynamic association between DNA, RNA, and TF on chromatin. TF-RNA interactions are a conserved feature important for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA, and protein is a general property of many TFs and is fundamental to their gene regulatory function.

Microenvironmental control of hematopoietic stem cell fate via CXCL8 and protein kinase C.

Altered hematopoietic stem cell (HSC) fate underlies primary blood disorders but microenvironmental factors controlling this are poorly understood. Genetically barcoded genome editing of synthetic target arrays for lineage tracing (GESTALT) zebrafish were used to screen for factors expressed by the sinusoidal vascular niche that alter the phylogenetic distribution of the HSC pool under native conditions. Dysregulated expression of protein kinase C delta (PKC-δ, encoded by prkcda) increases the number of HSC clones by up to 80% and expands polyclonal populations of immature neutrophil and erythroid precursors. PKC agonists such as cxcl8 augment HSC competition for residency within the niche and expand defined niche populations. CXCL8 induces association of PKC-δ with the focal adhesion complex, activating extracellular signal-regulated kinase (ERK) signaling and expression of niche factors in human endothelial cells. Our findings demonstrate the existence of reserve capacity within the niche that is controlled by CXCL8 and PKC and has significant impact on HSC phylogenetic and phenotypic fate.

PGE2 alters chromatin through H2A.Z-variant enhancer nucleosome modification to promote hematopoietic stem cell fate.

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler et al. Blood 122, 3074-3081(2013); W. Goessling et al. Cell Stem Cell 8, 445-458 (2011); W. Goessling et al. Cell 136, 1136-1147 (2009)]. Here, we report that PGE2-induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE2 stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE2, lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation.

Transplantation-based screen identifies inducers of muscle progenitor cell engraftment across vertebrate species.

Stem cell transplantation presents a potentially curative strategy for genetic disorders of skeletal muscle, but this approach is limited by the deleterious effects of cell expansion in vitro and consequent poor engraftment efficiency. In an effort to overcome this limitation, we sought to identify molecular signals that enhance the myogenic activity of cultured muscle progenitors. Here, we report the development and application of a cross-species small-molecule screening platform employing zebrafish and mice, which enables rapid, direct evaluation of the effects of chemical compounds on the engraftment of transplanted muscle precursor cells. Using this system, we screened a library of bioactive lipids to discriminate those that could increase myogenic engraftment in vivo in zebrafish and mice. This effort identified two lipids, lysophosphatidic acid and niflumic acid, both linked to the activation of intracellular calcium-ion flux, which showed conserved, dose-dependent, and synergistic effects in promoting muscle engraftment across these vertebrate species.

Transcription factor induction of vascular blood stem cell niches in vivo.

The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses in zebrafish, we define a conserved gene expression signature and cis-regulatory landscape that are unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code that involves members of the Ets, Sox, and nuclear hormone receptor families and is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance, and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.

Oncogenic CDK13 mutations impede nuclear RNA surveillance.

RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 (CDK13) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease.

Identification of unique α4 chain structure and conserved antiangiogenic activity of α3NC1 type IV collagen in zebrafish.

BACKGROUND: Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1-α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. RESULTS: We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during the zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved antiangiogenic activity in human endothelial cells. CONCLUSIONS: Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain.

Clonal hematopoiesis and inflammation - the perpetual cycle.

Acquired genetic or cytogenetic alterations in a blood stem cell that confer clonal fitness promote its relative expansion leading to clonal hematopoiesis (CH). Despite a largely intact hematopoietic output, CH is associated with a heightened risk of progression to hematologic malignancies and with non-hematologic health manifestations, including cardiovascular disease and overall mortality. We focus on the evidence for the role of inflammation in establishing, maintaining and reciprocally being affected by CH. We describe the known pro-inflammatory signals associated with CH and preclinical studies that elucidated the cellular mechanisms involved. We review the evolving literature on early-onset CH in germline predisposition conditions and the possible role of immune dysregulation in this context.

The effects of intensive feeding on reproductive performance in laboratory zebrafish (Danio rerio).

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.