There are at least three distinct MAP kinase signaling modules in mammalian cells, distinguished by the family of kinases (Erk, SAPK/JNK, or p38) that is ultimately activated. Many input signals ...activate multiple MAP kinase cascades, and the mechanisms that control the specificity of signal output are not well understood. We show that SEK1/MKK4, a MAP kinase kinase proposed to activate SAPK/JNK, is a very potent inhibitor of p54 SAPK beta/JNK3 both in vitro and in vivo if present at equimolar or higher ratios. In contrast SEK can activate SAPK when present in substoichiometric amounts, but this activation is slow, consistent with the rate-limiting step in activation being the dissociation of an inactive SEK:SAPK complex. The N-terminal unique region of SEK is both necessary and partially sufficient for inhibition of SAPK, and is also necessary for activation of SAPK by SEK in vitro. We have also used the p38 MAP kinase and its activator MKK6 to examine the regulatory relationships among different kinases involved in stress responses. We show using purified kinases that inhibitory activity is specific for the combination of SEK and SAPK: SEK can activate but not inhibit p38, and MKK6 can activate but not inhibit SAPK beta and p38. These results reveal a potential mechanism for regulating stress-activated kinases, adding to a growing body of evidence suggesting that MAP kinases are controlled by relatively stable interactions with their activators.
We have isolated a previously unknown human homeobox-containing cDNA, VENT-like homeobox-2 (VENTX2), using PCR with a bone marrow cDNA library and primers designed from the VENTX1 (alias HPX42) ...homeobox sequence. Here we describe the molecular cloning, chromosomal localization to 10q26.3, and functional analysis of this gene. The 2.4-kb human VENTX2 cDNA encoded a protein with a predicted molecular weight of 28 kDa containing a homeodomain with 65% identity to the Xenopus laevis ventralizing gene Xvent2B. VENTX2 antisera detected a 28-kDa protein in cells transfected with a VENTX2 expression construct, in a human erythroleukemic cell line and in bone marrow samples obtained from patients in recovery phase after chemotherapy. The similarity of the homeodomains from VENTX2 and the X. laevis Vent gene family places them in the same homeodomain class. Consistent with this structural classification, overexpression of VENTX2 in zebrafish embryos led to anterior truncations and failure to form a notochord, which are characteristics of ventralization.
Recent studies have shown that members of the angiopoietin-like protein (angptl) family can help stimulate ex vivo expansion of mouse and human hematopoietic stem cells (HSCs). The requirement of ...angptl for HSC development has yet to be determined. Here, the effects of morpholino (MO) knockdown of angptl1, angptl2 or the combination of both, were assessed during zebrafish development. Single MO knockdown of either angptl1 or angptl2 resulted in a decrease in c-myb positive HSCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis at 36 hours post-fertilization (hpf). Embryos injected with combined angptl1 and angptl2 MOs (double morphants) had an even more severe phenotype, in which both c-myb and runx1 positive HSCs were almost completely absent. In an effort to discern the onset of the phenotype, early HSC markers were evaluated. The double morphants had decreased expression of scl and lmo2 at 8–10 somite stages (ss) in the bilateral stripes of the lateral mesoderm region from which hematopoietic and vascular progenitors are thought to arise. To determine if angptl are sufficient to expand HSCs, angptl1 and angptl2 mRNA were injected into one-cell stage embryos and HSC markers were evaluated. Angptl1 and angptl2 mRNA significantly increased c-myb and runx1 positive HSCs in the AGM. Interestingly, addition of thrombopoietin, a potent stem cell growth factor, with angptl1 and angptl2 further increased HSCs in the AGM, implying a synergistic effect of these growth factors during HSC development. Angptl regulation of HSC development may occur by stimulating the production of the hemogenic endothelium. MO knockdown of either angptl1 or angptl2 alone did not alter vascular development significantly, while MO knockdown of both angptl1 and angptl2 resulted in a disruption of intersegmental blood vessel sprouting at 28 hpf. The arterial and venous specification in these double morphants was also disrupted at 28 hpf, with decreased expression of the arterial marker ephrinB2 but increased ectopic expression of the venous marker, flt4. These results strongly suggest an early downstream effect of angptl signaling on hemogenic endothelium. Furthermore, since Vegf signaling is indispensable for proper endothelial specification and definitive HSC formation, we found that in the double morphants, somite expression of vegf165 mRNA was decreased early in development. Although angptl1 mRNA expression was undetectable at this stage, angptl2 mRNA expression was localized in the yolk sac extension and posterior spinal cord, appearing in close proximity to the vegf positive somites. This suggests that in these locations, angptl may stimulate Vegf production in the embryo to participate in the induction of hemogenic endothelium and subsequently lead to HSC formation. Taken together, our data demonstrates that the angptl are required for HSC development by coordinating the production of a functional hemogenic endothelium that can lead to the formation of HSCs.
Abstract
Within a group of cancer-prone cells that harbor a shared oncogenic mutation, only rare clones transition to a malignant state. These rare and transient events occurring during cancer ...initiation remain incompletely understood, and we thus sought to visualize and molecularly characterize this crucial early step in oncogenesis of melanoma. We previously found that the zebrafish crestin gene, which specifically marks embryonic neural crest and normally turns off at three days of life, is specifically re-expressed in BRAFV600E/p53 mutant melanoma tumors in adult zebrafish. We cloned the crestin promoter/enhancer and developed an EGFP reporter that recapitulates the embryonic expression pattern of crestin mRNA and, crucially, marks de novo melanomas in living BRAFV600E/p53 mutant zebrafish. Remarkably, we also found that crestin:EGFP becomes active when lesions are only a few cells in number, potentially in the first cell of the melanoma. These crestin:EGFP positive patches are transplantable, and these precursor lesions are enriched for expression of neural crest progenitor (NCP) genes including the transcription factors sox10 and dlx2a, among other melanoma- and NCP-associated genes. In order to favor the readoption of an NCP-state, we forced misexpression of sox10 in melanocytes of BRAFV600E/p53 melanoma-prone zebrafish, and this led to enhanced melanoma formation. We then analyzed the chromatin landscape of both human and zebrafish melanoma cells using ChIP-Seq and ATAC-Seq and identified super-enhancers at crestin (in zebrafish), sox10, dlx2a, and other NCP/melanoma loci, which together described an overall chromatin signature consistent with features of the NCP state. These data support a model in which 1) an NCP program stochastically reactivates, as read out by crestin expression, in rare BRAFV600E/p53 mutated melanocytes at the initiation of melanoma formation and 2) reemergence of this NCP state is an important and potentially rate-limiting event in melanoma initiation. We anticipate that progenitor identity reemergence will prove to be a general feature of cancer initiation.
This abstract is also presented as Poster B19.
Citation Format: Charles K. Kaufman, Christian Mosimann, Andrew Thomas, Zi Peng Fan, Song Yang, Justin Tan, Rachel D. Fogley, Ellen van Rooijen, Elliott Hagedorn, Christie Ciarlo, Richard White, Dominick Matos, Ann-Christin Puller, Cristina Santoriello, Eric Liao, Richard A. Young, Leonard I. Zon. The reemergence of neural crest progenitor identity is a key event in the initiation of melanoma from a field of cancer-prone melanocytes. abstract. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR06.
1 Howard Hughes Medical Institute, Boston, Massachusetts 02115
2 Childrens Hospital, Division of Hematology/Oncology, Boston, Massachusetts 02115
3 Dana-Farber Cancer Institute, Boston, ...Massachusetts 02115
4 Harvard Medical School, Boston, Massachusetts 02115
5 Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403
6 Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan
Vertebrate hematopoiesis is regulated by distinct cell-specific transcription factors such as GATA-1 and SCL. Mammalian p45-NFE2 was characterized for its ability to bind the hypersensitive sites of the globin locus control region. NFE2 is a member of a capncollar (CNC) and basic zipper (BZIP) superfamily that regulates gene transcription. It has been implicated in diverse processes such as globin gene expression, oxidative stress, and platelet lineage differentiation. Here, we have isolated the zebrafish ortholog of NFE2. The gene is highly homologous, particularly in the DNA-binding domain. Mapping the zebrafish NFE2 to linkage group 23 establishes a region of chromosomal synteny with human chromosome 12, further suggesting evolutionary conservation. During embryogenesis, the zebrafish gene is expressed specifically in erythroid cells and also in the developing ear. NFE2 expression is lacking in zebrafish mutants that have no hematopoietic cells. An analysis of the sauternes mutant, which carries a mutation in the ALAS-2 gene and thus has defective heme synthesis, demonstrates higher levels of NFE2 expression than normal. This further establishes the block to erythroid differentiation in the sauternes mutant. Our studies demonstrate conservation of the vertebrate genetic program for the erythroid lineage.
hematopoiesis; transcription factors; erythroid lineage; sauternes mutant
In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this ...process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II Beta binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.
Abstract
CXCL8 (IL-8) is a chemokine with pleiotropic roles in host defense, angiogenesis and tumor metastasis. CXCL8 and its specific receptor, CXCR1, are broadly expressed within the hematopoietic ...and vascular systems. In an effort to identify novel secreted factors with effects on hematopoietic stem and progenitor cell (HSPC) function, we have recently identified CXCL8/CXCR1 signaling as a positive regulator of HSPC colonization of the zebrafish caudal hematopoietic territory (CHT). The CHT is a vascular niche that serves as the primary site of hematopoiesis from 36 hours post fertilization (hpf) to 6 days post fertilization (dpf). This observation raised the question whether CXCR1 signaling might induce dynamic changes in the CHT that favor HSPC colonization. CXCR1 was expressed at high levels in endothelial cells using a kdrl(VEGFR2):CXCR1;kdrl:mCherry double transgenic line. The CHT was imaged by fluorescence confocal microscopy, reconstructed in 3 dimensions and the volume measured using digital image analysis software. Overexpression of CXCR1 within the endothelial cells of these animals increased the volume of the CHT by 28% (p = 0.02). To understand how CXCR1 affects the dynamics of niche development, we globally overexpressed CXCR1 beginning at 36 hpf using a heat shock induction system and performed time lapse confocal microscopy from 52 to 72 hpf. This revealed that overexpression of CXCR1 consistently increased the CHT volume from 53 to 72 hpf compared to control (21% increase at 72 hpf, p = 0.004). These studies did not show whether CXCR1 acted directly on the vascular niche or whether CXCR1 expression in endothelial cells might induce expression of soluble factors or activate circulating cells that then cause expansion of the niche through indirect mechanisms. To address this, we created parabiotic zebrafish by fusing kdrl:mCherry embryos at 4 hpf. One half of each parabiotic animal was modified by DNA microinjection to globally overexpress CXCR1 or GFP as a control via heat shock induction at 36 and 48 hpf. The volume of the CHT was measured in each half of each parabiotic animal at 72 hpf. In control parabiotics overexpressing GFP, there was no difference in CHT volume between modified and unmodified sides of the organism. However, in parabiotics overexpressing CXCR1, the CHT of the modified side was 27% larger compared with the unmodified side (p = 0.012), consistent with our previous results and suggesting that CXCR1 acts directly on the niche in this system. We then asked whether this volume change could affect HSPC engraftment. Parabiotic animals were created using Runx1:mCherry embryos that carry an HSPC-specific reporter transgene as “donors” and WT embryos as “recipients”. The recipient niche was modified as before to overexpress CXCR1 or GFP as a control. At 72 hpf there was no difference in HSPC colonization of donor and recipient niches when the recipient niche expressed GFP. However, when the recipient niche expressed CXCR1, there was a significant increase in HSPC colonization of the recipient niche compared to the donor niche (11.4+/-2.4 vs 19.8+/-3.5 HSPCs per CHT, p = 0.02). Taken together, these results identify a novel role for CXCL8/CXCR1 signaling in angiogenesis and HSPC biology and they provide a new example of how innate immune signaling pathways are important for stem cell function. Administration of CXCL8 to hematopoietic stem cell transplant recipients may therefore improve HSPC engraftment and clinical outcomes in patients who are being treated for hematologic malignancies.
Citation Format: Bradley W. Blaser, Jessica L. Moore, Elliott Hagedorn, Brian Li, Vera Binder, Owen Tamplin, Leonard I. Zon. CXCL8/CXCR1 signaling promotes angiogenesis and hematopoietic stem and progenitor cell function abstract. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B035.
Abstract 850
The molecular mechanisms governing HSC homeostasis and potency are incompletely understood. Runx1-CBFb is required for HSC formation in the embryo and for maintaining normal HSC and ...progenitor cell frequencies in the adult. To identify chromatin regulatory proteins that function with Runx1-CBFb during hematopoiesis, we performed co-immunoprecipitation and mass spectrometry using a FLAG-tagged CBFb protein, and as a control a FLAG-CBFb (G61A/N104A) mutant that cannot bind Runx1. Multiple unique peptides of the ATP-dependent chromatin remodeling enzyme Chromodomain Helicase DNA binding protein 7 (CHD7) were immunoprecipitated specifically with FLAG-CBFb. Autosomal dominant CHD7 mutations are associated with CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). Several subunits of the nucleosome remodeling and histone deacetylase (NuRD) complex were also identified, including p66a, p66b, MTA2, and histone binding protein RBBP4. Hence Runx1-CBFb may associate with distinct CHD7 and NuRD containing complexes to regulate HSC function.
The CHD7 interaction with Runx1 requires the Runx1 transactivation domain, which is critical for all stages of hematopoiesis. The interaction of CHD7 with Runx1 does not require heterodimer formation with CBFb. Runx1+/−; Chd7+/− double heterozygous mice are born in a sub-Mendelian ratio, indicating that Runx1 and Chd7 interact genetically. Knockdown of chd7 in zebrafish embryos increased the number of runx1 expressing cells specified in the AGM, and this effect is cell autonomous as determined by blastula transplantation. Downstream lineages such as myeloid and erythroid cells were also expanded. Consistent with these results, conditional pan-hematopoietic deletion of Chd7 in mice with Vav1-Cre did not cause mature lineage differentiation defects. Methylcellulose colony forming assays of CHD7 deficient cells resulted in increased granulocyte/monocyte progenitors. Although CHD7 deficient bone marrow had a normal frequency of CD48− CD150+ Lin−Sca-1+c-kit+(LSK) phenotypic long term repopulating HSCs (LT-HSCs), it has a three fold higher frequency of functional LT-HSCs as determined by whole bone marrow limiting dilution transplants. By serial transplantation, a higher percentage of tertiary recipient mice were engrafted with CHD7 deficient bone marrow cells compared to wild type. In summary, our study identifies CHD7 as a novel epigenetic regulator of HSC and progenitor function that functions with Runx1 and members of the NuRD complex to negatively regulate HSC numbers and potency.
Zon:Fate Therapeutics: Founder Other.
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