Antisense Targeting of CXXC Finger Protein 1 Inhibits Genomic Cytosine Methylation and Primitive Hematopoiesis in Zebrafish

Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2007; 281(48):37034-44. DOI: 10.1074/jbc.M604546200
Source: PubMed
CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is a component of the Set1 histone methyltransferase complex. Mice lacking CFP1 suffer a peri-implantation lethal phenotype, and CFP1-deficient embryonic stem cells are viable but unable to differentiate and exhibit a 60-80% decrease in genomic cytosine methylation. A zebrafish homolog of CFP1 has been identified, is approximately 70% similar to murine CFP1, and is widely expressed during development. Zebrafish embryos treated with a zCFP1 antisense morpholino oligonucleotide had little or no circulating red blood cells and exhibited abnormal yolk sac morphology at 48 h post-fertilization. Many of the antisense-treated zebrafish also exhibited cardiac edema, and 14% were dead at 24 h post-fertilization. Morphant zebrafish also exhibited elevated levels of apoptosis, particularly in the intermediate cell mass, the site of primitive erythropoiesis, as well as aberrations in vascular development. Genomic DNA isolated from morphant embryos exhibited a 60% reduction of global genomic cytosine methylation. A similar phenotype was observed with an independent zCFP1 antisense morpholino oligonucleotide, but not following injection of an unrelated control oligonucleotide. The morphant phenotype was rescued when mRNA encoding murine CFP1 was co-injected with the antisense oligonucleotide. Genomic data base analysis reveals the presence of a second version of zebrafish CFP1 (zCFP1b). However, the morphant phenotype observed following specific depletion of zCFP1 indicates that these related genes have nonredundant functions controlling normal zebrafish hematopoiesis and epigenetic regulation. These findings establish the importance of CFP1 during postgastrulation development.

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    • "For instance, osr1 is required for the development of hemangioblast [35] . Specific depletion of cxxc1, which was identified as a commonly down-regulated genes among the four DBA zebrafish models, inhibited genomic cytosine methylation and primitive hematopoiesis in zebrafish [36]. It has been demonstrated that deficiency of dkc1 in zebrafish led to reduced definitive hematopoiesis mediated by P53 [37]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Diamond–Blackfan anemia (DBA) was the first ribosomopathy associated with mutations in ribosome protein (RP) genes. The clinical phenotypes of DBA include failure of erythropoiesis, congenital anomalies and cancer predisposition. Mutations in RPL5 are reported in approximately 9 ~ 21 % of DBA patients, which represents the most common pathological condition related to a large-subunit ribosomal protein. However, it remains unclear how RPL5 downregulation results in severe phenotypes of this disease. Results In this study, we generated a zebrafish model of DBA with RPL5 morphants and implemented high-throughput RNA-seq and ncRNA-seq to identify key genes, lncRNAs, and miRNAs during zebrafish development and hematopoiesis. We demonstrated that RPL5 is required for both primitive and definitive hematopoiesis processes. By comparing with other DBA zebrafish models and processing functional coupling network, we identified some common regulated genes, lncRNAs and miRNAs, that might play important roles in development and hematopoiesis. Conclusions Ribosome biogenesis and translation process were affected more in RPL5 MO than in other RP MOs. Both P53 dependent (for example, cell cycle pathway) and independent pathways (such as Aminoacyl-tRNA biosynthesis pathway) play important roles in DBA pathology. Our results therefore provide a comprehensive basis for the study of molecular pathogenesis of RPL5-mediated DBA and other ribosomopathies. Electronic supplementary material The online version of this article (doi:10.1186/s12920-016-0174-9) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016 · BMC Medical Genomics
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    • "Furthermore, treatment of HSCs with inhibitors of DNA methyltransferases (azacytidine) and histone deacetylases (trichostatin A, valproic acid) leads to altered cell fate and an expansion ex vivo of HSCs that retain bone marrowrepopulating potential [51, 52, 53, 54, 55]. Cfp1 physically interacts with Dnmt1 [10] and facilitates maintenance cytosine methylation, and a decline in global genomic cytosine methylation is the most obvious molecular defect observed in ES cells depleted of Cfp1 [9, 19]. However, apoptosis of hematopoietic cells following Cfp1 depletion occurs without a change in global cytosine methylation levels. "
    [Show abstract] [Hide abstract] ABSTRACT: CXXC finger protein 1 (Cfp1), encoded by the Cxxc1 gene, binds to DNA sequences containing an unmethylated CpG dinucleotide and is an epigenetic regulator of both cytosine and histone methylation. Cxxc1-null mouse embryos fail to gastrulate, and Cxxc1-null embryonic stem cells are viable but cannot differentiate, suggesting that Cfp1 is required for chromatin remodeling associated with stem cell differentiation and embryogenesis. Mice homozygous for a conditional Cxxc1 deletion allele and carrying the inducible Mx1-Cre transgene were generated to assess Cfp1 function in adult animals. Induction of Cre expression in adult animals led to Cfp1 depletion in hematopoietic cells, a failure of hematopoiesis with a nearly complete loss of lineage-committed progenitors and mature cells, elevated levels of apoptosis, and death within two weeks. A similar pathology resulted following transplantation of conditional Cxxc1 bone marrow cells into wild type recipients, demonstrating this phenotype is intrinsic to Cfp1 function within bone marrow cells. Remarkably, the Lin-Sca-1+c-Kit+ population of cells in the bone marrow, which is enriched for hematopoietic stem cells and multi-potential progenitor cells, persists and expands in the absence of Cfp1 during this time frame. Thus, Cfp1 is necessary for hematopoietic stem and multi-potential progenitor cell function and for the developmental potential of differentiating hematopoietic cells.
    Full-text · Article · Dec 2014 · PLoS ONE
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    • "Mice lacking CFP1 exhibit peri-implantation lethality (4.5– 6.5 days postcoitus) (Carlone and Skalnik, 2001 ). CFP1 is additionally required for postgastrulation development and survival, because zebrafish embryos injected with antisense morpholino oligonucleotides directed against CFP1 exhibit defective primitive hematopoiesis and decreased survival (Young et al., 2006). In addition, siRNA-mediated depletion of CFP1 in human leukemia cell lines leads to defects in cell proliferation and terminal myeloid differentiation (Young and Skalnik, 2007 ). "
    [Show abstract] [Hide abstract] ABSTRACT: CXXC finger protein 1 (CFP1) is a component of the Setd1A and Setd1B methyltransferase complexes, localizes to euchromatic regions of the genome, and specifically binds unmethylated CpG dinucleotides in DNA. Murine embryos lacking CFP1 exhibit peri-implantation lethality, a developmental time that correlates with global epigenetic reprogramming. CFP1-deficient embryonic stem (ES) cells exhibit a 70% reduction in global cytosine methylation and a 60% decrease in maintenance DNA methyltransferase (DNMT1) activity. DNMT1 protein level is reduced 50% in CFP1-deficient ES cells. Experiments were performed to investigate the role of CFP1 in regulating maintenance cytosine methylation. Coimmunoprecipitation experiments reveal that endogenous DNMT1 and CFP1 interact in vivo. Protein regions required for the interaction between DNMT1 and CFP1 were mapped. Amino acids 169-493 and 970-1617 of DNMT1 are each sufficient for interaction with CFP1. Three regions spanning the CFP1 protein, amino acids 1-123, 103-367, and 361-656, are each sufficient for interaction with DNMT1. Interestingly, a single-point mutation (C375A) within CFP1 that abolishes the interaction with the Setd1A and Setd1B histone H3K4 methyltransferase complexes does not disrupt the interaction between CFP1 and DNMT1. This result indicates that CFP1 intersects the cytosine methylation machinery independently of its association with the Setd1 complexes.
    Preview · Article · Sep 2008 · DNA and cell biology
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