The Chromatin Remodeling Protein Bptf Promotes Posterior Neuroectodermal Fate by Enhancing Smad2-Activated wnt8a Expression.

During vertebrate embryogenesis, the neuroectoderm is induced from dorsal ectoderm and then partitioned into anterior and posterior neuroectodermal domains by posteriorizing signals, such as Wnt and fibroblast growth factor. However, little is known about epigenetic regulation of posteriorizing gene expression. Here, we report a requirement of the chromatin remodeling protein Bptf for neuroectodermal posteriorization in zebrafish embryos. Knockdown of bptf leads to an expansion of the anterior neuroectoderm at the expense of the posterior ectoderm. Bptf functionally and physically interacts with p-Smad2, which is activated by non-Nodal TGF-ß signaling, to promote the expression of wnt8a, a critical gene for neural posteriorization. Bptf and Smad2 directly bind to and activate the wnt8a promoter through recruiting NURF remodeling complex. When bptf function or TGF-ß signal transduction is inhibited, the nucleosome density on the wnt8a promoter is increased. We propose that Bptf and TGF-ß/Smad2 mediate nucleosome remodeling to regulate wnt8a expression and hence neural posteriorization.

The E3 ligase RNF185 negatively regulates osteogenic differentiation by targeting Dvl2 for degradation.

Osteoblast plays a pivotal role in bone metabolism and bone remodeling by mediating bone formation and regulating the activity of osteoclast. Clarifying the regulators and regulation mechanisms of osteogenic differentiation of mesenchymal stem cells (MSCs) and pre-osteoblasts will provide tremendous promise for bone repair and bone regeneration. RNF185 was identified as a candidate of endogenous suppressors of osteogenic specification in human mesenchymal stem cells (hMSCs). Here we show that RNF185 down regulates osteogenic differentiation of mouse calvaria-derived MC3T3-E1 cells, confirmed by quantitative real-time-PCR (qRT-PCR) and alkaline phosphatase (ALP) activity. Further we confirm that RNF185 interacts with dishevelled2 (Dvl2), a key mediator of Wnt signaling pathway. Overexpression of RNF185 decreases the exogenous and endogenous level of Dvl2, promotes the ubiquitination and degradation of Dvl2 and inhibits Wnt signaling, which is evident from the down-regulation of ß-catenin mediated transcriptional activity. And Dvl2 reverses the effect of RNF185 on osteogenic differentiation of MC3T3-E1 cells. Taken together, our results indicate that RNF185 negatively regulates osteogenesis through the degradation of Dvl2 and down-regulation of canonical Wnt signaling pathway and suggest a possible therapeutic target in osteoporosis.

Decode-seq: a practical approach to improve differential gene expression analysis.

Many differential gene expression analyses are conducted with an inadequate number of biological replicates. We describe an easy and effective RNA-seq approach using molecular barcoding to enable profiling of a large number of replicates simultaneously. This approach significantly improves the performance of differential gene expression analysis. Using this approach in medaka (Oryzias latipes), we discover novel genes with sexually dimorphic expression and genes necessary for germ cell development. Our results also demonstrate why the common practice of using only three replicates in differential gene expression analysis should be abandoned.

The guanine nucleotide exchange factor Net1 facilitates the specification of dorsal cell fates in zebrafish embryos by promoting maternal ß-catenin activation.

Wnt/ß-catenin signaling is essential for the initiation of dorsal-ventral patterning during vertebrate embryogenesis. Maternal ß-catenin accumulates in dorsal marginal nuclei during cleavage stages, but its critical target genes essential for dorsalization are silent until mid-blastula transition (MBT). Here, we find that zebrafish net1, a guanine nucleotide exchange factor, is specifically expressed in dorsal marginal blastomeres after MBT, and acts as a zygotic factor to promote the specification of dorsal cell fates. Loss- and gain-of-function experiments show that the GEF activity of Net1 is required for the activation of Wnt/ß-catenin signaling in zebrafish embryos and mammalian cells. Net1 dissociates and activates PAK1 dimers, and PAK1 kinase activation causes phosphorylation of S675 of ß-catenin after MBT, which ultimately leads to the transcription of downstream target genes. In summary, our results reveal that Net1-regulated ß-catenin activation plays a crucial role in the dorsal axis formation during zebrafish development.

The coiled-coil domain containing protein Ccdc136b antagonizes maternal Wnt/ß-catenin activity during zebrafish dorsoventral axial patterning.

The coiled-coil domain containing protein CCDC136 is a putative tumor suppressor and significantly down-regulated in gastric and colorectal cancer tissues. However, little is known about its biological functions during vertebrate embryo development. Zebrafish has two CCDC136 orthologs, ccdc136a and ccdc136b, but only ccdc136b is highly expressed during early embryonic development. In this study, we demonstrate that ccdc136b is required for dorsal-ventral axial patterning in zebrafish embryos. ccdc136b morphants display strongly dorsalized phenotypes. Loss- and gain-of-function experiments in zebrafish embryos and mammalian cells show that Ccdc136b is a crucial negative regulator of the Wnt/ß-catenin signaling pathway, and plays a critical role in the establishment of the dorsal-ventral axis. We further find that Ccdc136b interacts with APC, promotes the binding affinity of APC with ß-catenin and then facilitates the turnover of ß-catenin. These results provide the first evidence that CCDC136 regulates zebrafish dorsal-ventral patterning by antagonizing Wnt/ß-catenin signal transduction and suggest a potential mechanism underlying its suppressive activity in carcinogenesis.

Identification and characterization of alternative promoters, transcripts and protein isoforms of zebrafish R2 gene.

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5' termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress.

Molecular analysis and functions of p53R2 in zebrafish.

p53R2 is a newly identified small subunit of ribonucleotide reductase and plays a pivotal role in the supply of dNTPs for genomic DNA repair and mitochondrial DNA synthesis, but little is known about its functions in zebrafish. Herein, we obtained the cDNA of zebrafish p53R2 that shares 72.8% and 72.5% amino acid identities with human p53R2 and zebrafish R2, respectively. Residues crucial for enzymatic activity are highly conserved among p53R2 proteins from different species. p53R2 in zebrafish was maternally expressed, its transcripts were detected in developing embryos and all adult tissues examined. A 250-bp minimal promoter upstream of the translational initiation site was identified to drive basal expression of p53R2 in a p53-independent manner. Expression of p53R2 was induced by DNA-damaging reagents CPT or MMS, but suppressed by p53-knockdown in zebrafish embryos. Moreover, p53R2 was mainly distributed in the cytoplasm of cells under normal condition and upon DNA damage. Furthermore, overexpression of p53R2 attenuated apoptosis of embryonic cells caused by CPT or MMS treatment and protected developing embryos from death. Therefore, functions of p53R2 in zebrafish are closely associated with its activity in DNA repair and synthesis.