Anming Meng

PPM1A Functions as a Smad Phosphatase to Terminate TGFβ Signaling

TGFbeta signaling controls diverse normal developmental processes and pathogenesis of diseases including cancer and autoimmune and fibrotic diseases. TGFbeta responses are generally mediated through transcriptional functions of Smads. A key step in TGFbeta signaling is ligand-induced phosphorylation of receptor-activated Smads (R-Smads) catalyzed by the TGFbeta type I receptor kinase. However, the potential of Smad dephosphorylation as a regulatory mechanism of TGFbeta signaling and the identity of Smad-specific phosphatases remain elusive. Using a functional genomic approach, we have identified PPM1A/PP2Calpha as a bona fide Smad phosphatase. PPM1A dephosphorylates and promotes nuclear export of TGFbeta-activated Smad2/3. Ectopic expression of PPM1A abolishes TGFbeta-induced antiproliferative and transcriptional responses, whereas depletion of PPM1A enhances TGFbeta signaling in mammalian cells. Smad-antagonizing activity of PPM1A is also observed during Nodal-dependent early embryogenesis in zebrafish. This work demonstrates that PPM1A/PP2Calpha, through dephosphorylation of Smad2/3, plays a critical role in terminating TGFbeta signaling.

Smad7 Antagonizes Transforming Growth Factor β Signaling in the Nucleus by Interfering with Functional Smad-DNA Complex Formation

ABSTRACT Smad7 plays an essential role in the negative-feedback regulation of transforming growth factor β (TGF-β) signaling by inhibiting TGF-β signaling at the receptor level. It can interfere with binding to type I receptors and thus activation of receptor-regulated Smads or recruit the E3 ubiquitin ligase Smurf to receptors and thus target them for degradation. Here, we report that Smad7 is predominantly localized in the nucleus of Hep3B cells. The targeted expression of Smad7 in the nucleus conferred superior inhibitory activity on TGF-β signaling, as determined by reporter assay in mammalian cells and by its effect on zebrafish embryogenesis. Furthermore, Smad7 repressed Smad3/4-, Smad2/4-, and Smad1/4-enhanced reporter gene expression, indicating that Smad7 can function independently of type I receptors. An oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad-responsive element via its MH2 domain, and DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-β target gene, by chromatin immunoprecipitation. Finally, we provide evidence that Smad7 disrupts the formation of the TGF-β-induced functional Smad-DNA complex. Our findings suggest that Smad7 inhibits TGF-β signaling in the nucleus by a novel mechanism.

Allelic reprogramming of the histone modification H3K4me3 in early mammalian development

Histone modifications are fundamental epigenetic regulators that control many crucial cellular processes. However, whether these marks can be passed on from mammalian gametes to the next generation is a long-standing question that remains unanswered. Here, by developing a highly sensitive approach, STAR ChIP–seq, we provide a panoramic view of the landscape of H3K4me3, a histone hallmark for transcription initiation, from developing gametes to post-implantation embryos. We find that upon fertilization, extensive reprogramming occurs on the paternal genome, as H3K4me3 peaks are depleted in zygotes but are readily observed after major zygotic genome activation at the late two-cell stage. On the maternal genome, we unexpectedly find a non-canonical form of H3K4me3 (ncH3K4me3) in full-grown and mature oocytes, which exists as broad peaks at promoters and a large number of distal loci. Such broad H3K4me3 peaks are in contrast to the typical sharp H3K4me3 peaks restricted to CpG-rich regions of promoters. Notably, ncH3K4me3 in oocytes overlaps almost exclusively with partially methylated DNA domains. It is then inherited in pre-implantation embryos, before being erased in the late two-cell embryos, when canonical H3K4me3 starts to be established. The removal of ncH3K4me3 requires zygotic transcription but is independent of DNA replication-mediated passive dilution. Finally, downregulation of H3K4me3 in full-grown oocytes by overexpression of the H3K4me3 demethylase KDM5B is associated with defects in genome silencing. Taken together, these data unveil inheritance and highly dynamic reprogramming of the epigenome in early mammalian development.

The Fused/Smurf Complex Controls the Fate of Drosophila Germline Stem Cells by Generating a Gradient BMP Response

In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. Here, we show that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. We observed similar roles for Fu during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism.

Inhibition of Severe Acute Respiratory Syndrome Virus Replication by Small Interfering RNAs in Mammalian Cells

ABSTRACT Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease that spread worldwide in early 2003. The cause was determined as a novel coronavirus (CoV), SARS-associated CoV (SARS-CoV), with a single-stranded, plus-sense RNA. To date, no effective specific treatment has been identified. To exploit the possibility of using RNA interference as a therapeutic approach to fight the disease, plasmid-mediated small interfering RNAs (siRNAs) were generated to target the SARS-CoV genome. The expression of siRNAs from two plasmids, which specifically target the viral RNA polymerase, effectively blocked the cytopathic effects of SARS-CoV on Vero cells. These two plasmids also inhibited viral replication as shown by titer assays and by an examination of viral RNA and protein levels. Thus, our results demonstrated the feasibility of developing siRNAs as effective anti-SARS drugs.

The evolutionally conserved activity of Dapper2 in antagonizing TGF-β signaling

Dapper1 and Dapper2, two divergent members of the Dapper family, have been suggested to modulate Wnt and TGF‐ß/Nodal signaling in Xenopus and zebrafish. To get a better understanding of Dapper function in mammals, we have cloned the mouse or‐tholog of zebrafish Dapper2, mDpr2 and investigated its function in regulating TGF‐ß signaling activity. Here, we showed that, like zebrafish Dapper2, overexpression of mDpr2 inhibited the TGF‐ß‐induced expression of the Smad‐responsive reporters and targeted TGF‐ß type I receptor ALK5 for degradation in mammalian cells. Overexpression of mDpr2 in the zebrafish embryos led to a decrease in expression of the meso‐derm marker no tail and goosecoid at the shield stage and eye fusion later, implying that mDpr2 may have an intrinsic in vivo activity similar to fish Dapper2 activity. The expression of mDpr2 was detected throughout the epiblast around the onset of gastrulation and in somites, the neural tube and gut at later stages in mouse embryos, implying a role in early embryonic development. Our data indicate that the function of Dpr2 as a negative regulator of the TGF‐ß/Nodal signal pathway is evolutionally conserved, at least in part, in fish and mammals.—Su, Y., Zhang, L., Gao, X., Meng, F., Wen, J., Zhou, H., Meng, A., Chen, Y.‐G. The evolutionally conserved activity of Dapper2 in antagonizing TGF‐β signaling. FASEB J. 21, 682–690 (2007)

Zebrafish sox9b is an early neural crest marker.

Abstract. Sox9 is a transcription factor related to campomelic dysplasia and sex reversal in human patients. Earlier studies in zebrafish led to the identification of two homologues of mammalian sox9, sox9a and sox9b. The present study represents the first evaluation of expression patterns of zebrafish sox9b during early embryogenesis. Our analyses reveal that sox9b transcripts are present throughout the life-cycle of the zebrafish, but exhibit tissue-specific distribution during embryogenesis. Zygotic expression of sox9b occurs in the anterolateral margins and the midline of the prospective dorsal neuroectoderm during late gastrulation. During early segmentation, the transcript is expressed in pairs of longitudinal bands in the prospective midbrain, hindbrain, and trunk, which identify the cranial and trunk neural crest progenitors. Neural crest cells cease expression of sox9b during migration, but some of their derivatives resume sox9b expression. Sox9b can serve as a marker for neural crest precursors.

Dapper1 Is a Nucleocytoplasmic Shuttling Protein That Negatively Modulates Wnt Signaling in the Nucleus

Wnt signaling, via the activation of the canonical β-catenin and lymphoid enhancer factor (LEF)/T-cell factor pathway, plays an important role in embryogenesis and cancer development by regulating the expression of genes involved in cell proliferation, differentiation, and survival. Dapper (Dpr), as a Dishevelled interactor, has been suggested to modulate Wnt signaling by promoting Dishevelled degradation. Here, we provide evidence that Dpr1 shuttles between the cytoplasm and the nucleus. Although overexpressed Dpr1 was mainly found in the cytoplasm, endogenous Dpr1 was localized over the cell, and Wnt1 induced its nuclear export. Treatment with leptomycin B induced nuclear accumulation of both endogenous and overexpressed Dpr1. We further identified the nuclear localization signal and the nuclear export signal within Dpr1. Using reporter assay and in vivo zebrafish embryo assay, we demonstrated that the forced nuclearly localized Dpr1 possessed the ability to antagonize Wnt signaling. Dpr1 interacted with β-catenin and LEF1 and disrupted their complex formation. Furthermore, Dpr1 could associate with histone deacetylase 1 (HDAC1) and enhance the LEF1-HDAC1 interaction. Together, our findings suggest that Dpr1 negatively modulates the basal activity of Wnt/β-catenin signaling in the nucleus by keeping LEF1 in the repressive state. Thus, Dpr1 controls Wnt/β-catenin signaling in both the cytoplasm and the nucleus.

smad2 and smad3 Are Required for Mesendoderm Induction by Transforming Growth Factor-β/Nodal Signals in Zebrafish

The transforming growth factor-β ligands Nodal, activin, and Vg1 play important roles in mesendoderm induction and patterning during vertebrate embryogenesis. These ligands are believed to transduce the signal through the receptor-activated transcription factors Smad2 and Smad3. However, the roles of smad2/3 genes in development of zebrafish embryos are largely unknown because the presence of multiple smad2/3 genes and their maternal expression have hampered the investigation of their developmental roles. We generated potent and specific dominant-negative forms of zebrafish Smad2, Smad3a, and Smad3b by mutating multiple amino acids. Overexpression of these mutants abolished mesendoderm induction by ectopic Nodal signaling in zebrafish embryos. Expression of dominant-negative smad2/3 abrogated Smad2/3 activities in wild-type embryos and caused various mesendodermal defects similar to those in Nodal-deficient embryos. Smad2/3-deficient cells transplanted into the blastodermal margin of wild-type hosts preferentially differentiated into ectodermal tissues rather than mesendodermal tissues, supporting the idea that response of cells to mesendoderm inducers requires Smad2/3 activities. Interference with Smad2/3 activities in Zoep, Moep, and MZoep mutant embryos resulted in more severe mesendodermal defects. Thus, our data reveal that Nodal signaling and mesendoderm induction depend on Smad2/3 and suggest that transforming growth factor-β signals other than Nodal also contribute to Smad2/3 signaling and embryonic patterning.

Tob genes in development and homeostasis

Members of the Btg/Tob protein family share a conserved N‐terminal region that confers the activity to inhibit cell proliferation. Tob1 and Tob2 proteins, which constitute a Tob subfamily, have a longer C‐terminal region than BTG proteins. Apparently, genomes of invertebrates and teleost species contain only a single Tob locus, whereas genomes of mammalian, avian, and amphibian species contain two Tob loci (Tob1 and Tob2). Tob genes are expressed in oocytes, sperm, early embryos, and various adult tissues, depending on the species. Recent reports indicate that Tob proteins play important roles in spermatogenesis, embryonic dorsoventral patterning, osteogenesis, T‐cell activation, and learning and memory. Accumulating evidence supports the hypothesis that Tob proteins act primarily as transcriptional repressors in several signaling pathways. Developmental Dynamics 236:913–921, 2007.