Wenyan Mei

DUX4, a candidate gene for facioscapulohumeral muscular dystrophy, causes p53-dependent myopathy in vivo

Facioscapulohumeral muscular dystrophy (FSHD) is associated with D4Z4 repeat contraction on human chromosome 4q35. This genetic lesion does not result in complete loss or mutation of any gene. Consequently, the pathogenic mechanisms underlying FSHD have been difficult to discern. In leading FSHD pathogenesis models, D4Z4 contractions are proposed to cause epigenetic changes, which ultimately increase expression of genes with myopathic potential. Although no gene has been conclusively linked to FSHD development, recent evidence supports a role for the D4Z4‐encoded DUX4 gene in FSHD. In this study, our objective was to test the in vivo myopathic potential of DUX4.

Regulation of nuclear-cytoplasmic shuttling and function of Family with sequence similarity 13, member A (Fam13a) by B56-containing PP2As and Akt

FAM13A is a novel human lung disease–associated gene. Fam13a is dispensable but is capable of inducing Wnt signaling. Nuclear localization of Fam13a is important for its function in the Wnt pathway. Akt/PP2A-dependent reversible phosphorylation on Ser-322 is a molecular switch that controls nuclear–cytoplasmic shuttling of Fam13a.

The 48-kDa Alternative Translation Isoform of PP2A:B56ε Is Required for Wnt Signaling during Midbrain-Hindbrain Boundary Formation

Alternative translation is an underappreciated post-transcriptional regulation mechanism. Although only a small number of genes are found to be alternatively translated, most genes undergoing alternative translation play important roles in tumorigenesis and development. Protein phosphatase 2A (PP2A) is involved in many cellular events during tumorigenesis and development. The specificity, localization, and activity of PP2A are regulated by B regulatory subunits. B56ϵ, a member of the B56 regulatory subunit family, is involved in multiple signaling pathways and regulates a number of developmental processes. Here we report that B56ϵ is alternatively translated, leading to the production of a full-length form and a shorter isoform that lacks the N-terminal 76 amino acid residues of the full-length form. Alternative translation of B56ϵ occurs through a cap-dependent mechanism. We provide evidence that the shorter isoform is required for Wnt signaling and regulates the midbrain/hindbrain boundary formation during Xenopus embryonic development. This demonstrates that the shorter isoform of B56ϵ has important biological functions. Furthermore, we show that the N-terminal sequence of B56ϵ, which is not present in the shorter isoform, contains a nuclear localization signal, whereas the C terminus of B56ϵ contains a nuclear export signal. The shorter isoform, which lacks the N-terminal nuclear localization signal, is restricted to the cytoplasm. In contrast, the full-length form can be localized to the nucleus in a cell type-specific manner. The finding that B56ϵ is alternatively translated adds a new level of regulation to PP2A holoenzymes.

Maternal Dead-End1 is required for vegetal cortical microtubule assembly during Xenopus axis specification.

Vertebrate axis specification is an evolutionarily conserved developmental process that relies on asymmetric activation of Wnt signaling and subsequent organizer formation on the future dorsal side of the embryo. Although roles of Wnt signaling during organizer formation have been studied extensively, it is unclear how the Wnt pathway is asymmetrically activated. In Xenopus and zebrafish, the Wnt pathway is triggered by dorsal determinants, which are translocated from the vegetal pole to the future dorsal side of the embryo shortly after fertilization. The transport of dorsal determinants requires a unique microtubule network formed in the vegetal cortex shortly after fertilization. However, molecular mechanisms governing the formation of vegetal cortical microtubule arrays are not fully understood. Here we report that Dead-End 1 (Dnd1), an RNA-binding protein required for primordial germ cell development during later stages of embryogenesis, is essential for Xenopus axis specification. We show that knockdown of maternal Dnd1 specifically interferes with the formation of vegetal cortical microtubules. This, in turn, impairs translocation of dorsal determinants, the initiation of Wnt signaling, organizer formation, and ultimately results in ventralized embryos. Furthermore, we found that Dnd1 binds to a uridine-rich sequence in the 3′-UTR of trim36, a vegetally localized maternal RNA essential for vegetal cortical microtubule assembly. Dnd1 anchors trim36 to the vegetal cortex in the egg, promoting high concentrations of Trim36 protein there. Our work thus demonstrates a novel and surprising function for Dnd1 during early development and provides an important link between Dnd1, mRNA localization, the microtubule cytoskeleton and axis specification.

The antagonistic action of B56-containing protein phosphatase 2As and casein kinase 2 controls the phosphorylation and Gli turnover function of Daz interacting protein 1

Background: The stability of Gli proteins is important for the outcome of Hedgehog signaling. Results: Dzip1 is involved in a novel Gli turnover mechanism. This function of Dzip1 is regulated by CK2 and PP2A-dependent reversible phosphorylation. Conclusion: The antagonistic action of PP2A and CK2 controls the phosphorylation and Gli turnover function of Dzip1. Significance: Our studies have identified a novel Gli regulatory mechanism. The Hedgehog (Hh) pathway is evolutionarily conserved and plays critical roles during embryonic development and adult tissue homeostasis. Defective Hh signaling has been linked to a wide range of birth defects and cancers. Hh family proteins regulate the expression of their downstream target genes through the control of proteolytic processing and the transcriptional activation function of Gli transcription factors. Although Hh-dependent regulation of Gli has been studied extensively, other Gli regulatory mechanisms remain relatively unappreciated. Here we report our identification of a novel signaling cascade that controls the stability of Gli proteins. This cascade consists of Daz interacting protein 1 (Dzip1), casein kinase 2 (CK2), and B56 containing protein phosphatase 2As (PP2As). We provide evidence that Dzip1 is involved in a novel Gli turnover pathway. We show that CK2 directly phosphorylates Dzip1 at four serine residues, Ser-664/665/706/714. B56-containing PP2As, through binding to a domain located between amino acid residue 474 and 550 of Dzip1, dephosphorylate Dzip1 on these CK2 sites. Our mutagenesis analysis further demonstrates that the unphosphorylatable form of Dzip1 is more potent in promoting Gli turnover. Consistently, we found that the stability of Gli proteins was decreased upon CK2 inhibition and increased by inhibition of B56-containing PP2As. Thus, reversible phosphorylation of Dzip1, which is controlled by the antagonistic action of CK2 and B56-containing PP2As, has an important impact on the stability of Gli transcription factors and Hh signaling.

hnRNP I Inhibits Notch Signaling and Regulates Intestinal Epithelial Homeostasis in the Zebrafish

Regulated intestinal stem cell proliferation and differentiation are required for normal intestinal homeostasis and repair after injury. The Notch signaling pathway plays fundamental roles in the intestinal epithelium. Despite the fact that Notch signaling maintains intestinal stem cells in a proliferative state and promotes absorptive cell differentiation in most species, it remains largely unclear how Notch signaling itself is precisely controlled during intestinal homeostasis. We characterized the intestinal phenotypes of brom bones, a zebrafish mutant carrying a nonsense mutation in hnRNP I. We found that the brom bones mutant displays a number of intestinal defects, including compromised secretory goblet cell differentiation, hyperproliferation, and enhanced apoptosis. These phenotypes are accompanied by a markedly elevated Notch signaling activity in the intestinal epithelium. When overexpressed, hnRNP I destabilizes the Notch intracellular domain (NICD) and inhibits Notch signaling. This activity of hnRNP I is conserved from zebrafish to human. In addition, our biochemistry experiments demonstrate that the effect of hnRNP I on NICD turnover requires the C-terminal portion of the RAM domain of NICD. Our results demonstrate that hnRNP I is an evolutionarily conserved Notch inhibitor and plays an essential role in intestinal homeostasis.

An interferon regulatory factor-like binding element restricts Xmyf-5 expression in the posterior somites during Xenopus myogenesis.

The expression of myf‐5, a key component of myogenic regulatory genes, declines progressively in mature somitic cells during vertebrate myogenesis. Little is known about how this down‐regulation takes place. Here we provide evidence that an interferon regulatory factor binding element (IRF element) within the Xenopus myf‐5 promoter is responsible for the elimination of myf‐5 transcription in mature somitic mesoderm of Xenopus embryos. We show that this IRF element mediates the down‐regulation of Xmyf‐5 transcription in gastrula embryos, and can specifically interact with nuclear proteins of early neurula. Moreover, deletion of this IRF element results in the anterior expansion of reporter gene transcripts within somitic mesoderm in transgenic embryos. Our results, therefore, provide insight into how the negative control of Xmyf‐5 expression takes place.

Reversible optogenetic control of kinase activity during differentiation and embryonic development.

A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proof-of-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves light-induced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue light-dependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings. Summary: Light is used to modulate the timing of mitogen-activated protein kinase activity during PC12 cell differentiation and distinct developmental stages of Xenopus embryos.

Cloning and analysing of 5′ flanking region of Xenopus organizer gene noggin

ABSTRACTXenopus organizer specific gene Noggin possesses nearly all the characterestic properties of the action of organizer to specify the embryonic body axis. To analyze how the maternal inherited factors control its expression pattern, we cloned the 5′ regulatory region of noggin gene. The 1.5 kb upstream sequense could direct reporter gene to express in vivo and data from deletion analysis indicated that a 229 base pair fragmet is essential for activating noggin expression. We further demonstrated that the response elements within this regulatory region were indeed under the control of growth factor activin and Wnt signaling pathway components.

hnRNP I regulates neonatal immune adaptation and prevents colitis and colorectal cancer

The intestinal epithelium plays a critical role in host-microbe homeostasis by sensing gut microbes and subsequently initiating proper immune responses. During the neonatal stage, the intestinal epithelium is under immune repression, allowing the transition for newborns from a relatively sterile intra-uterine environment to one that is rich in foreign antigens. The mechanism underlying such immune repression remains largely unclear, but involves downregulation of IRAK1 (interleukin-1 receptor-associated kinase), an essential component of toll-like receptor-mediated NF-κB signaling. We report here that heterogeneous nuclear ribonucleoprotein I (hnRNPI), an RNA binding protein, is essential for regulating neonatal immune adaptation. We generated a mouse model in which hnRNPI is ablated specifically in the intestinal epithelial cells, and characterized intestinal defects in the knockout mice. We found that loss of hnRNPI function in mouse intestinal epithelial cells results in early onset of spontaneous colitis followed by development of invasive colorectal cancer. Strikingly, the epithelium-specific hnRNPI knockout neonates contain aberrantly high IRAK1 protein levels in the colons and fail to develop immune tolerance to environmental microbes. Our results demonstrate that hnRNPI plays a critical role in establishing neonatal immune adaptation and preventing colitis and colorectal cancer.