Chaocui Li

Xenopus RCOR2 (REST corepressor 2) interacts with ZMYND8, which is involved in neural differentiation

Regulation of neuronal gene expression is critical to nervous system development. REST (RE1-silencing transcription factor) regulates neuronal gene expression through interacting with a group of corepressor proteins including REST corepressors (RCOR). Here we show that Xenopus RCOR2 is predominantly expressed in the developing nervous system. Through a yeast two-hybrid screen, we isolated Xenopus ZMYND8 (Zinc finger and MYND domain containing 8) as an XRCOR2 interacting factor. XRCOR2 and XZMYND8 bind each other in co-immunoprecipitation assays and both of them can function as transcriptional repressors. XZMYND8 is co-expressed with XRCOR2 in the nervous system and overexpression of XZMYND8 inhibits neural differentiation in Xenopus embryos. These data reveal a RCOR2/ZMYND8 complex which might be involved in the regulation of neural differentiation.

Differential expression of the Brunol/CELF family genes during Xenopus laevis early development

The BRUNOL/CELF family of RNA-binding proteins plays important roles in post-transcriptional regulation and has been implicated in several developmental processes. In this study, we describe the cloning and expression patterns of five Brunol genes in Xenopus laevis. Among them, only Brunol2 is maternally expressed and the zygotic expression of the other four Brunol genes starts at different developmental stages. During Xenopus development, Brunol1, 4-5 are exclusively expressed in the nervous system including domains in the brain, spinal cord, optic and otic vesicles. Brunol2 and 3 are expressed in both the somatic mesoderm and the nervous system. Brunol2 is also extensively expressed in the lens. In transfected Hela cells, BRUNOL1, 2 and 3 proteins are localized in both the cytoplasm and the nucleus, while BRUNOL4 and 5 are only present in the cytoplasm, indicating their different functions.

The Ubiquitin Ligase RNF220 Enhances Canonical Wnt Signaling through USP7-Mediated Deubiquitination of β-Catenin

ABSTRACT Wnt/β-catenin signaling plays critical roles in embryonic development and disease. Here, we identify RNF220, a RING domain E3 ubiquitin ligase, as a new regulator of β-catenin. RNF220 physically interacts with β-catenin, but instead of promoting its ubiquitination and proteasomal degradation, it stabilizes β-catenin and promotes canonical Wnt signaling. Our analysis showed that RNF220 interacts with USP7, a ubiquitin-specific peptidase, which is required for RNF220 to stabilize β-catenin. The RNF220/USP7 complex deubiquitinates β-catenin and enhances canonical Wnt signaling. Interestingly, the stability of RNF220 itself is negatively regulated by Gsk3β, which is a key component of the β-catenin destruction complex and is inhibited upon Wnt stimulation. Accordingly, the RNF220/USP7 complex works as a positive feedback regulator of β-catenin signaling. In colon cancer cells with stimulated Wnt signaling, knockdown of RNF220 or USP7 impairs Wnt signaling and expression of Wnt target genes, suggesting a potentially novel role of RNF220 in Wnt-related tumorigenesis.

RNF220, an E3 ubiquitin ligase that targets Sin3B for ubiquitination

Modification of proteins by ubiquitination plays important roles in various cellular processes. During this process, the target specificity is determined by ubiquitin ligases. Here we identify RNF220 (RING finger protein 220) as a novel ubiquitin ligase for Sin3B. As a conserved RING protein, RNF220 can bind E2 and mediate auto-ubiquitination of itself. Through a yeast two-hybrid screen, we isolated Sin3B as one of its targets, which is a scaffold protein of the Sin3/HDAC (histone deacetylase) corepressor complex. RNF220 specifically interacts with Sin3B both in vitro and in vivo. Sin3B can be regulated by the ubiquitin-proteasome system. Co-expression of RNF220 promotes the ubiquitination and proteasomal degradation of Sin3B. Taken together, these results reveal a new mechanism for regulating the Sin3/HDAC complex.

Xenopus Claudin-6 is required for embryonic pronephros morphogenesis and terminal differentiation

Claudins are tetratransmembrane tight junction proteins and play important roles in regulating paracellular permeability of different nephron segments of the kidney. However, the roles of claudins in kidney development remain largely unknown. Here we studied the expression and functions of claudin-6 in Xenopus pronephros development. Xenopus claudin-6 is expressed in the developing pronephric tubule and duct but not glomus. Knockdown of claudin-6 by specific morpholino led to severe defects in pronephros tubular morphogenesis and blocked the terminal differentiation of the tubule cells. The claudin-6 morpholino targeted tubule cells showed failure of apical accumulation of actin and reduced lateral expression of tight junction protein Na/K-ATPase, suggesting an incomplete epithelization likely due to defected cell adhesions and apical-lateral polarity. Our work uncovered a novel role for claudin-6 in embryonic kidney development.

Xenopus Dbx2 is involved in primary neurogenesis and early neural plate patterning

The evolutionarily conserved Dbx homeodomain-containing proteins play important roles in the development of vertebrate central nervous system. In mouse, Dbx and Nkx6 have been suggested to be cross-repressive partners involved in the patterning of ventral neural tube. Here, we have isolated Xenopus Dbx2 and studied its developmental expression and function during neural development. Like XDbx1, from mid-neurula stage on, XDbx2 is expressed in stripes between the primary motoneurons and interneurons. At the tailbud stages, it is detected in the middle region of the neural tube. XDbx2 acts as a transcriptional repressor in vitro and over-expression of XDbx2 inhibits primary neurogenesis in Xenopus embryos. Over-expression of XDbx genes represses the expression of XNkx6.2 and vise versa. Knockdown of either XDbx1, XDbx2 or both by specific morpholinos induces lateral expansion of XNkx6.2 expression domains. These data reveal conserved roles for Dbx in primary neurogenesis and dorsoventral neural patterning in Xenopus.

Xenopus Nkx6.3 is a neural plate border specifier required for neural crest development.

In vertebrates, the neural plate border (NPB) is established by a group of transcription factors including Dlx3, Msx1 and Zic1. The crosstalk between these NPB specifiers governs the separation of the NPB region into placode and neural crest (NC) territories and also their further differentiation. Understanding the mechanisms of NPB formation and NC development is critical for our knowledge of related human diseases. Here we identified Nkx6.3, a transcription factor of the Nkx family, as a new NPB specifier required for neural crest development in Xenopus embryos. XNkx6.3 is expressed in the ectoderm of the neural plate border region at neurula stages, covering the epidermis, placode and neural crest territories, but not the neural plate. Inhibition of Nkx6.3 by dominant negative construct or specific morpholino leads to neural crest defects, while overexpression of Nkx6.3 induces ectopic neural crest in the anterior neural fold. In animal caps, Nkx6.3 alone is able to initiate the whole neural crest regulatory network and induces neural crest fate robustly. We showed that overexpression of Nkx6.3 affects multiple signaling pathways, creating a high-Wnt, low-BMP environment required for neural crest development. Gain- and loss-of-function of Nkx6.3 have compound effects on the expression of known NPB genes, which is largely opposite to that of Dlx3. Overexpression of Dlx3 blocks the NC inducing activity of Nkx6.3. The crosstalk between Nkx6.3, Dlx3 and Msx1 is likely crucial for proper NPB formation and neural crest development in Xenopus.

The effects of centrally administered fluorocitrate via inhibiting glial cells on working memory in rats

Although prefrontal and hippocampal neurons are critical for spatial working memory, the function of glial cells in spatial working memory remains uncertain. In this study we investigated the function of glial cells in rats’ working memory. The glial cells of rat brain were inhibited by intracerebroventricular (icv) injection of fluorocitrate (FC). The effects of FC on the glial cells were examined by using electroencephalogram (EEG) recordings and delayed spatial alternation tasks. After icv injection of 10 μL of 0.5 nmol/L or 5 nmol/L FC, the EEG power spectrum recorded from the hippocampus increased, but the power spectrum for the prefrontal cortex did not change, and working memory was unaffected. Following an icv injection of 10 μL of 20 nmol/L FC, the EEG power spectra in both the prefrontal cortex and the hippocampus increased, and working memory improved. The icv injection of 10 μL of 50 nmol/L FC, the EEG power spectra in both the prefrontal cortex and in the hippocampus decreased, and working memory was impaired. These results suggest that spatial working memory is affected by centrally administered FC, but only if there are changes in the EEG power spectrum in the prefrontal cortex. Presumably, the prefrontal glial cells relate to the working memory.

Elongator Protein 3 (Elp3) stabilizes Snail1 and regulates neural crest migration in Xenopus.

Elongator protein 3 (Elp3) is the enzymatic unit of the elongator protein complex, a histone acetyltransferase complex involved in transcriptional elongation. It has long been shown to play an important role in cell migration; however, the underlying mechanism is unknown. Here, we showed that Elp3 is expressed in pre-migratory and migrating neural crest cells in Xenopus embryos, and knockdown of Elp3 inhibited neural crest cell migration. Interestingly, Elp3 binds Snail1 through its zinc-finger domain and inhibits its ubiquitination by β-Trcp without interfering with the Snail1/Trcp interaction. We showed evidence that Elp3-mediated stabilization of Snail1 was likely involved in the activation of N-cadherin in neural crest cells to regulate their migratory ability. Our findings provide a new mechanism for the function of Elp3 in cell migration through stabilizing Snail1, a master regulator of cell motility.

Trip12 is an E3 ubiquitin ligase for USP7/HAUSP involved in the DNA damage response

The deubiquitinating enzyme, USP7/HAUSP (herpesvirus‐associated ubiquitin‐specific protease), is a key regulator of the tumor suppressor p53 and plays a major role in regulating genome stability. Here, we report that the protein stability of USP7 is regulated by the ubiquitin–proteasome pathway. We identified the thyroid hormone receptor interactor 12 (Trip12) as a ubiquitin E3 ligase for USP7. We also found that Trip12 affects USP7‐mediated stabilization of p53 and the checkpoint proteins 53BP1 and Chk1. Knockdown of Trip12 leads to an increased cell population in G1 phase, mimicking USP7 overexpression. In contrast, Trip12 overexpression increased the number of cells in intra‐S‐phase, phenocopying the USP7 knockdown phenotype. Therefore, our data reveal an important modulatory role for Trip12 in the USP7‐dependent DNA damage response.