Post-translational histone modifications have important regulatory roles in chromatin structure and function. One example of such modifications is histone ubiquitination, which occurs predominately on histone H2A and H2B. Although the recent identification of the ubiquitin ligase for histone H2A has revealed important roles for H2A ubiquitination in Hox gene silencing as well as in X-chromosome inactivation, the enzyme(s) involved in H2A deubiquitination and the function of H2A deubiquitination are not known. Here we report the identification and functional characterization of the major deubiquitinase for histone H2A, Ubp-M (also called USP16). Ubp-M prefers nucleosomal substrates in vitro, and specifically deubiquitinates histone H2A but not H2B in vitro and in vivo. Notably, knockdown of Ubp-M in HeLa cells results in slow cell growth rates owing to defects in the mitotic phase of the cell cycle. Further studies reveal that H2A deubiquitination by Ubp-M is a prerequisite for subsequent phosphorylation of Ser 10 of H3 and chromosome segregation when cells enter mitosis. Furthermore, we demonstrate that Ubp-M regulates Hox gene expression through H2A deubiquitination and that blocking the function of Ubp-M results in defective posterior development in Xenopus laevis. This study identifies the major deubiquitinase for histone H2A and demonstrates that H2A deubiquitination is critically involved in cell cycle progression and gene expression.

Neural patterning occurs soon after neural induction during early development. In Xenopus, several caudalizing factors transform anterior neural to posterior neural tissue at the open neural plate stages, while other factors are responsible for setting up mediolateral polarity which becomes the dorsoventral (D-V) axis after neural tube closure. Many Wnt ligands are expressed in the neural tube in distinct anteroposterior (A-P) and D-V domains, implying a function in neural patterning. Here we report the cloning of a full-length Xenopus Wnt7B gene. Xwnt7B induces neural crest markers Xslug and Xtwist in ectodermal explants coinjected with neural inducer noggin and in ectodermal cells neuralized by dissociation. In vivo, Xwnt7B expands the Xtwist expression domain when injected in the animal pole. Our results suggest that Wnt members are involved in dorsoventral patterning of the neural tube.

Post-translational histone modifications play important roles in regulating gene expression programs, which in turn determine cell fate and lineage commitment during development. One such modification is histone ubiquitination, which primarily targets histone H2A and H2B. Although ubiquitination of H2A and H2B has been generally linked to gene silencing and gene activation, respectively, the functions of histone ubiquitination during eukaryote development are not well understood. Here, we identified USP12 and USP46 as histone H2A and H2B deubiquitinases that regulate Xenopus development. USP12 and USP46 prefer nucleosomal substrates and deubiquitinate both histone H2A and H2B in vitro and in vivo. WDR48, a WD40 repeat-containing protein, interacts with USP12 and USP46 and is required for the histone deubiquitination activity. Overexpression of either gene leads to gastrulation defects without affecting mesodermal cell fate, whereas knockdown of USP12 in Xenopus embryos results in reduction of a subset of mesodermal genes at gastrula stages. Immunohistochemical staining and chromatin immunoprecipitation assays revealed that USP12 regulates histone deubiquitination in the mesoderm and at specific gene promoters during Xenopus development. Taken together, this study identifies USP12 and USP46 as histone deubiquitinases for H2A and H2B and reveals that USP12 regulates Xenopus development during gastrula stages.Eukaryotic development requires precise control of gene expression patterns that are essential for cellular identity and differentiation (1, 2). Genomic DNA in eukaryotic cells is organized into a chromatin structure by association with histone and non-histone proteins (3, 4), and the structure of chromatin is believed to play a critical role in regulating chromatin-templated nuclear processes such as transcription (5, 6). Post-translational modifications of histones represent a major mechanism by which cells control the structure and function of chromatin. An increasing list of histone-modifying enzymes and histone modifications has been shown to be critical for normal development and to play causal roles in the pathogenesis of certain human diseases (7-9).Of the vast variety of histone modifications, histone ubiquitination is unique, in which a 76-amino acid bulky protein is attached primarily to histone H2A and H2B (10, 11). The recent characterization of ubiquitin ligase hPRC1L and deubiquitinase Ubp-M (USP16) for histone H2A revealed critical functions for this modification in gene silencing, X inactivation, cell cycle progression, and DNA damage repair (12-15). In addition to Ubp-M, 2A-DUB (MYSM1) and USP21 were also identified as H2A-specific deubiquitinases (16,17). These enzymes might function in different cellular processes, for example, 2A-DUB in androgen receptor-mediated gene activation and USP21 in liver regeneration (16,17). Recently, the Drosophila PcG gene calypso was found to encode a ubiquitin C-terminal hydrolase BAP1, which specifically deubiquitinates histone H2A and regulates Hox gene r...

Vertebrate neural induction requires inhibition of bone morphogenetic protein (BMP) signaling in the ectoderm. However, whether inhibition of BMP signaling is sufficient to induce neural tissues in vivo remains controversial. Here we have addressed why inhibition of BMP/Smad1 signaling does not induce neural markers efficiently in Xenopus ventral ectoderm, and show that suppression of both Smad1 and Smad2 signals is sufficient to induce neural markers. Manipulations that inhibit both Smad1 and Smad2 pathways, including a truncated type IIB activin receptor, Smad7 and Ski, induce early neural markers and inhibit epidermal genes in ventral ectoderm; and co-expression of BMP inhibitors with a truncated activin/nodal-specific type IB activin receptor leads to efficient neural induction. Conversely, stimulation of Smad2 signaling in the neural plate at gastrula stages results in inhibition of neural markers, disruption of the neural tube and reduction of head structures, with conversion of neural to neural crest and mesodermal fates. The ability of activated Smad2 to block neural induction declines by the end of gastrulation. Our results indicate that prospective neural cells are poised to respond to Smad2 and Smad1 signals to adopt mesodermal and non-neural ectodermal fates even at gastrula stages, after the conventionally assigned end of mesodermal competence, so that continued suppression of both mesoderm-and epidermis-inducing Smad signals leads to efficient neural induction.KEY WORDS: Neural induction, BMP, Smad1, Nodal, Smad2, Xenopus Development 134, 3861-3872 (2007) DEVELOPMENT 3862 region of BMP-specific Smad1, resulting in cytoplasmic retention of Smad1 and suppression of BMP signaling (Pera et al., 2003). By contrast, early Wnt signals are active over the entire dorsal domain of the embryo as a result of cortical rotation, and act both to repress transcription of BMP4 in the dorsal ectoderm and to stimulate expression of the BMP antagonists noggin and chordin, thus ensuring the clearance of BMP ligands and inhibition of BMP signals in the neural field (Baker et al., 1999;Wessely et al., 2001). Although FGF and Wnt may have BMP inhibition-independent functions, the nature of such actions is unknown.One central piece of data arguing against the default model is that unlike in animal caps or explanted ventral ectoderm (Lamb et al., 1993), inhibition of BMP signaling by secreted antagonists, a truncated type I receptor or the inhibitory Smad6 is not sufficient to induce neural marker expression in prospective ventral epidermis of frog embryos or in the chick extra-embryonic epiblast (Linker and Stern, 2004; Delaune et al., 2005). Clearly other conditions need to be met in order for neural specification to occur in competent nonneural ectoderm in vivo. One such condition may be a low level of FGF/ras signaling, as it induces neural markers in combination with BMP inhibitors in Xenopus (Linker and Stern, 2004; Delaune et al., 2005;Wawersik et al., 2005). Indeed, even in the absence of localized BMP inhibitors...

Neural crest cells arise from the border of the neural plate and epidermal ectoderm, migrate extensively and differentiate into diverse cell types during vertebrate embryogenesis. Although much has been learnt about growth factor signals and gene regulatory networks that regulate neural crest development, limited information is available on how epigenetic mechanisms control this process. In this study, we show that Polycomb repressive complex 2 (PRC2) cooperates with the transcription factor Snail2/Slug to modulate neural crest development in Xenopus. The PRC2 core components Eed, Ezh2 and Suz12 are expressed in the neural crest cells and are required for neural crest marker expression. Knockdown of Ezh2, the catalytic subunit of PRC2 for histone H3K27 methylation, results in defects in neural crest specification, migration and craniofacial cartilage formation. EZH2 interacts directly with Snail2, and Snail2 fails to expand the neural crest domains in the absence of Ezh2. Chromatin immunoprecipitation analysis shows that Snail2 regulates EZH2 occupancy and histone H3K27 trimethylation levels at the promoter region of the Snail2 target E-cadherin. Our results indicate that Snail2 cooperates with EZH2 and PRC2 to control expression of the genes important for neural crest specification and migration during neural crest development.