Clara Collart

SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5'-CACCT sequences in candidate target genes.

Activation of transforming growth factor β receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes. We describe a novel Smad-interacting protein, SIP1, which was identified using the yeast two-hybrid system. Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast andin vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation. SIP1 is a new member of the δEF1/Zfh-1 family of two-handed zinc finger/homeodomain proteins. Like δEF1, SIP1 binds to 5′-CACCT sequences in different promoters, including the Xenopus brachyury promoter. Overexpression of either full-length SIP1 or its C-terminal zinc finger cluster, which bind to the Xbra2promoter in vitro, prevented expression of the endogenousXbra gene in early Xenopus embryos. Therefore, SIP1, like δEF1, is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways. The identification of this Smad-interacting protein opens new routes to investigate the mechanisms by which transforming growth factor β members exert their effects on expression of target genes in responsive cells and in the vertebrate embryo.

New mode of DNA binding of multi-zinc finger transcription factors: deltaEF1 family members bind with two hands to two target sites.

SIP1, a Smad‐interacting protein, and δEF1, a transcriptional repressor involved in skeletal and T‐cell development, belong to the same family of DNA binding proteins. SIP1 and δEF1 contain two separated clusters of zinc fingers, one N‐terminal and one C‐terminal. These clusters show high sequence homology and are highly conserved between SIP1 and δEF1. Each zinc finger cluster binds independently to a 5′‐CACCT sequence. However, high‐affinity binding sites for full‐length SIP1 and δEF1 in the promoter regions of candidate target genes like Xenopus Xbra2, and human α4‐integrin and E‐cadherin, are bipartite elements composed of one CACCT and one CACCTG sequence, the orientation and spacing of which can vary. Using transgenic Xenopus embryos, we demonstrate that the integrity of these two sequences is necessary for correct spatial expression of a Xbra2 promoter‐driven reporter gene. Both zinc finger clusters must be intact for the high‐affinity binding of SIP1 to DNA and for its optimal repressor activity. Our results show that SIP1 binds as monomer and contacts one target sequence with the first zinc finger cluster, and the other with the second cluster. Our work redefines the optimal binding site and, consequently, candidate target genes for vertebrate members of the δEF1 family.

Titration of four replication factors is essential for the Xenopus laevis midblastula transition.

Regulating the MBT It has been known for more than 30 years that a defined number of cell divisions in the frog embryo precede a crucial developmental event called the midblastula transition (MBT). Collart et al. (p. 893, published online 1 August) now elucidate a mechanism involved in the control of the MBT. DNA replication initiation factors are titrated out during early cell divisions, which controls the elongation of the cell cycle and the onset of zygotic transcription during the MBT. Increasing numbers of nuclei compared with the cytoplasmic volume promotes a key developmental step in frog embryos. The rapid, reductive early divisions of many metazoan embryos are followed by the midblastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the MBT. We show that four DNA replication factors—Cut5, RecQ4, Treslin, and Drf1—are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis. The levels of these factors regulate multiple events of the MBT, including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.

High-resolution analysis of gene activity during the Xenopus mid-blastula transition

The Xenopus mid-blastula transition (MBT) marks the onset of large-scale zygotic transcription, as well as an increase in cell cycle length and a loss of synchronous cell divisions. Little is known about what triggers the activation of transcription or how newly expressed genes interact with each other. Here, we use high-resolution expression profiling to identify three waves of gene activity: a post-fertilisation wave involving polyadenylation of maternal transcripts; a broad wave of zygotic transcription detectable as early as the seventh cleavage and extending beyond the MBT at the twelfth cleavage; and a shorter post-MBT wave of transcription that becomes apparent as development proceeds. Our studies have also allowed us to define a set of maternal mRNAs that are deadenylated shortly after fertilisation, and are likely to be degraded thereafter. Experimental analysis indicates that the polyadenylation of maternal transcripts is necessary for the establishment of proper levels of zygotic transcription at the MBT, and that genes activated in the second wave of expression, including Brachyury and Mixer, contribute to the regulation of genes expressed in the third. Together, our high-resolution time series and experimental studies have yielded a deeper understanding of the temporal organisation of gene regulatory networks in the early Xenopus embryo.

Defining Synphenotype Groups in Xenopus tropicalis by Use of Antisense Morpholino Oligonucleotides

To identify novel genes involved in early development, and as proof-of-principle of a large-scale reverse genetics approach in a vertebrate embryo, we have carried out an antisense morpholino oligonucleotide (MO) screen in Xenopus tropicalis, in the course of which we have targeted 202 genes expressed during gastrula stages. MOs were designed to complement sequence between −80 and +25 bases of the initiating AUG codons of the target mRNAs, and the specificities of many were tested by (i) designing different non-overlapping MOs directed against the same mRNA, (ii) injecting MOs differing in five bases, and (iii) performing “rescue” experiments. About 65% of the MOs caused X. tropicalis embryos to develop abnormally (59% of those targeted against novel genes), and we have divided the genes into “synphenotype groups,” members of which cause similar loss-of-function phenotypes and that may function in the same developmental pathways. Analysis of the expression patterns of the 202 genes indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into early vertebrate development and paves the way for a more comprehensive MO-based analysis of gene function in X. tropicalis.

The Midblastula Transition Defines the Onset of Y RNA-Dependent DNA Replication in Xenopus laevis

ABSTRACT Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery.

Xnrs and Activin Regulate Distinct Genes during Xenopus Development: Activin Regulates Cell Division

Background The mesoderm of the amphibian embryo is formed through an inductive interaction in which vegetal cells of the blastula-staged embryo act on overlying equatorial cells. Candidate mesoderm-inducing factors include members of the transforming growth factor type β family such as Vg1, activin B, the nodal-related proteins and derrière. Methodology and Principle Findings Microarray analysis reveals different functions for activin B and the nodal-related proteins during early Xenopus development. Inhibition of nodal-related protein function causes the down-regulation of regionally expressed genes such as chordin, dickkopf and XSox17α/β, while genes that are mis-regulated in the absence of activin B tend to be more widely expressed and, interestingly, include several that are involved in cell cycle regulation. Consistent with the latter observation, cells of the involuting dorsal axial mesoderm, which normally undergo cell cycle arrest, continue to proliferate when the function of activin B is inhibited. Conclusions/Significance These observations reveal distinct functions for these two classes of the TGF-β family during early Xenopus development, and in doing so identify a new role for activin B during gastrulation.

Transforming growth factor β signalling in vitro and in vivo: activin ligand–receptor interaction, Smad5 in vasculogenesis, and repression of target genes by the δEF1/ZEB-related SIP1 in the vertebrate embryo

The identification and characterization of components of the transforming growth factor beta (TGFbeta) signalling pathway are proceeding at a very fast pace. To illustrate a number of our activities in this field, we first summarize our work aiming at the selection from a large collection of single residue substitution mutants of two activin A polypeptides in which D27 and K102, respectively, have been modified. This work has highlighted the importance of K102 and its positive charge for binding to activin type II receptors. Activin K102E, which did not bind to high-affinity receptor complexes, may be a valuable beta chain, when incorporated in recombinant inhibin to unambiguously detect novel inhibin binding sites at the cell surface. We then illustrate how Smad5 knockout mice and an overexpression approach with a truncated TGFbeta type II receptor in the mouse embryo can contribute to the identification of a novel TGFbeta-->TbetaRII/ALK1-->Smad5 pathway in endothelial cells in the embryo proper and the yolk sac vasculature. We conclude with a summary of our results with a Smad-interacting transcriptional repressor but focus on its biological significance in the vertebrate embryo.

The novel Smad-interacting protein Smicl regulates Chordin expression in the Xenopus embryo

In this paper, we investigate the function of Smicl, a zinc-finger Smad-interacting protein that is expressed maternally in the Xenopus embryo. Inhibition of Smicl function by means of antisense morpholino oligonucleotides causes the specific downregulation of Chordin, a dorsally expressed gene encoding a secreted BMP inhibitor that is involved in mesodermal patterning and neural induction. Chordin is activated by Nodal-related signalling in an indirect manner, and we show here that Smicl is involved in a two-step process that is necessary for this activation. In the first, Smad3 (but not Smad2) activates expression of Xlim1 in a direct fashion. In the second, a complex containing Smicl and the newly induced Xlim1 induces expression of Chordin. As well as revealing the function of Smicl in the early embryo, our work yields important new insight in the regulation of Chordin and identifies functional differences between the activities of Smad2 and Smad3 in the Xenopus embryo.

Smicl is a novel Smad interacting protein and cleavage and polyadenylation specificity factor associated protein.

Ligand‐bound receptors of the Transforming Growth Factor‐β (TGF‐β) family promote the formation of complexes between Smad proteins that subsequently accumulate in the nucleus and interact there with other transcriptional regulators, leading to modulation of target gene expression. We identified a novel nuclear protein, Smicl, which binds to Smad proteins. Smicl and Smads cooperate and enhance TGF‐β mediated activation of a Smad‐responsive reporter gene. A domain with five CCCH‐type zinc fingers in Smicl is structurally and functionally, at least in vitro, similar to a domain in CPSF‐30, the 30 kDa subunit of Cleavage and Polyadenylation Specificity Factor (CPSF). Like CPSF‐30, Smicl can associate with some other CPSF subunits characterized previously. Its effect on the induction of a reporter gene for TGF‐β requires the cleavage/polyadenylation signal downstream of the coding sequence of that gene. Thus, Smicl is a novel protein that displays CPSF‐30‐like activities, interacts in the nucleus with activated Smads, and potentiates in TGF‐β stimulated cells Smad‐dependent transcriptional responses, possibly in conjunction with the activity of CPSF complexes.