No abstract

Regulation of Wnt transcriptional targets is thought to occur by a transcriptional switch. In the absence of Wnt signaling, sequence‐specific DNA‐binding proteins of the TCF family repress Wnt target genes. Upon Wnt stimulation, stabilized β‐catenin binds to TCFs, converting them into transcriptional activators. C‐terminal‐binding protein (CtBP) is a transcriptional corepressor that has been reported to inhibit Wnt signaling by binding to TCFs or by preventing β‐catenin from binding to TCF. Here, we show that CtBP is also required for the activation of some Wnt targets in Drosophila. CtBP is recruited to Wnt‐regulated enhancers in a Wnt‐dependent manner, where it augments Armadillo (the fly β‐catenin) transcriptional activation. We also found that CtBP is required for repression of a subset of Wnt targets in the absence of Wnt stimulation, but in a manner distinct from previously reported mechanisms. CtBP binds to Wnt‐regulated enhancers in a TCF‐independent manner and represses target genes in parallel with TCF. Our data indicate dual roles for CtBP as a gene‐specific activator and repressor of Wnt target gene transcription.

The pluripotent nature of human embryonic stem cells (hESCs) makes them convenient for deriving therapeutically relevant cells. Here we show using Wnt reporter hESC lines that the cells are heterogeneous with respect to endogenous Wnt signalling activity. Moreover, the level of Wnt signalling activity in individual cells correlates with differences in clonogenic potential and lineage-specific differentiation propensity. The addition of Wnt protein or, conversely, a small-molecule Wnt inhibitor (IWP2) reduces heterogeneity, allowing stable expansion of Wnthigh or Wntlow hESC populations, respectively. On differentiation, the Wnthigh hESCs predominantly form endodermal and cardiac cells, whereas the Wntlow hESCs generate primarily neuroectodermal cells. Thus, heterogeneity with respect to endogenous Wnt signalling underlies much of the inefficiency in directing hESCs towards specific cell types. The relatively uniform differentiation potential of the Wnthigh and Wntlow hESCs leads to faster and more efficient derivation of targeted cell types from these populations.

High-throughput DNA sequencing technologies have revolutionized genomic analysis, including the de novo assembly of whole genomes. Nevertheless, assembly of complex genomes remains challenging, in part due to the presence of dispersed repeats which introduce ambiguity during genome reconstruction. Transposable elements (TEs) can be particularly problematic, especially for TE families exhibiting high sequence identity, high copy number, or complex genomic arrangements. While TEs strongly affect genome function and evolution, most current de novo assembly approaches cannot resolve long, identical, and abundant families of TEs. Here, we applied a novel Illumina technology called TruSeq synthetic long-reads, which are generated through highly-parallel library preparation and local assembly of short read data and which achieve lengths of 1.5–18.5 Kbp with an extremely low error rate (0.03% per base). To test the utility of this technology, we sequenced and assembled the genome of the model organism Drosophila melanogaster (reference genome strain y; cn, bw, sp) achieving an N50 contig size of 69.7 Kbp and covering 96.9% of the euchromatic chromosome arms of the current reference genome. TruSeq synthetic long-read technology enables placement of individual TE copies in their proper genomic locations as well as accurate reconstruction of TE sequences. We entirely recovered and accurately placed 4,229 (77.8%) of the 5,434 annotated transposable elements with perfect identity to the current reference genome. As TEs are ubiquitous features of genomes of many species, TruSeq synthetic long-reads, and likely other methods that generate long-reads, offer a powerful approach to improve de novo assemblies of whole genomes.

Both transcriptional activation and repression have essential functions in maintaining proper spatial and temporal control of gene expression. Although Wnt signalling is often associated with gene activation, we have identified several directly repressed targets of Wnt signalling in Drosophila. Here, we explore how individual Wnt target genes are specified for signal‐induced activation or repression. Similar to activation, repression required binding of Armadillo (Arm) to the N terminus of TCF. However, TCF/Arm mediated repression by binding to DNA motifs that are markedly different from typical TCF‐binding sites. Conversion of the novel motifs to standard TCF‐binding sites reversed the mode of regulation, resulting in Wnt‐mediated activation instead of repression. A mutant form of Arm defective in activation was still functional for repression, indicating that distinct domains of the protein are required for each activity. This study suggests that the sequence of TCF‐binding sites allosterically regulates the TCF/Arm complex to effect either transcriptional activation or repression.

Accurate evaluation of microbial communities is essential for understanding global biogeochemical processes and can guide bioremediation and medical treatments. Metagenomics is most commonly used to analyze microbial diversity and metabolic potential, but assemblies of the short reads generated by current sequencing platforms may fail to recover heterogeneous strain populations and rare organisms. Here we used short (150-bp) and long (multi-kb) synthetic reads to evaluate strain heterogeneity and study microorganisms at low abundance in complex microbial communities from terrestrial sediments. The long-read data revealed multiple (probably dozens of) closely related species and strains from previously undescribed Deltaproteobacteria and Aminicenantes (candidate phylum OP8). Notably, these are the most abundant organisms in the communities, yet short-read assemblies achieved only partial genome coverage, mostly in the form of short scaffolds (N50 = ∼2200 bp). Genome architecture and metabolic potential for these lineages were reconstructed using a new synteny-based method. Analysis of long-read data also revealed thousands of species whose abundances were <0.1% in all samples. Most of the organisms in this “long tail” of rare organisms belong to phyla that are also represented by abundant organisms. Genes encoding glycosyl hydrolases are significantly more abundant than expected in rare genomes, suggesting that rare species may augment the capability for carbon turnover and confer resilience to changing environmental conditions. Overall, the study showed that a diversity of closely related strains and rare organisms account for a major portion of the communities. These are probably common features of many microbial communities and can be effectively studied using a combination of long and short reads.

Many Wnts influence cell behavior by a conserved signaling cascade that promotes the stabilization and nuclear accumulation of b-catenin (b-cat), which then associates with TCF family members to activate target genes. The histone acetyltransferase CREB binding protein (CBP) can bind to TCF and inhibit Wnt signaling in Drosophila. In contrast, studies in vertebrates indicate a positive role for CBP and the closely related protein p300 as b-cat binding transcriptional co-activators. We address this discrepancy by demonstrating that in addition to its negative role, CBP has an essential positive role in Wnt signaling in flies. CBP binds directly to the C-terminus of Armadillo (Arm, the fly b-cat) and is recruited to a Wnt-regulated enhancer (WRE) in a Wnt-and Arm-dependent manner. In a human colorectal cancer cell line, we show that CBP and p300 can inhibit Wnt signaling and demonstrate that human p300 can bind directly to TCF4 in vitro. Our results argue that CBP/p300 has an evolutionarily conserved role as a buffer regulating TCF-b-cat/Arm binding. Subsequent to this interaction, it also has an essential role in mediating the transactivation activity of b-cat/Arm.

The nitrogen-regulated genes and operons of the Ntr regulon of Escherichia coli are activated by the enhancer-binding transcriptional activator NRIϳP (NtrCϳP). Here, we examined the activation of the glnA, glnK, and nac promoters as cells undergo the transition from growth on ammonia to nitrogen starvation and examined the amplification of NRI during this transition. The results indicate that the concentration of NRI is increased as cells become starved for ammonia, concurrent with the activation of Ntr genes that have lessefficient enhancers than does glnA. A diauxic growth pattern was obtained when E. coli was grown on a low concentration of ammonia in combination with arginine as a nitrogen source, consistent with the hypothesis that Ntr genes other than glnA become activated only upon amplification of the NRI concentration.Escherichia coli contains six operons that are known to be part of the Ntr regulon (argThisJQMP, astCADBE, glnALG, glnHPQ, glnKamtB, and nac). These operons require 54 -RNA polymerase for expression and are activated by the phosphorylated form of enhancer-binding transcription factor NRI (NtrC) (reviewed in reference 16). A seventh operon,