Evolution of the mammalian transcription factor binding repertoire via transposable elements.

Identification of lineage-specific innovations in genomic control elements is critical for understanding transcriptional regulatory networks and phenotypic heterogeneity. We analyzed, from an evolutionary perspective, the binding regions of seven mammalian transcription factors (ESR1, TP53, MYC, RELA, POU5F1, SOX2, and CTCF) identified on a genome-wide scale by different chromatin immunoprecipitation approaches and found that only a minority of sites appear to be conserved at the sequence level. Instead, we uncovered a pervasive association with genomic repeats by showing that a large fraction of the bona fide binding sites for five of the seven transcription factors (ESR1, TP53, POU5F1, SOX2, and CTCF) are embedded in distinctive families of transposable elements. Using the age of the repeats, we established that these repeat-associated binding sites (RABS) have been associated with significant regulatory expansions throughout the mammalian phylogeny. We validated the functional significance of these RABS by showing that they are over-represented in proximity of regulated genes and that the binding motifs within these repeats have undergone evolutionary selection. Our results demonstrate that transcriptional regulatory networks are highly dynamic in eukaryotic genomes and that transposable elements play an important role in expanding the repertoire of binding sites.

Integration of external signaling pathways with the core transcriptional network in embryonic stem cells.

Transcription factors (TFs) and their specific interactions with targets are crucial for specifying gene-expression programs. To gain insights into the transcriptional regulatory networks in embryonic stem (ES) cells, we use chromatin immunoprecipitation coupled with ultra-high-throughput DNA sequencing (ChIP-seq) to map the locations of 13 sequence-specific TFs (Nanog, Oct4, STAT3, Smad1, Sox2, Zfx, c-Myc, n-Myc, Klf4, Esrrb, Tcfcp2l1, E2f1, and CTCF) and 2 transcription regulators (p300 and Suz12). These factors are known to play different roles in ES-cell biology as components of the LIF and BMP signaling pathways, self-renewal regulators, and key reprogramming factors. Our study provides insights into the integration of the signaling pathways into the ES-cell-specific transcription circuitries. Intriguingly, we find specific genomic regions extensively targeted by different TFs. Collectively, the comprehensive mapping of TF-binding sites identifies important features of the transcriptional regulatory networks that define ES-cell identity.

A machine learning strategy to identify candidate binding sites in human protein-coding sequence.

BACKGROUND: The splicing of RNA transcripts is thought to be partly promoted and regulated by sequences embedded within exons. Known sequences include binding sites for SR proteins, which are thought to mediate interactions between splicing factors bound to the 5' and 3' splice sites. It would be useful to identify further candidate sequences, however identifying them computationally is hard since exon sequences are also constrained by their functional role in coding for proteins. RESULTS: This strategy identified a collection of motifs including several previously reported splice enhancer elements. Although only trained on coding exons, the model discriminates both coding and non-coding exons from intragenic sequence. CONCLUSION: We have trained a computational model able to detect signals in coding exons which seem to be orthogonal to the sequences' primary function of coding for proteins. We believe that many of the motifs detected here represent binding sites for both previously unrecognized proteins which influence RNA splicing as well as other regulatory elements.

Genomewide expression profiling in the zebrafish embryo identifies target genes regulated by Hedgehog signaling during vertebrate development.

Hedgehog proteins play critical roles in organizing the embryonic development of animals, largely through modulation of target gene expression. Little is currently known, however, about the kinds and numbers of genes whose expression is controlled, directly or indirectly, by Hedgehog activity. Using techniques to globally repress or activate Hedgehog signaling in zebrafish embryos followed by microarray-based expression profiling, we have discovered a cohort of genes whose expression responds significantly to loss or gain of Hedgehog function. We have confirmed the Hedgehog responsiveness of a representative set of these genes with whole-mount in situ hybridization as well as real time PCR. In addition, we show that the consensus Gli-binding motif is enriched within the putative regulatory elements of a sizeable proportion of genes that showed positive regulation in our assay, indicating that their expression is directly induced by Hedgehog. Finally, we provide evidence that the Hedgehog-dependent spatially restricted transcription of one such gene, nkx2.9, is indeed mediated by Gli1 through a single Gli recognition site located within an evolutionarily conserved enhancer fragment. Taken together, this study represents the first comprehensive survey of target genes regulated by the Hedgehog pathway during vertebrate development. Our data also demonstrate for the first time the functionality of the Gli-binding motif in the control of Hedgehog signaling-induced gene expression in the zebrafish embryo.

The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells.

Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. Using the chromatin immunoprecipitation paired-end ditags method, we mapped the binding sites of these factors in the mouse ES cell genome. We identified 1,083 and 3,006 high-confidence binding sites for Oct4 and Nanog, respectively. Comparative location analyses indicated that Oct4 and Nanog overlap substantially in their targets, and they are bound to genes in different configurations. Using de novo motif discovery algorithms, we defined the cis-acting elements mediating their respective binding to genomic sites. By integrating RNA interference-mediated depletion of Oct4 and Nanog with microarray expression profiling, we demonstrated that these factors can activate or suppress transcription. We further showed that common core downstream targets are important to keep ES cells from differentiating. The emerging picture is one in which Oct4 and Nanog control a cascade of pathways that are intricately connected to govern pluripotency, self-renewal, genome surveillance and cell fate determination.