12 research outputs found
Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes
The paired-end ditagging (PET) technique has been shown to be efficient and accurate for large-scale transcriptome and genome analysis. However, as with other DNA tag-based sequencing strategies, it is constrained by the current efficiency of Sanger technology. A recently developed multiplex sequencing method (454-sequencing™) using picolitre-scale reactions has achieved a remarkable advance in efficiency, but suffers from short-read lengths, and a lack of paired-end information. To further enhance the efficiency of PET analysis and at the same time overcome the drawbacks of the new sequencing method, we coupled multiplex sequencing with paired-end ditagging (MS-PET) using modified PET procedures to simultaneously sequence 200 000 to 300 000 dimerized PET (diPET) templates, with an output of nearly half-a-million PET sequences in a single 4 h machine run. We demonstrate the utility and robustness of MS-PET by analyzing the transcriptome of human breast carcinoma cells, and by mapping p53 binding sites in the genome of human colorectal carcinoma cells. This combined sequencing strategy achieved an approximate 100-fold efficiency increase over the current standard for PET analysis, and furthermore enables the short-read-length multiplex sequencing procedure to acquire paired-end information from large DNA fragments
De-Novo Identification of PPARγ/RXR Binding Sites and Direct Targets during Adipogenesis
BACKGROUND: The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPARgamma have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARgamma binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPARgamma binding sites in 3T3-L1 preadipocyte cells. METHODOLOGY/PRINCIPAL FINDINGS: Coupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPARgamma and its heterodimeric partner RXR during the course of adipocyte differentiation. Our validation studies prove that the identified sites are bona fide binding sites for both PPARgamma and RXR and that they are functionally capable of driving PPARgamma specific transcription. Our results strongly indicate that PPARgamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARgamma/RXR association is enriched within the proximity of the 5' region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARgamma as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPARgamma/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPARgamma direct targets that have not been previously reported to promote adipogenic differentiation. CONCLUSIONS/SIGNIFICANCE: We have identified in a genome-wide manner the binding sites of PPARgamma and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPARgamma function in the context of adipocyte differentiation
Transcriptomics during maternal-to-zygotic transition in the zebrafish: An mRNA-seq approach
Genome Wide Analysis Reveals Zic3 Interaction with Distal Regulatory Elements of Stage Specific Developmental Genes in Zebrafish
<div><p>Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 <i>in vivo</i>, by applying a combination of two powerful genomics approaches – ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in <i>zic3</i>-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using <i>in vivo</i> enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.</p></div
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
Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition
Maternally deposited mRNAs direct early development before the initiation of zygotic transcription during mid-blastula transition (MBT). To study mechanisms regulating this developmental event in zebrafish, we applied mRNA deep sequencing technology and generated comprehensive information and valuable resources on transcriptome dynamics during early embryonic (egg to early gastrulation) stages. Genome-wide transcriptome analysis documented at least 8000 maternal genes and identified the earliest cohort of zygotic transcripts. We determined expression levels of maternal and zygotic transcripts with the highest resolution possible using mRNA-seq and clustered them based on their expression pattern. We unravel delayed polyadenylation in a large cohort of maternal transcripts prior to the MBT for the first time in zebrafish. Blocking polyadenylation of these transcripts confirms their role in regulating development from the MBT onward. Our study also identified a large number of novel transcribed regions in annotated and unannotated regions of the genome, which will facilitate reannotation of the zebrafish genome. We also identified splice variants with an estimated frequency of 50%–60%. Taken together, our data constitute a useful genomic information and valuable transcriptome resource for gene discovery and for understanding the mechanisms of early embryogenesis in zebrafish
Zic3 regulates neural-specific expression through CNEs.
<p>A, number of CNEs found among the Zic3 peaks. A large subset was conserved between zebrafish and <i>Tetraodon</i>, while a smaller subset was conserved between zebrafish and human. B, distribution of CNE peaks with regards to their distance from TSS of genes. Enrichment of biological process (C) and tissue-specific expression (D) terms among genes associated with CNE peaks. Light and dark grey bars represent expected and observed enrichments of functional categories according to DAVID GO terms. E–H, representative figure of 24 hpf F<sub>0</sub> embryos expressing <i>gfp</i> (left panel) driven by Zic3 CNE peaks shown in UCSC browser image (right panel, green arrows); black horizontal line at the top of each panel represents 100 kb. E*, H*, dorsal view; F*, G*, lateral view.</p
Distribution of Zic3 peaks as identified in ChIP-seq experiments according to GREAT algorithm.
<p>A, distribution of peaks located in promoter (within 5 kb upstream of TSS), intragenic, and intergenic regions. B, gene association rule of ‘basal plus 100 kb’ according to GREAT algorithm. C, percentage of peaks associated with none, one, or two genes based on the gene association rule in B. D, number of peaks present in each distance categories along the x-axis, with regards to TSS of associated gene. E, region map showing overlap between genomic locations of peaks in 8 hpf and 24 hpf datasets. F–I, list of biological processes and tissue specific expression terms enriched among Zic3-associated genes at 8 hpf (F, G) and 24 hpf (H, I) according to DAVID GO terms. Light and dark grey bars represent the expected and observed enrichments of functional categories indicated along the y-axis.</p
Candidate target genes regulated by Zic3 at 8 hpf and 24 hpf developmental stages.
<p>Target genes are grouped based on their signaling pathway or functions. Changes in expression in microarray are represented by red and green backgrounds for up- and down-regulation respectively.</p
Zic3 binding sites associated with genes from the Nodal and Wnt pathway genes.
<p>UCSC browser image depicting genomic locations of Zic3 peaks identified near Nodal (A) and Wnt (B) pathway genes at 8 hpf. Single black vertical bars below histogram - peaks called by QuEST algorithm, blue horizontal bars - annotated exons (tall boxes), UTRs (half-sized boxes), introns (lines, arrowheads denote transcript orientation); Zic3 binding sites with negative (red arrows) and positive (green arrows) enhancer activity. Scale bars are indicated by black horizontal line at the top of each panel. C, list of tested fragments associated with Nodal and Wnt pathway genes. Enhancer-driven expression was assayed by qRT-PCR of <i>gfp</i> at 8 hpf and through microscopic observation of GFP expression pattern at 24 hpf. Between 50 to 100 embryos were assayed in each experimental time point. D–G, representative figure of <i>gfp</i> expression driven by selected fragments of Zic3 binding sites in F<sub>0</sub> embryos at 24 hpf, immunostained with anti-GFP antibody. D*, F*, dorsal view; E*, G*, lateral view.</p