ChIP-seq Defined Genome-Wide Map of TGFβ/SMAD4 Targets: Implications with Clinical Outcome of Ovarian Cancer

Deregulation of the transforming growth factor-β (TGFβ) signaling pathway in epithelial ovarian cancer has been reported, but the precise mechanism underlying disrupted TGFβ signaling in the disease remains unclear. We performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) to investigate genome-wide screening of TGFβ-induced SMAD4 binding in epithelial ovarian cancer. Following TGFβ stimulation of the A2780 epithelial ovarian cancer cell line, we identified 2,362 SMAD4 binding loci and 318 differentially expressed SMAD4 target genes. Comprehensive examination of SMAD4-bound loci, revealed four distinct binding patterns: 1) Basal; 2) Shift; 3) Stimulated Only; 4) Unstimulated Only. TGFβ stimulated SMAD4-bound loci were primarily classified as either Stimulated only (74%) or Shift (25%), indicating that TGFβ-stimulation alters SMAD4 binding patterns in epithelial ovarian cancer cells. Furthermore, based on gene regulatory network analysis, we determined that the TGFβ-induced, SMAD4-dependent regulatory network was strikingly different in ovarian cancer compared to normal cells. Importantly, the TGFβ/SMAD4 target genes identified in the A2780 epithelial ovarian cancer cell line were predictive of patient survival, based on in silico mining of publically available patient data bases. In conclusion, our data highlight the utility of next generation sequencing technology to identify genome-wide SMAD4 target genes in epithelial ovarian cancer and link aberrant TGFβ/SMAD signaling to ovarian tumorigenesis. Furthermore, the identified SMAD4 binding loci, combined with gene expression profiling and in silico data mining of patient cohorts, may provide a powerful approach to determine potential gene signatures with biological and future translational research in ovarian and other cancers

Comprehensive Analysis of Transcript Start Sites in Ly49 Genes Reveals an Unexpected Relationship with Gene Function and a Lack Of Upstream Promoters

Comprehensive analysis of the transcription start sites of the Ly49 genes of C57BL/6 mice using the oligo-capping 5′-RACE technique revealed that the genes encoding the “missing self” inhibitory receptors, Ly49A, C, G, and I, were transcribed from multiple broad regions in exon 1, in the intron1/exon2 region, and upstream of exon -1b. Ly49E was also transcribed in this manner, and uniquely showed a transcriptional shift from exon1 to exon 2 when NK cells were activated in vitro with IL2. Remarkably, a large proportion of Ly49E transcripts was then initiated from downstream of the translational start codon. By contrast, the genes encoding Ly49B and Q in myeloid cells, the activating Ly49D and H receptors in NK cells, and Ly49F in activated T cells, were predominantly transcribed from a conserved site in a pyrimidine-rich region upstream of exon 1. An ∼200 bp fragment from upstream of the Ly49B start site displayed tissue-specific promoter activity in dendritic cell lines, but the corresponding upstream fragments from all other Ly49 genes lacked detectable tissue-specific promoter activity. In particular, none displayed any significant activity in a newly developed adult NK cell line that expressed multiple Ly49 receptors. Similarly, no promoter activity could be found in fragments upstream of intron1/exon2. Collectively, these findings reveal a previously unrecognized relationship between the pattern of transcription and the expression/function of Ly49 receptors, and indicate that transcription of the Ly49 genes expressed in lymphoid cells is achieved in a manner that does not require classical upstream promoters

Gene expression profiling integrated into network modelling reveals heterogeneity in the mechanisms of BRCA1 tumorigenesis

Background: gene expression profiling has distinguished sporadic breast tumour classes with genetic and clinical differences. Less is known about the molecular classification of familial breast tumours, which are generally considered to be less heterogeneous. Here, we describe molecular signatures that define BRCA1 subclasses depending on the expression of the gene encoding for oestrogen receptor, ESR1. Methods: for this purpose, we have used the Oncochip v2, a cancer-related cDNA microarray to analyze 14 BRCA1-associated breast tumours. Results: signatures were found to be molecularly associated with different biological processes and transcriptional regulatory programs. The signature of ESR1-positive tumours was mainly linked to cell proliferation and regulated by ER, whereas the signature of ESR1-negative tumours was mainly linked to the immune response and possibly regulated by transcription factors of the REL/NFκB family. These signatures were then verified in an independent series of familial and sporadic breast tumours, which revealed a possible prognostic value for each subclass. Over-expression of immune response genes seems to be a common feature of ER-negative sporadic and familial breast cancer and may be associated with good prognosis. Interestingly, the ESR1-negative tumours were substratified into two groups presenting slight differences in the magnitude of the expression of immune response transcripts and REL/NFκB transcription factors, which could be dependent on the type of BRCA1 germline mutation. Conclusion: this study reveals the molecular complexity of BRCA1 breast tumours, which are found to display similarities to sporadic tumours, and suggests possible prognostic implications

Deletion Hotspots in AMACR Promoter CpG Island Are cis-Regulatory Elements Controlling the Gene Expression in the Colon

Alpha-methylacyl-coenzyme A racemase (AMACR) regulates peroxisomal β-oxidation of phytol-derived, branched-chain fatty acids from red meat and dairy products — suspected risk factors for colon carcinoma (CCa). AMACR was first found overexpressed in prostate cancer but not in benign glands and is now an established diagnostic marker for prostate cancer. Aberrant expression of AMACR was recently reported in Cca; however, little is known about how this gene is abnormally activated in cancer. By using a panel of immunostained-laser-capture-microdissected clinical samples comprising the entire colon adenoma–carcinoma sequence, we show that deregulation of AMACR during colon carcinogenesis involves two nonrandom events, resulting in the mutually exclusive existence of double-deletion at CG3 and CG10 and deletion of CG12-16 in a newly identified CpG island within the core promoter of AMACR. The double-deletion at CG3 and CG10 was found to be a somatic lesion. It existed in histologically normal colonic glands and tubular adenomas with low AMACR expression and was absent in villous adenomas and all CCas expressing variable levels of AMACR. In contrast, deletion of CG12-16 was shown to be a constitutional allele with a frequency of 43% in a general population. Its prevalence reached 89% in moderately differentiated CCas strongly expressing AMACR but only existed at 14% in poorly differentiated CCas expressing little or no AMACR. The DNA sequences housing these deletions were found to be putative cis-regulatory elements for Sp1 at CG3 and CG10, and ZNF202 at CG12-16. Chromatin immunoprecipitation, siRNA knockdown, gel shift assay, ectopic expression, and promoter analyses supported the regulation by Sp1 and ZNF202 of AMACR gene expression in an opposite manner. Our findings identified key in vivo events and novel transcription factors responsible for AMACR regulation in CCas and suggested these AMACR deletions may have diagnostic/prognostic value for colon carcinogenesis

Adaptive Evolution and the Birth of CTCF Binding Sites in the Drosophila Genome

Changes in the physical interaction between cis-regulatory DNA sequences and proteins drive the evolution of gene expression. However, it has proven difficult to accurately quantify evolutionary rates of such binding change or to estimate the relative effects of selection and drift in shaping the binding evolution. Here we examine the genome-wide binding of CTCF in four species of Drosophila separated by between ~2.5 and 25 million years. CTCF is a highly conserved protein known to be associated with insulator sequences in the genomes of human and Drosophila. Although the binding preference for CTCF is highly conserved, we find that CTCF binding itself is highly evolutionarily dynamic and has adaptively evolved. Between species, binding divergence increased linearly with evolutionary distance, and CTCF binding profiles are diverging rapidly at the rate of 2.22% per million years (Myr). At least 89 new CTCF binding sites have originated in the Drosophila melanogaster genome since the most recent common ancestor with Drosophila simulans. Comparing these data to genome sequence data from 37 different strains of Drosophila melanogaster, we detected signatures of selection in both newly gained and evolutionarily conserved binding sites. Newly evolved CTCF binding sites show a significantly stronger signature for positive selection than older sites. Comparative gene expression profiling revealed that expression divergence of genes adjacent to CTCF binding site is significantly associated with the gain and loss of CTCF binding. Further, the birth of new genes is associated with the birth of new CTCF binding sites. Our data indicate that binding of Drosophila CTCF protein has evolved under natural selection, and CTCF binding evolution has shaped both the evolution of gene expression and genome evolution during the birth of new genes

A systematic review and meta-analysis to revise the Fenton growth chart for preterm infants

BACKGROUND: The aim of this study was to revise the 2003 Fenton Preterm Growth Chart, specifically to: a) harmonize the preterm growth chart with the new World Health Organization (WHO) Growth Standard, b) smooth the data between the preterm and WHO estimates, informed by the Preterm Multicentre Growth (PreM Growth) study while maintaining data integrity from 22 to 36 and at 50 weeks, and to c) re-scale the chart x-axis to actual age (rather than completed weeks) to support growth monitoring. METHODS: Systematic review, meta-analysis, and growth chart development. We systematically searched published and unpublished literature to find population-based preterm size at birth measurement (weight, length, and/or head circumference) references, from developed countries with: Corrected gestational ages through infant assessment and/or statistical correction; Data percentiles as low as 24 weeks gestational age or lower; Sample with greater than 500 infants less than 30 weeks. Growth curves for males and females were produced using cubic splines to 50 weeks post menstrual age. LMS parameters (skew, median, and standard deviation) were calculated. RESULTS: Six large population-based surveys of size at preterm birth representing 3,986,456 births (34,639 births < 30 weeks) from countries Germany, United States, Italy, Australia, Scotland, and Canada were combined in meta-analyses. Smooth growth chart curves were developed, while ensuring close agreement with the data between 24 and 36 weeks and at 50 weeks. CONCLUSIONS: The revised sex-specific actual-age growth charts are based on the recommended growth goal for preterm infants, the fetus, followed by the term infant. These preterm growth charts, with the disjunction between these datasets smoothing informed by the international PreM Growth study, may support an improved transition of preterm infant growth monitoring to the WHO growth charts

Integrated Expression Profiling and Genome-Wide Analysis of ChREBP Targets Reveals the Dual Role for ChREBP in Glucose-Regulated Gene Expression

The carbohydrate response element binding protein (ChREBP), a basic helix-loop-helix/leucine zipper transcription factor, plays a critical role in the control of lipogenesis in the liver. To identify the direct targets of ChREBP on a genome-wide scale and provide more insight into the mechanism by which ChREBP regulates glucose-responsive gene expression, we performed chromatin immunoprecipitation-sequencing and gene expression analysis. We identified 1153 ChREBP binding sites and 783 target genes using the chromatin from HepG2, a human hepatocellular carcinoma cell line. A motif search revealed a refined consensus sequence (CABGTG-nnCnG-nGnSTG) to better represent critical elements of a functional ChREBP binding sequence. Gene ontology analysis shows that ChREBP target genes are particularly associated with lipid, fatty acid and steroid metabolism. In addition, other functional gene clusters related to transport, development and cell motility are significantly enriched. Gene set enrichment analysis reveals that ChREBP target genes are highly correlated with genes regulated by high glucose, providing a functional relevance to the genome-wide binding study. Furthermore, we have demonstrated that ChREBP may function as a transcriptional repressor as well as an activator