The ELT-2 GATA-factor and the global regulation of transcription in the C. elegans intestine

AbstractA SAGE library was prepared from hand-dissected intestines from adult Caenorhabditis elegans, allowing the identification of >4000 intestinally-expressed genes; this gene inventory provides fundamental information for understanding intestine function, structure and development. Intestinally-expressed genes fall into two broad classes: widely-expressed “housekeeping” genes and genes that are either intestine-specific or significantly intestine-enriched. Within this latter class of genes, we identified a subset of highly-expressed highly-validated genes that are expressed either exclusively or primarily in the intestine. Over half of the encoded proteins are candidates for secretion into the intestinal lumen to hydrolyze the bacterial food (e.g. lysozymes, amoebapores, lipases and especially proteases). The promoters of this subset of intestine-specific/intestine-enriched genes were analyzed computationally, using both a word-counting method (RSAT oligo-analysis) and a method based on Gibbs sampling (MotifSampler). Both methods returned the same over-represented site, namely an extended GATA-related sequence of the general form AHTGATAARR, which agrees with experimentally determined cis-acting control sequences found in intestine genes over the past 20 years. All promoters in the subset contain such a site, compared to <5% for control promoters; moreover, our analysis suggests that the majority (perhaps all) of genes expressed exclusively or primarily in the worm intestine are likely to contain such a site in their promoters. There are three zinc-finger GATA-type factors that are candidates to bind this extended GATA site in the differentiating C. elegans intestine: ELT-2, ELT-4 and ELT-7. All evidence points to ELT-2 being the most important of the three. We show that worms in which both the elt-4 and the elt-7 genes have been deleted from the genome are essentially wildtype, demonstrating that ELT-2 provides all essential GATA-factor functions in the intestine. The SAGE analysis also identifies more than a hundred other transcription factors in the adult intestine but few show an RNAi-induced loss-of-function phenotype and none (other than ELT-2) show a phenotype primarily in the intestine. We thus propose a simple model in which the ELT-2 GATA factor directly participates in the transcription of all intestine-specific/intestine-enriched genes, from the early embryo through to the dying adult. Other intestinal transcription factors would thus modulate the action of ELT-2, depending on the worm's nutritional and physiological needs

Conserved elements associated with ribosomal genes and their trans-splice acceptor sites in Caenorhabditis elegans

The recent publication of the Caenorhabditis elegans cisRED database has provided an extensive catalog of upstream elements that are conserved between nematode genomes. We have performed a secondary analysis to determine which subsequences of the cisRED motifs are found in multiple locations throughout the C. elegans genome. We used the word-counting motif discovery algorithm DME to form the motifs into groups based on sequence similarity. We then examined the genes associated with each motif group using DAVID and Ontologizer to determine which groups are associated with genes that also have significant functional associations in the Gene Ontology and other gene annotation sources. Of the 3265 motif groups formed, 612 (19%) had significant functional associations with respect to GO terms. Eight of the first 20 motif groups based on frequent dodecamers among the cisRED motif sequences were specifically associated with ribosomal protein genes; two of these were similar to mouse EBP-45, rat HNF3-family and Drosophila Zeste transcription factor binding sites. Additionally, seven motif groups were extensions of the canonical C. elegans trans-splice acceptor site. One motif group was tested for regulatory function in a series of green fluorescent protein expression experiments and was shown to be involved in pharyngeal expression

Caenorhabditis elegans cisRED: a catalogue of conserved genomic elements

The availability of completely sequenced genomes from eight species of nematodes has provided an opportunity to identify novel cis-regulatory elements in the promoter regions of Caenorhabditis elegans transcripts using comparative genomics. We determined orthologues for C. elegans transcripts in C. briggsae, C. remanei, C. brenneri, C. japonica, Pristionchus pacificus, Brugia malayi and Trichinella spiralis using the WABA alignment algorithm. We pooled the upstream region of each transcript in C. elegans with the upstream regions of its orthologues and identified conserved DNA sequence elements by de novo motif discovery. In total, we discovered 158 017 novel conserved motifs upstream of 3847 C. elegans transcripts for which three or more orthologues were available, and identified 82% of 44 experimentally proven regulatory elements from ORegAnno. We annotated 26% of the motifs as similar to known binding sequences of transcription factors from ORegAnno, TRANSFAC and JASPAR. This is the first catalogue of annotated conserved upstream elements for nematodes and can be used to find putative regulatory elements, improve gene models, discover novel RNA genes, and understand the evolution of transcription factors and their binding sites in phylum Nematoda. The annotated motifs provide novel binding site candidates for both characterized transcription factors and orthologues of characterized mammalian transcription factors

A methodology for analyzing SAGE libraries for cancer profiling

Serial Analysis of Gene Expression (SAGE) has proven to be an important alternative to microarray techniques for global profiling of mRNA populations. We have developed preprocessing methodologies to address problems in analyzing SAGE data due to noise caused by sequencing error, normalization methodologies to account for libraries sampled at different depths, and missing tag imputation methodologies to aid in the analysis of poorly sampled SAGE libraries. We have also used subspace selection using the Wilcoxon rank sum test to exclude tags that have similar expression levels regardless of source. Using these methodologies we have clustered, using the OPTICS algorithm, 88 SAGE libraries derived from cancerous and normal tissues as well as cell line material. Our results produced eight dense clusters representing ovarian cancer cell line, brain cancer cell line, brain cancer bulk tissue, prostate tissue, pancreatic cancer, breast cancer cell line, normal brain, and normal breast bulk tissue. The ovarian cancer and brain cancer cell lines clustered closely together, leading to a further investigation on possible associations between these two cancer types. We also investigated the utility of gene expression data in the classification between normal and cancerous tissues. Our results indicate that brain and breast cancer libraries have strong identities allowing robust discrimination from their normal counterparts. However, the SAGE expressio

Regulatory elements of <it>Caenorhabditis elegans</it> ribosomal protein genes

Abstract Background Ribosomal protein genes (RPGs) are essential, tightly regulated, and highly expressed during embryonic development and cell growth. Even though their protein sequences are strongly conserved, their mechanism of regulation is not conserved across yeast, Drosophila, and vertebrates. A recent investigation of genomic sequences conserved across both nematode species and associated with different gene groups indicated the existence of several elements in the upstream regions of C. elegans RPGs, providing a new insight regarding the regulation of these genes in C. elegans. Results In this study, we performed an in-depth examination of C. elegans RPG regulation and found nine highly conserved motifs in the upstream regions of C. elegans RPGs using the motif discovery algorithm DME. Four motifs were partially similar to transcription factor binding sites from C. elegans, Drosophila, yeast, and human. One pair of these motifs was found to co-occur in the upstream regions of 250 transcripts including 22 RPGs. The distance between the two motifs displayed a complex frequency pattern that was related to their relative orientation. We tested the impact of three of these motifs on the expression of rpl-2 using a series of reporter gene constructs and showed that all three motifs are necessary to maintain the high natural expression level of this gene. One of the motifs was similar to the binding site of an orthologue of POP-1, and we showed that RNAi knockdown of pop-1 impacts the expression of rpl-2. We further determined the transcription start site of rpl-2 by 5’ RACE and found that the motifs lie 40–90 bases upstream of the start site. We also found evidence that a noncoding RNA, contained within the outron of rpl-2, is co-transcribed with rpl-2 and cleaved during trans-splicing. Conclusions Our results indicate that C. elegans RPGs are regulated by a complex novel series of regulatory elements that is evolutionarily distinct from those of all other species examined up until now.</p

Caenorhabditis elegans cisRED: a catalogue of

conserved genomic element