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Results: 8

1.
Figure 6

Figure 6. Chromosomal location and fold-change of each heme-responsive gene.. From: Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans.

hrgs were arranged on each of the six chromosomes, which are depicted as white or black bars. The transcript levels of genes placed on white bars were altered at 4 µM heme, while the expression of genes on black bars was significantly altered at 500 µM heme. Vertical bars are drawn to scale and represent genes that were up-regulated (red) or down-regulated (green).

Scott Severance, et al. PLoS Genet. 2010 July;6(7):e1001044.
2.
Figure 5

Figure 5. Depiction of the number of hrgs found on each chromosome relative to the number of megabases in that chromosome.. From: Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans.

The chromosome with the highest number of hrgs is chromosome V. Red and brown regions indicate that the expression of a gene was increased at 4 and 500 µM heme, respectively. Light blue and dark blue bars represent a decrease in gene expression at 4 and 500 µM heme, respectively.

Scott Severance, et al. PLoS Genet. 2010 July;6(7):e1001044.
3.
Figure 3

Figure 3. Heme-dependent changes in gene expression.. From: Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans.

1The 288 hrgs were arranged into eight categories based on whether they were up-regulated, down-regulated, or unchanged at 4 µM or 500 µM heme when compared to control data from 20 µM heme. 2C. elegans protein sequences for the 288 hrgs were used to perform reciprocal BLAST searches to identify putative homologs (E-value cut-off ≥10−4) in humans and protozoans.

Scott Severance, et al. PLoS Genet. 2010 July;6(7):e1001044.
4.
Figure 1

Figure 1. Profiling strategy for analysis of heme-responsive genes.. From: Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans.

Worms were grown in axenic liquid mCeHR-2 medium containing 4, 20, or 500 µM hemin chloride for two synchronized generations, and late L4 larvae were harvested to extract RNA for microarray analyses. cDNA was synthesized by reverse transcription and hybridized to Affymetrix C. elegans Genome Arrays containing 22,627 probe sets per chip. Affymetrix MAS 5.0 software and RMA were utilized to analyze the data. Data from worms grown at 4 and 500 µM heme were compared to control data from worms grown at 20 µM heme. The expression of 288 genes was either increased or decreased in response to heme by at least 1.6 fold.

Scott Severance, et al. PLoS Genet. 2010 July;6(7):e1001044.
5.
Figure 8

Figure 8. Characterization of the three candidate genes identified from the functional RNAi screens.. From: Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans.

(A) Heme-dependent expression. qRT-PCR was performed in triplicate for hrg-4, mrp-5, and F22B5.4 at the indicated heme concentrations, and the fold change (mean ± SEM) is reported for each gene at each concentration of heme. (B) Accumulation of ZnMP in worms depleted of candidate genes by RNAi. Synchronized wild-type strain worms in the L1 stage were transferred from mCeHR-2 medium containing 2 µM heme to plates seeded with a lawn of bacteria producing dsRNA against vector, hrg-4, mrp-5, or F22B5.4 and allowed to develop for ≈72 h to the late L4 stage. At this point, worms were incubated in mCeHR-2 medium containing 5 µM ZnMP overnight. Images of a region of worm intestine were captured using a Leica DMIRE2 microscope equipped with a Rhodamine filter and a CCD camera. (C) Expression of hrg-4 and mrp-5 GFP transcriptional reporters. Transgenic worms expressing GFP under the control of 3 kb of the hrg-4 (bottom) or mrp-5 (top) putative promoters. Transgenic worms were grown in mCeHR-2 medium containing 4 µM heme for one generation. Representative images of worms were obtained with a Leica DMIRE2 microscope using a CCD camera.

Scott Severance, et al. PLoS Genet. 2010 July;6(7):e1001044.
6.
Figure 2

Figure 2. Confirmation of quality of microarray results.. From: Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans.

(A) Gene expression profile for the 288 hrgs identified from the microarrays. Changes in expression for the 288 hrgs are plotted for each biological replicate (thin vertical lines) of the three growth conditions with respect to heme concentration (4 µM, 20 µM, and 500 µM) as visualized with GeneSpring software (v7.2). Signal intensity values for genes in each biological replicate and each growth condition (as indicated) were normalized to the median value across the array after setting values <0.01 to 0.01 using GeneSpring software and the resulting values plotted on a log10 scale. Values for a single gene are connected by lines with the slope indicating any change in value across samples; line color coding reflects direction of change relative to the mean (as indicated by the color bar above the graph) and hue intensity reflecting statistical confidence in the value (confidence increases with increased brightness). (B) Validation of microarray results. Microarray data were verified by qRT-PCR of RNA from 24 genes of worms grown in mCeHR-2 medium supplemented with 4, 20, or 500 µM heme. RNA from 20 µM heme was used as the reference sample. Data were compared to internal GAPDH (gpd-2) control and the fold change was obtained using the 2 (-ΔΔCt) method. The significance was determined using GraphPad Instat (v. 3.06). The values for both the qRT-PCR analysis and the microarray experiment are provided in Table S4.

Scott Severance, et al. PLoS Genet. 2010 July;6(7):e1001044.
7.
Figure 7

Figure 7. Functional validation of heme-responsive genes.. From: Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans.

(A) RNAi depletion in a transgenic heme-sensor strain. GFP quantification by COPAS BioSort in strain IQ6011 fed HT115(DE3) bacteria grown in 5 µM heme and induced to synthesize dsRNA. RNAi of 288 hrgs identified six genes which caused a 2-fold increase or 2-fold decrease in GFP levels. Each data point represents the mean ± SEM. Y-axis represents average GFP values of all the gravid worms (≥30 worms in each well) in duplicate wells normalized to the value of the GFP in worms that had fed on bacteria transformed with the empty vector. (B) RNAi depletion in the presence of a toxic heme analog. Forty-one candidate genes encoding proteins with TMD were screened by feeding RNAi to strain IQ6011 and assessing survival of progeny in the presence of the toxic heme analog GaPP. Both the total number of eggs and the number of viable larvae were counted after 5 days of exposure to 1.5 µM GaPP as described in Materials and Methods. Each data point represents the mean ± SEM of two separate experiments and is depicted as percentage of survival compared to control plates with no GaPP. Knockdown of 7 genes (shown) resulted in animal survival in the presence of GaPP. Asterisk indicates genes that also altered GFP levels in strain IQ6011 worms (Figure 7A). (C) Summary of overlap between genes identified in (A,B). Six of the 288 hrgs were identified as interfering with heme homeostasis, as evidenced by their altered GFP levels at 5 µM. Seven of the 41 hrgs that encode for proteins with putative TMD protect against GaPP toxicity, as evidenced by an increase in progeny survival. When knocked down by RNAi, only three hrgs both alter the ability to sense heme and protect against GaPP toxicity.

Scott Severance, et al. PLoS Genet. 2010 July;6(7):e1001044.
8.
Figure 4

Figure 4. Comparative analysis of the heme-responsive genes.. From: Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans.

(A) Summary of overlap between hrgs across human and protozoan genomes. The sequences of proteins encoded by the 288 hrgs were obtained from Wormbase and used to search for homologs in the human genome and genomes of Trypanosoma brucei, Tryoanosoma cruzi, and Leishmania major. (B) Orthologs of hrgs in the genomes of parasitic nematodes. The 288 C. elegans hrg gene products were used to identify homologs in available parasitic nematode sequences. Based on 18S rRNA sequences, the phylum Nematoda is divided in five major clades; all five clades include parasites. Homologs were identified for 69 genes using amino acid sequences (at BLAST cut-off of 35 bits and 55% identity), and summarized on per clade level. A total of 440,012 peptides from 29 parasitic nematode species was used (clade I 29,203 peptides; clade III 145,044; clade IVA 13,636; clade IVB 92,514 and clade V 159,615 peptides). Nematode sequences used for this analysis are available on the parasitic nematodes website (http://www.nematode.net). (C) Gene ontology (GO) enrichment analysis of hrgs upregulated at 4 µM heme. Genes upregulated at 4 µM heme were analyzed using the Fisher's exact test and the topGO package from R. The most significant GO terms and their associated parent terms were used to construct a hierarchical graph such that the specificity of the terms increased from top to bottom. The text in each rectangle provides the GO ID and the ratio of the number of genes annotated with the GO term in the tested subset to that in the total gene set. The shade of green of each rectangle corresponds to the significance of the GO result. The complete table of P-values can be found in Table S6. Full GO terms are provided solely for genes with P<0.005.

Scott Severance, et al. PLoS Genet. 2010 July;6(7):e1001044.

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