Heme utilization in the Caenorhabditis elegans hypodermal cells is facilitated by hemeresponsive

Supplemental Data Heme utilization in the Caenorhabditis elegans hypodermal cells is facilitated by hemeresponsive gene-2 Caiyong Chen 1, Tamika K. Samuel 1, Michael Krause 2, Harry A. Dailey 3, and Iqbal Hamza 1 1 From the Department of Animal & Avian Sciences and Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, Maryland 20742 2 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 3 Biomedical and Health Sciences Institute, Department of Microbiology and the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602 Table of Contents S-1 Cover page S-2 Supplemental Methods S-3 Table S1 S-4 Table S2 S-5 Figure S1 S-6 Figure S2 S-7 Figure S3 S-8 Figure S4 S-9 Figure S5 S-10 Figure S6 S-11 Figure S7 S-1

Supplemental Method: Microarray Analysis C. elegans whole genome expression array chips (Affymetrix) were probed and the data analyzed using both MAS5.0 and Robust Multichip Analysis (RMA). For MAS5.0 analysis, a pseudochip was created from the three wildtype broodmate controls and used as a baseline file to which each of the experimental hrg-2 mutant samples were compared. For both heme concentration growth conditions (4 µm and 20 µm), the MAS5.0 detection and change in expression calls were used to narrow the list of genes of interest. For 4 µm heme, a comparison between hrg-2 mutants and the wildtype control resulted in 1,050 probe sets with decreased and 49 probe sets with increased expression across all three biological replicates. For 20 µm heme, this same comparison resulted in 273 probe sets with decreased and 35 probe sets with increased expression across all replicates. All probe sets resulting in altered expression across all three biological replicates, the most stringent condition, were subjected to Gene Ontology (GO) analysis using the DAVID functional annotation tools (http://david.abcc.ncifcrf.gov/summary.jsp). That analysis was most informative for the downregulated genes in 4 µm heme for hrg-2 mutants. Biological processes highly overrepresented in that dataset were cuticle collagen/molting (enrichment score 7.01), locomotion/larval growth (enrichment score 6.31), growth (enrichment score 4.99), hexose metabolism (enrichment score 2.78), fatty acid/lipid biosynthesis (enrichment score 2.48), and ER/cellular stress (enrichment score 2.47). To identify all Affymetrix array probe sets related to genes encoding cytochromes, we searched the C. elegans Wormbase genome database (WS195) for PFAM motifs related to cytochrome c (PF00034) and p450 (PF00067) and recovered 81 Affymetrix probe sets representing 75 genes. Among these probe sets at 4 µm heme, a comparison between hrg-2 mutants and the wildtype control resulted in one gene (cyp-37b1) that was consistently downregulated and no genes upregulated across all three biological replicates. For 20 µm heme, this same comparison resulted in three genes downregulated (cyp-25a1, cyp-29a3, cyp-36a1) and no genes upregulated (supplemental Fig S7). S-2

Supplemental Table S1. Transgenic C. elegans strains used in this study Number of Strain Genotype Method analyzed lines IQ8021 hrg-2 1.5kb ::gfp, rol-6+ microinjection 7 IQ8023 hrg-2 0.5kb ::gfp, rol-6+ microinjection 2 unc-119(ed3) III; hrg-2 1.5kb ::hrg-2::yfp, microparticle IQ8122 5 unc-119+ bombardment IQ8123 4 : TRAM: translocating chain associated membrane protein. unc-119(ed3) III; hrg-2 1.5kb ::hrg-2::yfp, microparticle dpy-7::mcherry::tram a, unc-119+ bombardment S-3

Supplemental Table S2. Oxygen consumption rates of transformed wildtype W303 and hem1δ yeast grown in 5 μm heme. Strains μm/min/mg Wildtype vector 19.95 ± 1.05 hem1δ vector 2.52 ± 0.25 hem1δ cdr-1 3.46 ± 0.23 ns hem1δ hrg-2 6.60 ± 0.11 *** hem1δ hrg-4 38.83 ± 0.37 *** ns: not significant when compared to hem1δ vector ***: P<0.001 compared to hem1δ vector S-4

Supplemental Figure S1. Chen et al., Supplemental FIGURE S1. Multiple sequence alignment of HRG-2 proteins among C. elegans, C. briggsae, and C. remanei. C. briggsae has two putative HRG-2 orthologs, of which the candidate with higher homology (WormBase protein ID CBP20711) is shown in the alignment. The protein ID for C. remanei HRG-2 is RP30064. Transmembrane domains (TMD) are marked with gray filled boxes. GST-like domains were drawn based on the amino acid positions in C. elegans HRG-2 and are marked with unfilled boxes. The numbers at the end of alignment indicate the percentage identity between C. elegans HRG-2 and its homologs. S-5

Supplemental Figure S2. Chen et al., Supplemental FIGURE S2. Evolutionary relationships of HRG-2, CDRs, and their homologs in nematodes. Protein sequences were aligned using the ClustalW program, and the phylogenetic tree was constructed with the neighbor-joining method in MEGA 4. The branch lengths of the tree reflect the evolutionary distances, which are in the units of the number of amino acid substitutions. The scale bar represents 10% sequencee divergence. HRG-2 and CDRs in C. elegans are marked with a box or asterisks. Cel: C. elegans. CBP: C. briggsae. CN: C. brenneri. RP: C. remanei. PP: P. pacificus. S-6

Supplemental Figure S3. Chen et al., Supplemental FIGURE S3. Multiple sequence alignment of HRG-2 and the putative homolgs in vertebrate species. The accession numbers of the putative homologs are NP_115900 (human), NP 780443 (mouse), XP_426188 (chick), and XP_692250 (zebrafish). The numbers at the end of alignment indicate the percentage identities between C. elegans HRG-2 and its homologs. S-7

Supplemental Figure S4. Chen et al., Supplemental FIGURE S4. The 0.5-kb intergenic region is responsible for the spatial expression and heme response of hrg-2. A, Schematic representation of the hrg-2::gfp 0.5 reporter construct. Genomic structures of hrg-2 and cdr-7 are shown on the top. NLS: nuclear localization signal. B, hrg-2::gfpattern is similar to that of hrg-2::gfp 1.5. Arrows indicate GFP expression in hypodermal cells. C, 0.5 is predominantly expressed in hypodermal cells in C. elegans. This expression The hrg-2::gfp 0.5 reporter is not expressed when the worms were exposed to 20 µm heme. Asterisk indicates the non-specific autofluorescence of gut granules. Scale bars, 20 µm. S-8

Supplemental Figure S5. Chen et al., Supplemental FIGURE S5. Immunofluorescence microscopy of HEK-293 cells expressing HRG-2. Cells co-transfected with HA-tagged HRG-2 and the mitochondrial marker mitoyfp were subjected to epifluorescence (top) or confocal (bottom) microscopy using anti-ha primary antibody and an Alexa 568 conjugated secondary antibody. Scale bar indicates 10 μm. S-9

Supplemental Figure S6. Chen et al., Supplemental FIGURE S6. hrg-2 deletion does not affect the growth of C. elegans. Both hrg-2 (tm3798) and its wildtype broodmate worms were first grown at 2 μm heme for one generation in mcehr-2 medium. Synchronized L1 larvae were inoculated into the liquid medium containing 0, 4, and 20 μm heme. After 9 days of growth, worms in all treatments were counted, and the numbers were normalized to the actual input (0 μm).. The experiment was performed in duplicate. A total of 268 wildtype and 206 hrg-2 worms were scored for the number of their progeny at each heme concentration. No statistical difference was observed between wildtype and hrg-2 animals (P >0.05). S-10

Supplemental Figure S7. Chen et al., Normalized Fold Change 1 0 1 2 3 4 5 6 7 cyp 25A1 cyp 29A3 cyp 36A1 cyp 37B1 4 μm Heme 20 μm Heme Supplemental FIGURE S7. Changes in cytochrome gene expression in hrg-2 mutants. Microarrays performed on hrg-2 and wildtype broodmate controls showed that one gene at 4 µm heme (cyp-37b1) and three genes at 20 µm heme (cyp-25a1, cyp-29a3, cyp-36a1) were downregulated from the 75 cytochrome encoding genes represented on the expression arrays Consistent changes in expression for these four genes were observed across all three biological replicates. S-11