C. elegans mutant and transgenic strains
Strains were maintained at 20°C unless otherwise noted, using standard methods 
. The following mutants were used: LGI: daf-16(mu86)
Compound mutant strains and transgenes used are as follows:
CF1595: daf-16(mu86)I; daf-2(e1370)III, AGK138: zfp-1(ok554)III; pdk-1(sa709)X, AGK241: rde-4(ne299)III; pdk-1(sa709)X, AGK25: age-1(hx546)II; zfp-1(ok554)III, AGK264: age-1(hx546)II; rde-4(ne299)III, AGK72: daf-16(mgDf50)I; armEx5, TJ356: zIs356 IV, AGK30: zfp-1(ok554)III; zIs356 IV, AGK262: zfp-1(ok554)III; zIs356 IV; pdk-1(sa709)X, AGK377: rde-4(ne299)III; zIs356 IV, AGK 265: rde-4(ne299)III; zIs356 IV; pdk-1(sa709)X, AGK267: zfp-1(ok554) unc-119(ed3)III; armIs5, AGK248: rde-4(ne299) zfp-1(ok554) unc-119(ed3)III; armIs5, AGK260: zIs356 IV; pdk-1(sa709)X, SP940: unc-52(e444)II; unc-1(e538)X; mnDp11(II;X;f).
Transgenic worms were created by microparticle bombardment using a PDS-1000 Hepta Apparatus (Bio-Rad) 
. All strains were made by co-bombardment of both a fosmid of interest and plasmid pMM016b (AddGene) for unc-119(ed3)
III rescue. Strains created are as follows: AGK29: armIs2 Is[unc-119+] – control strain, AGK128: armIs5 Is[ZFP-1::FLAG,unc-119+], AGK26: armEx5 Ex[ZFP-1::GFP,unc-119+].
Recombinant fosmid construction
The WRM0629bD09 fosmid containing the ZFP-1 locus was obtained from the C. elegans fosmid library generated by C. elegans Reverse Genetics Core Facility, Vancouver, B.C., Canada.
We generated derivative fosmid constructs to express recombinant ZFP-1 protein tagged with GFP or FLAG at the C-terminal portion of the protein by a fosmid recombineering method as described by 
Oxidative stress assays (paraquat sensitivity)
Paraquat sensitivity assays were done essentially as described by 
. L4 animals were transferred from NGM agar plates into 24-well plates (10 per well) containing 300 µL of 100 mM paraquat dissolved in M9. Worms were then incubated at 20°C and scored for survival after 20 hours. Dead animals were scored by their continuous absence of swimming movements and pharyngeal pumping. A t-test between two means was used to calculate statistical significance.
Assays were performed as described by 
. Worms were kept at 20°C on NGM plates (10 animals per plate). Day of hatching was used as the first time point. Dead animals were scored as dead when they refused to move after repeated prodding with a pick. Animals that crawled away from the plate, exploded, or contained internally hatched worms were excluded from the analysis. Life spans were determined in parallel for all strains shown together on graphs. Statistical significance was determined by a log-rank analysis using Prizm software.
P. aeruginosa infection
survival assays were performed as described earlier 
. To avoid the confounding effects of varying brood sizes, egg laying rates and progeny hatching within the infected worms on worm mortality, we used worms rendered sterile by RNAi of pos-1
, loss of which results in inviable embryos 
. Worms that died due to desiccation on the walls of the Petri dish or due to bursting vulva were censored from further analysis. Statistical analysis was performed using Kaplan-Meier non-parametric survival analysis using the software Statview (Version 5.0.1 SAS Institute Inc.). P
<0.001 was considered significantly different than wild type.
Analysis of the nuclear localization of DAF-16::GFP
Since the addition of the DAF-16::GFP transgene to the zfp-1(ok554); pdk-1(sa709) double mutant strain led to a penetrant dauer phenotype at 20°C, all DAF-16::GFP strains were maintained at 16°C. L4 and adult stage worms were used for scoring nuclear localization. Worms were mounted on agarose pads and DAF-16::GFP localization was assessed in 10–20 worms at a time using 200X magnification on a Zeiss AxioImager Z1 immediately, higher magnification images of DAF-16::GFP localization in intestinal cells were done at 630X.
RNA extraction and RT-qPCR
Synchronous populations of animals were grown at 20°C on NGM plates seeded with OP50 E. coli at a density of approximately 100,000 animals per 15 cm Petri dish and harvested at specific stages of development. The harvested animals were washed three times with M9 buffer and the pellet was frozen in dry ice with TRI Reagent (MRC, Inc.). After five times of freeze and thaw, total RNA was isolated according to the TRI Reagent protocol. Ten micrograms of the total RNA sample was digested with 2U of Turbo DNase (Ambion) at 37°C for 1hr followed by phenol-extraction and ethanol-precipitation. cDNA was generated from 2 µg of total RNA, using oligo-dT primer and RevertAid Reverse Transcriptase (Fermentas). Quantitative PCR was performed on the Mastercycler ep realplex (Eppendorf) using the QuantiFast SYBR Green PCR Kit (Qiagen). Thermocycling was done for 40 cycles in a two-step cycling, according to the manufacturer's instructions, with 25 µl of reaction containing 12.5 µl SYBR master mix, 0.15 µl of 100 µM primers, 5 µl of diluted cDNA, and 7.2 µl dH2O. Each PCR reaction was performed in triplicate. We used the ΔΔCt method to quantify the change in mRNA expression in the mutant samples compared to wild type and act-3 mRNA was used as a reference gene. The primers used were as follows: Forward CACGAGACTTCTTACAACTCC and Reverse GCATACGATCAGCAATTCCT for act-3 mRNA detection, Forward AGCCATCAACACCGTCTAAC and Reverse CGAATTGGCGCGTGGTGC for pdk-1 mRNA detection, Forward GCTAGGATGTCAGGTGGTC and Reverse CCAAGAGAAGCCACGAAAGC for aqp-1 mRNA detection, Forward ATGCTCGTGCTCTTGCTGAG and Reverse GACTGACCGAATTGTTCTCCAT for gst-4 mRNA detection, Forward TACCGATGAGGAGTGGGAGA and Reverse CGAATTCCCGAGCAAGATAA for gst-38 mRNA detection, Forward TTTCAGAATCACAGAGCAACAC and Reverse TGCGATACATGTTCAGAAGAG for zfp-1 mRNA detection, Forward ACACTATTAAGCGCGACTTCG and Reverse AGTTGGCAATCTTCCAAATAGC for sod-3 mRNA detection, Forward pdk-1 ex2-ex3 junction CCTACAGCCAGGTATTCCG and Reverse pdk-1 intron 3 ACAAGTGGATTTTGATGGGTTC for detecting the mutant sa709 pdk-1 mRNA and pre-mRNA and Reverse pdk-1 ex3-4 junction GATCACGAAATAAATTCTAGCCTGG-for detecting the wild-type pdk-1 mRNA.
For detection of bi-directional transcription at the pdk-1 promoter the primers used were as follows. Region 1 RT primers: detecting (−) strand transcript CCGAGGTTATAATTTTGGCTAAACTT; detecting (+) strand transcript ATCAAGAGATACAGCGGGAG. Region 1 PCR primers:
Region 2 RT primers: detecting (−) strand transcript CTCCCGCTGTATCTCTTGAT
detecting (+) strand transcript GTACGGTTGTTATCGCTTTCAGG.
Region 2 PCR primers: forward - GAATGTTCAAAGCCTTAAAGC
reverse – AGGGATAATTGGAGTGACATGG.
Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation was performed following the modENCODE Protocol from the Lieb Lab with the following modification: 2.5–3mg of cross-linked extract from L3 or adult worms was incubated for 1h at 4°C with the specific antibody and the immune complexes were then incubated with 60 µl IgG Dynabeads (Invitrogen) for 1h at 4°C. DNA was cleaned up with the Qiagen PCR purification kit. For the FLAG ChIP, we incubated the cross-linked extract with ANTI-FLAG M2 Affinity Gel (Sigma) for 2h at 4°C and, after the washing steps, eluted with 300 µg/ml of FLAG peptide (Sigma) for 30min at 4°C. The other antibodies used were anti-ZFP-1 (generated by the Lieb Lab) and anti-Pol II 8WG16 (Covance).
The immunoprecipitated DNA was quantified by qPCR using the ΔΔCt method to calculate the percentage of immunoprecipitation relative to the input. We used the following specific primers: Forward AAACAACACATAGACTTGTGCC and Reverse GTACGGTTGTTATCGCTTTCAG to amplify the promoter region of the pdk-1 gene; Forward pdk-1 ex2 GCAAGTGAATCGGAGAACAG and Reverse pdk-1 ex2 TGAAGAAACATGAAGTGCTTGG to amplify the coding region of the pdk-1 gene; Forward TTTCAGAACTATCATGCCACG and Reverse TCTCTGAGCACACTTTGAGG to amplify the promoter region of the aqp-1 gene; Forward aqp-1 ex5 TTGCCAGTTATCCATCTCCA and Reverse aqp-1 ex5 CTCTCATCAATAACAACGCAG to amplify the coding region of the aqp-1 gene; Forward TTAGATAGAGAATTGGCGAGAG and Reverse CAAGTAGCAAAGCGATAAACC to amplify the promoter region of the gst-4 gene; Forward gst-4 ex4 TGAAGTTGTTGAACCAGCC and Reverse gst-4 ex4 CCCAAGTCAATGAGTCTCCA to amplify the coding region of the gst-4 gene.
To investigate the function of ZFP-1 with ChIP we first developed an antibody (termed JL00006_ZFP1) to the C-terminal portion of the protein. Alternative transcription start sites give rise to two ZFP-1 protein isoforms with identical C-terminal domains. As expected, both isoforms are recognized by the JL00006_ZFP1 antibody. The protocols used for generating ZFP-1 ChIP/chip data are described at http://www.modencode.org/Lieb.shtml
Determining genes bound by ZFP-1
genes (refSeq id) from genome build CE4 (ws170) were extracted from the UCSC genome browser's refGene table. A gene was called bound by ZFP-1 if the center base pair of a ZFP-1 peak overlapped the ORF or the 1,500 bp upstream region. Overlap calls were done using the Galaxy web tool. Of the total 24,901 genes, 3,598 were bound by ZFP-1. Genome-wide ZFP-1 localization data are available at modENCODE: http://intermine.modencode.org/