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Supporting Information Supporting Information Whittle et al. 10.1073/pnas.0812894106 SI Text range based on analysis on 1.5% agarose gels after reversal of In Vitro Genomic Selection. Recombinant NFI-1-GST fusion pro- cross-linking. DNA-protein complexes were precipitated with tein was immobilized on Glutathione Sepharose 4B (Amersham) anti-NFI immune serum (5 ␮l) or preimmune serum (5 ␮l) as a and C. elegans genomic DNA was used for in vitro genomic control and 5% of the input sample was set aside. Samples were selection. The fusion protein contained the NFI-1 DNA-binding processed using the ChIP assay kit (Upstate). Following reversal domain and a short downstream region. For NFI-1-GST Sepha- of the cross-links, RNase A and Proteinase K treatments, the rose preparation, 400 ml of induced DH5␣ cells expressing immunoprecipitated DNA was purified using phenol-chloro- NFI-1-GST (plasmid pGEXNFI-1) was extracted in buffer L (25 form, precipitated with ethanol, and dissolved in 50 ␮l of DNase, mM Hepes pH 7.5, 10% sucrose, 0.35 M NaCl, 5 mM DTT, 1 mM Rnase-free water. For ChIP-chip, samples were amplified by PMSF, 0.1% Nonidet P-40 and 2 mg/ml of lysozyme). The extract either ligation mediated PCR (5) or a modified Whole Genome (35 ml) was flash-frozen in 5-ml aliquots and stored at –70 °C. To Amplification protocol [Sigma; (6)] as previously described. immobilize NFI-1-GST protein on Glutathione Sepharose 4B, 5 ml of cell extract was incubated with 65 ␮l (bed volume) of Microarrays and Data Extraction. DNA microarrays (Agilent Tech- Sepharose 30 min at 4 °C, washed 3 times with L buffer minus nologies) covering the entire C. elegans genome with 185,000 lysoyme and sucrose, and 2 times with buffer B (25 mM Hepes, probes at an average start-to-start spacing of 600 bp were used pH 7.5, 5 mM MgCl2, 4 mM DTT, 150 mM NaCl and 0.05% for ChIP-chip (GEO accession no. GPL7776). Four NFI-1 ChIP Nonidet P-40). Approximately 10 ␮g was bound as estimated by biological replicates and 2 NFI-1 Preimmune Mock ChIP-chip SDS PAGE. In vitro genomic selection was performed as experiments were performed. Raw intensities for each ChIP described (1), with some modifications to optimize binding were normalized by median centering the log2(ChIP/Input). conditions. Briefly, 5 ␮g of genomic DNA from a mixed age Normalized log2 ratios from each experiment were converted to population of N2 worms was digested with Sau3A I and incu- standard Z-scores and combined by taking the median of bated with NFI-1-GST Sepharose in 200 ␮l of buffer B at 4 °C, experiments. Probes corresponding to known repetitive ele- followed by 3 washes. DNA was eluted with 10 mM of Gluta- ments were spatially sequestered and removed from subsequent thione in Buffer B, purified using phenol-chloroform and eth- analysis. Raw and processed data can be accessed at NCBI GEO anol precipitation, and dissolved in 12-␮lH2O. DNA was ligated accession number GSE13918. Significant binding peaks were to linkers, PCR amplified, and subjected to the second and third derived using a perl implementation of ChIPOTle (7) using a rounds of selection. After the last round of selection and window size of 1,800 bp, step size of 600 bp, at a Bonferroni Ϫ amplification, DNA was TOPO-cloned (Invitrogen) and all corrected P-value of 1 ϫ 10 12. For each of the 55 discovered clones were sequenced. binding peaks, the maximum probe within a peak was extracted and annotated to the nearest gene using a C. elegans implemen- DNA-Binding Assays. Gel mobility-shift assays and competition tation of Cis-element annotation software (8) and hand-checked analysis to assess enrichment for NFI binding sites during in vitro for accuracy (Wormbase release ws170). selection were performed as described previously (2). Worm extracts were prepared from dounce-homogenized mixed-age ChIP-chip Data Analysis. For motif analysis, a 1,500-bp window worms using the Nonidet P-40-based extraction buffer described centered on the peak maximum probe was used. Extracted previously (2). NFI-1-GST protein was purified on Glutathione sequences (genome release ws170/ce4) were masked for repet- Sepharose 4B from extracts of Escherichia coli as described itive elements using RepeatMasker (9). ChIP sequences were above for genomic selection. Microfiltration on Millipore 5000 ranked by maximum probe Z-score and MDscan (10) was used was used to remove Glutathione after elution and to concentrate for motif discovery. Matrixscan (5) was used to find motifs using protein. Next, 100 ng of recombinant GST-NFI-1 protein and the the MDscan-generated position weight matrix for the top- labeled 26-mer oligonucleotides containing a wild-type NFI scoring motif with a word size of 15 bp. Distance to nearest TSS binding site (wt) 5ЈAGGTCTGGCTTTGGGCCAAGAGC- mappings, random window generation, and perfect-match motif CGC or a site with a single point mutation (mut) 5ЈAG- finding were performed using custom Perl and Ruby scripts GTCTcGCTTTGGGCCAAGAGCCGC, shown previously to (available upon request). R was used for statistical analysis and abolish the binding of vertebrate NFI proteins (3), were used. A plotting. Genome browser visualizations were obtained using the 100-fold molar excess of unlabeled PCR amplified DNA from UCSC genome browser (http://genome.ucsc.edu), genome re- each round of selection was added to the indicated samples. lease ws170/ce4. Modeling of nucleosome occupancy and micrococcal nuclease Chromatin Immunoprecipitation. Rabbit polyclonal antiserum was mapping of nucleosome occupancy and position (Adjusted Nu- raised against recombinant NFI-1-GST fusion protein described cleosome Stringency) were derived from published datasets (11, above. Antibody recognition of native NFI-1 protein bound to 12) and extracted via the UCSC genome browser (http:// DNA was verified by gel mobility-shift assays (Fig. S9). ChIP was genome.ucsc.edu). Raw expression data were obtained from the performed on a mixed-age population of N2 worms. Worm Stanford Microarray Database (http://smd.stanford.edu/) for a culture was initiated with 20 young adult worms on 10-cm previously published time-course of the C. elegans lifecycle (13). NGM/DH10B plates (10–14 plates for one experiment). Ap- Raw intensities for each expression microarray channel (mixed proximately 0.5 ml of worm pellet was collected for each ChIP RNA reference or single stage) were percentile-ranked as a sample. Cross-linking conditions were as previously described measure of relative RNA abundance. (4). Cross-linked pellets (120–150 mg) were resuspended in 1 ml Precomputed blastp hits derived from wormbase release of ChIP lysis buffer (Upstate) and sonicated using a Branson ws170 were used to find orthologs of the C. elegans NFI-1 targets. Sonifier 250 (output 30 and DutyCycle 30% setting) with 15 sets The protein with the lowest e-value was chosen and 3kb (C. of 10 pulses (1 sec each) on ice with 1-min intervals between each briggsae) or 5 kb (mouse/human) upstream of the TSS was set for cooling. The sonicated fragments were Ϸ200- to 1,300-bp examined using Matrixscan (5) for motifs using the C. elegans Whittle et al. www.pnas.org/cgi/content/short/0812894106 1of12 derived position weight matrix. C. briggsae sequences and anno- using iQ SYBR Green Supermix (Bio-Rad) on a Bio-Rad iCycler tations were obtained for wormbase genome release ws190; and 1 ␮l of DNA precipitated with anti-NFI immune or preim- mouse (Ensembl50/NCBI m37), and human (Ensembl50/ mune serum. Input DNA sample was diluted Ϸ1,000-fold to NCBI36) sequences and annotations were obtained via Ensembl achieve a Ct value within the same range. All reactions were in (http://www.ensembl.org/) and the UCSC genome browser. duplicate. Primers designed within the coding region of ama-1, a locus negative for NFI-1 binding, were used as an internal qPCR Analysis of ChIP-chip Data. qPCR was used to determine control to normalized quantification in qPCR reactions. qPCR relative amount of specific loci in IP, Input, and Mock (Preim- primers are available on request. Data are expressed as IP/Input mune) samples. Amplicons of 100–200 bp were designed using where DDCT ϭ (CtIP࿝locusX Ϫ CtIP࿝ama-1) Ϫ (CtInput࿝locusX Ϫ Ct Macvector software for each loci to ensure a uniform assay Input࿝ama-1). As a negative control, Mock/Input was analyzed in performance under cycling conditions: 50 °C for 2 min, 95 °C for parallel. DDAverage Ct values for ama-1 and input were used in 8 min, 30 sec and 40 cycles 95 °C for 15 sec, and 60 °C for 1 min, calculations. Bars on the graphs represent corresponding DDCt following by melt-curve data collection. qPCR was performed values and their range. 1. Shostak Y, Van Gilst MR, Antebi A, Yamamoto KR (2004) Identification of C. elegans 8. Ji X, Li W, Song J, Wei L, Liu XS (2006) CEAS: cis-regulatory element annotation system. DAF-12-binding sites, response elements, and target genes. Genes Dev 18:2529–2544. Nucleic Acids Res 34(Web Server issue):W551–W554. 2. Lazakovitch E, et al. (2005) nfi-I affects behavior and life-span in C. elegans but is not 9. Chen N (2004) Current Protocols in Bioinformatics (Wiley, Hoboken, NJ), Chapter 4: essential for DNA replication or survival. BMC Dev Biol 5:24. Unit 4–10, 4.10.1–4.10.14. 3. Goyal N, Knox J, Gronostajski R (1990) Analysis of multiple forms of nuclear factor I in 10. Liu XS, Brutlag DL, Liu JS (2002) An algorithm for finding protein-DNA binding sites human and murine cell lines.
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