ChIP-chip analysis of Dosage Compensation complex members SDC-3 and DPY-27 in C elegans wild type embryos.

Among organisms with chromosome-based mechanisms of sex determination, failure to equalize expression of X-linked genes between the sexes is typically lethal. In C. elegans, XX hermaphrodites halve transcription from each X chromosome to match the output of XO males1. Here, we mapped the binding location of the condensin homolog DPY-27 and the zinc finger protein SDC-3, two components of the C. elegans dosage compensation complex (DCC)2,3. Strong foci of DCC binding were observed on X, around which broader regions of localization were centered. Binding foci, but not adjacent regions of localization, were distinguished by clusters of a stereotypic 10-bp DNA sequence, suggesting a recruitment-and-spreading mechanism for X recognition. In contrast to the Drosophila DCC, the C. elegans DCC was bound preferentially upstream of genes, suggesting modulation of transcriptional initiation and transcription-coupled spreading. A mechanism for tuning DCC activity at specific loci was indicated by stronger DCC binding upstream of genes with high transcriptional activity. These data provide a basis for understanding how proteins involved in higher-order chromosome dynamics can regulate transcription at individual loci. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Keywords: ChIP-chip dosage compensation complex For extract preparations, N2 adults were grown in standard S-liquid media and embryos were obtained by bleaching25. Embryos were fixed with 2% formaldehyde for 30 minutes at room temperature and washed with 100 mM Tris pH 7.5 once, M9 buffer twice and 10 ml FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate; 150 mM NaCl) with protease inhibitors (Calbiochem) and frozen at -80ºC. 500 ul of packed embryos were thawed and resuspended in 2 ml of FA buffer and dounce homogenized on ice with 30 strokes. Samples were sonicated with a Branson sonifier at 30% amplitude for 7 cycles of 12 pulses (0.9 sec on, 0.1 sec off) and were cooled in dry ice/ethanol bath for 2 seconds between cycles. Cellular debris was removed by microfuge centrifugation at 13,000 g for 15 minutes at 4ºC. Immunoprecipitation reactions contained approximately 3 mg of total protein with 3-5 ug of purified antibodies in 500 ul total volume, with 1% sarkosyl. Prior to addition of the antibody, 10% of the material was taken as input. Immunocomplexes were collected with Protein A-sepharose beads (Amersham Biosciences) and washed with 1 ml of the following buffers : FA buffer 2 X 5 min, FA-1M NaCl (FA buffer with 1 M NaCl) 5 min, FA-500 mM NaCl 10 min, TEL buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0) 10 min., TE pH 8.0 2 X 5 min. Complexes were eluted in 1% SDS in TE with 250 mM NaCl at 65ºC 30 minutes. Samples and inputs were treated with Proteinase K for 1 hour and crosslinks were reversed at 65ºC overnight. DNA was purified with Qiagen PCR purification columns and amplified by LM-PCR26. Microarrays and Data Extraction The C. elegans genome tiling arrays were designed and constructed by NimbleGen System Inc. using maskless array synthesis technology, with approximately 650,000 oligonucleotides per array27. Two arrays covered the C. elegans genome at ~86-bp spacing with 50mer oligonucleotides. We performed three biologically independent experiments for SDC-3, DPY-27 and No Antibody control ChIPs. Amplified samples were labeled and hybridized to high-resolution microarrays by the NimbleGen Service Laboratory as described previously. Two ChIPs (ChIP 1 and 3) were labeled with Cy5 and the reference materials (Input DNA) were labeled with Cy3, and for one (ChIP 2), the dyes were swapped. Raw intensity data was extracted from scanned images using NimbleScan 2.3 extraction software (NimbleGen Systems Inc.).