Genome-scale ChIP-chip analysis using 10,000 human cells

Abstract

The technique of chromatin immunoprecipitation (ChIP) is a powerful method for identifying in vivo DNA binding sites of transcription factors and for studying chromatin modifications. Unfortunately, the large number of cells needed for the standard ChIP protocol has hindered the analysis of many biologically interesting cell populations that are difficult to obtain in large numbers. New ChIP methods involving the use of carrier chromatin have been developed that allow the one-gene-at-a-time analysis of very small numbers of cells. However such methods are not useful if the resultant sample will be applied to genomic microarrays or used in ChIP-sequencing assays. Therefore, we have miniaturized the ChIP protocol such that as few as 10,000 cells (without the addition of carrier reagents) can be used to obtain enough sample material to analyze the entire human genome. We demonstrate the reproducibility of this MicroChIP technique using 2.1 million feature high-density oligonucleotide arrays and antibodies to RNA polymerase II and to histone H3 trimethylated on lysine 27 or lysine 9.

Figure 1. Flowchart diagram of the MicroChIP assay

We have modified the physical aspects of the ChIP assay (Step 1), incorporated an amplification method that allows linear representation of 10−100 pg of DNA (Step 2), and used high density oligonucleotide arrays to demonstrate that transcription factor binding and histone modifications can be studied on a genome-wide scale using only 10,000 human cells (Step 3).

Figure 2. Reproducibility of the MicroChIP assay

(A) Testing the WGA4 amplification method. ChIP assays using 10⁷ cells were performed, then 10 ng of each sample was amplified with WGA2 (left panel) and 50 pg was amplified with WGA4 (center panel). ChIP assays using 10⁵ cells were performed and 50 pg of each sample were amplified with WGA4 (right panel). Primers for the promoters of RNAPII and SOAT were used as the positive controls for RNAPII and H3me3K27 ChIP samples, respectively. IgG ChIP assays were performed as negative controls. (B) Comparison of two H3me3K27 MicroChIP-chip arrays. The Maxfour values from a promoter array for two H3me3K27 ChIP-chip experiments are plotted. (C) Comparison of RNAPII and H3me3K27 data. The Maxfour values from a promoter array for H3me3K27 vs RNAPII targets are plotted. (D) Comparison of top-ranked RNAPII targets. Two independent Standard ChIPs and MicroChIPs were performed, using antibodies to RNAPII. The overlap denotes the common targets in the top ranked 10%, 20%, and 30% of the promoters on the arrays (which represents ∼2,000, ∼4,000, or ∼8,000 promoters in each set) bound by each factor in the two independent Standard ChIPs, the two independent MicroChIPs, or in the averaged Standard vs MicroChIP experiments. Also shown is the overlap expected by random chance. A similar analysis for H3me3K27 is shown in Figure S1. (E) Comparison of binding patterns on Standard vs MicroChIP arrays. Each vertical bar represents the enrichment of a single probe as a log₂ ratio value between the enriched ChIP sample and the Input sample; the chromosomal position is indicated along the bottom axis. The top panel depicts a Histone1 cluster region in chromosome 6, which is enriched for RNAPII binding. The bottom panel shows the HoxB cluster region in chromosome 17, which is enriched for H3me3K27 binding.

Figure 3. ChIP-chip assays using 10,000 cells and 2.1 million feature arrays

ChIP assays using 10,000 cells per assay were performed using antibodies to H3me3K9, RNAPII, and H3me3K27. (A) Primers for the promoters of ZNF44 and ZNF333 were used as positive control for the H3me3K9 samples, primers for the RNAPII promoter was used as a positive control for the RNAPII sample, and primers for SOAT were used as a positive control for the H3me3K27 sample. (B) The enrichment values for all of the probes representing chromosome 19 from H3me3K9 sample B and C were compared; this represents ∼357,000 probes. See Supplementary Figure S2 for graphs of all three pairwise comparisons for the probes on chromosome 19 and Supplementary Table S2 for all three pairwise comparisons for the probes for the three subarrays on the 2.1 million feature array. (C) The probe enrichment values for the entire chromosome 19 are shown for the three independent H3me3K9 ChIP-chip experiments. (D) The binding patterns (using a 3 oligomer sliding window) for a 80,000 nt region of chromosome 18 are shown for the three independent H3me3K9 ChIP-chip experiments.