Supplementary Figure 2

Transcription

1 Supplementary Figure 2 a SBS-C1 SBS-C2 SBS-C3 SBS-C4 SBS-C5 SBS-C6 SBS-C7 SBS-C8 SBS-C9 LCR CNS-1 CNS-2 Il5 Rad50 Il13 Il4 Kif3a Sept kb Sau3AI BamHI/Bgl II A B C D E F G H I J K L M N O P Q R S Crosslink Urea Gradient un-crosslinked protein Digestion loop DNA 3C Assay ChIP-Loop Assay Ligation Reverse Crosslink ChIP Ligation non sepecific protein and DNA PCR

2 Supplementary Figure 2 b locus Sau3AI 0 S1 S2 500 S S bp * * BamHI/Bgl II B1 B2 0 B3 500 B bp * * Crosslinking frequency [ T 2] X= c H { [ } ] { [ *] [*] } A B C D X = A C B D SBS-C1 (Sau3AI fragment 2) Resting

3 Supplementary Figure 2 d SBS-C1 SBS-C2 SBS-C3 SBS-C4 SBS-C5 SBS-C6 SBS-C7 SBS-C8 SBS-C9 LCR CNS-1 CNS-2 Il5 Rad50 Il13 Il4 Kif3a Sept kb Sau3AI BamHI/Bgl II A B C D E F G H I J K L M N O P Q R S T H 2 Rad50 promoter SBS-C9 (fragment C) (fragment S) M A B C D E F G H I J K L M N O P Q R S A B C T H 2* *

4 Supplementary Fig. 2. ChIP loop assay and 3C assay to determine chromatin structure of the T H 2 cytokine locus. General principle) Chromatin loops that form in vivo can be trapped during formaldehyde crosslinking of cells. If remote DNA sequences are physically brought in close proximity by chromatin looping in vivo, they can be found in the same crosslinked chromatin fragment, and the DNA sequences at the stem of chromatin loops can be identified. Briefly, after digesting crosslinked chromatin with a restriction enzyme that removes the loop portion of chromatin, the two sequences initially located at the stem of loop can then be ligated intra-molecularly, after dilution of the chromatin sample to avoid inter-molecular ligation events. After reverse crosslinking (to remove proteins), purified genomic DNA that had been intra-molecularly ligated, excluding the loop portion of DNA, can be amplified by PCR, using primer pairs designed from the two distal sequences of interest. The ligation products detected by PCR indicates physical interactions between DNA fragments held together by higher-order chromatin structure. This method called the chromosome conformation capture (3C) assay (Dekker, J., Rippe, K., Dekker, M. & Kleckner, N., Science 295, , 2002). allows us to detect the frequency with which two remote genomic sequences interact in space in a given nucleus. The more recently devised ChIP-loop assay (Horike, S., Cai, S., Miyano, M., Cheng, J.F. & Kohwi-Shigematsu, T., Nat Genet 37, 31-40, 2005), a modified 3C analysis, studies the chromatin fragments that were immunoprecipitated with an antibody specific for a chromatin associated protein of interest. Although both assays examine long-range DNA interactions between regulatory elements, the advantage of the ChIP-loop assay is that it enables us to study a specific group of chromatin loops that are fastened at their base with a specific protein, or that associates with specific histone modification. For instance, using the ChIP-loop assay, we recently reported that methyl CpG-binding protein 2 (MeCP2) regulates higher-order chromatin organization and is responsible for chromatin looping of the transcriptionally-silent, but not active chromatin, at the locus of one of its target genes, Dlx5/6. The crosslinking frequencies of any two DNA fragments, as determined by the intensity of the PCR signals of a ligation product, depends on the relative proximity of the two DNA fragments to each other at a given time point. Relative ligation crosslinking

5 frequencies of any two DNA fragments were calculated as described below to normalize various parameters such as PCR amplification efficiencies, ligation and crosslinking efficiencies, the amounts of the template initially used. a, The experimental strategies of ChIP-loop and 3C assays. For these assays, we used Sau3AI restriction enzyme-digested fragments (1-20) as well as BamH1/BglII restriction enzyme-digested fragments (A-S), which were used for the verification purpose. Primers (forward and backward) were designed in each of these fragments. After formaldehyde crosslinking, we purified crosslinked chromatin by urea-gradient ultracentrifugation to remove un-crosslinked protein and proceeded to 3C assay as described in Methods. For ChIP-loop assay, we performed chromatin immunoprecipitation before intra-molecular ligation to identify the chromatin loop defined by specific protein. b, The genomic DNA prepared for either ChIP-loop or 3C assay as described above was subjected to PCR amplification using various combination of forward and backward primer pairs derived from the DNA fragments (1-20 or A-S). These PCR products derived from the 200-kb T H 2 cytokine locus using genomic DNA is designated T H 2. We used the b-actin locus (using primers S2 and S3 for Sau3AI digested/re-ligated templates, or B2 and B3, for BamH1/BglII digested/re-ligated templates) as an internal control to normalize any difference in the amounts of the genomic DNA, crosslinking and ligation efficiencies. Upon either Sau3AI or BamH1/BglII digestion and ligation, a PCR product of 149bp or 131bp is generated, respectively. The PCR products using S2 and S3 or B2 and B3 primers are indicated as. To correct for ligation efficiency and amplification efficiency of different primers, a mixture of plasmid DNA containing the b-actin locus between S1 and S4 primers or B1 and B4 primers and two BAC clones covering the 200-kb T H 2 cytokine locus (see Methods) was subjected to PCR amplification with the same series of primer pair combination from different DNA fragments. The PCR products derived from the BAC clones and plasmid DNA are indicated by T H 2* and *, respectively. We calculated relative crosslink frequency using the formula shown, which corrects for any differences in PCR amplification efficiencies, crosslinking and ligation efficiencies, the amounts of the template initially used and the size of the PCR products. c, We confirmed that premixing of primers from the T H 2 cytokine gene locus and the actin locus does not

6 affect the final results of our 3C assay. The top panels show data generated by premixing the primers, and the middle and bottom panels show data generated separately with either T H 2 primers or actin primers. d, Sau3AI digestion is blocked by overlapping CpG methylation. Therefore, we checked Sau3AI digestion sites (5 GATC3 ) at both ends of all 20 Sau3AI DNA fragments and identified 7 sites that have either 5 CGATC3 or 5 GATCG3. In our 3C data, we found evidence that Sau3AI successfully digested five of these seven sites, indicating that the overlapping CpGs were not methylated in five sites. However, for the remaining two sites, corresponding to fragment 3 and 4, our 3C data showed no evidence of cleavage at these sites. Therefore, we employed BamH1/BglII-digested chromatin to repeat the 3C analysis. The BglII fragment C did not crosslink with any other fragments, except for fragment D due to the short distance, confirming that this region was not involved in long range interaction with other sites at this locus. On the other hand, crosslinking of SBS-C9 (fragment S) with the Il5 promoter (fragment A) and SBS-C1 (fragment B) has been verified using the BamH1/BglII digested chromatin.