Supplementary Methods
Chromosome conformation capture (3C) analysis
The protocol used for this assay was developed by Dekker and coworkers15 for yeast cells
and modified by Tolhuis et al. for mammalian cells16. Single cell suspension of 1x107
cells were diluted in 10 ml of Bruff’s/10%FCS media and were cross-linked with 2%
formaldehyde, for 10 minutes at room temperature. The amount of formaldehyde used
was the one giving the strongest signal in preliminary experiments using 0.5%, 1% and
2% of formaldehyde and analyzing the crosslinking efficiencies of IL-4 and IL-13
promoter fragments in TH2 cells (Figure 1D). The reaction was quenched with 0.125M
glycine and cells were centrifuged for 8 minutes at 1300 rpm, at 4oC. The pellet was
washed once with ice cold PBS and the cell pellet was lysed with 5ml of ice cold lysis
buffer (10mM Tris-HCl pH:8.0, 10mM NaCl, 0.2% NP-40, protease inhibitors). Nuclei
were pelleted and resuspended in 0.5ml 1.2x BglII restriction buffer (NEB3). SDS was
added in 0.3% final concentration and nuclei were incubated for 1 hour at 37oC with
shaking. Then, TritonX-100 was added at a final concentration of 1.8% and nuclei were
incubated for 1 hour at 37oC with shaking. The sample was incubated overnight at 37oC
with shaking, upon the addition of 400 units of BglII. The percentage of digestion in each
different cell type was identified utilizing real time PCR analysis with primers spanning
the BglII cites of interest (Supplementary Fig. 3 online). The percentage of digestion was
similar between the different cell types. . The enzyme was inactivated with SDS (1.3%
final concentration) and shaking for 25 minutes at 65oC. 7ml of 1xligation buffer and
TritonX-100 (1% final concentration) were added to nuclei and incubated for 1 hour at
37oC. 100 Weiss units of ligase were added and the sample was incubated for 4 hours at
16oC and 30 minutes at room temperature. The DNA concentration in the ligation
reaction was kept very low (2.7ng/μl). In preliminary experiments we tested different
dilutions of the DNA and different concentrations of ligase used in order to detect
specific signals. Protein digestion and reverse cross-linking was performed with
overnight incubation at 65oC with 300μg ProteinaseK. RNA was degraded with RNAse
for 30-45 minutes at 37oC. DNA was phenol/chloroform extracted and ethanolprecipitated.
To generate control templates for the positive controls, BAC clones were used for
all the loci of interest. For the TH2 locus we used the BAC clone B182 (Genome systems
Inc.), for IFN- locus we used the BAC clone RPCI.24-352N22 (CHORI), for Gm-csf we
used the BAC clone RPCI.23-397C23 (CHORI), and for Gapd and  -globin loci we
used PCR to amplify the regions spanning the BglII sites of interest as before18. 30μg of
the BAC clone were digested with 300 units of BglII overnight at 37oC. DNA was phenol
chloroform extracted and ethanol precipitated. DNA fragments were ligated with T4
DNA ligase at a DNA concentration of 300ng/μl. Using a BAC clone to prepare the
control template all possible restriction fragments upon BglII digestion are present in
equimolar amounts and upon ligation at a high DNA concentration, all possible ligation
products are present in the sample. For the templates to function as positive controls for
the interchromosomal 3C analysis different BAC clones were mixed in equimolar
quantities, digested and ligated in high molecular concentration (300 ng/l) as before18.
The primers for the fragments on the TH2 cytokine locus as well as the Gapd locus were
used as previously described18. By normalizing, within a certain cell type, each cross-
linking frequency to the cross-linking frequency observed between the control fragments,
we can directly correct for differences in quality and amount of template.
The sizes of all BglII restriction fragments generated for the different genomic
loci that were used are indicated in Figure 1.. Using BAC clones to prepare positive
control templates, all the possible restriction fragments upon BglII digestion were present
in equimolar amounts and upon ligation at a high DNA concentration all possible ligation
products were present in the sample. We chose the GAPD locus to be used as a control
for this analysis as a constitutively expressed gene at similar levels in all cell types, and
also we utilized the  -globin locus as a non-expressed gene in the cell types used for this
analysis. By normalizing each cross-linking frequency to the cross-linking frequency
observed between the control GAPD fragments for a certain cell type, we can directly
correct for differences in quality and amount of template. The linear range of
amplification was determined for all the templates that were used for amplification by
serial dilutions. The amount of DNA within the linear range, usually 1ng for the control
templates and 20-60ng for the crosslinked templates, was used for the experiments
designed to establish quantitation. For the optimal comparison of the control templates
with the crosslinked templates, and having the same complexity in the PCR reactions, the
control templates were mixed with an amount of digested and ligated genomic DNA
similar to that used for the crosslinked templates (20-60ng). The control genomic
template was prepared by digesting (BglII) and ligating genomic DNA. The DNA
concentration upon ligation is determined. By comparing the signals obtained by
quantitative PCR for the crosslinked templates versus the control templates, we correct
for differences in amplification efficiency between primer sets and also for differences in
signal intensities due to the size of PCR products (all of the primers were designed to
give PCR products of 80-210bp). All primers were designed to have an annealing
temperature of 56-58oC, and they all yielded a product when used with the positive
control templates. The pair of primers of a given restriction fragment was combined with
the pair of primers of a second restriction fragment in all pairwise combinations. The
PCR conditions used were very stringent in order to detect only the specific signals. PCR
cycles used were: an initial denaturing step for 3 minutes at 94oC followed by 36 repeats
of a cycle with 30 seconds at 94oC, 20 seconds at 56oC, and 15 seconds at 72oC, followed
by a final step of 2 minutes at 72oC. 36 PCR cycles are enough to detect the specific PCR
fragments for a rare molecular phenomenon to happen in the nucleus after digestion and
ligation. For a 20μl PCR reaction, 20-60ng of each crosslinked template was used with
1xPCR buffer, 2.5mM MgCl2, 0.25mM dNTPs, 0.5pmol of each primer, 1.5Units of Tsg
DNA polymerase (Centaur) and 2.5% DMSO. PCR products were run in 2% agarose gels
and specific products were quantified using an AlphaInnotech Fluorchem 8800 image
analyzer and ScionImage analysis software. The templates were prepared 2 times from
independent experiments and the whole set of PCR reactions for the analysis of
interaction of one restriction fragment with all the rest were repeated at least three times.
Data presented are the average of results for all PCR reactions done.
A very important experimental control was introduced in our analysis. All PCR signals
that were considered as positive were isolated, gel extracted (Qiaquick, Qiagen) cloned in
a TA vector (TA cloning Kit, Invitrogen) and sequenced to identify unambiguously the
sequence of the chimeric BglII ligated fragments.
The primers for the 3C analysis of the loci were:
Ifn.Primer 1: 5’-AGAATGTGCCATTAGAAGTC-3’
Primer 2: 5’-CAGATGTAAGATGGGATCTC-3’
Primer 3: 5’-CATATCAGCTGGTGTATGC-3’
Primer 5: 5’-GTATATGGGAAGAGTCGGAT-3’
Primer 6: 5’-ATATGGGATAAGATGACTATG-3’
Primer 7: 5’-ATCTGTCTGTGTCATCTACC-3’
Primer 8: 5’-CAATCCAGAATTGGAAGGCTC-3’
Primer 9: 5’-GCTTTGATCATAGCTTCATTATC-3’
Primer 10: 5’-TGGCTACTGATTAATAAGCAGC-3’
Primer 11: 5’-CAGTCAAAATGGTCACCCTAC-3’
Primer 12: 5’-ACAGTCAACTATTGGATGGATC-3’
Primer 13: 5’-GGGGATTGCATTGAATCTGTA-3’
Primer 14: 5’-CAACCTGTGTTATTTTGTAATAGC-3’
Primer 15: 5’-GACATGTCCTGTGTTGTGTTC-3’
Gm-csf,
Primer 2: 5’-GGGCCTCAACAACTCTTAA-3’
Primer 3: 5’-CATGGCTCTGGACGATAAA-3’
 -globin, Primer 1: 5’-TCTGCTGTATCTGTGTCAGTG-3’
Primer 2: 5’-AATGGACAGAAGTCATGGACC-3’
Primer 3: 5’-CTCTTCAGCATCTTCTAGTTTTA-3’
Primer 4: 5’-CACTGTGCTTTTCTGTGCTAC-3’
Primer 5: 5’-GATTATCTCAGGTTGAGCATTAG-3’
Primer 6: 5’-AGTTCTGTGGTAGTCACTAAAAC-3’
Primer 7: 5’-AGATTTTTCCACTCCCTATTC-3’
Primer 8: 5’-TGGGTGTAAGAAACCTAAATTAG-3’