Supplementary Figure Legends
Supplementary Figure 1: The IFN- and TH2 loci are colocalized in the nucleus as
detected by 2D-fluorescence in situ hybridization. (a-d) Diagrammatic representation of
the percentage of cells that have all four alleles of the loci colocalized, or only one allele
of each locus colocalizes, or all four alleles are localized in separate spots. The FISH
experiments were performed in naïve T cells, TH1 and TH2 cells. The experiments were
done to determine the relative localization of the Ifn and the TH2 cytokine gene loci, or
the Ifn and Gapd gene loci, or the TH2 and Gapd gene loci. More than a hundred cells
for each category were scored blindly and slides were prepared at least three times from
different preparations of sorted naïve CD4+ T cells. To consider two signals as
colocalized the distance between them was ≤ 5 pixels.
Supplementary Figure 2: Immuno-DNA FISH experiments reveal that the colocalized
signals reside in euchromatin. 3D cell preparations of naïve CD4+ T cells from wt and
RHS7-/- mice were hybridized with a mouse monoclonal HP1 antibody and visualized
with an Alexa Fluor-350 secondary antibody (blue). DNA-FISH was performed with a
rhodamine labeled BAC clone for the TH2 locus and a spectrum Green BAC clone for the
IFN- locus. 7.1% of the co-localized signals in wt naïve T cells and 8.8% of the colocalized signals in RHS7-/- naïve T cells were colocalized with HP1 stained regions
and were considered as heterochromatinized.
Supplementary Figure 3: Digestion of chromatinized genomic DNA within the nuclei
of different cells types. Post-formaldehyde fixed nuclei of naive CD4+ T cells, TH1 cells,
TH2 cells, primary fibroblasts, B cells and NK cells as well as naked genomic DNA were
digested with BglII overnight at 37oC and, after reversing the crosslinks genomic DNA
was phenol-chloroform extracted and upon precipitation it was used for real time PCR
analysis using Sybr-Green and pairs of primers covering the BglII sites that were used to
generate the restriction fragments that were used for the 3C analysis. For correcting for
the input DNA used from each cell type, primers amplifying Rad50 promoter were used.
Supercoiled genomic DNA was used as an undigested control giving the maximum of
amplification. x axis represents the percentage of digestion of each BglII restriction
enzyme site checked. The scheme on the right indicates the genomic organization of the
loci with the number of BglII restrict ion enzyme sites that were checked.
Supplementary Figure 4: IFN and TH2 loci colocalize in a more loose manner in the T
cell nucleus. Cells with colocalized signals were analyzed for the distance of the IFN
and TH2 alleles in Naïve wt cells (n=34 alleles) and RHS7 ko naïve T cells (n=31 alleles)
as well as for the distance of the GM-CSF and TH2 cytokine loci in wt naïve T cells
(n=49 alleles). In wt naïve T cells the colocalized IFN and TH2 alleles had a distance of
1.88±1.97 pixels whereas in RHS7 ko naïve cells the distance was 4.45±2.37 pixels. For
the GM-CSF and the TH2 alleles which are linked on the same chromosome with a
relative distance of ~500-600kb, the distance between the signals as detected by FISH
was 0.187±0.49 pixels. One-way analysis of variance followed by Dunnett’s T3 post-hoc
testing revealed significant (p<0.001) differences between each of the experimental
groups. Although the alleles of the GM-CSF and the TH2 loci as detected by FISH appear
as overlapping no such physical interaction is detected by 3C analysis, implying that
FISH has the ability to identify large scale interactions whereas 3C is more accurate in
identifying precise genomic interactions.