Figure 1.
Schematic representations of Chromosome Conformation Capture (3C) and Complete-genome 3C by Vectorette amplification (4Cv).
A: 3C - The physical proximity between two DNA elements in cells is converted into a direct juxtaposition, which is detected by PCR using sequence-specific primers. B: The library of genomic interactions is digested with a second enzyme (NlaIII), vectorette linkers are added and the subset of interactions involving a specific sequence (e.g. β-globin, red line) is amplified. The resulting 4Cv library is analysed by sequencing cloned products or bands excised from a gel.
Figure 2.
3C results confirm the specific interaction between LCR and active gene.
A: 3C was performed in fetal liver and brain, and ligation products detected using the primer pairs shown. Primers for Calreticulin (CalR) were used as a control for cross-linking. Positive and negative controls were an equimolar mix of all PCR products and water respectively. Black squares represent genes, short arrows represent hypersensitive sites, horizontal purple arrows represent approximate primer positions (not to scale). B: Real time PCR using SYBR green was used to accurately quantify levels of Hbb-b1 ligated to HS2 and Hbb-y following 3C reactions on fetal liver and brain samples. Cross-linking frequency was determined relative to CalR (see materials and methods). HS2-Hbb-b1 cross-linking frequency is shown as a dark grey box, and Hbb-y-Hbb-b1 as a light grey box. Standard deviation from at least 3 independent experiments is shown.
Figure 3.
Typical result of the 4Cv procedure. 4Cv was performed by amplifying ligation products containing Hbb-b1 from an E18.5 brain (A) or liver (B) 3C library.
4Cv products separated on a 15% polyacrylamide gel next to a 10 bp ladder (M). Bands were excised, reamplified and sequenced, chromosomal locations of the Hbb-b1-ligated sequences are indicated on the right. Sequencing results showed that the band at approx 205 bp was the original ‘endogenous’ sequence at the Hbb-b1 gene (obtained when BglII fails to cut during the restriction digest stage of 3C).
Figure 4.
Distribution of 4Cv results on chromosome 7. Sequencing analysis of 4Cv experiments reveals the distribution of intrachromosomal interactions of Hbb-b1 in E18.5 liver (red lines, top) and brain (blue lines, bottom) cells.
The centromere and β-globin locus are marked by a black line and arrow respectively.
Figure 5.
Intra- vs. interchromosomal interactions of the β-globin locus.
Sequencing analysis of 4Cv experiments in fetal liver and brain reveals a greater percentage of interchromosomal (light grey) interactions when the gene is expressed (in liver). Interactions with other regions on chromosome 7 (i.e. intrachromosomal, dark grey) appear to be more prominent in the brain where Hbb-b1 is silent. Results represent at least two biological replicates and incorporate both band and colony sequences (endogenous sequences are omitted from the calculation); error bars show standard deviation.