Figure 5.

Intrachromosomal interactions involving mouse remote regulatory sequences and the mouse α2 globin promoter. (A) Chromosomal organisation of the mouse α globin locus. The following genes are annotated: 3, IL9RP3; 4, m-99; 5, Dist; 6, MPG; 7, c16orf35 gene. These are shown as boxes transcribed from the top (above the line) or bottom (below the line) DNA strand. Red numbers indicate the points (coordinates) analysed by 3C. 3C assays were performed using HindIII-digested, fixed chromatin from ES, uninduced MEL (U-MEL), induced MEL (I-MEL) and primary erythroblasts (Ter119+). The shaded area corresponds to the region containing all sequences that interact with the α globin gene (B). The bar chart (y-axis) shows the enrichment of PCR product (%) normalised to the enrichment within the Ercc3 gene (=100%). This provides an internal, genomic control for the crosslinking procedure and any general changes in nuclear or chromatin structure (de Laat and Grosveld, 2003). This, in turn, is a measure of the association between the points indicated to a fixed point (the mouse α2 globin promoter) in cells representing different stages of differentiation, from embryonic stem cells (ES) to mature erythroblasts (I-MEL and primary Ter119+ cells). We have not considered the α1 gene in this study because the HindIII fragment containing this gene is too large (14 kb) for appropriate 3C analysis. Data shown represent the average of at least two independent experiments using Taqman/real-time PCR. Error bars denote s.e.m. Each PCR was performed several times and averaged. Signals were normalised to the total amount of DNA used, estimated with an amplicon located within a HindIII fragment. Coordinates of the points analysed are indicated on the x-axis. As an illustration of the specificity of these interactions, the interaction between α globin and HS-26 (∼40 kb apart) is enriched in erythroid cells, whereas the interaction between α globin and a region at +174 (∼40 kb in the opposite direction) is not enriched.