Intrachromosomal interactions, between the human α-globin genes and conserved upstream elements. (A) Chromosomal organization of the human α-globin locus annotated as previously described.²³  Red numbers indicate the points (coordinates) analyzed by q3C. q3C assays were performed using HindIII-digested, fixed chromatin from primary T lymphocytes (gray bars) and primary erythroblasts (red bars), generating restriction fragments of 3 to 5 kb. This allowed us to use q3C to evaluate all interactions between the α-globin genes and a variety of points along the α-globin cluster, including all the remote upstream elements. Twelve restriction fragments located within (HS −48, HS −46, HS −40, HS −33, and HS −10 and the α-globin genes) or outside (fragments 53, 189, 218, 380, 401, and 441) the α-globin locus were chosen to determine the locus conformation (A). The shaded area corresponds to the region containing all sequences that interact with the α-globin genes (B-J). The bar chart (y-axis) shows the enrichment of PCR product (%) normalized to the enrichment within the human Ercc3 gene (= 100%). This provides an internal, genomic control for the cross-linking procedure and any general changes in nuclear or chromatin structure.²⁴  These independent graphs represent a measure of the association between the points indicated (x-axis) to the anchor points (B) “fragment 53,” (C) HS −48, (D) HS −46, (E) HS −40, (F) HS −33, (G) HS −10, (H) α-globin genes, (I) “fragment 189,” and (J) “fragment 380.” For each new anchor point, a new Taqman probe and reverse primer were designed and thus results can be compared only within, but not between, each set of graphs. The resolution of this 3C assay can be estimated by comparing “positive” and “negative” restriction fragments located at a similar distance to their target. For example, the locus position 189 is located 26 kb downstream of α-globin and does not interact, whereas HS −10, located 26 kb upstream of α-globin, shows a 3-fold enrichment in erythroid cells (H). Data shown represent the average of a least 3 independent experiments using Taqman/real-time PCR. Error bars denote SEM. Each PCR was performed several times and averaged. Signals were normalized to the total amount of DNA used, estimated with an amplicon located within a HindIII fragment (Table 1). Coordinates of the points analyzed are indicated on the x-axis.