Figure 2. 6C results and validation.

(A and B) Interaction frequencies of SOX9 (A) and PTHLH (B) 6C data. In SOX9 6C, 4 CREs were detected; 9 were found in PTHLH 6C (black symbols). re70373, re18527, and re52431 were the most abundant, and further characterized by high mammalian conservation, H3K4me1 enrichment (ENCODE), and ESPERR regulatory potential (denoted by “+”) (positions in bp; UCSC assembly hg18). re52431 interacted with SOX9 in trans. (C) New 6C libraries validated prior 6C results; control was a LCL. A direct PCR approach amplified re70373, re18527, and re52431. In the LCL, the C28/I2 CRE interactions were significantly reduced; only for re70373 were interactions in C28/I2 and LCL detected equally. (D) Distances of cis- and trans-regulatory elements identified in PTHLH 6C and SOX9 6C. re18527 (18,527,200 bp) and re52431 (52,431,500 bp) interacted with PTHLH, re70373 (70,373,400 bp) interacted in cis and re52431 in trans with SOX9. re52431 was further named CISTR-ACT, due to its cis and trans interactions. (E) Luciferase reporter assays of re70373, CISTR-ACT^F (full-length regulatory sequence), and CISTR-ACT^S (containing the most conserved part) elements were placed in front of the SOX9 promoter; all 3 enhanced transcription. (F) re18527, CISTR-ACT^F, and CISTR-ACT^S, either alone or in combination, controlled the PTHLH promoter and enhanced luciferase transcription (n = 6). ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05.