Fig. 2: Igh locus function is dependent on a VH promoter, an EVH and CBE in Abl-t pro-B cells.
From: An Igh distal enhancer modulates antigen receptor diversity by determining locus conformation
Source data are provided as a Source Data file. All p values are from two-tail Student’s t test. a (Upper panel) The Igh locus from the VH14-2 gene to EVH2. (Lower panel) Biallelic identical deletions constructed for the VH14-2 promoter (VH14-2 Pr, yellow rectangle), F.6 CBE (F.6_CBE, purple triangle), EVH1 (red square), and EVH2 (white square) in the Abl-t 445.3.11 cell line. b ChIP assays for CTCF binding at the F.6 CBE in the control (n = 3), Site I.1- (n = 3) and F.6 CBE (n = 3) KO lines. c 3C looping assays. (Upper panel) Map of genomic distances. (Lower panel) Normalized 3C crosslinking frequencies from four experiments in which the 455.3.11 (n = 10) or 445.3.8 (n = 2) matched controls were set to 1.0 and represented once. The crosslinking frequencies were averaged for chromatins from each KO type (EVH1 KO, n = 9; EVH2 KO, n = 6; F.6_CBE KO (n = 6); F.6_CBE KO (n = 8); VH14-2 Pr KO (n = 6) and normalized to the control. d QRT-PCR assays for the VH14-2 gene using 18 S RNA as a loading control. Samples were from control (n = 14) VH14-2 Pr KO (n = 8), EVH1 KO (n = 6), F.6_CBE KO (n = 9). e RT-PCR of VH81X and VH2-5 GLTs and the 18 S RNA loading control were harvested at 32 and 28 cycles, respectively. Cells were STI-571 (2.5 mM) treated for 48 h. f Schematic of the DHJH clusters and proximal VH gene segments (vertical bars), with primer positions (colored arrows) used to analyze D-J and V-DJ rearrangements. g, h Lines were stably complemented with RAG1 and STI-517 treated. g D-J rearrangements in gDNA were analyzed by qPCR with DFL16.1 and JH1 primers. Samples were from 455.3.11 (n = 4), 455.3.8 (n = 5), VH14-2 Pr KO (n = 4), EVH1 KO (n = 6), F.6_CBE KO (n = 4). h Normalized qRT-PCR data for V- > DJ rearrangement using VH81X and VH7183 primers in combination with JH1. VH81X-JH1 analyses were from control 455.3.11 (n = 4), 455.3.8 (n = 3), EVH1 KO (n = 6),VH14-2 Pr KO (n = 3), F.6_CBE KO (n = 4). Samples numbers for VH7183-JH1 analyses were identical to that of VH81X-JH1 except for 455.3.11 (n = 12), VH14-2 Pr KO.1 (n = 5) and F.6_CBE KO.2 (n = 4).
