Ki67 deficiency reduces accessibility at the Rag locus, decreases mRNA expression of Rag1, and impairs V(D)J gene rearrangement in B cell progenitors. (A) Accessibility of the Rag locus (chr2:101,550,000–111,655,000) in the pro-B and small pre-B cells from WT and Mki67−/− mice. The putative enhancer region at the Rag locus annotated by ImmGen is highlighted. ENCODE candidate cis-regulatory elements (cCRE) are shown at the bottom. A zoomed-in image of the putative enhancer region at the Rag locus is shown. Data are representative of four mice from each group. (B) Quantification of the read counts of the putative enhancer region at the Rag locus in pre-pro-B, pro-B, and small pre-B cells from WT (n = 4) and Mki67−/− (n = 4) mice. Statistical differences were determined by Mann–Whitney tests with P values shown. ns, P > 0.05. (C) mRNA expression of Rag1 and Rag2 in pre-pro-B, pro-B, and small pre-B cells from WT (n = 6) and Mki67−/− (n = 6) mice by RT-qPCR. For each gene, CT values of each sample were first normalized by the CT values generated by a housekeeping assay (ΔCT) before being further normalized using one of the samples from the WT pro-B cells (ΔΔCT). All data points were finally normalized by fold-change against the mean of the 2−ΔΔCT values of the WT pro-B cells samples in each experiment. Data are merged from two independent experiments. Statistical differences were determined by multiple Mann–Whitney tests (corrected for multiple comparisons using the Holm–Sidak method) with adjusted P values shown. ns, P > 0.05. (D) Illustration of V(D)J rearrangement process of Igh locus in WT condition. V-D-J, Igh allele containing non-recombined germline V, D, and J gene segments; V-DJ, Igh allele containing non-recombined germline V gene segments; VDJ, successfully rearranged Igh allele; VDJ*, non-productively rearranged Igh allele. (E) Relative abundance of germline sequences between DH and JH segments in WT (n = 3) or Mki67−/− (n = 3) pro-B cells measured by qPCR. CT values of each sample were first normalized by the CT values generated by a housekeeping assay (ΔCT) before being further normalized using one of the samples from the WT condition (ΔΔCT). (F) Relative abundance of germline sequences between VH and DH segments in WT (n = 3) or Mki67−/− (n = 3) large pre-B cells measured by qPCR. ΔΔCT was calculated as described in E. Schematic diagrams showing the positions of primers used for amplifying germline sequences are illustrated in E and F. Data in E and F are representative of two independent experiments. Statistical differences were determined in E and F by two-tailed unpaired t test with P values shown.
Figure 4.

Ki67 deficiency reduces accessibility at the Rag locus, decreases mRNA expression of Rag1, and impairs V(D)J gene rearrangement in B cell progenitors. (A) Accessibility of the Rag locus (chr2:101,550,000–111,655,000) in the pro-B and small pre-B cells from WT and Mki67−/− mice. The putative enhancer region at the Rag locus annotated by ImmGen is highlighted. ENCODE candidate cis-regulatory elements (cCRE) are shown at the bottom. A zoomed-in image of the putative enhancer region at the Rag locus is shown. Data are representative of four mice from each group. (B) Quantification of the read counts of the putative enhancer region at the Rag locus in pre-pro-B, pro-B, and small pre-B cells from WT (n = 4) and Mki67−/− (n = 4) mice. Statistical differences were determined by Mann–Whitney tests with P values shown. ns, P > 0.05. (C) mRNA expression of Rag1 and Rag2 in pre-pro-B, pro-B, and small pre-B cells from WT (n = 6) and Mki67−/− (n = 6) mice by RT-qPCR. For each gene, CT values of each sample were first normalized by the CT values generated by a housekeeping assay (ΔCT) before being further normalized using one of the samples from the WT pro-B cells (ΔΔCT). All data points were finally normalized by fold-change against the mean of the 2−ΔΔCT values of the WT pro-B cells samples in each experiment. Data are merged from two independent experiments. Statistical differences were determined by multiple Mann–Whitney tests (corrected for multiple comparisons using the Holm–Sidak method) with adjusted P values shown. ns, P > 0.05. (D) Illustration of V(D)J rearrangement process of Igh locus in WT condition. V-D-J, Igh allele containing non-recombined germline V, D, and J gene segments; V-DJ, Igh allele containing non-recombined germline V gene segments; VDJ, successfully rearranged Igh allele; VDJ*, non-productively rearranged Igh allele. (E) Relative abundance of germline sequences between DH and JH segments in WT (n = 3) or Mki67−/− (n = 3) pro-B cells measured by qPCR. CT values of each sample were first normalized by the CT values generated by a housekeeping assay (ΔCT) before being further normalized using one of the samples from the WT condition (ΔΔCT). (F) Relative abundance of germline sequences between VH and DH segments in WT (n = 3) or Mki67−/− (n = 3) large pre-B cells measured by qPCR. ΔΔCT was calculated as described in E. Schematic diagrams showing the positions of primers used for amplifying germline sequences are illustrated in E and F. Data in E and F are representative of two independent experiments. Statistical differences were determined in E and F by two-tailed unpaired t test with P values shown.

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