Fig. 6. The noncoding enhancer RNA, lncRNA CSR^IgA, regulates the pivotal function of CTCF^lncCSR.

(A) IgA CSR deficiency in lncCSR^IgA−/− CH12F3 cells can be rescued by overexpression of lentivirally transduced lncRNA-CSR ^IgA. FACS plots of cytokine-stimulated CH12F3 B cells that are parental clones (CH12WT), CH12/lncCSR^IgA−/−, and rescued either by an inverted sequence of lncCSR (lncRNA-CSRInv) or by the physiological lncRNA-CSR^IgA (representative of three independent clones) are shown. (B) Plot summarizing multiple experiments that evaluate CSR rescue of lncCSR^IgA−/− CH12 cells transduced with lncCSR-RNA^IgA. The number of independently performed experiments is shown in the figure by black circles. **P ≤ 0.01 and ***P ≤ 0.001, by Student’s t test. (C) Quantitation by qRT-PCR of lncCSR^IgA expression in lncCSR^IgA−/− cells after lentiviral infection of the lncCSR^IgA−/− cells. Note the logarithmic scale on the vertical axis. (D) Degradation of lncRNA-CSR^IgA with a Cas13a/gRNA approach reduces IgA CSR efficiency. gRNAs specific to the lncRNA-CSR^IgA were constructed to specifically knock down the lncRNA-CSR^IgA (right) but did not alter the AID expression levels (left) or IgSμ transcription (center). **P ≤ 0.01 and ***P ≤ 0.001, by Student’s t test (E) Decreased IgA CSR in CH12F3 cells that express two different gRNA pairs that knocked down lncRNA-CSR^RIgA expression: FACS plots (left), percent CSR decrease (middle), and ratio of CSR decrease in comparison with Cas13 with empty gRNA vector control (right). (F) Coimmunoprecipitation of a stem-loop–associated lncRNA-CSR^IgA after expression in 293 T cells validates interaction with SMC3 (top blot), PARP1 (middle blot), and SUPT16H (bottom blot). (G) CTCF protein association to CTCF binding sites flanking locus A (CTCF^LocusA), lncCSR^IgA (CTCF^lncCSRIgA), and locus B (CTCF^LocusB) was analyzed by ChIP-seq experiments from B cells isolated from lncCSR^IgAWT/WT and lncCSR^IgA−/− littermate mice. (H) ChIP experiment to confirm that the recruitment of lncRNA-CSR^IgA interacting factors (identified by mass spectrometry) SMC3, SUPT16H, and PARP1 to CTCF^lncCSRIgA is strongly decreased. Reintroduction of lncRNA-CSR^IgA in the lncCSR^IgA−/− CH12F3 cells partly rescues the recruitment of the three factors to CTCF^lncCSRIgA.