Figure 5: WASH is required for Arid1a to activate AHR expression.
From: WASH maintains NKp46+ ILC3 cells by promoting AHR expression
(a) Arid1a mRNA levels in the indicated cell subsets were examined through RT-PCR. (b,c) ChIP analysis of Ahr promoters in NKp46+ ILC3 cells from Arid1aflox/flox and Arid1aflox/floxRorc-Cre mice with antibodies against H3K9K14ac (b) or H3K4me3 (c). (d) Nuclei isolated from Arid1aflox/flox and Arid1aflox/floxRorc-Cre NKp46+ ILC3 cells were digested by 1 unit of DNase I for the indicated times, followed by DNA extraction for PCR analysis of Ahr promoter. For b–d, n=6. (e) WASH silenced NK92 cells were transfected with full-length (FL) or truncated Arid1a (Δ968-1484 and Δ1935-2283), followed by luciferase assay (upper panel). Endogenous expression levels of AHR were examined by immunoblotting with anti-AHR antibody (lower panel). (f) NKp46+ ILC3 cells from Washflox/flox and Washflox/floxNcr1-Cre mice were in situ hybridized with probes against Ahr promoter, followed by staining with antibodies against WASH and Arid1a (upper panel). White arrow head indicates Ahr promoters colocalized with Arid1a. Percentages of cells with Arid1a colocalized Ahr promoter were calculated (lower panel). At least 200 NKp46+ ILC3 cells were counted. Scale bar, 5 μm. (g) NKp46+ ILC3 cells from Washflox/flox and Washflox/floxNcr1-Cre mice were subjected to ChIP assay with antibody against Arid1a, followed by PCR assay of Ahr promoter. n=5. (h) NKp46+ ILC3 cells from Washflox/flox and Washflox/floxNcr1-Cre mice were subjected to ChIP assay with antibodies against the indicated BAF components, followed by PCR assay of Ahr promoter. n=5. (i) Control and WASH silenced NK92 cells were transfected with the indicated Arid1a plasmids, followed by ChIP of Ahr promoter with antibody against Flag. Data are shown as means±s.d. *P<0.05; **P<0.01; ***P<0.001. Data are representative of at least three independent experiments.
