I?B? is a dual-use coactivator of NF-?B and POU transcription factors
OCA-B, OCA-T1, and OCA-T2 belong to a family of transcriptional coactivators that bind to POU transcription factors (TFs) to regulate gene expression in immune cells. Here, we identify IkBz (encoded by the NFKBIZ gene) as the fourth member of the OCA protein family. While originally discovered as an inducible regulator of NFkB, we show here that IkBz shares a microhomology with OCA proteins and uses this segment to simultaneously bind to POU transcription factors and octamer motif-containing DNA. Our functional reporter assays suggest that IkBz requires its interaction with POU TFs to coactivate immune-related genes. This finding is reinforced by our epigenomic analysis of MYD88 L265P-mutant lymphoma cells, which revealed colocalization of IkBz, the POU transcription factor OCT2, and NFkB:p50 at hundreds of DNA elements harboring octamer and kB motifs. These results suggest that IkBz is a transcriptional coactivator that integrates and amplifies the output of NFkB and POU transcription factors at inducible genes in immune cells. Overall design: For each ChIP, 20?million cells were used. Cells were cross-linked with 1% formaldehyde for 10?min at room temperature with agitation and quenched with 0.125?M glycine for 5?min at room temperature. After washing twice with PBS, cells were incubated in 1?ml cell lysis buffer (10?mM Tris-HCl (pH?8.0), 10?mM NaCl, 0.2% NP-40 with protease inhibitor) for 15?min on ice. Nuclei were isolated by centrifugation at 600×g for 30?s, resuspended in 1?ml nuclear lysis buffer (50?mM Tris-HCl (pH?8.0), 10?mM EDTA, 1% SDS with protease inhibitor) and sonicated using a Bioruptor Pico (Diagenode) (30s on/off, 10 cycles). In all cases, the chromatin was centrifuged at maximum speed in a tabletop centrifuge for 15?min at 4?°C. The supernatant was mixed with 7?ml IP dilution buffer (20?mM Tris-HCl (pH?8.0), 2?mM EDTA, 150??mM NaCl, 1% Triton X-100), and incubated with the 2.5?µg of the indicated antibody for 2?h. Protein A/G magnetic beads (25?µl, Dynabeads, Thermo Fisher Scientific) were washed twice with PBS and added to the antibody–chromatin mixture at 4?°C overnight. The beads were washed once with IP wash 1 buffer (20?mM Tris-HCl pH?8.0, 2?mM EDTA, 50?mM NaCl, 1% Triton X-100, 0.1% SDS), twice with high salt buffer (20?mM Tris-HCl (pH?8.0), 2?mM EDTA, 500?mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP wash 2 buffer (10?mM Tris-HCl pH?8.0, 1?mM EDTA, 250?mM LiCl, 1% NP-40, 1% sodium deoxycholate) and twice with TE (pH?8.0). Chromatin DNA was eluted, and cross-linking was reversed in 200?µl nuclear extraction buffer with 12?µl of 5?M NaCl and 1?µg?/mL RNase A at 65?°C overnight. The beads were then discarded. The DNA-containing supernatant was treated with 4?µg?/mL proteinase K at 56?°C for 20?min and purified using the QIAquick PCR purification kit (QIAGEN) in 60?µl water.