Fig 2: BCL6 polymerization enhances SIAH1 interaction and ubiquitination.a, Correlation of p-values for two genome-wide CRISPR-Cas9 knockout screens: x-axis - reporter screen for eGFPBCL6 stability in HEK293TCas9 cells upon BI-3802 treatment, and y-axis - BI-3802 resistance screen in SuDHL4Cas9 cells. Guides were collapsed to gene level (n = 3; 4 guides/gene; two-sided empirical rank-sum test-statistics). b, Flow cytometry analysis of HEK293TCas9 cells expressing the indicated BCL6-BTB domain fusion construct treated with DMSO or 1 μM BI-3802 for 7 hours (bars represent mean, n = 3). c, Immunoblots of eGFP immunoprecipitation in the presence of 2μM BI-3802 or DMSO from HEK293TCas9 cells transduced with indicated eGFPBCL6 constructs and V5SIAH144C>S (n = 2). d, Immunoblots of StrepBCL65−360 in vitro ubiquitination by SIAH1FL in the presence of DMSO or 1 μM BI-3802 (n = 2). e, BodipyBCL65−360 was titrated to 0.2 μM BiotinSIAH1SBD in DMSO, 2 μM BI-3812, or 2 μM BI-3802, and the signal was measured by TR-FRET. Lines represent standard four parameter log-logistic curve fit (n = 3). f, HEK293TCas9 cells expressing the eGFPBCL61−275 reporter and V5SIAH1 were treated with 0.5 μM MLN7243 for 2 hours, and 1 μM BI-3802 for 1 hour. Cells were imaged by indirect immunofluorescence as indicated (n = 2). Scale bar is 5 μm.

Fig 3: SIAH1 induces degradation of BCL6 via VxP motif.a, Flow cytometry analysis of HEK293TCas9 cells expressing full length eGFPBCL6 stability reporter and vectors expressing no-insert control, SIAH1 or SIAH1C44S, exposed to DMSO or BI-3802 for 2 hours. Bars represent the mean (n = 3). b, Alignment of the BCL6 SIAH1 recognition site with previously published peptide sequences recognized by SIAH1 with inferred consensus SIAH1 binding site. c, CRISPR-Cas9 knockout screen with the Bison library for eGFPBCL6AA1−129+241−260 stability in HEK293TCas9 cells after 16 hours of treatment with 1 μM BI-3802 or DMSO. Guides were collapsed to gene level (n = 1; 4 guides/gene; two-sided empirical rank-sum test-statistics). d, Flow cytometry analysis of HEK293TCas9 cells expressing eGFPBCL6FL or eGFPBCL6FL VSP>GSA treated with DMSO or 1 μM BI-3802 for 7 hours (bars represent mean, n = 3).

Fig 4: Representative images of hematoxylin and eosin staining (HE) and immunohistochemistry against CD20 (B cells), CD3 (T cells), MECA79 (high endothelial venules; HEVs), CD40 (follicular dendritic cells), and BCL6 (germinal center) in a small-sized aggregation of lymphocytes in goat mammary gland tissues. Scale bar, 100 μm or 25 μm (clippings).

Fig 5: SATB1-dependent transcriptional regulation of Bcl6 impacts Tfh development.a Two significant SATB1-dependent loops connect Bcl6 (same loop anchor for both loops; chr16:23985000–23990000; mm10) with its super-enhancer regions (SE1 – chr16:24245000–24250000, SE2 – chr16:24505000–24510000). In Satb1 cKO, these interactions were diminished as seen in the Hi-C heatmap. Legend: th – thymocytes, DP – CD4+CD8+ T cells, RKO – Rad21fl/flCd4-Cre+ and SKO – Satb1fl/flCd4-Cre+. b Computational 3D modeling utilizing the WT and Satb1 cKO Hi-C data, to visualize the proximity between the Bcl6 gene body and its two super-enhancer regions. The black beads represent the edge of super-enhancer regions demarcated by the SATB1 loop anchors (SE1 and SE2 as in a). Color gradient represents linear genomic position along the locus. c Western blot analysis for BCL6 expression of whole cell protein extracts prepared from WT and Satb1 cKO thymocytes. Actin serves as a loading control (upon pixel analysis of the images a 45% reduction of BCL6 expression in Satb1 cKO thymocytes was calculated). Two biological replicates were performed with similar results. The Western blot analysis was performed three times with similar results. d H&E staining of mouse spleen sections. In WT sections, Red Pulp (RP), lymphoid Nodules of the white pulp (N), the Hilus (H) and Trabecular Vein (TV) are labeled. Arteries (black arrows) are present in each nodule. The nodule structure is clear with extensive periarteriolar lymphocyte sheath (PALS), rich in round, dark stained T lymphocytes surrounding the arterioles (WT2). The distinct marginal zone surrounding the marginal sinus (MZ and magenta arrow, respectively) is shown. Dotted lines mark lighter stained B-lymphocyte rich follicle regions within the nodule. In Satb1 cKO (SKO), there is disturbed spleen structure with few apparent small sized lymphoid nodules with the periarteriolar region depleted of T lymphocytes. The follicular region has accumulated large phagocytic cells and displays many foci of phagocytosis of apoptotic cells (green arrows). The red pulp contained higher numbers of haemopoietic cell clusters and megakaryocytes (yellow arrows) suggesting increased haemopoietic activity. Scale bar WT1 & SKO1 100 µm and WT2 & SKO2 50 µm. The experiment was repeated four times with similar results. e WT pancreas sections were incubated with serum from either WT or Satb1 cKO animals to detect the presence of autoantibodies in the Satb1 cKO serum. Scale bar 100 µm. The experiment was repeated 2 times with similar results.