Blood, Vol. 112, Issue 3, 644-651, August 1, 2008

Reversible disruption of BCL6 repression complexes by CD40 signaling in normal and malignant B cells
Blood Polo et al. 112: 644

Supplemental materials for: Polo et al

Files in this Data Supplement:

• Figure S1. Centroblast preparation resulted in more than a 95 percent homogeneous population (JPG, 178 KB) -
  Discarded human tonsillectomy specimens were subjected to a purification protocol to enrich for centroblasts, as described in the methods section. The purity of primary centroblasts thus obtained was determined by flow cytometry using the CD77 and CD38 surface markers. A representative analysis is shown.
  
  
  
  
  
  
• Figure S2. CD40L signaling can disrupt the BCL6 repression complex in primary human centroblasts (JPG, 95.8 KB) -
  QChIP was performed in primary centroblasts at the indicated timepoints using antibodies for BCL6 (A), SMRT and HDAC3 (B), histone 3 and histone 4 acetyl (C), and RNA polymerase II (D). The Y axis of panels A and B represent the % of BCL6, SMRT or HDAC3 binding relative to negative control antibody (actin) to the CD23b promoter after CD40L treatment vs. time zero. The Y axis of panels C and D show the fold difference in enrichment of the CD23b promoter (in C), or the CD23b promoter, exon 1 and exon 4 fragments (in D) vs. the negative control antibody (actin). The data show that in primary germinal center centroblasts, CD40L signaling can rapidly eject SMRT and HDAC3 from the BCL6 repression complex without affecting BCL6 binding. This leads to histone acetylation and RNA polymerase II processivity through the coding sequence of the gene. These experiments were ech performed twice, each time in triplicate.