GSE122752
KS-associated herpesvirus Rta and cellular POU proteins regulate DNA binding of the Notch signaling protein RBP-Jk/CSL broadly to the virus genome
Interaction of the Kaposi's sarcoma-associated herpesvirus (KSHV) Rta protein with the cellular Notch signaling effector, Recombination Signaling Protein (RBP)-Jk (aka CSL and CBF-1), is essential for viral reactivation from latency.  We previously showed that Rta binds to a DNA motif repeated in the viral Mta promoter (called “CANT” or Rta-c) to stimulate RBP-Jk DNA binding, and distinguished Rta from the activated Notch-1 protein.  To determine whether Rta's mechanism would apply generally to other viral promoters, we employed chromatin immunoprecipitation/deep sequencing (ChIP/Seq) to identify Rta and RBP-Jk binding sites across the KSHV genome.   We show that RBP-Jk binds nearly exclusively to unique genome sites during latency and reactivation.   Many, but not all, reactivation-specific RBP-Jk peaks were associated with Rta bound to single Rta-c motifs.   Other motifs that were over-represented with stimulated RBP-Jk DNA binding including those for the cellular DNA binding proteins BCL11A, MNT, MAF B, and TCF12.  Four of the top seven motifs that were most over-represented with inhibition of RBP-Jk DNA binding were putative binding sites for the Pit/Oct/Unc (POU) family of proteins, including POU3F3 (Oct-8) and POU5F1 (Oct-4).   Interestingly, two other POU motifs, including one for the POU2F1 (Oct-1) protein, are associated with inhibition of RBP-Jk DNA binding unless Rta bound to a nearby Rta-c motif.   The relative distances between the Rta-c, POU, and RBP-Jk motifs were conserved at reactivation-specific RBP-Jk peaks in three promoters that Rta transactivated, and the Rta-c and Oct-1 motifs overlapped in two of those.  The proximity of the Rta-c/Oct-1 motif to an RBP-Jk motif is critical for Rta transactivation of the ORF50AS/KbZIP promoter, and knockdown of Oct-1 protein debilitated reactivation and production of infectious virus.  Our data suggest a broad role for POU proteins in regulating DNA binding of RBP-Jk and its associated transactivators. Overall design: ChIP/Seq using anti-Rta and anti-RBP-Jk antibodies on chromatin from B cells latently infected with KSHV or induced to reactivate by treating with valproic acid (VPA).  Rabbit total IGG is the control antibody.