Dysregulation of Pax5 Activity Contributes to the Extinction of the B-Cell Phenotype in Reed-Sternberg Cells
Authors: Collins Graham P , Paterson Jennifer C , May Gillian E , Gupta Rajeev , Marafioti Teresa , Mason David Y , Enver Tariq 
Published: April 2020

Abstract
Hodgkin/Reed-Sternberg cells (HRS cells) are thought to be derived from post-germinal centre B-cells and yet have down-regulated the B-cell phenotype. The B-cell transcription factor Pax5 is important in the maintenance of B-cell identity and we demonstrate that it is down-regulated in HRS cells lines and in HRS cells of the majority of primary classical Hodgkin Lymphoma (cHL) cases. Specifically, 3/30 cases were negative for Pax5, 16/30 were weakly positive, 10/30 cases were moderately positive and 1/30 showed Pax5 staining of equivalent intensity to infiltrating, polyclonal B-cells. In order to functionally test the relevance of a reduced Pax5 expression level, the cHL cell lines L428 and L1236 were stably transfected with Pax5 using a lentiviral transfection system. Transfection of L1236 resulted in up-regulation of CD79a protein expression. However, CD79a was not upregulated in L428 and expression of the Pax5 target genes Cd19 and Blnk was unaffected by Pax5 transfection in both cell lines. Chromatin immunoprecipitation demonstrated that Pax5 failed to bind the high affinity binding site within the Cd19 promoter in the cHL lines despite high levels of Pax5 expression, appropriately localised to the nucleus. Pax5 could, however, bind synthetic oligonucleotide corresponding to this site (as shown by electrophoretic mobility shift assays) raising the possibility that epigenetic modification in vivo may be responsible for the failure to bind DNA. Bisulphite genome sequencing confirmed that in cHL cell lines, the region surrounding the Pax5 binding site in the Cd19 promoter was extensively methylated. Moreover, histone modification analysis also demonstrated an absence of markers of accessible, active chromatin (di- and trimethylated H3K4) and an enrichment of a marker indicating closed, repressive chromatin (trimethylated H3K27). Within the Cd79a promoter, previous studies have implicated the methylation status of a single cytosine residue within the binding site for a Pax5-Ets1 complex to be an important determinant of activation of the Cd79a gene. Interestingly, this residue was shown to be largely methylated in L428 cells but largely unmethyated in L1236 cells, providing a likely mechanism for the differential activation of this gene by transfected Pax5 protein. To investigate whether the observed epigenetic changes were responsible for preventing Pax5 binding and activity at the Cd19 and Cd79a promoters, Pax5 transfected cHL cell lines were cultured in the presence of the demethylating agent 5-aza-2-deoxycytidine. Up-regulation of Cd19 and Cd79a expression was significantly greater in Pax5 transfected cells than in control transfected cells. To conclude: our data suggests that dysregulation of Pax5 activity (at the levels of protein expression and epigenetic modification of the Pax5 binding sites) is important in mediating the extinction of the B-cell programme in HRS cells.