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SOX11 is an SRY-related HMG-box transcription factor (TF) family member aberrantly expressed in the majority of mantle cell lymphoma (MCL) cases. The transcriptional program regulated by SOX11 and its potential oncogenic mechanisms in MCL pathogenesis have recently started to be elucidated. However, the mechanisms underlying its aberrant expression in MCL remain unclear. No mutations, genetic aberrations or direct correlations with differential DNA methylation at the promoter region related to its expression have been found in MCL. As it is becoming increasingly clear that remote enhancers may regulate gene expression, we hypothesized that distant regulatory regions might be involved in regulating aberrant SOX11 expression in MCL.

In this study, we aimed to identify and characterize distal regulatory elements associated with aberrant SOX11 expression in MCL.

We analyzed the 3-dimensional structure of the SOX11 genomic region and its chromatin architecture by combining 4C- and ChIP-sequencing in Z-138 and JVM-2, a SOX11-positive and a SOX11-negative MCL cell line, respectively. Additionally, we analyzed the DNA methylation status of distant enhancers that potentially regulate SOX11 expression by bisulfite pyrosequencing in normal naive B cells, SOX11-positive and SOX11–negative primary MCL cases. Finally, we performed TF motif enrichment analysis to identify TFs that potentially regulate aberrant SOX11 expression in MCL.

By 4C-sequencing in the SOX11-positive cell line Z-138, we identified two genomic regions that show high contact frequencies with the SOX11 locus in 3-dimensional space. These regions were located between 500KB and 1MB from the SOX11 locus. In Z-138, these regions were enriched for histone marks H3K4me1 and H3K27ac, indicative of enhancer activity. In the SOX11-negative MCL cell line JVM-2, neither high contact frequencies with the SOX11 locus, nor enrichment of H3K4me1 and H3K27ac were observed at these loci.

Next, we investigated whether enhancer activity and DNA methylation levels are inversely associated at the identified distant regulatory regions, as described for other loci. We observed that DNA methylation levels in these regions were significantly lower in SOX11-positive (n=12, average methylation levels 13-21%) than in SOX11-negative primary MCL cases (n=10, average methylation levels 51-85%) (P-value <0.01). Furthermore, in normal naive B cells these regions were highly methylated (n=4, average methylation levels 76-92%). These data support our 4C-seq findings in MCL cell lines and suggest that these regions harbour active enhancers only in SOX11-positive primary MCL cases.

Next, we investigated the binding of 161 TFs (as identified by the ENCODE project) at the SOX11 locus and its potential distant enhancers. In total we identified binding sites of 13 TFs bound both to the SOX11 locus and to one or both potential distant enhancers. None of these were significantly higher expressed in SOX11-positive compared to SOX11-negative primary MCL cases. Surprisingly, one of the distant enhancer regions is bound by many TFs involved in B-cell development. However, it remains to be elucidated how these B-cell related TFs play a role in SOX11 expression in MCL, as in normal B cells SOX11 is not expressed.

Altogether, our data suggest a model in which SOX11 expression in MCL is associated with looping of the SOX11 locus to two newly-identified distant enhancer regions. Further experiments, however, are necessary to elucidate the mechanisms underlying their aberrant activation in SOX11-positive MCL cases.