ALBERT

Description: Introduction SOX11 is a transcription factor (TF) aberrantly expressed in the majority of mantle cell lymphomas (MCLs), which is generally associated with aggressive clinical behaviour. No mutations, genetic aberrations or direct correlations with differential DNA methylation at the promoter related to its expression have been found in MCL. Deeper insights into its regulation can be found by considering the three-dimensional (3D) chromatin structure. It is becoming clear that the genome can be partitioned into 3D building blocks, topologically associated domains (TADs) and that enhancer regions likely regulate genes within their TADs by 3D contacts, but do not affect genes outside their own TADs. By mapping the 3D chromatin structure, we previously identified a distant putative SOX11 enhancer showing enhancer activity and 3D contacts with the SOX11 gene in the SOX11-positive MCL cell line Z-138, but not in the SOX11-negative MCL cell line JVM-2. Aims We aimed to deepen our understanding of the differential 3D contacts and enhancer activity previously observed at the putative SOX11 enhancer in SOX11-positive versus SOX11-negative MCL cell lines by addressing the following questions: (i) Do TAD boundaries around the SOX11 locus change between SOX11-positive and -negative MCLs? (ii) How do the 3D contacts and chromatin states at this region behave in primary MCL cases and normal B cells? (iii) Is the putative SOX11 enhancer involved in SOX11 expression in other tissues? Methods We have extended our experimental analyses of the putative SOX11 enhancer by performing (i) HiC-sequencing and 3D fluorescence in situ hybridization (3D FISH) in MCL cell lines Z-138 and JVM-2, (ii) 4C-sequencing, chromatin inmmunoprecipiation followed by deep sequencing (ChIP-seq) of 6 histone marks, an Assay for Transposase-Accessible Chromatin with deep sequencing (ATAC-seq) and chromatin state modeling by chromHMM (using the 6 histone marks) in primary MCL cases and normal naive and memory B-cells. Furthermore, we have explored the activity of this region in other SOX11 expressing cell lines studied within the ENCODE Consortium. Results HiC-sequencing in the cell lines Z-138 (SOX11-positive) and JVM-2 (SOX11-negative) showed that the SOX11 locus and its putative enhancer are located within the same TAD in both samples. Hence, shifts in TAD boundaries do not seem to underlie the differential 3D chromatin interactions between the SOX11 locus and its putative enhancer in these two cell lines. By ChIP-seq and chromatin state modeling we observed that the promoter of SOX11 is poised, i.e., carrying histone marks H3K4me3 and H3K27me3, in normal naive and memory B-cells and the SOX11-negative MCL primary case. Furthermore, we observed weak enhancer activity at the putative SOX11 enhancer in normal naive and memory B-cells and the SOX11-negative MCL primary case, but strong enhancer activity, marked by the presence of H3K27ac, only in SOX11-positive samples. In addition, by ATAC-seq we identified two specific chromatin accessible regions that potentially represent the transcription factor binding sites responsible for activation of this enhancer region in SOX11-positive MCLs. By 4C-sequencing we observed that the SOX11 locus and its putative enhancer show high 3D contacts in two other SOX11-positive MCL cell lines (GRANTA-519 and JEKO-1) and in a SOX11-positive primary MCL case, but not in a SOX11-negative primary MCL case. Furthermore, the differential 3D contacts at these regions in Z-138 and JVM-2 were confirmed by 3D FISH, which is currently being performed in primary MCL cases. Interestingly, no 3D contacts were observed in normal naive and memory B cells, indicating that although the SOX11 promoter is poised within these normal B-cell subpopulations, primed looping at these regions does not exist and seems not to explain the 3D contacts we observed in SOX11-positive MCL cell lines and primary cases. When investigating chromatin states in cell lines studied by ENCODE with an active SOX11 promoter (H1-hESC, HSMM and NHLF) none of them show activity in the identified region, suggesting that the putative SOX11 enhancer is de novo activated only in the context of MCL lymphomagenesis. Conclusions We provide new evidence that the activation of a distant SOX11 putative enhancer and its 3D contacts to the SOX11 gene, is a de novo event in SOX11-positive MCL cell lines and primary cases that is likely specific for this malignancy. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Modulation of the DNA methylation landscape during cell differentiation is a well-established phenomenon. The B-cell lineage represents a paradigmatic cellular model to study the dynamic epigenome during cell development and specification because major B-cell maturation stages are well defined and display differential phenotypic and gene expression features. Furthermore, different B-cell subpopulations show different proliferation abilities, microenvironmental influences and life spans, providing a window of opportunity to study the epigenome in the context of multiple processes. Methods: We performed whole-genome bisulfite sequencing (WGBS), high-density methylation microarrays and gene expression profiling of ten purified human B-cell subpopulations spanning the entire differentiation program, ranging from uncommitted progenitors to terminally-differentiated plasma cells. Results: The results of both WGBS and methylation microarrays indicate that B-cell ontogenesis involves an extensive and gradual reconfiguration of the DNA methylome. We uncovered that non-CpG methylation at CpApC trinucleotides is present in progenitor cells and disappears upon B-cell commitment independently of CpG demethylation. CpG methylation, in contrast, changed extensively during the entire B-cell maturation program, with one quarter of all measured CpGs showing dynamic methylation. B-cell enhancers suffered more extensive methylation changes than promoter regions, especially in the early differentiation steps up to the germinal center B-cell (gcBC) stage, and their demethylation seemed to be mediated by binding of lineage-specific transcription factors. Enhancers with dynamic methylation were related to genes involved in a large B-cell network that showed high gene expression variability throughout differentiation. In highly proliferative gcBCs, we observed a shift of dynamic methylation from regulatory towards non-functional elements; gcBCs start to undergo global demethylation of late-replicating heterochromatic regions and methylation of polycomb-repressed regions. This signature becomes particularly extensive in long-lived memory B cells and plasma cells, indicating that these changes start in highly proliferative cells and then accumulate in non-proliferative cells with extended lifespan. Conclusion: Our epigenomic analysis of the B-cell differentiation program extends our knowledge on how the DNA methylome is modulated during cell specification and maturation and offers a resource for researchers in the field, both at global and single gene levels. Disclosures No relevant conflicts of interest to declare.

Description: Mantle cell lymphoma (MCL) is an aggressive mature B-cell neoplasm molecularly characterized by the presence of the t(11;14) and expression of the transcription factor SOX11 in most of the patients. A subset of tumors do not express SOX11 and are frequently associated with hypermutated IGHV, a predominantly non-nodal leukemic disease and an indolent clinical course. To gain insight into the potential role of epigenetic alterations in the pathogenesis of MCL, we have extensively characterized the DNA methylomes of 86 tumors by high density DNA methylation arrays and whole-genome bisulfite sequencing (WGBS) of selected samples, using naïve and memory B cells (NBC and MBC, respectively) as controls. An unsupervised principal component analysis showed the presence of two major MCL subgroups associated with different molecular features such as SOX11 expression levels and IGHV mutational status. The major subgroup (C1) comprised mostly SOX11 positive MCLs with low levels of IGHV somatic hypermutation (SHM), although some SOX11 positive cases with high levels of SHM also belonged to C1. Interestingly, this group also contained few SOX11 negative cases, all presenting low levels of SHM (IGHV germline identity ≥98%). The second, smaller subgroup (C2), was comprised almost entirely by SOX11 negative cases, all having high levels of SHM (IGHV germline identity

Description: Analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed regional DNA hypermethylation embedded in extensive global hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM as compared to normal plasma cells were located outside CpG islands and were unexpectedly associated with intronic enhancer regions active in normal B cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with downregulation of its host genes. ChIP-seq and DNAseI-seq further revealed that DNA hypermethylation in these regions was related to enhancer decommissioning. Hypermethylated enhancer regions overlap with binding sites of B-cell specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation suggesting that MM cells reacquire epigenetic features of undifferentiated cells upon loss of expression of B-cell specific TFs. Overall, we have identified DNA hypermethylation of developmentally-regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM. Disclosures No relevant conflicts of interest to declare.