Elucidation of the epigenetic code of the chromosomal region 13q14.3 in Chronic Lymphocytic Leukemia (CLL)

B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world with a median age of 72 years of CLL patients at diagnosis. The most common genomic aberration in CLL is the deletion of a critical region in chromosomal band 13q14.3, which is deleted in more than 50 % of patients. The high frequency of deletions of 13q14.3 in CLL and other tumors points to a tumor suppressor mechanism localized in the critical region. The candidate genes localized in the critical region in 13q14.3 have been shown to be monoallelically expressed. Towards elucidation of the tumor suppressor mechanism in 13q14.3, the epigenetic modifications of genes and CpG-islands in the region were analyzed in this thesis. Monoallelic expression of almost all genes in the critical region in 13q14.3 in non-malignant cells was detected to be marked by epigenetic modifications at promoter and exonic regions of these genes. A chromatin pattern specific for monoallelically expressed and imprinted genes, which is promoter-restricted enrichment of dimethylated lysine 4 of histone H3 (H3K4me2), was detected at the genes DLeu2/BCMSUN and C13ORF1. Detection of this specific chromatin pattern led to identification of a novel monoallelically expressed gene in the region: C13ORF1. Here, C13ORF1 was in addition to RFP2 and DLeu2/BCMSUN, shown to be monoallelically expressed in B- and T-cells from healthy donors. The chromatin immunoprecipitation (ChIP) methodology was established in order to quantify two chromatin modifications at the critical region. ChIP was technically established and optimized to analyze enrichment of the histone mark H3K4me2 that is correlated with an active transcription state, and of the histone variant macroH2A, which was shown to be allelically enriched at monoallelically silenced loci, at 13q14.3. In addition, also DNA-methylation of the CpG-islands was analyzed. Thus, an epigenetic code of the candidate genes that correlates with their transcription status was determined in non-malignant hematopoietic and CLL cells. In non-malignant cells, the epigenetic code identifies one active and one inactive copy of the critical region in 13q14.3. Thereby, the genes DLeu2/BCMSUN and C13ORF1 are monoallelically expressed from the epigenetically active allele and their expression status is marked by a distinct chromatin pattern. In contrast, the distinct chromatin pattern could not be detected at the gene RFP2 that was earlier shown to be monoallelically expressed. The quantification of H3K4me2 and macroH2A occurrence at RFP2 points to allelic differences of enrichment at the gene locus, although this could not be further defined by the chosen methods. However, allelic enrichment of chromatin marks could be detected at two distinct loci in the 5’-regions of the large noncoding RNA genes, BCMS and DLeu2/BCMSUN, in samples derived from healthy probands. Thereby, in this study, two candidate locus control regions (LCRs) were identified that are located in the CpG-islands D and E in the promoter region of the two large ncRNAs and showed differential DNA-methylation and histone modifications. The differential epigenetic code and the localization of the candidate LCRs suggest their involvement in the regulation of expression of the two large ncRNA genes that span the critical region in 13q14.3. In summary these findings suggest a model of the epigenetically regulated tumor suppressor mechanism in 13q14.3 that leads to monoallelic expression of the candidate genes in the region. This model consists of the features that i) 13q14.3 is present in one active and one silent chromosome copy in non-malignant healthy cells, ii) the large ncRNAs BCMS and BCMSUN are likely involved in the regulation (in cis) of expression of the other genes in 13q14.3, and iii) differential epigenetic modifications of candidate LCRs control transcription of the large ncRNA genes. Accordingly, the epigenetic status of the LCRs is critical for transcription of the ncRNAs that in turn regulate the expression of the candidate genes in the critical region. Surprisingly, the most evident epigenetic aberration at 13q14.3 was the generally higher enrichment of the active chromatin mark at the region in CLL cells. However, also the inactive chromatin mark was increased at 13q14.3 in CLL cells. Significant differences of enrichment of H3K4me2 and macroH2A between non-malignant and CLL cells, were detected at CpG-islands B, C and E and at the exonic region of DLeu2/BCMSUN, which clearly showed that the aberrant modifications in CLL are most evident at the promoter regions of the genes C13ORF1, RFP2 and DLeu2/BCMSUN, respectively. The chromatin pattern that marks monoallelic expression at C13ORF1 and DLeu2/BCMSUN remained unchanged in the analyzed CLL cells. However, in CLL cells derived from patients with different genomic aberrations, the deletion of genomic material from 13q14.3 and loss of active chromatin marks could be correlated. This suggests that in del(13q) CLL patients, indeed the active copy of 13q14.3 is lost, which would explain downregulation of expression of the genes in the critical region observed in these patients. In line with the proposed model of the tumor suppressor mechanism in 13q14.3, the most evident aberrant epigenetic modifications of 13q14.3 in CLL cells were detected at the two candidate LCRs, where distinct epimutations were detected. Thus, an epimutation in 13q14.3 was shown that could be responsible for the inactivation of the epigenetic regulatory mechanism of 13q14.3 in CLL cells. Further, these findings suggest that the regulatory impact of the ncRNAs, the LCR or another yet unidentified part of the critical region in 13q14.3 is affected by this epimutation, which leads to deregulation and inactivation of the tumor suppressive function of 13q14.3. In conclusion, a specific epigenetic code at the genes in the critical region in 13q14.3 was determined that is correlated with their transcriptional state. Evidence was obtained for a model of epigenetic regulation that implies differential chromatin packaging of the two copies of 13q14.3 and results in monoallelic expression of the candidate genes in healthy B- and T-cells. Furthermore, two candidate LCRs were identified in the critical region in 13q14.3 in non-malignant cells that showed distinct epimutations in CLL cells. The localization and the detected epimutations of the LCRs in CLL point to their role in regulation of the ncRNA genes that most likely regulate expression of the other genes in the critical region. On the basis of these findings, further characterization of epigenetic features at 13q14.3 will help to fully elucidate the complex epigenetic regulatory network that controls the tumor suppressor mechanism in 13q14.3. The findings presented here will help to understand the pathogenicity of (epigenetic) inactivation of the tumor suppressor mechanism in CLL and thereby provide the basis for development of more efficient and specific therapies for CLL in the future