In neuroblastoma, genomic rearrangements of the TERT locus put TERT expression under the control of super-enhancers elements as demonstrated in cohorts of mixed stage neuroblastoma (Peifer, 2015; Valentijn, 2015). Recent discoveries in neuroblastoma demonstrated that deregulated TERT expression is also achieved by integration of genomic circle DNA proximal to the TERT gene, resulting in transcriptional upregulation of TERT (Koche, 2020). The neuroblastoma cell lines GI-ME-N, CLB-GA, Kelly and possibly LAN-2 were identified to harbor a TERT rearrangement (Gartlgruber, 2018; Peifer, 2015). Rearrangements of the TERT locus were shown to occur in a region 50 kb upstream of the TERT gene in patient tumors (Gartlgruber, 2018; Peifer, 2015). In contrast, this study identified the breakpoint in GI-ME-N
cells to be located within the TERT 5’-UTR, adding more complexity to TERT gene regulation.
Genomic TERT rearrangements resulting in high TERT expression and telomerase activity were described in several cancer entities including neuroblastoma, glioblastoma and meningioma (Diplas, 2018; Juratli, 2018; Peifer, 2015; Valentijn, 2015). Future studies applying chromosome confirmation capture technologies or ChIP sequencing are necessary to understand how the TERT gene is regulated and which factors drive its expression in the corresponding cellular context of a genomic TERT rearrangement (Gartlgruber, 2018).
Besides activating mutations in the TERT gene body or in the gene promotor, epigenetic modifications were identified to upregulate TERT expression across tumor entities (Zhao, 2009). In this study, ChIP sequencing of solvent-treated cells confirmed the epigenetic profile indicative of an active chromatin state (Gartlgruber, 2018; Peifer, 2015; Valentijn, 2015). After panobinostat treatment, this profile revealed genome-wide alterations in the analyzed histone marks, but no major changes of the epigenetic environment of the rearranged TERT region were detected after panobinostat treatment. The few significantly altered histone modifications in the rearranged TERT region are not likely to severely influence the expression of TERT, and cannot explain the repression of TERT mRNA levels after panobinostat treatment. Comparing TERT-rearranged tumors and cell lines with samples without a TERT rearrangement, histone marks H3K27ac, H3K4me1 and H3K4me3 were enriched at the transcriptional start site of TERT, which are all associated with actively transcribed protein-coding genes (Gartlgruber, 2018;
Peifer, 2015; Valentijn, 2015). Histone mark H3K36me3 was enriched across the TERT gene body, an indication of actively transcribed genes (Gartlgruber, 2018; Peifer, 2015). Repressive chromatin marks H3K27me3 and H3K9me3 showed a more divergent pattern, also being enriched at TERT gene body and the transcriptional start site (Gartlgruber, 2018; Peifer, 2015).
The parallel occurrence of activating and repressive chromatin marks at the same locus is termed a bivalent state, allowing timely gene activation or repression upon stimulation (Voigt, 2013). This epigenetic profile indicative of an actively transcribed TERT locus was confirmed here in solvent-treated cells and the efficacy of panobinostat treatment was ensured by technical validation of samples prior to sequencing. Sample analysis applying qRT-PCR confirmed decreasing TERT levels after panobinostat treatment, and pull-down efficacy of proteins was exemplarily analyzed at GRHL1 and MIR183 loci, demonstrating technical accuracy of sample preparation. Induction of GRHL1 and MIR183 was previously demonstrated in neuroblastoma cells after panobinostat treatment (Fabian, 2014; Lodrini, 2013). In this study, the six histone modifications from the publication of Peifer et al. were used for the analysis of histone modifications in the TERT region after panobinostat treatment. These histone modifications
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represent prominent histone marks playing major roles in gene regulation and are commonly used for ChIP sequencing analyses (Kimura, 2013). There are additional histone marks like H3K4me2 and H3K4ac that were shown to be involved in TERT transcription in multiple cancers (Li, 2016a; Stern, 2015). It would be worthwhile investigating whether these histone modifications are differentially regulated after panobinostat treatment at the TERT locus. In addition, this study investigated chromatin marks 18 h after panobinostat treatment. This period might have been too short to induce changes in the selected chromatin marks at the TERT locus, although TERT levels are reduced to about 40% at that time point. Regulatory mechanisms such as miRNA-mediated degradation or post-transcriptional modifications mediating the early repression of TERT could explain the clear reduction of TERT mRNA level early after onset of panobinostat treatment. Looking at histone modifications at later time points might unravel an additional participation of histone marks in the regulation of TERT and contribute to our understanding of the epigenetic landscape at the TERT locus after HDACi treatment. Therefore, histone modifications participating in the regulation of the TERT region induced by HDACi panobinostat cannot completely be ruled out.
Global increase of histone acetylation is a well-described effect of HDACi treatment (Dias, 2018; Glick, 1999). In normal human fibroblasts and epithelial cells, HDAC inhibition induces expression of TERT by changing the acetylation status of nucleosomal histones resulting in an open chromatin state, but not in cancer cells (Takakura, 2001). In contrast, in breast cancer cells and other cancers, genome-wide hyperacetylation of histones was shown to be increased after panobinostat treatment (Singh, 2016; Tate, 2012). Global increase in histone mark H3K27ac was also reported here after panobinostat treatment, but the few significantly altered acetylated sites in the rearranged TERT-region are unlikely to explain the strong repression of TERT.
Additional so far unknown hyperacetylated sites may participate to indirectly regulate the expression of TERT after panobinostat treatment.
Many oncogenes acquire close proximity to transcriptional strong enhancer elements, a process called super-enhancer hijacking (Northcott, 2014). This results in an induction of massive oncogene expression and increased expression of subsequent target genes. In TERT-rearranged neuroblastoma, several strong enhancer regions like EBF Transcription Factor 1 (EBF1), Membrane Associated Ring-CH-Type Finger 11 (MARCH11) and Neuropeptide Y (NPY) juxtaposed the TERT gene (Gartlgruber, 2018; Peifer, 2015). Further analysis of ChIP sequencing data of the TERT-rearranged cell line models focusing on patterns of H3K27ac and H3K4me1 histone modifications, which are known to mark active enhancers, could identify the
responsible enhancer elements driving TERT expression (Ernst, 2011; Heintzman, 2007). It would be interesting to assess whether panobinostat treatment influences the interaction of acquired enhancer elements and TERT. The TERT locus is transcriptionally silenced in most somatic cells and becomes (re)activated during tumorigenesis (Low, 2013). HDAC inhibition reverses this repression in normal cells (Cong, 1999; Takakura, 2001; Wang, 2003). ATAC sequencing in CLB-GA and Kelly neuroblastoma cells revealed an enrichment of peaks at the TERT locus and neighboring genes, indicative of open chromatin (Gartlgruber, 2018).
Circularized chromosome conformation capture (4-C) sequencing analysis identifying possible regulatory elements and super-enhancers driving TERT expression are worthwhile investigating in TERT-rearranged neuroblastoma. Further investigations possibly applying ATAC-seq or DNase-footprinting could help to illuminate the regulation of the TERT locus after panobinostat treatment.
In addition to histone modification, DNA methylation of CpG dinucleotides can influence TERT gene transcription (Jie, 2019; Zhu, 2010). HDACi were demonstrated to indirectly influence DNA methylation, by downregulation of DNA methyltransferase 3 beta (DNMT3B) mRNA and protein expression in human endometrial adenocarcinoma cells (Xiong, 2005). The study presented here demonstrates that methylation is globally reduced after panobinostat treatment. The TERT locus was less methylated at few CpG sites after panobinostat treatment, but generally showed no major alterations at individual CpG dinucleotides. Few alternately methylated CpG sites were distributed spatially within the TERT region and might not be sufficient to result in transcriptional repression of TERT (Bird, 2004; Deaton, 2011).
Methylation of CpG dinucleotides is generally considered to silence gene expression in somatic tissue (Long, 2017), and HDAC inhibition indirectly reduces methylation at CpG sites (Zopf, 2012). Further, it was shown in healthy mice that panobinostat treatment induced DNA hypomethylation (Al-Hamamah, 2019). Cancer cells display a dysregulation of CpG methylation patterns, resulting in oncogene activation (Gal-Yam, 2008). There is controversy discussion about how methylation regulates the expression of TERT (Jie, 2019). Increased methylation of CpG sites at the TERT promotor was demonstrated to repress TERT in teratocarcinoma cells, normal oral fibroblasts and senescent normal oral keratinocytes (Lopatina, 2003; Shin, 2003). In contrast, across tumor entities including colon, breast, prostate and brain cancer, hypermethylation of the THOR region proximal to TERT was described to result in upregulation of TERT gene expression (Lee, 2019; Seynnaeve, 2017). In non-small cell lung cancer cells, treatment with HDACi vorinostat induced reduction of telomerase activity and diminished methylation of the THOR region and expression of
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methyltransferases DNMT1 and DNMT3b (Li, 2011). Methylation array profiling of mixed-stage neuroblastoma revealed that MYCN-amplified tumors showed the highest levels of CpG methylation in the TERT region, followed by TERT-rearranged neuroblastoma (Gartlgruber, 2018; Peifer, 2015). Strongly enriched methylation of CpG site cg11625005 (chr5:1295737) close to the TERT promotor was observed in neuroblastoma tumors with high TERT expression (Gartlgruber, 2018; Peifer, 2015). In this study, the rearranged TERT region of the GI-ME-N cell line reveals a distinct genomic environment that is lacking the native sequence proximal to TERT, including the gene promotor itself. Therefore, the published epigenetic regulation of TERT cannot be transferred to the data presented in this study. It remains to be investigated whether structural motifs such as E-boxes, GC or ETS binding sites are proximal to TERT, and if they are regulated by HDACi treatment.
In telomerase-positive cells, it was shown that methylation of the TERT promoter was necessary for active gene transcription and that CCCTC-Binding Factor (CTCF) binding to the first exon repressed TERT transcription, which was abrogated when methylation of its recognition sequence was increased (Renaud, 2005; Renaud, 2007). Applying ChIP sequencing, it would be interesting to assess whether binding of the transcriptional insulator CTCF to TERT is increased after panobinostat treatment, possibly mediating TERT repression. In addition, changes in methylation of CpG dinucleotides are indirect effects mediated by HDAC inhibitors and the investigated time point of 18 h after treatment might have been too early to reveal clear changes of the methylation status in the rearranged TERT region. Looking at methylation patterns at later time points might unravel a possible differential methylation of CpG sites regulating TERT expression, and contribute to our understanding of the methylation status at the TERT locus after panobinostat treatment in TERT-rearranged neuroblastoma cells. Further analyses of the methylation status of the TERT region applying bisulfite cloning and sequencing might help to uncover potential changes in the methylation status of the TERT locus (Zhang, 2009). Therefore, regulation of the TERT region by alternatively methylated CpG sites induced by panobinostat treatment might possibly contribute to the repression of TERT at later time points. Focusing on the TERT locus, further analyses to investigate the chromatin state after panobinostat treatment are necessary to unravel the regulation of the locus after panobinostat treatment. This study demonstrates that panobinostat treatment revealed no major changes in the investigated epigenetic marks at the TERT locus in TERT-rearranged cells.