Complex context-relationships between DNA methylation and accessibility, histone marks, and hTERT gene expression in acute promyelocytic leukemia cells: perspectives for all-trans retinoic acid (ATRA) in cancer therapy (original) (raw)
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The expression of hTERT gene, encoding the catalytic subunit of telomerase, is a feature of most cancer cells. Changes in the chromatin environment of its promoter and binding of transcriptional factors have been reported in differentiating cells when its transcription is repressed. However, it is not clear whether these changes are directly involved in this repression or only linked to differentiation. In a maturation-resistant acute promyelocytic leukemia (APL) cell line (NB4-LR1), we have previously identified a new pathway of retinoid-induced hTERT repression independent of differentiation. Using a variant of this cell line (NB4-LR1 SFD), which resists to this repression, we show that although distinct patterns of histone modifications and transcription factor binding at the proximal domain of hTERT gene promoter could concur to modulate its expression, this region is not sufficient to the on/off switch of hTERT by retinoids. DNA methylation analysis of the hTERT promoter led to the identification of two distinct functional domains, a proximal one, fully unmethylated in both cell lines, and a distal one, significantly methylated in NB4-LR1 SFD cells, whose methylation was further re-enforced by retinoid treatment. Interestingly, we showed that the binding to this distal domain of a known hTERT repressor, WT1, was defective only in NB4-LR1 SFD cells. We propose that epigenetic modifications targeting this distal region could modulate the binding of hTERT repressors and account either for hTERT reactivation and resistance to retinoid-induced hTERT downregulation.
Cancer Research, 2007
hTERT, which encodes the catalytic subunit of telomerase and is expressed in most immortalized and cancer cells, has been reported to have increased DNA methylation in its promoter region in many cancers. This pattern is inconsistent with observations that DNA methylation of promoter CpG islands is typically associated with gene silencing. Here, we provide a comprehensive analysis of promoter DNA methylation, chromatin patterns, and expression of hTERT in cancer and immortalized cells. Methylation-specific PCR and bisulfite sequencing of the hTERT promoter in breast, lung, and colon cancer cells show that all cancer cell lines retain alleles with little or no methylation around the transcription start site despite being densely methylated in a region 600 bp upstream of the transcription start site. By real-time reverse transcription-PCR, all cancer cell lines express hTERT. Chromatin immunoprecipitation (ChIP) analysis reveals that both active (acetyl-H3K9 and dimethyl-H3K4) and inactive (trimethyl-H3K9 and trimethyl-H3K27) chromatin marks are present across the hTERT promoter. However, using a novel approach combining methylation analysis of ChIP DNA, we show that active chromatin marks are associated with unmethylated DNA, whereas inactive marks of chromatin are associated with methylated DNA in the region around the transcription start site. These results show that DNA methylation patterns of the hTERT promoter (À150 to +150 around the transcription start) are consistent with the usual dynamics of gene expression in that the absence of methylation in this region and the association with active chromatin marks allow for the continued expression of hTERT.
Journal of Ovarian Research, 2019
Background: All-trans retinoic acid (ATRA) is currently being used to treat hematological malignancies, given the ability to inhibit cell proliferation. This effect seems to be related to epigenetic changes of the TERT (Telomerase Reverse Transcriptase) promoter. When hypomethylated, ATRA-inducible TERT repressors can bind the promoter, repressing transcription of TERT, the rate-limiting component of telomerase. Ovarian carcinomas are heterogeneous tumors characterized by several aberrantly methylated genes among which is TERT. We recently found a hypomethylation of TERT promoter in about one third of serous carcinoma, the most lethal histotype. Our aim was to investigate the potential role of ATRA as an anticancer drug in a subgroup of ovarian carcinoma where the TERT promoter was hypomethylated. Methods: The potential antiproliferative and cytotoxic effect of ATRA was investigated in seven serous ovarian carcinoma and one teratocarcinoma cell lines and the results were compared to the methylation status of their TERT promoter. Results: The serous ovarian carcinoma cell line OVCAR3, harboring a hypomethylated TERT promoter, was the best and fastest responder. PA1 and SKOV3, two cell lines with an intermediate methylated promoter, revealed a weaker and delayed response. On the contrary, the other 5 cell lines with a highly methylated promoter did not respond to ATRA, indicative of ATRA-resistant cells. Conclusions: Our results demonstrate an inverse correlation between the methylation level of TERT promoter and ATRA efficacy in ovarian carcinoma cell lines. Although these results are preliminary, ATRA treatment could become a new powerful, personalized therapy in serous ovarian carcinoma patients, but only in those with tumors harboring a hypomethylated TERT promoter.
Studies on the Role of hTERT – a Telomerase Regulator of DNA Methylation in Cervical Cancer
Madridge Journal of Case Reports and Studies, 2020
Telomerase is a cellular reverse transcriptase which stabilizes telomere length by adding hexameric TTAGGG repeats onto the telomeric ends of chromosomes. Expression of telomerase occurs in germ cells and stem cells but not in normal (somatic) cells. However, in many tumors, telomerase is activated, which is one of the immortalization steps. In humans, telomerase expression is regulated by a telomerase subunit called hTERT. The promoter region of the gene that encodes hTERT is located in a CpG island and was shown to be regulated, at least in part, by DNA methylation. In this study, we have analyzed the methylation status of the hTERT gene in primary squamous cell carcinomas, SCC-1 and SCC-2, and HPV+ cervical tissue scrapes, moderate dyskaryosis and mild dyskaryosis. By using the bisulfite genomic sequencing assay, the region between +90 bp and +566 bp within the gene was analyzed. We found that the primary squamous cell carcinoma SCC-1, which consists of 70% carcinoma cells, was more methylated than SCC-2, which consisted of 40% carcinoma cells. However, the number of methylated CpGs in cervical scrapes classified as moderate dyskaryosis and mild dyskaryosis were higher than in SCC-2. In the present study, we demonstrated the importance of DNA methylation profile at hTERT promoter site as a promising biomarker for cervical cancer. Further, it also provides a possible mechanism into the epigenetic regulation of hTERT in various other cancer developments.
Nucleic Acids Research, 2011
Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci.
Scientific Reports
Acute promyelocytic leukemia (APL) is characterized by arrested differentiation of promyelocytes. patients treated with all-trans retinoic acid (AtRA) alone experience relapse, while patients treated with AtRA and arsenic trioxide (Ato) are often relapse-free. this suggests sustained changes have been elicited by the combination therapy. To understand the lasting effects of the combination therapy, we compared the effects of ATRA and ATO on NB4 and ATRA-resistant NB4-MR2 APL cells during treatment versus post treatment termination. After treatment termination, NB4 cells treated with ATRA or ATO reverted to non-differentiated cells, while combination-treated cells remained terminally differentiated. This effect was diminished in NB4-MR2 cells. This suggests combination treatment induced more permanent changes. Combination treatment induced higher expression of target genes (e.g., transglutaminase 2 and retinoic acid receptor beta), which in NB4 cells was sustained post treatment termination. to determine whether sustained epigenetic changes were responsible, we quantified the enrichment of histone modifications by chromatin immunoprecipitation, and CpG methylation by bisulfite-pyrosequencing. While ATRA and combination treatment induced similar histone acetylation enrichment, combination treatment induced greater demethylation of target genes, which was sustained. therefore, sustained demethylation of target genes by AtRA and Ato combination treatment is associated with lasting differentiation and gene expression changes. Acute promyelocytic leukemia (APL) accounts for approximately 10-15% of adult acute myeloid leukemias 1 and until recently was associated with high mortality and poor patient outcomes 2. The malignancy is characterized by arrested promyelocyte differentiation in the myeloid lineage of hematopoietic stem cells, resulting in the absence of mature granulocytes and an over accumulation of promyelocyte precursors 3. The deficiency of this population of cells manifests in patients as severe coagulation defects that can culminate in fatal disseminated intravascular coagulation and systemic hemorrhaging 4. Uniquely, the cause in 98% of APL cases is attributed to a single chromosomal translocation event between chromosome 15 and chromosome 17 5-7. This results in aberrant fusion between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes located on the respective chromosomes. The resultant PML-RARα chimeric protein behaves as an altered RAR nuclear receptor, which changes the DNA binding specificity, and represses the transcriptional programs normally controlled by RAR-retinoid-X-receptor heterodimers, through enhanced interactions with corepressors 8-10. Consequently, differentiation is abrogated and immortalization of the promyelocytes is promoted. Similar to other malignancies, APL cells are characterized by aberrant epigenetic changes to DNA methylation and histone modifications which interfere with normal transcriptional programs and contribute to blocked
Expression of hTERT is the major limiting factor for telomerase activity. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. In this study, we used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5 0 -regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. This study underlines the dual role of DNA methylation in hTERT transcriptional regulation. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.
Telomerase is a cellular reverse transcriptase which stabilizes telomere length by adding hexameric TTAGGG repeats onto the telomeric ends of chromosomes. Expression of telomerase occurs in germ cells and stem cells but not in normal (somatic) cells. However, in many tumors, telomerase is activated, which is one of the immortalization steps. In humans, telomerase expression is regulated by a telomerase subunit called hTERT. The promoter region of the gene that encodes hTERT is located in a CpG island and was shown to be regulated, at least in part, by DNA methylation. In this study, we have analyzed the methylation status of the hTERT gene in primary squamous cell carcinomas, SCC-1 and SCC-2, and HPV+ cervical tissue scrapes, moderate dyskaryosis and mild dyskaryosis. By using the bisulfite genomic sequencing assay, the region between +90 bp and +566 bp within the gene was analyzed. We found that the primary squamous cell carcinoma SCC-1, which consists of 70% carcinoma cells, was more methylated than SCC-2, which consisted of 40% carcinoma cells. However, the number of methylated CpGs in cervical scrapes classified as moderate dyskaryosis and mild dyskaryosis were higher than in SCC-2. In the present study, we demonstrated the importance of DNA methylation profile at hTERT promoter site as a promising biomarker for cervical cancer. Further, it also provides a possible mechanism into the epigenetic regulation of hTERT in various other cancer developments.
Blood, 2013
The origin of aberrant DNA methylation in cancer remains largely unknown. In the present study, we elucidated the DNA methylome in primary acute promyelocytic leukemia (APL) and the role of promyelocytic leukemia-retinoic acid receptor α (PML-RARα) in establishing these patterns. Cells from APL patients showed increased genome-wide DNA methylation with higher variability than healthy CD34(+) cells, promyelocytes, and remission BM cells. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score, and Flt3-mutation status characterized methylation subtypes. Transcription factor-binding sites (eg, the c-myc-binding sites) were associated with low methylation. However, SUZ12- and REST-binding sites identified in embryonic stem cells were preferentially DNA hypermethylated in APL cells. Unexpectedly, PML-RARα-binding sites were also protected from aberrant DNA methylation in APL cells. Consistent with this, myeloid cells from preleukemic PML-RARα ...