Characterization of human telomerase reverse transcriptase promoter methylation and transcription factor binding in differentiated thyroid cancer cell lines.

Telomerase reverse transcriptase (TERT) activation plays an important role in cancer development by enabling the immortalization of cells. TERT regulation is multifaceted, and its promoter methylation has been implicated in controlling expression through alteration in transcription factor binding. We have characterized TERT promoter methylation, transcription factor binding, and TERT expression levels in five differentiated thyroid cancer (DTC) cell lines and six normal thyroid tissue samples by targeted bisulfite sequencing, ChIP-qPCR, and qRT-PCR. DTC cell lines express varying levels of TERT and exhibit TERT promoter methylation patterns similar to patterns seen in other telomerase positive cancer cell lines. The minimal promoter immediately surrounding the transcription start site is hypomethylated, while further upstream portions show dense methylation. In contrast, the TERT promoter in normal thyroid tissue is largely unmethylated throughout and expresses TERT minimally. Transcription factor binding is also affected by TERT mutation status. The E-twenty-six (ETS) factor GABPA exhibits TERT binding in the TERT mutant DTC cells only, and allele-specific methylation patterns at the minimal promoter were observed as well, which may indicate allele-specific factor recruitment at the minimal promoter. Furthermore, we identified binding sites for activators MYC and GSC in the hypermethylated upstream region, pointing to its possible importance in TERT regulation. Overall, TERT expression and telomerase activity depend on the interplay of multiple regulatory mechanisms including TERT promoter methylation, mutation status, and recruitment of transcription factors. This work explores of the interplay between these regulatory mechanisms and offers insight into cellular control of active telomerase in human cancer.

Insight into the RNA Exosome Complex Through Modeling Pontocerebellar Hypoplasia Type 1b Disease Mutations in Yeast.

Pontocerebellar hypoplasia type 1b (PCH1b) is an autosomal recessive disorder that causes cerebellar hypoplasia and spinal motor neuron degeneration, leading to mortality in early childhood. PCH1b is caused by mutations in the RNA exosome subunit gene, EXOSC3 The RNA exosome is an evolutionarily conserved complex, consisting of nine different core subunits, and one or two 3'-5' exoribonuclease subunits, that mediates several RNA degradation and processing steps. The goal of this study is to assess the functional consequences of the amino acid substitutions that have been identified in EXOSC3 in PCH1b patients. To analyze these EXOSC3 substitutions, we generated the corresponding amino acid substitutions in the Saccharomyces cerevisiae ortholog of EXOSC3, Rrp40 We find that the rrp40 variants corresponding to EXOSC3-G31A and -D132A do not affect yeast function when expressed as the sole copy of the essential Rrp40 protein. In contrast, the rrp40-W195R variant, corresponding to EXOSC3-W238R in PCH1b patients, impacts cell growth and RNA exosome function when expressed as the sole copy of Rrp40 The rrp40-W195R protein is unstable, and does not associate efficiently with the RNA exosome in cells that also express wild-type Rrp40 Consistent with these findings in yeast, the levels of mouse EXOSC3 variants are reduced compared to wild-type EXOSC3 in a neuronal cell line. These data suggest that cells possess a mechanism for optimal assembly of functional RNA exosome complex that can discriminate between wild-type and variant exosome subunits. Budding yeast can therefore serve as a useful tool to understand the molecular defects in the RNA exosome caused by PCH1b-associated amino acid substitutions in EXOSC3, and potentially extending to disease-associated substitutions in other exosome subunits.

Human telomerase reverse transcriptase regulation by DNA methylation, transcription factor binding and alternative splicing (Review).

The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), plays an essential role in telomere maintenance to oppose cellular senescence and, is highly regulated in normal and cancerous cells. Regulation of hTERT occurs through multiple avenues, including a unique pattern of CpG promoter methylation and alternative splicing. Promoter methylation affects the binding of transcription factors, resulting in changes in expression of the gene. In addition to expression level changes, changes in promoter binding can affect alternative splicing in a cotranscriptional manner. The alternative splicing of hTERT results in either the full length transcript which can form the active telomerase complex with hTR, or numerous inactive isoforms. Both regulation strategies are exploited in cancer to activate telomerase, however, the exact mechanism is unknown. Therefore, unraveling the link between promoter methylation status and alternative splicing for hTERT could expose yet another level of hTERT regulation. In an attempt to provide insight into the cellular control of active telomerase in cancer, this review will discuss our current perspective on CpG methylation of the hTERT promoter region, summarize the different forms of alternatively spliced variants, and examine examples of transcription factor binding that affects splicing.