PITX1 protein interacts with ZCCHC10 to regulate hTERT mRNA transcription

Abstract

Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase ( hTERT ), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, suggesting that this enzyme activity contributes to an unlimited replication potential of cancer cells via regulation of telomere length. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 ( PITX1 ) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is the functional unit that suppresses hTERT transcription, and may play a crucial role as a novel tumor suppressor complex.

Human chromosome 3p21.3 carries TERT transcriptional regulators in pancreatic cancer

“…Recent studies have shown that TSGs can cooperate with many nearby genes that are deleted together, which results in a more invasive disease 43 . We have previously reported that PITX1 , a negative regulator of hTERT , interacts with zinc finger CCHC-type containing 10 ( ZCCHC10 ) to regulate hTERT transcription, and both genes are encoded together in the 5q31.1 region 44 . Therefore, chromosome engineering technologies that can transfer an entire chromosome or chromosomal fragments, which contain the essential genomic context for regulation of gene transcription, may enable elucidation of tumor suppressor functions by specific chromosomal domains 45 .…”

Human Chromosome 3p21.3 Carries TERT  Transcriptional Regulators in Pancreatic Cancer

“…Recent studies have shown that TSGs can cooperate with many nearby genes that are deleted together, which results in a more invasive disease 41 . We have previously reported that PITX1, a negative regulator of hTERT, interacts with zinc nger CCHC-type containing 10 (ZCCHC10) to regulate hTERT transcription, and both genes are encoded together in the 5q31.1 region 42 . Therefore, chromosome engineering technologies that can transfer an entire chromosome or chromosomal fragments, which contain the essential genomic context for regulation of gene transcription, may enable elucidation of tumor suppressor functions by speci c chromosomal domains 43 .…”

PITX1 inhibits the growth and proliferation of melanoma cells through regulation of SOX family genes

“…Cell culture was performed as described previously 37 . A2058 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).…”

“…Analysis of apoptosis was performed as described previously 37 . Apoptotic cells were measured by Annexin V staining using an APC Annexin V apoptosis detection kit with 7-AAD according to the manufacturer's (BioLegend, San Diego, CA, USA) instructions.…”

“…Western blotting was performed as described previously 37 . Membranes were blotted with rabbit polyclonal antibody against human PITX1 antigen (ab70273, 1:2,000; Abcam, Cambridge, MA, UK), rabbit monoclonal antibody against human SOX9 antigen (ab185966, 1:2000; Abcam), rabbit monoclonal antibody against human SOX10 antigen (ab155279, 1:2000; Abcam), mouse monoclonal antibody against FLAG antigen (F1804, 1:2000; Sigma), or with polyclonal antibody against α-tubulin (PM054-7, 1:5000; MBL, Tokyo, Japan) and the appropriate standard peroxidase-labeled anti-mouse IgG and anti-rabbit IgG secondary antibodies, according to the manufacturer's instructions (GE Healthcare, Piscataway, NJ, USA).…”