DNA methylation mediated up-regulation of TERRA non-coding RNA is coincident with elongated telomeres in the human placenta.
What factors regulate elongated telomere length in the human placenta?



Hypomethylation of TERRA promoters in the human placenta is associated with high TERRA expression, however, no clear mechanistic link between these phenomena and elongated telomere length in the human placenta was found.



Human placenta tissue and trophoblasts show longer telomere lengths compared to gestational age-matched somatic cells. However, telomerase (hTERT) expression and activity in the placenta is low, suggesting a role for an alternative lengthening of telomeres (ALT). While ALT is observed in 10-15% of human cancers and in some mouse stem cells, ALT has never been reported in non-cancerous human tissues.



Human term placental tissue and matched cord blood mononuclear cells (CBMCs) were collected as part of the Peri/Postnatal Epigenetic Twins study (PETS). In addition, first trimester placental villi, purified cytotrophoblasts, choriocarcinoma cell lines and a panel of ALT-positive cancer cell lines were tested. Telomere length was determined using the Terminal Restriction Fragment (TRF) assay and a relative quantitative PCR method. DNA methylation levels at several CpG rich subtelomeric TERRA promoters were determined using bisulfite conversion and the SEQUENOM EpiTYPER platform. Expression of TERRA and hTERT was determined using quantitative RT-PCR. ALT was assessed using the C-circle assay (CCA).



The human placenta tissue and purified first trimester trophoblasts showed low subtelomeric (TERRA) DNA methylation compared to matched CBMCs and other somatic cells. Interestingly placental TERRA methylation was lower than ALT-cancer cell lines, previously reported to be hypomethylated at these loci. Low TERRA methylation was associated with higher expression of TERRA RNA in placenta compared to matched CBMCs. Detectable levels of C-circles were observed in first trimester placental villi, but not term placenta, suggesting that the ALT mechanism may be active in specific placental cells in early gestation. C-circle analysis of purified first trimester trophoblasts and ALT-associated PML bodies (APB) staining of first trimester villi cross-sections failed to identify this specific cell type population.



While first trimester villi showed detectable levels of C-circles, these levels were very low compared with those observed in ALT-positive tumours and cell lines. This is consistent with a small sub-population of ALT-positive cells but this requires further investigation. Finally, no mechanistic link was established between TERRA DNA methylation, the presence of C-circles and longer telomere length.



Given the previously described role of TERRA ncRNA as a negative regulator of telomerase, the finding of elevated TERRA and long telomeres is counterintutive. ALT as a mechanism for telomere length maintenance has only been reported in certain human cancers, and recently in mouse embryonic stem cells and embryos. As with many aspects of cancer, it appears that ALT activity in tumours may be the inappropriate activation of a pathway found in very specific cell types in human development. Our data are the first supportive evidence for ALT in a non-cancerous human tissue, a result that requires further investigation and replication. The level of TERRA methylation in the human placenta is significantly lower than found in ALT cancer cell lines and somatic cells, raising the possibility of a novel mechanism in maintaining low methylation at subtelomeric regions.



Not applicable.



This study was supported by NHMRC early career fellowship (B.N.), NHMRC Senior Research Fellowship (R.S.) and the Victoria Government Infrastructure Grant. R.R. holds a patent for the C-circle assay. No other conflicts declared.
Murdoch Childrens Research Institute-Cancer and Disease Epigenetics, Royal Children's Hospital Flemington Road, Parkville, Melbourne, Victoria 3052, Australia.
Boris
B
Novakovic
Cancer Research Unit, Children's Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia.
Christine E
CE
Napier
Murdoch Childrens Research Institute-Cancer and Disease Epigenetics, Royal Children's Hospital Flemington Road, Parkville, Melbourne, Victoria 3052, Australia.|Department of Paediatrics, University of Melbourne, Parkville VIC 305 2.
Regan
R
Vryer
Embryo Implantation Laboratory, Hudson Institute for Medical Research, Monash University, Clayton VIC 3168, Australia.
Eva
E
Dimitriadis
Pregnancy Research Centre, Department of Perinatal Medicine, Royal Women's Hospital , Parkville, Victoria 3052, Australia.|Centre for Genetic Diseases, Hudson Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia.
Ursula
U
Manuelpillai
Department of Pathology and Centre for Trophoblast Research, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
Andrew
A
Sharkey
Department of Paediatrics, University of Melbourne, Parkville VIC 305 2.|Early Life Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital , Parkville VIC 3052, Australia.
Jeffrey M
JM
Craig
Cancer Research Unit, Children's Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia.
Roger R
RR
Reddel
Murdoch Childrens Research Institute-Cancer and Disease Epigenetics, Royal Children's Hospital Flemington Road, Parkville, Melbourne, Victoria 3052, Australia richard.saffery@mcri.edu.au.|Department of Paediatrics, University of Melbourne, Parkville VIC 305 2.
Richard
R
Saffery
D002460:Cell Line; D019175:DNA Methylation / Q000235:genetics / Q000502:physiology*; D005260:Female; D006801:Humans; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000235:genetics; D022661:RNA, Untranslated / Q000235:genetics*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D016615:Telomere / Q000235:genetics / Q000378:metabolism*; D059505:Telomere Homeostasis / Q000235:genetics / Q000502:physiology; D014327:Trophoblasts / Q000378:metabolism
D022661:RNA, Untranslated; D019098:Telomerase
ALT; DNA methylation; TERRA; alternative lengthening of telomeres; placenta; promoter methylation; telomerase; telomeres; trophoblast