hTERT transcription is repressed by Cbfa1 in human mesenchymal stem cell populations.
Human BMSSCs lose telomerase activity in vitro, which leads to chromosomal instability and cellular senescence. We observed an inverse expression pattern between the osteogenic master regulatory gene, CBFA1, and the stem cell-associated gene, hTERT. We showed that Cbfa1 acts as a partial repressor of TERT, which may facilitate cellular differentiation.


BACKGROUND
The absence of telomerase activity by cultured human bone marrow stromal stem cells (BMSSCs) causes critical shortening of chromosomal telomeres, leading eventually to cellular senescence. Ex vivo expansion of BMSSCs correlates to an increase in osteogenic lineage associated markers such as alkaline phosphatase, bone sialoprotein, and osteocalcin that are regulated by the master regulatory transcription factor, Cbfa1 (Runx2). This study examined whether Cbfa1 was capable of regulating the promoter of the early stem cell-associated gene, telomerase reverse transcriptase (TERT).


METHODS
Human BMSSCs were isolated by fluorescence-activated cell sorting. Telomerase activity was determined using the telometric repeat amplification protocol. CBFA1 and TERT gene expression was assessed by real-time PCR. The functional capacity of Cbfa1 to bind to the hTERT promoter was performed using a modified electrophoretic mobility shift assay (EMSA). Chromatin immunoprecipitation (ChIP) analysis was used to examine Cbfa1 binding to the hTERT promoter in vivo. Functional analysis of CBFA-1 wildtype and mutant DNA binding sites on TERT promoter fragments was assessed using the promoterless green fluorescence protein (GFP) reporter vector, pEGFP-1, after transfection into HOS cells.


RESULTS
This study showed an inverse expression pattern between the osteogenic master regulatory gene, CBFA1, and the stem cell-associated gene, hTERT. The data showed that BMSSCs undergo osteogenic commitment after the loss of hTERT expression, with concomitant elevated levels of CBFA1 transcripts. In addition, two unique Cbfa1 DNA binding sites were identified on the hTERT proximal promoter by EMSA supershift assay. Mutated forms of the putative Cbfa1 binding sites, created by site-directed mutagenesis, were able to abolish this interaction. ChIP analysis showed that Cbfa1 interacted directly with the hTERT promoter in vivo. Functional studies using GFP reporter constructs, driven by 2- and 3-kbp hTERT proximal promoter fragments, showed significantly lower levels of transcriptional activity compared with corresponding constructs with mutated Cbfa1 binding site Oligo 2.


CONCLUSIONS
These studies suggest that Cbfa1 may act as a repressor of early stem cell markers such as hTERT as one possible mechanism for facilitating cellular differentiation.
Isenmann
Sandra
S
Mesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and Veterinary Science/University of Adelaide, South Australia, Australia.
Cakouros
Dimitrios
D
Zannettino
Andrew
A
Shi
Songtao
S
Gronthos
Stan
S
D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D000077214:Becaplermin; D001665:Binding Sites; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000502:physiology*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020574:Proto-Oncogene Proteins c-sis; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
D000954:Antigens, Surface; D050659:Core Binding Factor Alpha 1 Subunit; D016895:Culture Media, Serum-Free; D009838:Oligodeoxyribonucleotides; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D053773:Transforming Growth Factor beta1; D000077214:Becaplermin; C509186:TERT protein, human; D019098:Telomerase; D000469:Alkaline Phosphatase