The proximal promoter region of mTert is sufficient to regulate telomerase activity in ES cells and transgenic animals

Abstract

Background: The reverse transcriptase of telomerase (Tert) controls telomerase activity maintaining the end of linear chromosomes in eukaryotic cells. Telomerase function is highly active in undifferentiated multipotent stem cells, decreases with cell differentiation and is generally absent from most somatic cells in the adult. Its absence is responsible of telomeres shortening in such somatic cells. Using an in vivo transgenic model and an in vitro culture differentiation of adult stem cells, we examined the elements of the mouse Tert (mTert) promoter that control telomerase activity.

Results: Three constructs comprising 1, 2 or 5 kb of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (EGFP) were electroporated into embryonic stem (ES) cells. Transformed ES cells were able to mimic the expected mTert expression, which was associated to green fluorescence. One and 5 kb promoter produced the higher expression of EGFP, on ES cells. When ES cells were allowed to differentiate to embryoid bodies and to other cell types, they lost gradually the expression of mTert-EGFP as consequence of differentiation. No differences were found among the three constructs analyzed. We then generated transgenic mice with the three constructs. Expression of the reporter gene was monitored by reverse transcription-PCR analysis and EGFP visualization. The mRNA expression of the three constructs was lower than the endogenous mTert, but mimicked the endogenous mTert transcription pattern; however, no fluorescent expression of EGFP was detected in adult tissues. EGFP expression of the three constructs was visualized at the blastocysts stage and in new ES cells generated from them; in the germinal ring of E13 dpc foetuses; in ES-like colonies and in germinal stem cells generated from neonatal and adult testis cells; and in neuroesferes generated from E14 dpc foetuses' brain cells.

Conclusion: The 1 kb promoter upstream of the initiating ATG codon of mTert contains all the regulatory elements to control telomerase expression in ES cells during in vitro loss of pluripotency. The transgenic mouse lines generated represent an appropriate system to analyze the expression of mouse Tert gene under physiological condition and during establishment of stem cell lines generated from embryonic or adult tissues.

Figure 1

Promoter and regulators elements of the transcriptional of human and mouse TERT. CpG island in human and mouse TERT promoter identify using the CpG Island Explorer Program at are indicated in red line (A). Construct 5 k contain 4.5 Kb of the mouse promoter region, construct 1 k contains the proximal region of the promoter where all the transcription activator elements of the promoter reside (4 regions of c-Myc binding and two regions recognized by proteins from the sp1 family) and construct 2 k, includes, in addition to this proximal region, two of the three regions of MZF2 (myeloid zinc finger protein) binding, that reduces the transcriptional activity of the promoter (B).

Figure 2

mTert promoter activity in R1 ES cells. On the left, the size of the mTert-pormoter-EGFP reporter construct is shown. Cells transformations with an EGFP plasmid without promoter are used as controls for each transfection. On the right, the relative GFP expression of the three promoter construct, and the standard deviation is indicated by solid bars. The GFP intensity produced by the promoterless construct was normalized to value of 100; GFP intensity of other constructs is shown relative to this control. Results are expressed as the mean of at least three independent experiments. A, b, c indicate p < 0.05 in a one-tailed unpaired X²-test.

Figure 3

Sequence of microphotograph of ES cells transformed with 5 k-mTert-EGFP during differentiation induced by LIF removal. (A) ES cell expressing EGFP; (B) three days after LIF removal the ES cells form embryonic bodies (EBs); (C) Differentiation of EBs showing a diminishes on fluorescence intensity; and (D) next stage of differentiation showing a loss of fluorescence; in some wells groups of EGFP expressing cells remain immersed into differentiated colonies.

Figure 4

Levels of GFP expression (none, low, medium or high) in the different 5 k-mTert-GFP cellular clones derived from B6D2 and R1 ES cell lines. (A) 5 kmTert-GFP expression in 76 B6D2 clones, and (B) 5 kmTert-GFP expression in 85 R1 clones. GFP expression increased during 2–3 d after removal of LIF and subsequently diminished in parallel to EBs differentiation.

Figure 5

Expression of 1 k-mTert-EGFP transgene at blastocyst stage (A, B) and ES-like colonies (C, D) and germinal stem cells (E, F) generated from neonatal testis cells.

Figure 6

Fluorescence and contrast phase images of 10DIV cultures of forebrain foetus from 1 k-mTert-EGFP (A) and 5 k-mTert-EGFP (B) transgenic mice.