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Vol. 18, Issue 2, 669-677, February 2007
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Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033
Submitted September 19, 2006;
Revised November 13, 2006;
Accepted November 27, 2006
Monitoring Editor: John Cleveland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The process of differentiation involves switching on or off genes that are expressed in specific types of cells and at specific times. This programming of gene transcription occurs in the context of their native chromatin environment and is often stably maintained throughout the life span of an organism. Many genes essential for proper development of the embryo have been discovered and most of them encode proteins that regulate transcription (Arney and Fisher, 2004; Chambers and Smith, 2004). However, the dynamic interactions between these proteins and chromatin are still areas of intensive investigation. Detailed studies of gene transcription regulatory mechanisms during cell differentiation are essential not only for understanding the fundamental mechanisms of development and differentiation, but also for harnessing the differentiation and proliferation capacity that make stem cells invaluable for regenerative medicine.
Telomerase, an enzyme synthesizing TTAGGG telomere repeats, is pertinent to self-renewal potential of stem cells and is required for long-term cellular proliferation and survival (Morrison et al., 1996; Lee et al., 1998). Telomerase is tightly regulated throughout development (Kim et al., 1994; Prowse and Greider, 1995). In early embryonic tissues and stem cells, telomerase is highly expressed and telomeres are stably maintained (Wright et al., 1996; Sharpless and DePinho, 2004). Although telomerase is detected in many adult stem and progenitor cells, its level in these cells is not sufficient to maintain telomere length (Roy et al., 2004). Furthermore, telomerase is undetectable in the majority of human adult somatic cells, which are programmed for a limited number of cell divisions (Wright et al., 1996). These cells undergo senescence and/or apoptosis after their telomeres are depleted (Bodnar et al., 1998; Zhu et al., 1999), which is likely a molecular basis for the replicative senescence checkpoint in human cells, known as the Hayflick limit (Verfaillie et al., 2002).
Telomerase consists of a catalytic subunit of telomerase reverse transcriptase (TERT) and a template RNA subunit (TER). The TERT subunit is the limiting subunit of telomerase (Liu et al., 2000) and its gene transcription correlates with telomerase activity in most cells and tissues that have been examined (Greenberg et al., 1998; Günes et al., 2000; Wang and Zhu, 2003). Ectopic expression of the hTERT gene is sufficient for cellular immortalization (Bodnar et al., 1998; Zhu et al., 1999); yet normal human cells rarely undergo spontaneous immortalization. It has therefore been suggested that repression of the hTERT gene is very tight and stable throughout development (Wright and Shay, 2000; Rangarajan and Weinberg, 2003). However, molecular mechanisms of this transcriptional repression in somatic cells remain to be elucidated. We have recently shown that the endogenous hTERT gene is embedded in a condensed chromatin environment in several immortal human cell lines and normal human fibroblasts (Wang and Zhu, 2004), suggesting that epigenetic mechanisms play a key role in the developmental regulation of hTERT expression. Thus, repression of the hTERT gene may be a model to understand how transcriptional programming is established and maintained during cell differentiation and development.
Embryonic stem cells (ESCs) are pluripotent cells that have potential to develop into all somatic cell types, providing a powerful system for studying mechanisms involved in differentiation and development. To understand the molecular mechanisms involved in hTERT repression, we utilized the mouse ESC system and asked how the human genomic DNA containing all the regulatory regions of the hTERT gene was programmed during differentiation of ESCs. Here, we report the creation of a novel chromosomally integrated reporter of hTERT transcription using a bacterial artificial chromosome (BAC) containing three consecutive human loci, hCRR9, hTERT, and hXtrp2. Using Firefly and Renilla luciferases (Fluc and Rluc) to monitor the hTERT and hCRR9 promoters, respectively, we showed that the hTERT promoter in stably integrated reporter lines was progressively down-regulated during differentiation of the mouse ESCs into embryoid bodies, whereas the hCRR9 promoter was constitutively transcribed at a high level throughout the differentiation process. The hTERT promoter underwent complete silencing upon further differentiation into osteogenic cells and their derivatives, a process dependent on the deacetylation of histones H3 and H4 at the hTERT promoter. In addition, the human loci in the integrated BAC reporter in the differentiated cells adopted chromatin structures similar to their respective endogenous counterparts in human cells. Inhibition of histone deacetylases by trichostatin A (TSA) resulted in chromatin opening at the hTERT promoter and a partial activation of hTERT promoter in differentiated cells. Thus, our results demonstrated that the ectopic human TERT loci underwent their designated developmental programming during differentiation of the mouse ESCs, providing a model for studying genetic and epigenetic regulation of hTERT transcriptional silencing.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and manuscript in preparation). First, a 2.7-kb BstEII fragment from pYF10 plasmid (Wang and Zhu, 2003) was recombined with the original BAC, resulting in the insertion of the Fluc open reading frame (ORF) and a SV40 poly-A site into the translational initiation codon of the hTERT gene. Next, a 1.5-kb HindIII-BamHI Rluc cassette from pRL-SV40 (Promega, Madison, WI) was inserted into the hCRR9 initiation codon. Finally, the BspHI-ClaI fragment containing PGK promoter/puromycin gene from pLZRSpBMN was inserted into the SfoI site of SacB gene in the BAC vector backbone sequence (pBACe3.6). The resulting products were confirmed by restriction enzyme digestions and sequencing the recombination junctions.
Cell Culture, Transfection, and ESC Differentiation
The mouse ESC line HM-1 was cultured on 0.1% gelatin-coated dishes in DMEM with 20% fetal bovine serum (FBS) and 1000 U/ml LIF (Chemicon, Temecula, CA). The modified BAC construct 117B23-cRtFsP was transfected into HM-1 ESCs using FuGene6 (Roche, Indianapolis, IN) and selected with 0.5 µg/ml puromycin for 10–14 d. The colonies exhibiting both Fluc and Rluc activities were then isolated. Data obtained from three clones, C1, F2-1, and F2-9, are presented here. ESC differentiation into EBs and osteogenic cultures were as described previously (Woll and Bronson, 2006).
Chromatin Analyses
DNase I sensitivity assays were performed as described previously (Wang and Zhu, 2003, 2004). Full-length genomic bands were quantified by phosphoimaging and generalized DNase I sensitivities were calculated by the equation S = log(Xd/Xi/log(Cd/Ci)) x T, where X and C are test and control band intensities for the initial (i) or digested (d) samples and T is the size ratios of the control to test fragments (Saitoh et al., 2000). All probes specific to human genomic DNA with the exception of CpG1 have been published (Wang and Zhu, 2004). The CpG1 and mouse probes were amplified by PCR from genomic DNA using primers listed in Table 1.
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) and hCRR9 promoters (Table 1).
Gene Expression Analyses
RT-PCR was performed as described previously (Wang and Zhu, 2004). Equal amount of total RNAs from various samples was used for RT-PCR reactions. All the experiments were repeated at least once. Primers for osteocalcin (OCN), type I collagen (COL1), and Runx2 were the same as previously published (Woll and Bronson, 2006). The amplifications were 28 cycles for OCN and CoL1 and 32 cycles for Runx2. Other transcripts were amplified for 29 cycles using primers listed in Table 1.
Luciferase activities were measured using the Dual Luciferase Reporter assay system (Promega). For imaging, Fluc substrate D-luciferin (0.312 mg/ml, Prolume, Pinetop, AZ) or Rluc substrate Coelenterazine (12 µg/ml, Prolume) were added into culture media. Bioluminescent images were acquired using an IVIS 50 Imaging System and analyzed with Living Image software Version 2.5 (Xenogen Biosciences, Hopkinton, MA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wang and Zhu, 2003, 2004). Therefore, these two genes can serve as ideal internal controls for analyzing the hTERT transcriptional regulation. The BAC DNA was modified using a new recombination procedure that was based on a BAC recombineering method published by the Copeland laboratory (Lee et al., 2001). The procedure involved a two-step positive and negative selection scheme, which allowed precise modifications of the BAC sequences without leaving behind any unwanted sequences (unpublished results). Figure 1A shows the schematic representation of the final BAC construct, in which Fluc and Rluc cassettes were inserted into the hTERT and hCRR9 translational initiation codons, respectively. Transcription from the hTERT and hCRR9 promoter can thus be monitored by luciferase activities. In addition, a puromycin-resistant marker was inserted into the middle of the SacB gene within the vector backbone. The modified BAC construct was transfected into murine ESCs, HM-1. A total of 12 puromycin-resistant colonies that expressed both Fluc and Rluc activities were isolated. The majority of clones contained most, if not all, of the BAC sequences, as demonstrated by Southern blot analyses (see data in Figures 4 and 5). Three independent lines, C1, F2-1, and F2-9, were used in this report.
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Although EBs contained a range of differentiated cells, there were also undifferentiated and partially differentiated cells in these structures (Keller, 1995). These less differentiated cells might still express significant levels of TERT. To study the repression of the hTERT promoter in fully differentiated cells, the developing 2-d EBs were dispersed and cultured under a previously published osteogenic culture condition (Woll and Bronson, 2006). Bone nodules formed after 3–4 wk in osteogenic cultures. Most of these colonies stained positive by silver nitrate, indicating that the differentiated cells secreted mineralized bone matrix, a characteristic of osteoblast differentiation (Figure 2A). Cells of the osteogenic lineage express a number of molecular markers. For example, Runx2/cbfa1 plays a critical role in osteoblastic differentiation and regulates the expression of osteoblast-specific genes, such as osteocalcin and type I collagen. To examine the expression of these osteogenic markers in bone colonies, total RNAs were extracted from individual colonies and analyzed by semiquantitative RT-PCR. Compared with the mCRR9 RNA levels, which were similar in ESCs and bone colonies, levels of osteocalcin, type I collagen, and Runx2/Cbfa1 mRNAs were all significantly increased in bone colonies (Figure 2B), confirming that the colonies indeed contained differentiated osteogenic cells (zur Nieden et al., 2003; Woll and Bronson, 2006).
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Epigenetic Regulation of the Transgenic hTERT Promoter in Differentiated Mouse Cells
To obtain a large number of differentiated cells for epigenetic analyses, the bone colonies were trypsinized and passaged in MEM medium containing 10% FBS. Relatively homogenous populations of proliferative and morphologically differentiated cells, F2-1d and F2-9d, were derived from ESC clones F2-1 and F2-9, respectively, after passaging for 2 months (Figure 3A). Luciferase activities in F2-1d and F2-9d were determined by bioluminescent imaging and compared with parental ESCs (Figure 3B). The quantified Rluc imaging signal from the hCRR9 promoter did not change before or after differentiation. However, the Fluc signal dramatically decreased by up to five orders of magnitude in both F2-1d and F2-9d cells compared with their respective parental ESCs (Figure 3C). In fact, little Fluc signal above background was detected in F2-1d and F2-9d cells, indicating that the hTERT promoter in the transgenic BAC construct was completely silenced in these differentiated cells.
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3.9 kb), corresponding to a DHS near the core hTERT promoter, was detected in undifferentiated F2-1 cells (Figure 4B). This DHS disappeared in differentiated F2-1d cells, correlating with silencing of the hTERT promoter in differentiated mouse cells. The same DHS was also detected in F2-9 cells but not in F2-9d cells (data not shown). Because this DHS was also present at the endogenous hTERT promoter in human cells expressing telomerase, the data supported our earlier conclusion that its appearance correlated with hTERT transcription (Wang and Zhu, 2004).
Previous studies suggested that the physical location of hTERT gene occurred within a condensed chromatin environment, which might contribute to the repression of hTERT gene in human somatic cells (Wang and Zhu, 2004). To determine whether the hTERT locus in the transgenic BAC DNA had a similar chromatin conformation upon integration in ESCs, generalized DNase I sensitivity of the three loci, hCRR9, hTERT, and hXtrp2, in the transgenic BAC reporter was measured. Genomic DNAs isolated from DNase I–treated nuclei of F2-1 and F2-1d cells were digested with various combinations of restriction enzymes and subjected to Southern analyses (Figure 5). In the absence of any DHSs within a chromatin fragment, generalized DNase I sensitivity can be measured as disappearing rate of the restriction fragment as a function of DNase I concentration (Saitoh et al., 2000; Wang and Zhu, 2004). Thus, fragments with no detectable DHSs were chosen for experiments in Figure 5. Because the kinetics of digestion also depends on fragment sizes, restriction fragments with similar sizes were compared wherever possible, and the digestion rates were normalized to their length. In addition, fragment a at the 5' half of the hCRR9 locus was used as an internal control because the hCRR9 promoter was constitutively active and thus was likely to maintain an open chromatin conformation throughout cell differentiation.
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-globin loci, which were associated with pericentric heterochromatin regions in nonerythroid cells (Brown et al., 2001). There was also a trend that the hTERT locus was more nuclease-resistant than the upstream hCRR9 locus in differentiated F2-1d cells. However, for most chromatin fragments, the difference between F2-1 ESCs and F2-1d cells were not significant in these experiments by statistical analyses. As a control, the 
-globin loci were highly sensitive to DNase I treatment in differentiated F2-1d cells, as was in most somatic cells (Vyas et al., 1992). Therefore, in both undifferentiated and differentiated cells, the integrated BAC reporter formed a chromatin environment that was similar to the corresponding native loci in human cells (Wang and Zhu, 2004).
Transcriptional silencing is often accompanied by CpG methylation. In fact, a CpG island is present at the hTERT promoter (Wick et al., 1999). It was reported that the hTERT promoter was often methylated in human cell lines and cancer cells; however, the extent of CpG methylation was not directly correlated with hTERT gene expression (Devereux et al., 1999; Dessain et al., 2000; Liu et al., 2004a). To determine whether silencing of the transgenic hTERT promoter is associated with CpG methylation, genomic DNAs from F2-1 ESCs and F2-1d cells were subjected to digestion by methylation-sensitive enzyme HpaII or methylation-insensitive MspI. The digested DNAs were analyzed by Southern analysis using a probe (CpG1) hybridized to a human genomic sequence, –1080 to +31 base pairs relative to the hTERT transcription start site. Both HpaII and MspI recognize sequence CCGG, which represents 17 of 99 CpG sites within the region recognized by this probe. As a control, the same blot was also hybridized to the Rluc sequence, which was integrated at the hCRR9 promoter and constitutively expressed. As shown in Figure 6, the ratio of HpaII- to MspI-digested low-molecular-weight DNA in the CpG1 region was identical to that of the Rluc fragment in both F2-1 and F2-1d cells, as determined by phosphoimaging analysis. As a positive control, the HpaII-digested low-molecular-weight CpG1 signal was almost completely absent in human cancer line A431, in which the hTERT promoter was heavily methylated (Guilleret et al., 2002). In addition, the same blot was also probed with another CpG-rich hTERT genomic sequence + 78 to +1682, which included most of exon 1, intron 1, and most of exon 2, and no significant CpG methylation was detected either (data not shown). Furthermore, treatment with 5 µM of 5-aza-2'-deoxycytidine or 5-aza-cytidine for 3–6 d did not induce Fluc expression in either F2-1d or F2-9d cells (data not shown). Therefore, our results indicated that the CpG methylation status of the hTERT promoter might not be required for hTERT silencing during in vitro differentiation of ESCs.
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). It has been demonstrated that the endogenous hTERT transcription in human cells could be induced by inhibition of histone deacetylases in several cell types (Cong and Bacchetti, 2000; Wang and Zhu, 2003). To determine the role of histone deacetylation in the repression of transgenic hTERT promoter, histone acetylation at the hTERT promoter was examined by chromatin immunoprecipitation (ChIP; Figure 7A, top panel). In F2-9 ESCs, the N-terminal tails of histones H3 and H4 were acetylated (lanes 1 and 2). In contrast, such acetylation was undetectable in F2-9d differentiated cells. Likewise, binding of RNA polymerase II to the hTERT promoter was detected in F2-9 ESCs but not in F2-9d cells (lane 3). As a control, no significant changes in acetylation of histones H3 and H4 and PolII binding were detected at the hCRR9 promoter (Figure 7A, bottom panel). Similar results were obtained with F2-1 and F2-1d cells (data not shown). These results demonstrated that silencing of the transgenic hTERT promoter was associated with histone deacetylation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Developmental silencing of hTERT gene is likely a multistep process. Indeed, repression of hTERT promoter was progressive during differentiation of ESCs. The Fluc activity decreased about 10-fold during the 2-wk period of in vitro differentiation into EBs. In fully differentiated cells derived from bone colonies, the hTERT promoter was completely silenced. This was reminiscent of the endogenous hTERT gene in human cells, which has been shown to be under much tighter repression than the mTERT gene in mouse cells (Wright et al., 1996). The native hTERT gene is located near the tip of human chromosome 5p. It was previously suggested that the regulation of hTERT transcription might be under influence of the telomere due to telomere positional effect (Shay and Wright, 2000; Baur et al., 2001). Our data indicated that neither the transcriptional silencing nor the native chromatin conformation of hTERT locus required its physical position near telomere.
Regulation of hTERT transcription involves multiple trans-acting factors. Switch of promoter occupancy from c-Myc/Max to Mad1/Max complexes at the hTERT promoter was shown to correlate with hTERT down-regulation in differentiating HL60 and U937 cells (Günes et al., 2000; Xu et al., 2001). Other factors may also be involved in hTERT repression. For example, it has been reported that transcription factor E2F1, tumor suppressors Menin and Wilms' tumor 1 (WT1), and the TGF- signaling pathway all mediated hTERT transcriptional repression in various cell types (Oh et al., 1999; Crowe et al., 2001; Lin and Elledge, 2003). The roles of these factors in hTERT repression during development remain to be elucidated.
Besides trans-acting factors, the hTERT locus may also subject to epigenetic regulation (Liu et al., 2004a). Recruitment of histone deacetylases by negative regulators and local histone deacetylation at the hTERT promoter were associated with hTERT repression (Xu et al., 2001; Won et al., 2002). Consistently, histones H3 and H4 at the transgenic hTERT promoter were acetylated in ESCs and deacetylated in differentiated cells. Inhibition of histone deacetylases by TSA led to a more than 100-fold induction of Fluc expression from the hTERT promoter, accompanied by the opening of chromatin configuration at the hTERT promoter as demonstrated by DNase I assays. However, this TSA-induced expression was well below the level of Fluc expression in ESCs, suggesting that histone deacetylation and chromatin condensation at the hTERT promoter are necessary but not sufficient for the repression of hTERT promoter in differentiated cells. Additional mechanisms might also contribute to the silencing of hTERT promoter in these cells.
Methylation of the CpG island at the 5' end of the hTERT gene was proposed to play a role in hTERT repression (Devereux et al., 1999; Dessain et al., 2000). However, the transgenic hTERT promoter was not extensively methylated at CpG sites in either ESCs or differentiated cells (Figure 6). Apparently, extensive CpG methylation, as it occurs in many tumor-derived cell lines, was not required for the silencing of hTERT promoter in this ESC in vitro differentiation model. Although the role of CpG methylation in hTERT regulation may vary in different cell types, it is possible that most de novo CpG methylation at the hTERT promoter was acquired during tumor growth and/or in vitro passaging of oncogene-transformed cells (Dessain et al., 2000). Thus, promoter methylation may not be directly involved in hTERT repression during normal development. Consistent with this notion, the CpG island at the hTERT promoter was more extensively methylated in SV40 transformed human fibroblasts than in parental normal human fibroblasts, even though neither cells expressed hTERT (data not shown).
Our recent data indicated that the native hTERT locus is located in a large condensed chromosomal environment in human cells (Wang and Zhu, 2004). In mouse ESCs and their differentiated derivatives, the integrated human sequence adopted a chromatin configuration that was similar to the native loci in human cells. A strong DHS appeared at the transcription start site in undifferentiated cells and disappeared upon differentiation, as was the case in many human somatic cells (Wang and Zhu, 2004). The generalized DNase I sensitivity of integrated human loci was also similar to their native counterparts in human cells: the hCRR9 gene was sensitive to DNase I digestion, whereas the hXtrp2 gene was resistant to the nuclease. A possible difference between the transgenic sequence in mouse cells and the native sequence in human cells was found in the hCRR9/hTERT intergenic region. In mouse F2-1 ESCs and F2-1d differentiated cells, this region was relatively sensitive to DNase I digestion, compared with the hXtrp locus. However, this region was highly resistant to nuclease digestion in most human somatic cells (Wang and Zhu, 2004). This difference might be attributable to the presence of abundant Alu-elements and other human-specific repetitive sequences, which accounted for about half of the 23-kb sequence in intergenic region. It would be reasonable to speculate that such human-specific repetitive elements are more prone to form repressive chromatin structure in human cells than in mouse cells. Studies on transgenic mouse lines using an 8-kb hTERT promoter fragment controlling a LacZ reporter revealed that the reporter expression was overall similar to hTERT expression, albeit with significant variations between transgenic lines (Ritz et al., 2005). More recently, Horikawa et al. (2005) reported a transgenic mouse strain with a BAC fragment containing the hTERT gene as well as an 11-kb upstream sequence and a 1.2-kb downstream sequence. Although hTERT gene expression in this transgenic mouse line was similar to the endogenous hTERT expression in most human tissues, the transgene expression was also detected in a few tissues such as brain, in which native hTERT expression was not detectable. These results suggested either that the 8- and 11-kb upstream sequences were not sufficient to maintain tight repression in some tissues or that the human sequence might behave differently in mouse cells. Although the role of repetitive sequences in regulating hTERT transcription remains to be determined, the overall resemblance between chromatin structures of the transgenic BAC DNA in mouse cells and the native counterparts in human cells rendered the transgenic human reporter in mouse ESCs a suitable model to study the developmental regulation of the hTERT gene in a relevant chromatin context.
It was previously reported that a 4.5-kb mTERT promoter fragment was able to control green fluorescence protein (GFP) expression in a way that was similar to the endogenous mTERT expression during the early differentiation of an ESC line (Armstrong et al., 2000). The GFP expression decreased <5-fold in DMSO or all-trans retinoic acid induced differentiation of ESCs. Recently, GFP reporters controlled by shorter mTERT promoter fragments (1, 2, or 5 kb) were integrated into mouse ESCs. Down-regulation of GFP expression was also observed during differentiation of mESCs (Pericuesta et al., 2006). These experiments suggested that short fragments of the mTERT promoter contained elements involved in repression of mTERT transcription. However, complete silencing of the mTERT promoter was not reported during in vitro differentiation of ESCs. One possibility is that the mTERT promoter is not as tightly repressed as the hTERT promoter. Alternatively, short mTERT promoter fragments might not contain all regulatory elements required for the complete silencing of the TERT promoter.
Although the established ESC lines contained randomly integrated BAC reporters and had multiple copies of BAC reporters (data not shown), regulation of luciferase expression was virtually identical in all three ESC lines that have been examined. Future studies using Cre-mediated recombination through the loxP and lox511 sites within the BAC vectors (pBACe3.6 or pTARBAC1) would allow targeted chromosomal integration of BAC constructs. Targeted single-copy integration, combined with mutagenesis of BAC reporters using BAC recombineering, would form the foundation of a genetically amendable system for dissecting cis-regulatory elements responsible for genetic and epigenetic regulation of hTERT silencing during differentiation and development. Furthermore, using dual luciferase measurements, the BAC reporter would be a suitable model to screen for potential therapeutic targets involved in telomerase regulation.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Jiyue Zhu (joz1{at}psu.edu)
Abbreviations used: BAC, bacterial artificial chromosome; hTERT and mTERT, human and mouse telomerase reverse transcriptase; CRR9, cisplatin-resistance related gene; Xtrp2, a gene encoding an orphan transporter protein; DHS, DNase I hypersensitive site; HDAC, histone deacetylase; TSA, trichostatin A; ESC, embryonic stem cell; EB, embryoid body; Fluc, Firefly luciferase; Rluc, Renilla luciferase
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