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. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: J Psychiatr Res. 2020 Nov 17;143:543–549. doi: 10.1016/j.jpsychires.2020.11.027

Analysis of telomere length variation and Shelterin complex subunit gene expression changes in ethanol-exposed human embryonic stem cells

Muhammad Moazzam a,+, Terrence Yim b,+, Vidhya Kumaresan c, David C Henderson d, Lindsay A Farrer e,f,g, Huiping Zhang d,e,*
PMCID: PMC8126580  NIHMSID: NIHMS1647244  PMID: 33243459

Abstract

Telomeres protect chromosome ends from degradation. Telomere length (TL) can be altered by aging and environmental stress. Shortened TL has been observed in peripheral blood leukocytes of alcohol dependent subjects and ethanol-exposed somatic cells. To understand the impact of ethanol on telomeres in pluripotent stem cells, we investigated the influence of ethanol on TL and the expression of six Shelterin complex subunit or telomere-regulating genes (POT1, RAP1, TIN2, TPP1, TRF1, and TRF2) in human embryonic stem cells (hESCs), which were exposed to 0, 25, 50, or 100 mM of ethanol for 3, 7, or 14 days. Ethanol-induced TL and Shelterin complex subunit gene expression changes were examined by quantitative polymerase chain reactions. Two-way ANOVA tests indicated that TL variation and expression changes of four associated Shelterin complex subunit genes (POT1, TPP1, TIN2, and TRF2) were mainly dependent on the length of ethanol exposure, while TRF1 and RAP1expression was influenced by ethanol concentration, exposure time, and the interaction of ethanol concentration and exposure time. Tukey’s multiple comparison tests showed that TL and the expression of POT1, RAP1, TIN2, TPP1, and TRF1 were decreased after a 7-day (versus a 3-day) ethanol exposure. However, the decreased expression of all six Shelterin complex subunit genes was recovered and TL was not further shortened after a 14-day (versus a 7-day) ethanol exposure, likely due to the adaptation of hESCs to ethanol-induced stress. Our study provided further evidence that TL is regulated and maintained by telomere-regulating genes in stem cells under ethanol stress.

Keywords: telomere length, Sheltering complex subunit genes, ethanol exposure, human embryonic stem cells, quantitative polymerase chain reaction, two-way ANOVA

1. Introduction

Human telomeres consist of tandem repeats (TTAGGG) at the end of chromosomes and protect the integrity of the genome by preventing chromosomal rearrangement or fusion. Specifically, telomeres play important roles in (1) sheltering genetic information (O’Sullivan and Karlseder, 2010), (2) distinguishing and protecting the ends of chromosomes from DNA damaging (Smith et al., 2020), (3) serving as a docking site for DNA repair proteins (Chen et al., 2008), and (4) providing information on the history of a cell’s proliferation (Murillo-Ortiz et al., 2013). Telomere length (TL) can reflect the biological age of cells since the telomere degrades with each replication. There is evidence that shortened TL leads to senescence, apoptosis, and oncogenic transformation of somatic cells (Crompton, 1997; Shammas, 2011). Shortened TL has been associated with a shorter lifespan (Whittemore et al., 2019) as well as a number of aging-related diseases such as cardiovascular disease (Zhan and Hagg, 2019), diabetes (Gurung et al., 2019), dementia (Guo and Yu, 2019; Hagg et al., 2017), and hypertension (Liu et al., 2019; Zgheib et al., 2018). On the other hand, environmental factors, such as alcohol use (Kang et al., 2017; Martins de Carvalho et al., 2019; Yamaki et al., 2019) and posttraumatic stress disorder (PTSD) (Avetyan et al., 2019; Meier et al., 2019), may lead to accelerated cellular aging by altering the length of telomeres.

Telomeres are regulated by telomerase, but the activity of telomerase is progressively shut off when embryonic stem cells are differentiated into somatic cells (Greenberg et al., 1998). Additionally, telomeres are maintained by six associated subunit proteins of the Shelterin complex (Fig. 1). Three Shelterin subunit proteins [telomeric repeat binding factor 1 (TRF1 or TERF1), telomeric repeat binding factor 2 (TRF2 or TERF2), and protection of telomeres 1 (POT1)] directly recognize telomeric TTAGGG repeats. They are interconnected by three additional Shelterin subunit proteins [TIN2 or TINF2 (TRF1 interacting nuclear factor 2), ACD (an alternative name is TPP1) (ACD Shelterin complex subunit and telomerase recruitment factor), and RAP1 (TRF2 interacting protein or repressor/activator protein 1 homolog)]. These six subunit proteins form the Shelterin complex that protect telomeres from being recognized by DNA damage surveillance and processed by DNA repair pathways (de Lange, 2005, 2018).

Fig. 1.

Fig. 1.

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Schematic of the Shelterin complex on telomeric DNA.

TRF1: telomeric repeat binding factor 1; TRF2: telomeric repeat binding factor 2; POT1: protection of telomeres 1; TIN2: TRF1 interacting nuclear factor 2; ACD (or TPP1): ACD Shelterin complex subunit and telomerase recruitment factor (or POT1 and TIN2-interacting protein); and RAP1: repressor/activator protein 1 homolog.

Aging and environmental stress (such as alcohol exposure) may disrupt telomere homeostasis. Shortened TL has been observed in subjects with alcohol use disorder (AUD) (Kang et al., 2017; Martins de Carvalho et al., 2019; Pavanello et al., 2011; Strandberg et al., 2012; Yamaki et al., 2019). The causal relationship between alcohol exposure and TL (or the molecular mechanisms underlying the relationship) is not well understood. Using yeast as a model organism, Romano et al. found that alcohol and acetic acid elongated telomeres whereas caffeine and high temperature shortened telomeres (Romano et al., 2013). Using human somatic cells (including foreskin fibroblast and hepatocellular carcinoma cells) as models, Harpas et al. reported that exposure to a moderate concentration (25 mM) of ethanol for one week moderately shortened telomeres in cells (Harpaz et al., 2018). Further studies are needed to confirm these findings. It is also necessary to understand the mechanism behind ethanol-induced TL changes.

The present study aimed to elucidate the relationship of ethanol exposure with TL and the expression of six telomere-regulating or Shelterin complex subunit genes (POT1, RAP1, TIN2, TPP1, TRF1, and TRF2). Human embryonic stem cells (hESCs) were chosen as cellular models to investigate the impact of ethanol concentration and exposure time on TL and the expression of these six Shelterin complex subunit genes. A cellular model controls the influence of environmental factors, thus allowing us to detect TL and telomere-regulating gene expression changes mainly due to ethanol exposure. Mounting evidence suggests that maternal alcohol consumption during pregnancy can result in embryo-fetal defects or intrauterine growth retardation (Coll et al., 2018). The perturbation of alcohol on the activity and differentiation of hESCs in the developing fetus may result in fetal alcohol spectrum disorders (FASD), which is classified as a “stem cellopathy” (Mahnke et al., 2018). Adult stem cells function similarly to hESCs. They are undifferentiated cells but retain the ability of self-renewal and differentiation into a variety of specialized cells. Adult stem cells contribute to tissue homeostasis and repair throughout life span (Clevers, 2015). Exposure to alcohol can influence the function of different types of adult progenitor/stem cells, such as neural stem cells, liver stem/progenitor cells, intestinal stem cells, bone-marrow-derived mesenchymal stromal cells, dental pulp stem cells, adventitial progenitor cells, hematopoietic stem cells, and cancer stem cells (Di Rocco et al., 2019). Based on these findings, we assumed that alcohol exposure of stem cells in embryos or adult tissues or organs could lead to altered TL and expression changes of telomere-regulating genes. Using hESCs as a cellular model, we examined ethanol-induced TL variation and expression changes of telomere-regulating genes. Our study showed that ethanol exposure-associated TL variation and gene expression changes were dependent mainly on the duration of ethanol exposure.

2. Material and methods

2.1. Cell culture

H1 human embryonic stem cells (hESCs) (obtained from the WiCell Research Institute, Madison, USA) were maintained at 37°C in the Essential 8™ Medium (Thermo Fisher Scientific, Waltham, MA, USA) at an atmosphere of 5% CO2 and a relative humidity of 100%. Cells were grown on Vitronectin (Thermo Fisher Scientific, Waltham, MA USA) in a feeder-free culture system. The cell culture medium was changed every day to prevent stem cell differentiation. Cells were passaged at ~80% confluency with the use of ReLeSR™ (StemCell Technologies, Vancouver, Canada) and split in a ratio of ~1:6. ROCK Inhibitor Y-27632 (STEMCELL Technologies, Vancouver, Canada) was added to the newly passaged cells at a concentration of 10 μM for one day to prevent dissociation-induced apoptosis. H1 hESC colonies were characterized by immunostaining [using antibodies reacting with stage-specific embryonic antigen-3 (SSEA3) and antigen-4 (SSEA-4)], nuclear staining [using 4’,6-diamidino-2-phenylindole (DAPI) which is a fluorescent stain that binds strongly to A-T rich regions in DNA], and alkaline phosphatase (AP) staining (using a fluorescent substrate for AP for characterizing pluripotent stem cells) (Supplemental Fig. S1).

Ethanol was added to the culture medium during days of treatment. H1 hESCs were cultured in the medium containing 0 mM, 25 mM [equivalent to blood ethanol levels (BEL) in social drinkers (Weiner et al., 2007)], 50 mM [equivalent to BEL in heavy drinkers (Preedy and R.R, 2003)], and 100 mM [equivalent to BEL in intoxicated drinkers (Soderberg et al., 2003)] of ethanol for 3, 7, or 14 days followed by a 24-hour withdrawal period. Cells were harvested in Dulbecco’s Phosphate-Buffered Saline (DPBS) (Thermo Fisher Scientific, Waltham, MA, USA) and pelleted by centrifugation (1,200 g for 3 minutes). The experiment was conducted in triplicate for each of 12 conditions (4 ethanol concentrations × 3 exposure times). Thirty-six (4×3×3) hESC pellets were collected for genome DNA and total RNA extraction.

2.2. Genomic DNA and total RNA extraction from hESCs

Each of the 36 hESC pellets were split into two parts, one for genomic DNA extraction and another for total RNA extraction. Genomic DNA was extracted from hESCs using the DNeasy® Blood & Tissue Kit (QIAGEN, Germantown, MD, USA). Total RNA was extracted from hESCs using the miRNeasy kit (QIAGEN, Germantown, MD, USA). The concentration and purity of extracted DNA and RNA samples were determined using the NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

2.3. Determination of ethanol-induced TL changes by quantitative polymerase chain reactions (qPCRs)

The relative TL of hESCs (exposed to different concentrations of ethanol for different durations) was quantified using the Relative Human Telomere Length Quantification qPCR Assay Kit (Sciencell, Carlsbad, CA, USA). The kit provided two primer sets. The first primer set (i.e., the telomere primer set) amplified telomere sequences. They were designed to ensure high efficiency and no non-specific amplification of the telomeric sequence. The second primer set [i.e., the single copy reference (SCR) primer set] amplified a 100-bp long region on human chromosome 17 that served as the reference for data normalization. Telomere length (TL) was examined by qPCR using the FastStart Essential DNA Green Master Kit (Roche, Mannheim, Germany) and the QuantStudio™ 12K Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA USA). Genomic DNAs (20 ng) extracted from each of the 36 hESC samples were included as templates in the qPCR mixture (20 ul). The cycling conditions were: 95°C for 10 minutes, followed by 32 three-step cycles at 95°C for 20 seconds, 52°C for 20 seconds, and then 72°C for 45 seconds. A melting curve analysis was performed after qPCRs. The threshold cycle (Ct) of TL and SCR was analyzed using the QuantStudio™ 12K Flex Software.

2.4. Analysis of ethanol-induced Shelterin complex subunit gene expression changes by reverse-transcription and qPCR (RT-qPCR)

PCR primers for six Shelterin complex subunit genes and the beta-actin gene (ACTB; a house-keeping gene) were designed using the NCBI Primer BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast/). cDNA was synthesized from 200 ng of total RNAs using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Waltham, MA, USA). The reverse-transcription (RT) reaction was performed in a TC-4000 thermocycler (Techne, Burlington, NJ, USA) at 37°C for 1 hour and then at 95°C for 5 minutes for inactivation of the reverse transcriptase. Shelterin complex subunit genes and ACTB were amplified using the Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) under the following PCR conditions: an initial denaturation stage at 98°C for 30 seconds, followed by 30 cycles of the following parameters: 98°C for 10 seconds, then 55–63°C for 30 seconds, and finally 72°C for 30 seconds. The final stage was the extension step (5 minutes at 72°C). Agarose gel electrophoresis was used to visualize the size of PCR products obtained from the amplification of the six Shelterin complex subunit genes and the house-keeping gene ACTB. Agarose gel electrophoresis analysis showed that the RT-PCR product size was 354 bp for TRF2, 269 bp for POT1, 253 bp for TIN2, 480 bp for TRF1, 299 bp for ACD (or TPP1), 578 bp for RAP1, and 266 bp for ACTB (Supplemental Fig. S2). The size of RT-PCR products of six Shelterin complex subunit genes and ACTB was the same as that calculated based on their cDNA sequences. Optimized primer sets and annealing temperatures were used in qPCRs to determine the expression levels of Shelterin complex subunit genes. Primer sequences, optimized annealing temperatures, and amplicon sizes are presented in Supplemental Table S1.

Expression levels of six Shelterin complex subunit genes and ACTB were analyzed using reagents from the QuantiTect SYBR Green PCR Kit (QIAGEN, Mannheim, Germany). The qPCR was performed in the QuantStudio™ 12K Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA USA). The cycling conditions were: 95°C for 15 minutes, followed by 40 three-step cycles at 94°C for 15 seconds, then at the optimized annealing temperature (Supplemental Table S1) for 30 seconds, and finally at 72°C for 30 seconds. A melting curve analysis was performed after qPCRs. The threshold cycle (Ct) of six Shelterin complex subunit genes and ACTB was analyzed using the QuantStudio™ 12K Flex Software.

2.5. Statistical analysis

The length of telomeres and the expression levels of six Shelterin complex subunit genes in the 36 hESC samples (4 ethanol concentrations × 3 exposure times × 3 experiments) were measured by qPCRs. The relative TL of a DNA sample was calculated as ΔCt(TL) = Ct(TL) − Ct(SCR). Similarly, the relative expression level of a Shelterin complex subunit gene was calculated as ΔCt(Shelterin gene) = Ct(Shelterin gene) − Ct(ACTB). Ct (threshold cycle) was the intersection between an amplification curve and a threshold line. A larger ΔCt meant a shorter length of telomeres or a lower expression level of Shelterin complex subunit genes. We performed analysis of variance (ANOVA) tests in R using the aov() function (Chambers et al., 1992). First, we checked whether the values of ΔCt(TL) and ΔCt(Shelterin gene) fit a normal distribution (bell curve) using histograms. Second, we performed two-way ANOVA F tests to model ΔCt(TL) or ΔCt(Shelterin gene) as a function of ethanol concentration and exposure time as well as the interaction of ethanol concentration with exposure time. Finally, we used the Tukey’s Honestly Significant Difference (HSD) post-hoc test to perform pairwise comparisons. All analyses were conducted with R3.6.3 statistical software (R.Core.Team, 2013).

3. Results

3.1. TL shortening was associated with ethanol exposure time

As shown in Supplemental Table S2, two-way ANOVA tests did not reveal a significant difference in average TL by ethanol concentration [(F(3) = 0.61, P = 0.613] or the interaction of ethanol concentration and exposure time [(F(6) = 0.27, P = 0.946]. However, a statistically-significant difference in average TL by exposure time was observed [(F(2) = 5.81, P = 0.009]. Tukey’s HSD post-hoc tests also did not show a significant difference in average TL between groups of hESCs treated with different concentrations of ethanol. Nevertheless, a 7- or 14-day treatment led to a significantly shorter TL compared to a 3-day treatment (7-day vs. 3-day: diff = 0.34, Padj = 0.003; 14-day vs. 3-day: diff = 0.23, Padj = 0.05). The impact of treatment duration (3-, 7-, or 14-day) on TL of hESCs exposed or unexposed to ethanol was shown in Figure 2.

Fig. 2.

Fig. 2.

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The effect of treatment duration on TL in hESCs exposed and unexposed to ethanol.

X-axis: hESCs treated with or without ethanol for 3, 7, or 14 days.

Y-axis: Relative telomere length (TL) calculated as ΔCt(TL) = Ct(TL) − Ct(SCR).

Without ethanol exposure: hESCs cultured in triplicate for 3, 7, or 14 days without ethanol exposure.

With ethanol exposure: hESCs cultured in triplicate for 3, 7, or 14 days with the exposure of ethanol at concentrations of 25, 50, or 100mM.

*: P-value < 0.05.

3.2. POT1, TPP1, TIN2, and TRF2 expression was influenced by ethanol exposure time

Two-way ANOVA tests did not reveal a significant effect of ethanol concentration or the interaction of ethanol concentration and exposure time on the expression of four associated Shelterin complex subunit genes (POT1, TPP1, TIN2, and TRF2) (Supplemental Tables S3–S6). However, a statistically-significant difference in the expression levels of these four genes by exposure time was observed [POT1: F(2) = 34.94, P = 7.8×10−8; TPP1: F(2) = 54.60, P = 1.2×10−9; TIN2: F(2) = 29.66, P = 3.3×10−7; and TRF2: F(2) = 21.73, P = 4.1×10−6]. Tukey’s HSD post-hoc tests also did not show a significant difference in the expression levels of these four genes between groups of hESCs treated with different concentrations of ethanol. However, there were statistically-significant differences in average expression levels of these four genes between groups of hESCs exposed to ethanol for different time periods. The expression levels of POT1, TPP1, and TIN2 were downregulated after a 7-day (versus a 3-day) treatment [diff(POT1) = 1.25, Padj(POT1) = 2.0×10−7; diff(TPP1) = 1.42, Padj(TPP1) = 0.000; and diff(TIN2) = 1.24, Padj(TIN2) = 2.9×10−6] but upregulated after a 14-day (versus a 7-day) treatment [diff(POT1) = −1.43, Padj(POT1) = 0.000; diff(TPP1) = −1.73, Padj(TPP1) = 0.000; and diff(TIN2) = −1.34, Padj(TIN2) = 7.0×10−7]. No significant difference in TRF2 expression was observed between the 3-day and 7-day treatment groups, but an extended (or 14-day) treatment led to a significantly higher expression level of TRF2 [14-day vs. 3-day: diff(TRF2) = −1.80, Padj(TRF2) = 5.6×10−6; 14-day vs. 7-day: diff(TRF2) = −1.85, Padj(TRF2) = 3.5×10−6]. The effect of ethanol exposure time (3-, 7-, or 14-day) on POT1, TPP1, TIN2 and TRF2 expression in hESCs was shown in Figure 3.

Fig. 3.

Fig. 3.

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The effect of ethanol exposure time on POT1, TPP1, TIN2, and TRF2 expression in hESCs.

X-axis: hESCs cultured in triplicate for 3, 7, or 14 day with the exposure to ethanol of 25, 50, or 100 mM.

Y-axis: Relative expression levels of POT1, TPP1, TIN2, and TRF2 estimated as ΔCt(Shelterin subunit gene) = Ct(Shelterin subunit gene) − Ct(ACTB).

***: P-value < 0.001.

3.3. TRF1 and RAP1 expression was influenced by both ethanol concentration and exposure time

Two-way ANOVA tests revealed statistically-significant differences in average expression levels of two other Shelterin complex subunit genes in hESCs by ethanol concentration [TRF1: F(3) = 10.28, P = 1.5×10−4; RAP1: F(3) = 4.08, P = 0.018] and exposure time [TRF1: F(2) = 14.68, P = 6.9×10−5; RAP: F(2) = 26.64, P = 8.0×10−7] as well as the interaction of ethanol concentration and exposure time [TRF1: F(6) = 4.17, P = 5.2×10−3; RAP1: F(6) = 3.49, P = 0.013] (Supplemental Tables S7 and S8). Tukey’s HSD post-hoc tests showed that the expression level of TRF1 was significantly higher in hESCs exposed to ethanol of 50 mM (50 mM vs. 0 mM: diff = −1.29, Padj = 0.001) or 100 mM (100 mM vs. 0 mM: diff = −0.87, Padj = 0.034). Similar to POT1, TPP1, and TIN2, the expression level of TRF1 was decreased after a 7-day (versus a 3-day) ethanol exposure (diff = 0.84, Padj = 8.4×10−3) but elevated after a 14-day (versus a 7-day) ethanol exposure (diff = −1.05, Padj = 1.1×10−3) (Supplemental Tables S7). The effect of ethanol exposure time (3-, 7-, or 14-day) and concentration (25, 50, or 100 mM) on TRF1 expression in hESCs was shown in Figure 4. Furthermore, Tukey’s HSD post-hoc tests showed that there was a trend of elevated RAP1 expression when hESCs were exposed to 100 mM of ethanol (diff = −0.89, Padj = 0.052). Consistent with TL variation, the expression level of RAP1 was decreased after a 7- or 14-day (versus 3-day) ethanol exposure (7-day vs. 3-day: diff = 1.69, Padj = 5.1×10−6; 14-day vs. 3-day: diff = 1.03, Padj = 0.003) (Supplemental Tables S8). The effect of ethanol exposure time (3-, 7-, or 14-day) and concentration (25, 50, or 100 mM) on RAP1 expression in hESCs was shown in Figure 5.

Fig. 4.

Fig. 4.

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The effect of ethanol exposure time and concentration on TRF1 expression in hESCs. X-axis: hESCs cultured in triplicate for 3, 7, or 14 day with the exposure to ethanol of 25, 50, or 100 mM.

Y-axis: Relative expression levels of TRF1 estimated as ΔCt(TRF1) = Ct(TRF1) − Ct(ACTB).

*: P-value < 0.05.

Fig. 5.

Fig. 5.

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The effect of ethanol exposure time and concentration on RAP1 expression in hESCs. X-axis: hESCs cultured in triplicate for 3, 7, or 14 day with the exposure to ethanol of 25, 50, or 100 mM.

Y-axis: Relative expression levels of RAP1 estimated as ΔCt(RAP1) = Ct(RAP1) − Ct(ACTB).

*: P-value < 0.05; ***: P-value < 0.001.

4. Discussion

The present study investigated TL variation and expression changes of six Shelterin complex subunit (or telomere-regulating) genes in hESCs that were exposed to ethanol of three different concentrations (equivalent to BELs in social, heavy, or intoxicated drinkers) for three different time periods (3-, 7-, and 14-days). Under the exposure of ethanol at these three physiologically-relevant concentrations, the variation of TL and the expression changes of six Shelterin complex subunit genes in hESCs were mainly dependent on the length of ethanol exposure, although we also observed the interactive effect of ethanol concentration and exposure time on the expression of two Shelterin complex subunit genes (TRP1 and RAP1).

First, the length of telomeres was mainly influenced by ethanol exposure time irrespective of the three physiologically-relevant ethanol concentrations. Ethanol exposure for a certain period of time (or 7 days) resulted in a shortened TL (Supplemental Table S2 and Fig. 2). This result is consistent with the findings from a previous study that TL was moderately shortened in human somatic cells exposed to 25 mM of ethanol for one week (Harpaz et al., 2018). As described in Supplemental Tables S3–S8 and Figures 3–5, the expression levels of Shelterin complex subunit genes (except TRF2) were also reduced after a 7-day (versus a 3-day) ethanol exposure. Hence, we postulated that telomere shortening might be resulted from downregulated expression of telomere-regulating genes. Of interest, the length of telomeres was not shortened further after an extended or 14-day (versus a 7-day) ethanol exposure. This is likely due to the upregulated expression of all six Shelterin complex subunit genes after a 14-day (versus a 7-day) ethanol exposure (Fig. 3–5). Our study provided further evidence that TL is likely maintained and regulated by telomere-regulating genes including these six Shelterin complex subunit genes.

Second, similar to ethanol-induced TL variation, the expression levels of four Shelterin complex subunit genes (POT1, TPP1, TIN2, and TRF2) were also mainly influenced by the duration of ethanol exposure regardless of the three physiologically-relevant ethanol concentrations (Supplemental Tables S3–S6 and Fig. 3). As shown in Figure 1, these four genes are functionally associated. The POT1 protein contains two oligonucleotide/oligosaccharide binding (OB) fold domains: OB1 (recognizing 5’-TTAGGG) and OB2 (bound to the downstream TTAG-3’) (Lei et al., 2004). POT1 helps forming the telomere-stabilizing D-loop, preventing the degradation of single-stranded DNA by nucleases, and sheltering the 3’ G-overhang of telomeres. The TPP1 (or ACD) protein is associated with POT1 and promotes the binding of POT1 to single-stranded telomeric DNAs. The TPP1 (or ACD)-POT1 heterodimer enhances telomere elongation by recruiting telomerase to telomeres and increasing its processivity (Abreu et al., 2010; Wang et al., 2007). The TIN2 protein is important for assembly of the Shelterin complex as it interacts with three DNA-binding proteins (TPP1, TRF1, and TRF2) of the Sheleterin complex (Kim et al., 1999; O’Connor et al., 2006). The TRF2 protein binds the telomeric double-stranded 5’-TTAGGG-3’ repeat and enhances the folding of telomeres into loops to prevent unwanted DNA repair and chromosome end-joining (Necasova et al., 2017). TRF2 also recruits Shelterin complex subunit proteins RAP1 and TIN2 for the protection of telomeres. The close interaction of these four Sheleterin complex subunit genes was reflected by the covariation of their expression under the influence of ethanol. A 7-day (versus a 3-day) ethanol exposure led to downregulated expression of POT1, TPP1, TIN2, while a 14-day (versus a 7-day) ethanol exposure resulted in increased expression of all four genes (POT1, TPP1, TIN2, and TRF2). These results implied that after a certain period (or a 7-day) of ethanol exposure, the expression of these genes was inhibited by ethanol exposure-induced stress. However, an extended (or a 14-day) ethanol exposure may lead to adaptation of hESCs to ethanol or ethanol-induced stress. As a result, the decreased expression levels of these Shelterin complex subunit genes were recovered, and thus the length of telomeres was not shortened further. These findings suggest that when ethanol-induced stress is present, the cellular machinery is reprogramed to produce more POT1, TPP1, TIN2, and TRF2 to counteract telomere degradation.

Third, different from TL and the above four Shelterin complex subunit genes (POT1, TPP1, TIN2, and TRF2), the expression of two other Shelterin complex subunit genes (TRF1 and RAP1) was influenced by both ethanol concentration and ethanol exposure time as well as the interaction of ethanol concentration and ethanol exposure time (Supplemental Tables S7 and S8 and Fig. 4 and 5). The function of TRF1 and RAP1 in TL regulation was different from the above four Shelterin complex subunit genes (POT1, TPP1, TIN2, and TRF2). The TRF1 protein binds to the telomeric double-stranded 5’-TTAGGG-3’ repeat and functions as an inhibitor of telomerase, thus negatively regulating the length of telomeres (Shen et al., 1997). A long-term overexpression of TRF1 resulted in telomere shortening, while the expression of less active TRF1 mutants led to telomere elongation (Okamoto et al., 2008; van Steensel and de Lange, 1997). The present study showed that ethanol exposure at 50 or 100 mM resulted in upregulated expression of TRF1 (Supplemental Table S7 and Fig. 4). This may partially explain the shortening of TL in ethanol-exposed hESCs. The RAP1 protein does not bind DNAs directly. It is recruited to telomeric 5’-TTAGGG-3’ sites via its interaction with TRF2 or other factors. RAP1 does not participate in the protection of telomeres against non-homologous end-joining (NHEJ)-mediated repair. Instead, it is required for negatively regulating telomere recombination and affects TL by repressing homology-directed repair (HDR) (Li et al., 2000). We found that RAP1 expression varied along with ethanol exposure time in the same way as ethanol-induced TL changes. Thus, these results further support that TRF1 and RAP1, like the above four Shelterin complex subunit genes, may play important roles in telomere regulation and maintenance.

Some limitations of this study should be noted. First, we did not examine the effect of ethanol metabolites (i.e., acetaldehyde) on TL and the expression of Shelterin complex subunit genes in hESCs. Although a significant association between heavy drinking and shortened TL in peripheral blood leukocytes (PBLs) has been reported (Pavanello et al., 2011), it is controversial whether ethanol or its metabolites accelerates TL shortening. As we know, it is acetaldehyde (rather than ethanol) that causes facial flushing and other physical symptoms, and acetaldehyde is implicated in various types of cancer such as upper aerodigestive tract cancer (Seitz and Stickel, 2007). Therefore, in our follow-up studies, we should further examine the effect of ethanol metabolites particularly acetaldehyde on TL and the expression of telomere-regulating genes. Second, we did not include other cell lines in this study, and thus we do not know if ethanol-induced TL variation and expression changes of Shelterin complex subunit genes in hESCs appeared in other cell lines as well. We should have included other cell lines particularly neuronal cell lines as well as tissues from addiction- or reward-related regions of postmortem brains of subjects with alcohol use disorder in this study. Third, we only assessed ethanol-induced expression changes of a limited number of telomere-regulating genes (i.e., 6 Shelterin complex subunit genes). It is necessary to use transcriptome profiling approaches (e.g., RNA-seq) to evaluate ethanol-induced expression changes of genes involved in the telomere interactome. Additionally, cell passaging may reduce the pluripotency of hESCs, even though we passaged hESCs using an enzyme-free reagent (ReleSR) for cell dissociation and removal of differentiated hESCs.

Taken together, our study demonstrated that ethanol-induced TL variation and expression changes of Shelterin complex subunit genes were mainly determined by ethanol exposure duration when hESCs were exposed to ethanol of physiologically-relevant concentrations. Shelterin complex subunit proteins that interact with each other exhibited a similar trend of ethanol-induced expression changes of their genes. The maintenance of TL in hESCs under ethanol-caused stress is likely due to the coordinated regulation of Shelterin complex subunit genes.

Supplementary Material

1

Acknowledgements

This study was supported by the Boston University Faculty Start-up Fund and the grant (R01AA025080) from the National Institute on Alcohol Abuse and Alcoholism (NIAAA).We are grateful to the WiCell Research Institute, Madison, USA for proving the H1 human embryonic stem cells (hESCs) for the study.

Footnotes

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Conflicts of Interest

The authors report no biomedical financial interests or potential conflicts of interest.

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