Protecting a transgene expression from the HAC-based vector by different chromatin insulators

Abstract

Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoid(tetO)-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoid(tetO)-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors.

Fig. 1

a Diagram of the alphoid^tetO-HAC with a unique gene loading loxP site [20]. The HAC contains <6,000 copies of the 42-bp tetracycline operator (tetO) sequence incorporated into every second alphoid DNA monomer of the 1.1 mega-base size alphoid DNA array [19, 21]. Because tetO sequences are bound with very high affinity and specificity by the tet repressor (tetR), the tetO sequences in the HAC can be targeted efficiently in vivo with tetR chromatin modifier fusion proteins. This can result in HAC elimination from cell populations by inactivation of its conditional kinetochore [19]. A gene-loading site is inserted into a transcriptionally permissive centrochromatin domain that is flanked by heterochromatin domains essential for centromere function. A repositioning of a heterochromatin domain(s) may potentially lead to silencing of genes loaded into the HAC. b Diagrams of the vectors used in this study. All vectors contain an EGFP color marker under a control of CAG promoter and a 3′ HPRT-loxP module. In three vectors (#2, #3, and #4), EGFP is flanked either by cHS4, gamma-satellite DNA or tDNA sequences, correspondingly. Vector #1 has no insulator sequence. The vectors were inserted into the loxP site of the alphoid^tetO-HAC propagated in HPRT-deficient hamster CHO cells

Fig. 2

Schematic diagram of the generation of isogenic hamster and human cell lines containing the alphoid^tetO-HACs carrying four different constructs. a Steps of MMCT to transfer a HAC into hamster CHO and then to human HeLa cells. The alphoid^tetO-HAC was engineered in human HT1080 cells [19]. The loxP gene loading site was inserted into the HAC by homologous recombination in chicken DT40 cells [20]. This modified HAC was transferred to hamster CHO cells. In those cells, four vectors containing the EGFP transgene with or without flanking insulator elements and a 3′ HPRT-loxP module were inserted into the loxP site of the HAC by Cre-loxP mediated recombination, producing isogenic hamster cell lines. From CHO cells, autonomously propagated HACs carrying four different vectors were transferred to HeLa cells, producing isogenic human cell lines. b Loading of the vectors into the loxP site of the HAC propagated in HPRT-deficient hamster CHO cells is accompanied by reconstitution of the HPRT gene allowing cell selection on HAT medium. Lanes 1, 2, 3, and 4 correspond to PCR products obtained with the genomic DNA isolated from HAC-containing clones (n4, c11, g2 and t2) of CHO cells that were chosen for MMCT transfer to human HeLa cells. c PCR control for cross contamination of human HeLa cells by hamster chromosomes. Lanes 5–10 correspond to randomly chosen clones of HeLa cells containing the HAC (G10, T5, C6); clones were checked by PCR using furin-F/furin-R hamster specific primers (lanes 5–7) or by PCR using dog-F1/dog-R1 human specific primers (lanes 8–10). Lanes 11, 12 correspond to the HAC-containing clone of HeLa cells (N5) checked by PCR using either hamster (lane 11) or human (lane 12) specific primers. FISH analysis of d the hamster clones (n4, c11, g2, t2) carrying the autonomously propagated HACs and e human clones (N5, C6, G10, T5) carrying the autonomously propagated HACs. FISH analysis in human cells was performed using PNA labeled probes for telomeric (red) and tetO-alphoid sequences (green). FISH analysis in hamster cells was performed using the BAC probe specific for the HAC-vector sequence (red) (see details in "Materials and methods"). Chromosomal DNA was counterstained with DAPI (blue). The HACs are indicated by arrows

Fig. 3

Long-term EGFP transgene expression in hamster CHO cells. a Relative mean EGFP fluorescence determined by FACS of cells carrying different HAC constructs grown under BS selection (clones n4, c11, g1, g2, t1, t2). Error bars represent standard deviation. EGFP-positive cells were sorted and analyzed by FACS over the course of 3 months, with measurements taken every 2 weeks. The measurement for each clone is the average of three experiments. b Flow cytometry histograms of levels of EGFP fluorescence in the population at the beginning of the experiment (0 weeks) and after 12 weeks of culture under BS selection. The x-axis represents the intensity of the fluorescence, the y-axis the number of cells. c Fluorescence images of CHO cells with the alphoid^tetO-HAC carrying the different EGFP constructs after 8 weeks (n4, c11, g2, t2) of culture. Cells were cultured in BS⁺ medium to select for retention of the HAC. d FISH analysis of representative CHO clones (n4, c11, g2, t2) carrying the autonomously propagated HACs after 12 weeks of culture under selection in BS⁺ medium. Chromosomal DNA was counterstained with DAPI (blue). The HACs are indicated by arrows

Fig. 4

Long-term EGFP transgene expression in human HeLa cells. a Relative mean EGFP fluorescence determined by FACS of cells carrying different HAC constructs grown under BS selection similar to that described in Fig. 3 for hamster CHO cells. Each data point represents the mean ± SD from three independent experiments. Three clones for the HAC construct without insulator (N1, N5, N7), two clones for the HAC construct with chicken insulator cHS4 (C4, C6), three clones for the HAC construct with gamma-satellite DNA (G1, G3, G10) and three clones for the HAC construct with tDNA (T3, T5, T10) were cultured during 12 weeks. Two clones for each HAC construct (C4, C6, G1, G10, T5, T10) were grown for 22 weeks. Cells were cultured with HAC selection (in BS⁺ medium). b Flow cytometry histograms of the levels of EGFP fluorescence in the population at the beginning of the experiment (0 weeks) and after 20 weeks of culture. The x-axis represents the intensity of the fluorescence, the y-axis the number of cells. c Fluorescence images of HeLa cells with the alphoid^tetO-HAC carrying the different EGFP transgenes after 8 weeks of culture (N5, C6, G10, T5 clones). d FISH analysis of representative HeLa clones (N5, C6, G10, T5) carrying the autonomously propagated HACs after 20 weeks of culture under selection in BS⁺ medium. Chromosomal DNA was counterstained with DAPI (blue). The HACs are indicated by arrows

Fig. 5

Temporal analysis of the geomentric mean EGFP intensity of clones of CHO (a) and HeLa (b) cells with the alphoid^tetO-HAC carrying different EGFP transgenes (brown no insulator; yellow with cHS4; green with gamma-satellite DNA; blue with tDNA; purple untransformed cells) during 12 weeks of culture with selection in BS⁺ medium. c EGFP expression undergoes a progressive decline over the course of 12 weeks if the gene is not flanked by insulator sequences in HeLa cells. The error bars represent the pooled standard deviation of all clones from each insulator type

Fig. 6

ChIP analysis of H3K4me3 chromatin in the transgene cassettes on the alphoid^tetO HAC propagated in HeLa cells. a A map of the transgenes within the HAC; HSV-TK, Hygro, HPRT genes plus an EGFP color marker flanked on both sides by different insulators. The bsr gene is repeated 30–40 times within the 1.1 Mb tetO-alphoid array of the alphoid^tetO-HAC [19, 21]. Arrows indicate the direction of transcription of the transgenes. b ChIP analysis was performed for clones C6, G1, T5, and N5. Enrichment is shown relative to the 5S rRNA control locus. Satellite 2 sequence, Sat2, from endogenous pericentromeric repeats was included as a negative control. c The n4 clone of CHO and N1, N5, and N7 clones of HeLa cells were treated either with TSA or SAHA. EGFP intensity is shown relative to the non-treated cultures. Fluorescence images of cells with and without drug treatment are shown. In the CHO control cell line (n4), a significant increase in GFP intensity was observed upon treatment with either TSA or SAHA [one-way ANOVA, F (2, 15) = 589.0; TSA, p < 0.0001; SAHA, p < 0.0001]. There was no significant difference in GFP intensity between TSA- and SAHA-treated cells (p = 0.4523). A similar pattern was observed in HeLa control cell lines N1, N5, and N7. GFP expression was significantly increased in HeLa cells upon drug treatment. [one-way ANOVA, F (8, 27) = 1287; N1-TSA, p < 0.0001; N1-SAHA, p < 0.0001; N5-TSA, p < 0.0001; N5-SAHA, p < 0.0001; N7-TSA, p < 0.0001; N7-SAHA, p < 0.0001]. No significant difference was detected between TSA- and SAHA-treated cells (N1, p = 0.4615; N5, p = 0.9597; N7, p = 0.0502)