Molecular characterization of a human matrix attachment region that improves transgene expression in CHO cells

    Molecular characterization of a human matrix attachment region that improves transgene expression in CHO cells Qiu-li Sun, Chun-peng Zhao, Shao-nan Chen, Li Wang, Tian-yun Wang PII: DOI: Reference:

S0378-1119(16)30049-X doi: 10.1016/j.gene.2016.02.009 GENE 41166

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Please cite this article as: Sun, Qiu-li, Zhao, Chun-peng, Chen, Shao-nan, Wang, Li, Wang, Tian-yun, Molecular characterization of a human matrix attachment region that improves transgene expression in CHO cells, Gene (2016), doi: 10.1016/j.gene.2016.02.009

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ACCEPTED MANUSCRIPT Molecular characterization of a human matrix attachment region that improves transgene expression in CHO cells

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Running title: Matrix attachment region and CHO cells

Qiu-li Sun1, Chun-peng Zhao1, Shao-nan Chen1, Li Wang1 and Tian-yun Wang1,2*

Department of Biochemistry and Molecular Biology, Xinxiang Medical University,

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Henan 453003 China;

Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory

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Correspondence to:

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Medicine, Xinxiang, 453003, Henan, China

Tianyun Wang,

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Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Jinsui Road,

Xinxiang 453003, Henan P.R. China Tel: 86-373-3831899 E-mail: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT Chinese hamster ovary (CHO) cells offer many advantages for recombinant gene

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expression, including proper folding and post-translational modification of the

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recombinant protein. However, due to positional effects resulting from the neighboring chromatin, transgenes are often expressed at low levels in these cells. While previous studies demonstrated that matrix attachment regions (MARs) can be

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utilized to increase transgene expression by buffering transgene silencing, the

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mechanism by which this occurs is poorly understood. We therefore performed a deletion analysis of the human β-globin MAR sequence to characterize the regions

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that are necessary to enhance transgene expression in CHO cells. Our results indicate

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that of the six β-globin MAR fragments tested (MAR-1-6; nucleotides 1-540,

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420-1020, 900-1500, 1380-1980, 1860-2460, and 2340-2999, respectively), MAR-2, followed by MAR-3, was the most effective region for promoting stable and elevated

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transgene expression. Meanwhile, bioinformatic analyses demonstrated that these fragments encode a MAR-like motif and several transcription factor binding sites, including special AT-rich binding protein 1 (SATB1), CCAAT-enhancer-binding proteins (C/EBP), CCCTC-binding factor (CTCF), and Glutathione (GSH) binding motifs, indicating that these elements may contribute to the MAR-mediated enhancement of transgene expression. In addition, we found that truncated MAR derivatives yield more stable transgene expression levels than transgenes lacking the MAR. We concluded that the MAR-mediated transcriptional activation of transgenes requires a specific AT-rich sequence, as well as specific transcription factor-binding

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Keywords:

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Chinese hamster ovary; Gene expression; Matrix attachment region; Molecular characterization; Transgene silencing

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1. Introduction

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Since the emergence of the use of recombinant proteins, the production of proteins with therapeutic applications has become an important part of the biopharmaceutical

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industry. Indeed, the value of therapeutic recombinant proteins, including

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recombinant antibodies, has surpassed $1,00 billion per year worldwide. Notably,

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nearly 70% of these proteins are produced using Chinese hamster ovary (CHO) cells [1-3], making CHO cells the preferred mammalian cell line for the production of

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recombinant therapeutic proteins. Xu et al.investigated the genomic sequence of the CHO-K1 cell , and found that the assembly comprises 2.45Gb genomic sequence with 24,383 predicted genes. In additon homologs for most human glycosylation-associated genes and many important viral entry genes are present, but some not expressed in the genome of CHO-K1 cells [4]. Upon introduction and integration into the host cell genome, efficient transgene expression is highly dependent on the site of integration [5-7]. Specifically, integration into heterochromatin, a transcriptionally inactive region, often results in silencing of the transgenic sequence. As such, developing methods to buffer this silencing effect and

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ACCEPTED MANUSCRIPT to enhance transgene expression is of great interest from a biotechnology and gene therapy perspective.

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Matrix attachment regions (MARs), also referred to as scaffold attachment

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regions (SARs), are genomic DNA sequences that serve as attachment points to facilitate the anchoring of the chromatin structure to the nuclear matrix during interphase [8]. The most consistent features of MARs include the prevalence of

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AT-rich sequences, origins of replication, binding sites for DNA topoisomerase II,

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special AT-rich binding protein 1 (SATB1) motifs, kinked DNA, and curved DNA [9]. Recently, the use of MARs for the production of recombinant proteins has raised

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considerable interest. Several studies demonstrated that these sequences enable

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increased production of recombinant proteins of biotechnological interest in

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mammalian cell lines [8, 10-15]. Furthermore, multiple studies demonstrated that a human MAR, termed β-globin MAR, increases transgene expression and decreases

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the probability of transgene silencing in CHO cells [12, 14, 16]. Although the function of MARs appears to be evolutionarily conserved, defining the discrete elements or consensus sequences that may mediate MAR activity has been unsuccessful. In this study, we therefore attempted to identify functional elements of MARs, and to assess their effects on chromatin structure. Our results imply that binding sites for transcriptional activating proteins contribute to the MAR-mediated increase in transgene expression. These results provide a mechanistic basis for the epigenetic insulator and transcriptional augmentation activities of MARs.

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ACCEPTED MANUSCRIPT 2. Methods 2.1. Plasmids and constructs

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As shown in Figure 1A, the human β-globin MAR (No: L22754) sequence were

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cloned upstream of the Simian virus 40 (SV40) early promoter, which drives CTA reporter gene expression, within the pCATG vector [9, 11], thereby producing pCAMM(Figure 1B). Six sub-fragments [from 5´-3´; nucleotides 1-540, 420-1020,

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900-1500, 1380-1980, 1860-2460, and 2340-2999] were amplified from the flanking

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regions of the β-globin MAR sequence using the primers listed in Table 1. The fragments were each designed to overlap with adjacent sub-fragments by 120

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nucleotides. Bidirectional cloning was achieved by digesting the resulting PCR

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fragments with KpnI and BglII restriction enzymes, and subsequently inserting these

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sequences into linearized pCATG plasmid treated with the same restriction enzymes

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to generate the following six plasmids: pCAM-1, 2, 3, 4, 5, and 6, respectively.

2.2. CHO cell culture and transfection CHO cells (Institute of Laboratory Animal Sciences, Beijing, China) were cultivated in Dulbecco’s modified Eagle’s (DMEM) medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) at 37°C in a humidified incubator with 5% CO2. The cells were then seeded into six-well plates at approximately 3 × 106 cells/well. Prior to transfection, the plasmid DNA was linearized by treating with ScaI restriction enzyme, which specifically targets a unique site within the ampicillin resistance gene encoded by the plasmid.

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ACCEPTED MANUSCRIPT Cells were divided into the following eight treatment groups: pCAM-1–6, pCAMM, and pCATG, which were used as a negative control. Transfections were performed

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using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA), according to the

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manufacturer’s instructions. Forty-eight hours post-transfection, 800 μg/ml G418 (Calbiochem, Billerica, MA, USA) was added to the culture medium, and cells were incubated for 14 days until single colonies appeared. Subsequently, cell populations

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exhibiting stable transgene integration were cultured in the presence of 500 μg/ml

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G418 for 10 to 14 days, and then subjected to enzyme-linked immunosorbent assay (ELISA) analysis for detection of chloramphenicol acetyltransferase (CAT)

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2.3. CAT assays

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production.

CAT assay analysis was performed as in previous studies [12, 14, 15]. Briefly,

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G418-resistant transfected cells were collected, adjusted to a density of 1 × 106 cells/ml, and rinsed three times with precooled phosphate buffered saline (PBS). Cells were then treated with 1 ml of lysis buffer, mixed gently by shaking, incubated at room temperature for 30 min, and centrifuged at 12,000 rpm for 15 min at 4°C. The levels of CAT in the resulting supernatants were measured using a CAT ELISA Kit (Roche, Basel, Switzerland), according to the manufacturer’s instructions. Experiments were performed in triplicate.

2.4. Reverse transcription quantitative PCR (RT-qPCR)

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ACCEPTED MANUSCRIPT Total RNA was harvested from approximately 5 × 106 transfected CHO cells using an RNeasy Kit (Qiagen, Venlo, Netherlands), according to the manufacturer’s

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instructions. One milligram of total RNA from each sample was then reverse

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transcribed with oligo (dT) primers using the First Strand cDNA Synthesis Kit (GE Healthcare, Little Chalfont, United Kingdom), according to the manufacturer’s instructions, and RT-qPCR was performed using a Bio-Rad CFX Connect™ system

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(Bio-Rad, Hercules, CA, USA), a Power SYBR® Green PCR Master Mix (Cat:

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4367659; Applied Biosystems, Waltham, MA, USA), and the following primers: CAT, 5´-GGTGAGCTGGTGATATGGGA-3´ and 5´-AGGGATTGGCTGAGACGAAA-3´;

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β-actin (internal control), 5´-CCTCTATGCCAACACAGTGC-3´ and 5´-CCT

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GCTTGCTGATCCACATC-3´. The reaction conditions were as follows: 95°C for 3

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min followed by 40 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s, and a final extension at 60°C for 10 min. β-actin expression was used as the reference.

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Mean Ct values were calculated using the relative quantification method (ΔΔCt).

2.5. Bioinformatics analyses Allele-specific transcription factor binding sites were identified using MatInspector software (http://www.genom atix.de/products/index.html) [17]. CpG islands were analyzed with CPGPLOT [18]. Other motifs previously associated with MARs included unwinding motifs (AATATATT and AATATT), polyAs and polyTs, poly Gs and polyCs, topoisomerase II (GTNWAYATTNATTNATNNR), and a motif thought to relieve the superhelical stress of DNA [19-21]. Lastly, structural motifs,

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ACCEPTED MANUSCRIPT including nucleosome-binding and nucleosome-disfavoring sites (green and red boxes,

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respectively), were identified using GeneExpress software [22].

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3. Results 3.1. Characterization of MAR elements

We cloned the human β-globin MAR (No: L22754) and sequenced, the results

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showed that ten mutations occurred in the cloned sequence, including point mutation,

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deletion and addition (Supplementary Table S1). As described above, MAR elements are typically AT-rich and contain a MAR consensus sequence. They also often bear

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topoisomerase-II-binding sites [23]. As shown in Table 2, the MAR-2, MAR-3, and

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MAR-4 sub-fragments each contained a MAR-like motif (AATATATTT; nucleotides

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840-848, 1149-1157, and 1636-1644, respectively). Furthermore, both the MAR-3 and MAR-4 fragments encoded a topoisomerase-II-binding site (nucleotides

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1252-1266 and 1456-1470, respectively). Meanwhile, the 3´ end of the MAR-6 fragment contained both a beta-globin cluster and an Alu element (nucleotides 2687-2998 and 2804-2988, respectively). We next utilized bioinformatic approaches to identify putative transcription factor-binding motifs within the β-globin MAR coding sequence (Fig. 1C~D, Supplementary Table S2, S3). As shown in Table 3, we detected two SATB1 motifs and one CCCTC-binding factor (CTCF) motif within the (+) strand of the MAR-2 sub-fragment, and one SATB1 motif within the (+) strand of the MAR-3 sub-fragment. Meanwhile, the (−) strand of the MAR-2 and MAR-3 sub-fragments

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ACCEPTED MANUSCRIPT contained two CCAAT-enhancer-binding protein (C/EBP) and two GSH motifs, and

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one C/EBP and two GSH motifs, respectively (Table 3).

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3.2. Chloramphenicol acetyltransferase assays

After G418 selection, stably-transfected CHO cells were screened and RT-qPCR was utilized to assess CAT mRNA expression levels. The highest level of

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MAR-mediated CAT expression (5.20-fold greater than that of the control vector;

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P<0.05) was observed in the cells transfected with the construct encoding the full-length human β-globin MAR sequence (Fig. 2A). Of the deletion constructs,

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MAR-2 and MAR-3 (nucleotides 420-1020 and 900-1500, respectively) yielded the

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highest levels of transgene expression, increasing CAT mRNA levels to 3.15- and

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1.90-fold higher than that of the control construct, respectively. MAR-6 did not show the enhancing function on transgene expression. In contrast, the MAR-1, MAR-4, and

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MAR-5 (nucleotides 1-540, 1380-1980, and 1860-2460, respectively) constructs failed to enhance transgene expression. Similar results were obtained by ELISA. The highest levels of CAT protein production (2.37-fold increase compared to that of the control; P<0.05) were observed in the CHO cells containing the construct encoding the full-length β-globin sequence. Furthermore, while the MAR-2 and MAR-3 deletion constructs exhibited enhanced transgene expression compared to that of the control, the MAR-4, MAR-5 and MAR-6 sequences were not sufficient to promote transgene expression.

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ACCEPTED MANUSCRIPT 3.3. Identification of MAR elements necessary for transgene expression To determine which MAR motifs are responsible for enhancing transgene

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expression, we first analyzed the relationship between specific MAR sequences and

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the level of CAT expression. The MAR-2, -3, and -4 fragments each encoded a MAR-like motif. Meanwhile, MAR-2 and MAR-3 yielded the highest levels of transgene expression, indicating that the MAR-like motif may contribute to the

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observed MAR-mediated increase in CAT expression. Conversely, the topoisomerase

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II site, the β-globin cluster, and the Alu elements appeared to be dispensable for this effect, as the fragments that encoded these elements failed to promote CAT

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expression. Furthermore, while transcription factor-binding motif analyses indicated

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that the SATB1, C/EBP, CTCF, and GSH sites may contribute to the MAR-mediated

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effect on gene expression, they also indicated that sites such as the FAST-1 and GSH

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sites are not essential for this activity.

3.4. MAR fragments enable stable, elevated expression of CAT in CHO cells The stably-transfected CHO cells were cultured as cell suspensions in a fully synthetic medium in the absence of antibiotic selection, and CAT expression was estimated at regular intervals. We observed more stable CAT expression in the cells encoding the MAR constructs than in the control population during continuous culturing (>150 days; Fig. 3). Moreover, the MAR-2 and -3 fragments yielded CAT expression levels that were similar to that mediated the full-length MAR element, and which were significantly higher than those observed in the cell populations encoding

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ACCEPTED MANUSCRIPT the other MAR fragments. These findings demonstrate that the MAR-2- and MAR-3-mediated increases in transgene expression can be stably maintained during

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prolonged culturing.

4. Discussion

The inconsistent transgene expression levels observed within CHO cells is a

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major drawback to using this cell line for the production of recombinant proteins. As a

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result, labor-intensive selection and screening approaches are necessary to generate cell lines that stably express the exogenous gene at high levels. Recently, the use of

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MARs for enhancing transgene expression, particularly for the production of proteins

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of therapeutic value and for gene therapy, has been extensively investigated. Indeed

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multiple MAR sequences, such as the human β-globin MAR, have been shown to exert anti-silencing and transcriptional augmentation effects [12, 14, 16, 24]; however,

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not all MARs exhibit these characteristics. While bioinformatic analyses have identified putative genetic elements within the human MAR coding sequence, including an AT-rich core sequence and a flanking region enriched in transcription factor binding motifs [10, 23], attempts to identify consensus sequences within MAR sequences or to determine which genetic elements mediate the anti-silencing activities have failed [25]. MARs consist of AT-rich regions that are several hundred bases in length, and often contain short AT-rich boxes that exhibit strong potential for causing base-unpairing strain. As such, previous studies have proposed that the secondary

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ACCEPTED MANUSCRIPT structure of MARs is largely responsible for their functional activities [26, 27]. However, studies of the chicken lysozyme MAR found that binding of the MAR to

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the nuclear matrix was not sufficient to enhance transgene expression [10, 28]. In this

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study, we performed a deletion analysis of the β-globin MAR to identify the core elements that are necessary for enhancing gene expression in vitro. Our results indicate that this activity is dependent, at least in part, on a combination of two types

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of DNA elements: AT-rich sequences and transcription factor-binding sites such as

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SATB1, C/EBP, and CTCF sites.

AT-rich sequences, which are essentially comprised of uninterrupted rows of

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alternating adenine (A) and thymine (T) residues, were proposed to be essential for

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the MAR-mediated increase in transgene expression [29]. Notably, certain AT cores

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function as binding sites for transcription factors that specifically target AT-rich sequences [29, 30]. Arope et al. found that the core region of human X-68 MAR

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mediates the full anti-silencing activity of the MAR, whereas it is unable to provide a high transcriptional augmentation effect. However, the mere presence of the AT dinucleotide repeats from MARs was insufficient for mediating the anti-silencing effect [31]. In this study, among the six sub-fragments tested, we found that the fragment encoding nucleotides 420-1020 (MAR-2), followed by that encoding nucleotides 900-1500 (MAR-3), of the β-globin MAR sequence yielded the highest levels of CAT mRNA and protein expression. Transcription factor binding motifs, such as nuclear matrix protein 4 (NMP4), SATB1, FAST-1, and C/EBP binding sites, are thought to play an important role in

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ACCEPTED MANUSCRIPT promoting transgene expression [31]. Consistent with these findings, in this study, bioinformatic analyses detected a MAR-like motif, as well as SATB1, C/EBP, CTCF,

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and GSH transcription factor binding sites within the MAR-2 and -3 regions,

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indicating that these elements may contribute to the observed enhancement of CAT expression mediated by the MAR sequence. Specifically, these results suggest that the mechanism by which MARs enhance transgene expression may be, at least partially,

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dependent on the positioning of certain transcription factor binding sites within the

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MAR. In addition, we found that these truncated MAR derivatives provided more stable transgene expression than the construct lacking an MAR sequence. MAR-2 and

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-3 regions have some advantages and significance in theoretical research and practical

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value, the truncated DNA fragments are easily performed and highly transfection

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efficiency,and the mechanism of MAR’s regulation function may be elaborated through the subfragments of MAR.

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During the process of heterochromatic spreading, certain silencing proteins, such as histone deacetylases (HDAC) or DNA methyltransferases, are recruited. As such, the integration of transgenes into heterochromatic regions results in gene silencing or low levels of expression. Meanwhile, MAR sequences enable the recruitment of transcription factors, such as histone acetyltransferases (HATs) and other remodeling proteins, which restrain heterochromatic spreading and modify the state of the chromatin, thereby promoting transgene expression. Notably, the permissive chromatic structure of MARs might also contribute to homologous recombination [32], which could explain the increased transgenic copy numbers observed in

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ACCEPTED MANUSCRIPT recombinant host cells. Epigenetic regulation of gene expression influences all aspects of genetic transfer in

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eukaryotes [33]. MAR-binding proteins such as SAT B1 and CTCF are associated

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with the formation of DNA loops, which can result in the migration of epigenetic regulatory sequences to within close proximity of the enhancer and promoter sequences that drive the expression of their target genes,[34,35].Otherwise,the MAR

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elements may bring the nuclear matrix,consisting mainly of transcription factors and

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RNA processing enzymes, such as C/EBP, GSH in the proximity of transcriptional initiation sites at promoters[36]. Indeed, a previous study demonstrated that the MAR

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sequence can facilitate the migration of the nuclear matrix, which contains a variety of

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transcription factors and RNA processing enzymes, to within proximity of certain

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promoter sequences, thereby enabling the binding of RNA polymerases and subsequent transcriptional expression of the downstream gene(s) [31]. The use of

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miRNAs to engineer Chinese hamster ovary (CHO) cells is an emerging strategy to improve recombinant protein production. Jadhav et al. provided valuable evidence for miR-17 as a cell engineering target to enhance CHO cell productivity[37]. In conclusion, our results indicate that MAR-mediated transcriptional activation requires specific AT-rich sequences and transcription factor binding motifs. However, a more extensive and systematic study of these elements is needed to fully understand the nature and organization of the motifs that are essential for this activity.

Conflict of interest

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ACCEPTED MANUSCRIPT The authors confirm that this article content has no conflict of interest.

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Acknowledgement

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This work was partly supported by the grants from the National Natural Science Foundation of China (No. 31371332) and and the Grant from Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, China

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(No.XTCX-2015-ZD1).

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ACCEPTED MANUSCRIPT Figure legends Fig. 1 Schematic representation of β-globin MAR subfragment and illustration of

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its transcription factor binding motifs. A) Structures of deletion mutant constructs

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of the human β-globin MAR element cloned in the forward direction. B) Schematic diagram representing the full-length human β-globin MAR cloned upstream of a minimal SV40 promoter and CAT reporter gene. C) Schematic illustration of various

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transcription factors binding motifs within the (+) strand of the full-length human

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β-globin MAR. D) Schematic illustration of various transcription factor binding

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motifs within the (-) strand of the full-length human β-globin MAR.

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Fig. 2 Effects of the full length and the deletion constructs of the human human

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β-globin MAR element on CAT expression in G418-resistant transfectants. A) The CAT expression level was detected by fluorescence quantitative RT-qPCR in

AC

G418-resistant transfectants with either the pCATG control vector or a MAR-containing vector were measured; B) The CAT expression level was detected by ELISA in G418-resistant transfectants with either the pCATG control vector or a MAR-containing vector. Lane 1 indicates the control, 2~8 represent the the CAT expression vector containing full length MAR, and deleted human β-globin MAR (1-6) of the human β-globin MAR element.

Fig. 3 Analysis of the stably of high-producing CHO cell lines. MAR-2, MAR-3, full length MAR and the control were transfected into CHO cells and the CAT

21

ACCEPTED MANUSCRIPT amount was determined by ELISA at regular time as described in methods. Lane 1~3 indicates the the CAT expression vector containing full length MAR, and deleted

IP

T

human β-globin MAR (1-2) of the human β-globin MAR element, respectively and

AC

CE P

TE

D

MA

NU

SC R

lane 4 represents the control.

22

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

Figure 1

23

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

Figure 2a

24

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

Figure 2b

25

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

Figure 3

26

ACCEPTED MANUSCRIPT Table 1

The PCR primers used in this study

Position

Primer sequence(5′-3′)

MAR-1

1-540

P1:ATCGGTACCAAGCTTCTGACAAATTATTC TTCCT

T

Name

420-1020

IP

P2:TGA AGATCTCACTCTTTCATCAGTAATCACTCAG P1: ATC GGTACC GGTTCACTCT GCTATAGCAA TTTCA

MAR-2 900-1500

SC R

P2: TGA AGATCT ACATTGAATC AAAGGTAATG TTGCC P1: ATC GGTACC TAGTTAGTAA GACATCACCT TGCAT

MAR-3

P2: TGA AGATCT GTAAGAATCC TTTCAATGTG TGTGT P1: ATC GGTACC ACTCAAATAA ATACCTGCTT CATAG

NU

1380-1980 MAR-4

P2: TGA AGATCT TCATCCACTT ATTTATACAT TTAAA P1: ATC GGTACC AGTAGAATTA TACTCCCACT TTAGT

MA

1860-2460 MAR-5

P2: TGA AGATCT GTCCTTGTCT ATTTTCCCTG AAGTT

2340-2999

P1: ATC GGTACC CAAAGGAGAA AAGTTTGTTG GCCTC

D

MAR-6

AC

CE P

TE

P2: ATC GGTACC CAAAGGAGAA AAGTTTGTTG GCCTC-

27

ACCEPTED MANUSCRIPT Table 2 Molecular sequence characteristic of six sub-fragments of β-globin MAR Motif

MAR-1

MAR-2

MAR-3

MAR-4

MAR-5

MAR-6

Position

0

0

1

1

1

0

Topo II

0

0

1

1

0

beta-globin cluster

0

0

0

0

0

1

840~848, 1149~1157, 1636~1644 1252~1266 1456~1470 2687~2998

Alu element

0

0

0

0

0

1

2804~2998

AT content (%)

61.85

60.33

67.50

66.83

62.83

56.15

IP

SC R

NU MA D TE CE P AC

28

T

MAR- like motif

0

ACCEPTED MANUSCRIPT

Table 3. Locations of various transcription factor binding motifs within the six sub-fragments of β-globin MAR MAR-1

MAR-2

MAR-3

MAR-4

MAR-5

CEBP

1 0 0 0 2 3 0

1 2 2 1 0 3 1

1 1 1 2 1 0 0

1 0 0 0 0 0 1

1 0 0 0 1 0 0

0

0

0

1 1 0 0

1 2 0 0

1 2 0 0

0 0 0 1 1 0 0

+ + + +

IP

SC R

NU 29

Strand

0

0

0

-

1 0 0 0

0 0 0 0

0 1 0 1

+ + -

MA

FAST

D

GSH

TE

CTCF

CE P

NFAT

AC

SATB1

MAR-6

T

Matrix Family

ACCEPTED MANUSCRIPT

Abbreviations Chinese hamster ovary(CHO); matrix attachment regions(MARs);AT-rich binding proteins(C/EBP);CCCTC-binding

T

1(SATB1);CCAAT-enhancer-binding

IP

protein

factor(CTCF);Glutathione(GSH);scaffold attachment regions(SARs);special AT-rich protein

1(SATB1);

Fluorescence

AC

CE P

TE

D

MA

NU

(RT-qPCR); CCCTC-binding factor (CTCF).

quantitative

SC R

binding

30

reverse

transcription

ACCEPTED MANUSCRIPT Highlights

IP

T

Characterize the regions of MAR sequence that are necessary to enhance transgene

SC R

expression.

MAR-like motif contribute to the MAR-mediated enhancement of transgene

MA

NU

expression.

Truncated MAR derivatives yield more stable transgene expression levels than

TE

D

lacking the MAR.

AC

sequence.

CE P

MAR-mediated transcriptional activation of transgenes requires a specific AT-rich

31