An activation‐independent role of transcription factors in insulator function

Fourel, Geneviève; Boscheron, Cécile; Revardel, Émanuelle; Lebrun, Eléonore; Hu, Yan; Simmen, Katia Carmine; Müller, Karin H.; Li, Rong; Mermod, Nicolas; Gilson, Éric

doi:10.1093/embo-reports/kve024

Cited by 77 publications

(101 citation statements)

References 22 publications

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“…This effect is consistent with the inhibition of the propagation of heterochromatin along the chromosomal domain by the boundary formed by an interposed MAR element, thereby preventing the silencing of the transgene. Such a mechanism has also been proposed for mammalian DNA binding proteins acting as boundary elements in lower eukaryotic cells (Fourel et al, 2001). Alternatively, the MAR may actively reconfigure chromatin around its chromosomal integration site and thereby prevent transgene silencing, for instance by mediating histone modifications or changes in subnuclear localization.…”

Section: Discussionmentioning

confidence: 93%

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Girod

Zahn-Zabal

Mermod

2005

Biotechnol. Bioeng.

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104

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Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/ MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.

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Section: Discussionmentioning

confidence: 93%

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Girod

Zahn-Zabal

Mermod

2005

Biotechnol. Bioeng.

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104

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“…Interestingly, one of the strongest boundary activities detected in this organism consisted of the transcriptional activation domain of CTF-1, a member of the CTF/NF-I family of mammalian transcription and replication factors previously shown to interact directly with histone H3 (35,36). Furthermore, CTF-1 was shown to remodel reconstituted chromatin in vitro (36) and native chromatin at yeast origins of replication in vivo (37,38).…”

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confidence: 88%

“…1A and Ref. 35). To investigate the possibility that CTF-1 histone interaction might be involved in its boundary activity, we first evaluated whether the histone-binding portion of CTF-1 might bear the boundary activity.…”

Section: Chromatin Boundary Activity Of the Ctf-1 Transcriptionalmentioning

confidence: 99%

“…In yeast, transcriptional activation domains of various species were shown to efficiently protect reporter genes against telomeric silencing (35). These were shown to prevent the silencing of telomere-proximal genes, when interposed via a heterologous GAL4 DNA-binding domain between the gene and the VII-L telomere, and to generate an apparent discontinuity in silent chromatin propagation (35).…”

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confidence: 99%

“…These were shown to prevent the silencing of telomere-proximal genes, when interposed via a heterologous GAL4 DNA-binding domain between the gene and the VII-L telomere, and to generate an apparent discontinuity in silent chromatin propagation (35). Transcriptional activity per se appears not to be involved in these effects.…”

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confidence: 99%

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Chromatin Domain Boundaries Delimited by a Histone-binding Protein in Yeast

Ferrari

Simmen

Dusserre

et al. 2004

Journal of Biological Chemistry

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When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres toward more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one such transcription factor, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to the CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino acid substitutions that similarly inhibited the boundary and histone binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains.The expression state of a eukaryotic gene depends in part on its location in the chromosome. This position effect results from the organization of eukaryotic genomes into discrete functional domains, defined by local differences in chromatin structure. The expression of genes within each domain appears to be defined and maintained by the concerted action of regulatory elements such as promoters, enhancers, silencers, and locus control regions. Individual domains may be bordered by boundary elements that separate regions of permissive and silent chromatin. Experimentally, boundary elements have been defined functionally by their ability to protect against position effect when flanking the assayed gene. Examples of boundary elements in metazoans include the subtelomeric anti-silencing regions at yeast telomeres, the scs and scsЈ elements flanking the 87A1 hsp70 locus in Drosophila, and the chicken ␤-globin boundary elements (1-4). Several proteins have been associated with barrier activities in yeast, Drosophila, and mammalian cells (5).Transcriptional repression of the yeast Saccharomyces cerevisiae subtelomeric regions is one of the best-studied examples of position-dependent gene expression. This phenomenon is referred to as telomere position effect and is similar to the position effect variegation (PEV) initially described in Drosophila (6). Transcriptional silencing at yeast telomeres is associated with a heterochromatin-like structure (7,8). DNA wrapped in transcriptionally silent chromatin replicates late in S phase and is refractory to some modifying agents. In addition, nucleosomes have reduced acetylation compared with nucleosomes from active region of the genome (9, 10).Transcriptional silencing at subtelomeric regions is mediated by a multiprotein nucleosome binding complex called the SIR complex, composed of the Sir2, Sir3, and...

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Braking the silence: How heterochromatic gene repression is stopped in its tracks

Donze

Kamakaka

2002

BioEssays

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Eukaryotic DNA is assembled into nucleosomes, which are further packaged into higher order chromatin structures containing many non-histone chromosomal proteins. The details of this packaging have profound effects on gene expression and other cellular processes involving the genetic material. Heterochromatic domains of the genome are usually transcriptionally repressed, while euchromatic regions are transcriptionally competent. Current models of gene activation postulate the existence of boundary elements that either prevent inappropriate activation of genes by distal enhancers (enhancer blockers), or sequences that block the propagation of heterochromatin into euchromatic regions (barriers). While numerous boundary sequences have been identified, little is known with regard to the molecular mechanisms used to punctuate the genome. This review will focus on recent data that provide insight into the mode of action of barrier elements.

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An activation‐independent role of transcription factors in insulator function

Cited by 77 publications

References 22 publications

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Chromatin Domain Boundaries Delimited by a Histone-binding Protein in Yeast

Braking the silence: How heterochromatic gene repression is stopped in its tracks

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