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

Abstract

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...

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

“…This observation is consistent with previous studies where CTF/NF1 proteins act as barrier elements that block the propagation of silent chromatin structures, and thus protect transgenes from silencing effects mediated by repressive chromatin structures. 31,32,43 Hence, CTF/NF1 binding sites act both as enhancer-blockers and as barrier insulator elements. Maintenance of a euchromatic status of the provirus should favor the production of retroviral vectors.…”

“…16,32 Nevertheless, whether these binding sites may also function as barrier elements upon transgene integration at internal chromosomal loci has not been assessed. The CTF/NF1 adeno (5-bp spacing) or CTCF nat binding sites were sub-cloned on each side of an SV40 promoter/GFP gene cassette (Figure 4a), to address the potential barrier properties of these sequences at random chromosomal locations.…”

“…30 The CAAT box-binding transcription factor/nuclear factor-1 (NF1, also called CTF/NF1) consists of a family of widely expressed transcription factors that possess a barrier function. 31,32 This family comprises NF1-A, NF1-B, NF1-C and NF1-X subtypes that share the same DNA-binding domain. 33,34 Of note, the NF1-C regulatory domain prevents the propagation of repressive chromatin structures that stem from the telomere, and thereby prevent transgene silencing at mammalian and yeast cells telomeres.…”

“…33,34 Of note, the NF1-C regulatory domain prevents the propagation of repressive chromatin structures that stem from the telomere, and thereby prevent transgene silencing at mammalian and yeast cells telomeres. 31,32 NF1-C regulates DNA replication by promoting the recruitment of the DNA polymerase to the adenovirus and SV40 origins of replication, and is also involved in promoter regulation. [35][36][37] Thus, CTF/NF1 proteins were described as barrier elements that act to control DNA transcription and replication, yet a potential enhancer-blocking function had not been assessed.…”

L’effet de position télomérique

“…Une autre observation intéressante a été que le recrutement expérimental, entre les télomères et un gène rapporteur, de CTF1 (CCAAT-box-binding transcription factor 1), un facteur de transcription doté d'activité de remodelage de la chromatine, était capable de protéger le transgène de la répression transcriptionnelle induite par l'HC télomérique [17]. Des observations similaires avaient été faites au préalable chez la levure [18], suggérant à nouveau une conservation des mécanismes de régulation du TPE. La démonstration de l'existence d'un TPE sur des transgènes avait-elle la moindre signification physiologique ?…”