Long-Range Communication between the Silencers of HMR

Abstract

Gene regulation involves long-range communication between silencers, enhancers, and promoters. In Saccharomyces cerevisiae, silencers flank transcriptionally repressed genes to mediate regional silencing. Silencers recruit the Sir proteins, which then spread along chromatin to encompass the entire silenced domain. In this report we have employed a boundary trap assay, an enhancer activity assay, chromatin immunoprecipitations, and chromosome conformation capture analyses to demonstrate that the two HMR silencer elements are in close proximity and functionally communicate with one another in vivo. We further show that silencing is necessary for these long-range interactions, and we present models for Sir-mediated silencing based upon these results.Gene activation and gene repression are central to the proper development and differentiation of organisms. DNA elements such as promoters, enhancers, and silencers play a central role in eukaryotic gene regulation. These elements are separated from each other by several kilobase pairs of DNA but are able to communicate with one another to regulate the activation or repression of genes. The exact mechanism by which distally located elements communicate with one another is not clear and is one of the key questions in gene regulation. Long-range communication between distantly located elements in chromosomes is thought to occur by one of two principal mechanisms (8). One class of models postulate that a signal emanating from a distal regulatory element spreads along the DNA fiber until it encounters a proximal regulatory element. A second class of models postulate that distal and proximal regulatory elements interact with one another directly, with the intervening DNA forming a loop. Both mechanisms must function within the context of the global chromosome structure, which appears to be composed of large chromosome loops that attach to a proteinaceous superstructure (11). The nucleus appears to be divided further into distinct chromatin compartments, with heterochromatic domains being present in regions near the nuclear periphery while euchromatic domains are found mainly in the interior of the nucleus, although a significant portion of euchromatin is located near nuclear pores.It has been suggested that the functionally and structurally defined chromatin domains may be coincident (36). Enhancers and locus control regions (LCRs) are long-range regulatory elements that activate promoters in a distance-and orientation-independent manner, and recent studies indicate that enhancers and LCRs often cluster together in three-dimensional space to form an "active chromatin hub" (23,49,58,59).Similarly, in yeast the promoters and terminators of genes are in close proximity to one another (3, 48) and tethered to the nuclear pore (10, 52). The consequence of this spatial organization is that the DNA between these regulatory elements is looped out. It is thought that the formation of these nuclear substructures aids in transcription activation.Silencers are negative regulatory element...

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…Later studies suggested that the HM loci also fold back upon themselves, consistent with earlier findings that the DNA supercoiling of the silenced domains was altered (Bi and Broach 1997;Cheng et al 1998;Valenzuela et al 2008). Molecular genetic studies showed that distant silencers synergize one another, as if they interact physically Fourel et al 1999;Pryde and Louis 1999;Cheng and Gartenberg 2000;Oki et al 2004;Valenzuela et al 2008). Even silent chromatin domains separated by great distances, like the HM loci at the ends of chromosome III, have been shown to interact ( Figure 2C); in essence by folding back upon one another (Lebrun et al 2003;Miele et al 2009;Kirkland and Kamakaka 2013).…”

“…However, the notion of a silent chromatin-mediated fold back was supported by the use of novel, transcriptional-reporter constructs that detected long-range interactions within silenced subtelomeric regions (de Bruin et al 2001). Later studies suggested that the HM loci also fold back upon themselves, consistent with earlier findings that the DNA supercoiling of the silenced domains was altered (Bi and Broach 1997;Cheng et al 1998;Valenzuela et al 2008). Molecular genetic studies showed that distant silencers synergize one another, as if they interact physically Fourel et al 1999;Pryde and Louis 1999;Cheng and Gartenberg 2000;Oki et al 2004;Valenzuela et al 2008).…”

Rpd3-dependent boundary formation at telomeres by removal of Sir2 substrate

“…4C), because it causes ADE2 derepression while maintaining URA3 repression. This effect of tethered Rpd3 is the same as that of Sas2 at the two loci (32) and shows that Rpd3 is not only a "desilencer" but can be classified as a "true barrier" factor. These results show that targeting of the HDAC Rpd3 disrupts heterochromatin spreading.…”

The Ku Complex in Silencing the Cryptic Mating-Type Loci ofSaccharomyces cerevisiae

“…The process repeats until Sir2-4 protein complexes bind and modify the nucleosomes that compose the silent loci (reviewed in Rusche et al 2003;Moazed et al 2004). The binding of the Sir2-4 protein complexes may directly repress transcription and/or lead to higher-order looping and compaction that renders the underlying DNA less accessible (Vazquez and Schedl 1994;Cheung et al 2000;Sekinger and Gross 2001;Valenzuela et al 2008).…”