Mechanism of regulation of 'chromosome kissing' induced by Fob1 and its physiological significance.

“Chromosome kissing” refers to protein-mediated and physiologically relevant interactions between two sites located on either different chromosomes or the same chromosome. Intrachromosomal kissing interactions are also called chromosome looping (Schleif 1992). Chromosome kissing facilitates three-dimensional mechanisms of control of different DNA transactions. For example, Reb1-mediated termination of DNA replication in fission yeast (Singh et al. 2010) and forkhead protein-dependent control of the timing of replication initiation (Knott et al. 2012) are modulated by chromosome kissing. Differentiation of naive T helper cells into the Th1 and Th2 forms (Spilianakis et al. 2005) and X-chromosome inactivation (Xu et al. 2006) are some of the other examples of biological activities controlled by kissing interactions. There are several examples of biological regulation by chromosome looping in prokaryotes (Hochschild and Ptashne 1988; Mukherjee et al. 1988a,b; Lobell and Schleif 1990; Miron et al. 1992; Schleif 1992) and eukaryotes (Tolhuis et al. 2002). The principal objective of this study was to unravel a mechanism of regulation of “chromosome kissing” and investigate the DNA transactions regulated by it. Recent methods designed to detect interchromosomal and intrachromosomal interactions have made it possible to map interacting sites in eukaryotic nuclei on a genome-wide scale (Dekker et al. 2002; Splinter et al. 2004; Simonis et al. 2007; de Laat et al. 2008). However, the proteins responsible for promoting most of these interactions and their physiological significance are known in only very few cases. There is also little or no information available on the mechanisms of regulation of kissing interactions. In mammalian cells, a complex of cohesin and CTCF (CCCTC-binding factor) promotes formation of topological domains of potentially interacting chromosomal loops (Zuin et al. 2014). Cohesin has also been implicated in chromosomal loop stabilization by interaction with a transcription mediator presumably at the stem of a DNA stem–loop structure (Kagey et al. 2010). Within the topological domains, specific loop-to-loop interaction by chromosome kissing probably involves sequence-specific DNA-binding proteins that form protein bridges between the interacting sites by oligomerization (Singh et al. 2010; this study). Recombination (e.g., translocation) at a distance promoted by long-range protein–DNA interactions is potentially significant because, among other things, it can give rise to fused oncogenes or place a quiescent oncogene under the control of a strong promoter, resulting in its pathogenic overexpression. Chromosome translocations are caused not just by random DNA breakage and stochastic rejoining of the broken ends. Rather, breaks located in close proximity to each other in the nucleoplasm tend to undergo preferential rejoining (Roix et al. 2003; Roukos and Misteli 2014). The Fob1 protein binds to two tandem terminator (Ter) sites called Ter1 and Ter2 (also called replication fork barriers [RFBs]) (Brewer 1988; Brewer et al. 1992), located in the nontranscribed spacer 1 (NTS1) between the end of the DNA encoding the 35S precursor rRNA and that of the 5S RNA-encoding DNA in each of the rDNA repeats. Fob1–Ter interaction causes polar replication fork arrest (Kobayashi et al. 2004; Mohanty and Bastia 2004; for reviews of replication termination, see Bastia and Mohanty 1996, 2006; Dalgaard et al. 2011; Bastia and Zaman 2014). The fork arrest at Ter sites in rDNA prevents head on collision between the leftward moving fork and the rightward moving 35S transcript (Brewer 1988; Takeuchi et al. 2003). Transcription replication interference can cause genetic instability (Liu and Alberts 1995; Mirkin and Mirkin 2007; Aguilera and Garcia-Muse 2012; Bermejo et al. 2012; Lin and Pasero 2012) that significantly increases the “genetic load,” which can presage induction of cancer. In the absence Sir2 (a NAD-dependent histone deacetylase), Fob1-mediated fork arrest is known to promote recombination between rDNA repeats (Kobayashi et al. 2004; Kobayashi and Ganley 2005). The presence of Sir2 in the nucleolar milieu suppresses recombination and transcription by RNA polymerase II (Pol II) but not by RNA Pol I or Pol III. Intrachromatid recombination generates rDNA circles, and their excision reduces the number of rDNA repeats. Although it is known that intrachromatid recombination in mother cells causes a decrease in their replicative life span (RLS) (Sinclair and Guarente 1997; Defossez et al. 1999), little is known about the molecular mechanisms that regulate the process. Sir2 is loaded onto rDNA as a part of the complex called RENT1 that consists of Net1, Cdc14, Tof2, Lrs4, and Csm1 proteins (Straight et al. 1999; Moazed 2001; Huang and Moazed 2003; Huang et al. 2006). There are two foci at which RENT1 gets loaded onto rDNA. Loading at the Ter sites requires Fob1–Net1 interaction, whereas that near the promoter region of 35S RNA requires RENT1–RNA Pol I interaction (Huang et al. 2006). We reported previously that silencing by RENT1 and fork arrest are two independent functions of Fob1 (Bairwa et al. 2010). Sir2 is known to suppress intrachromatid but not interchromatid recombination in the rDNA array (Kobayashi et al. 2004; Kobayashi and Ganley 2005). Using Saccharomyces cerevisiae as a model system, the principal focus of this study was to determine whether and how chromosome kissing is regulated and whether it promoted recombination between two geographically separated sites in rDNA. Incidentally, we also studied the effect of the control mechanism on RLS that was used as an additional biological readout. Here, we show that oligomerization of Fob1 promotes chromosome kissing at Ter sites of the NTS1 of rDNA. The kissing interaction triggers intrachromatid recombination that is known to control the RLS. We show further that the C-terminal domain of Fob1 (C-Fob1) is inhibitory and interacts with its N-terminal domain (N-Fob1) to antagonize not only Fob1 oligomerization but also its interaction with other proteins. Consistent with the proposition that phosphorylation of C-Fob1 is the trigger that breaks its intramolecular interaction with N-Fob1, we discovered that certain Thr/Ser residues of C-Fob1, when replaced by Ala, caused constitutive C-Fob1–N-Fob1 interaction, whereas phosphomimetic Asp substitutions at the same locations counteracted this effect, thereby illuminating the mechanism of control of chromosome kissing, recombination, and RLS.