DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

Abstract

Bacteriophage λ stably maintains its dormant prophage state but efficiently enters lytic development in response to DNA damage. The mediator of these processes is the λ repressor protein, CI, and its interactions with λ operator DNA. This λ switch is a model on the basis of which epigenetic switch regulation is understood. Using single molecule analysis, we directly examined the stability of the CI-operator structure in its natural, supercoiled state. We marked positions adjacent to the λ operators with peptide nucleic acids and monitored their movement by tethered particle tracking. Compared with relaxed DNA, the presence of supercoils greatly enhances juxtaposition probability. Also, the efficiency and cooperativity of the λ switch is significantly increased in the supercoiled system compared with a linear assay, increasing the Hill coefficient. The decision between the dormant state (the lysogenic state) and the vegetative state (the lytic state) made by bacteriophage λ upon infection of an Escherichia coli was the first epigenetic switch to be deciphered (1) and continues to provide insights into biological processes (2, 3). Once the lysogenic state is established, it is exceptionally stable and departure in the absence of the DNA damage sensing system is nearly always caused by mutation (4-6). Maintenance of lysogeny is mediated by the λ repressor protein, CI, and its formation of a complex with phage DNA. The CI protein binds at two regulatory regions called operator right (OR) and operator left (OL), located about 2.3 kbp apart on the phage genome. Each operator is a constellation of three adjacent subsites that each bind dimers of CI in a hierarchical manner (1, 7). In a lysogenic cell, CI prevents lytic growth by directly repressing the lytic promoters pR and pL. This repression is enhanced by longrange cooperative interactions between CI dimers bound to the OL1-OL2 and OR1-OR2 regions, thus looping the DNA that lies between them (8) (Fig. 1 A and B). CI-mediated DNA looping brings OR3 in proximity to OL3. This juxtaposition allows a CI dimer bound to the strong OL3 to facilitate another CI dimer to bind to the intrinsically weak OR3, thus reducing the concentration of CI necessary to occupy OR3. In this manner CI can autoregulate its own expression by binding to OR2 (activation) or by binding to OR3 (repression). This autoregulation is crucial for the phage to maintain repression while preventing excessive accumulation of CI. When DNA in the lysogen is damaged, the bacterial DNA-damage-sensing system is triggered, leading to CI inactivation by self-cleavage (9) and to an efficient switch to the lytic state, eventually leading to production of progeny phage and cell death (1).The switch from the lysogenic to the lytic state must be decisive. Partial entry into vegetative growth may kill the host without producing a full burst of progeny phage. Because the in vivo state of DNA is supercoiled (10), we sought to examine protein-mediated DNA looping on native, supercoiled DNA. In vitro single molecule exper...

DNA supercoiling during transcription

“…Using magnetic tweezers, Ding et al showed that (−) DNA supercoiling can encourage the formation of λ repressor (CI)-mediated loops in DNA (Ding et al 2014), which in turn prevent over-expression of this repressor protein so that the cell can still preserve sensitivity to conditions that trigger virulence (lysis). Norregaard et al (Norregaard et al 2013) developed a unique peptide nucleic acid (PNA)-based assay with a tethered particle tracking method (Norregaard et al 2014) and showed that the presence of DNA supercoils can greatly enhance the juxtaposition probability of λ repressor (CI) and, hence, significantly increase the efficiency and cooperativity of a λ epigenetic switch.…”

Supercoiling Effects on Short-Range DNA Looping in E. coli

“…Under these conditions, extensive DNA bending may be particularly facilitated by energy stored in superhelical strain. Comparing facilitation of small and large DNA loops by negative supercoiling will be interesting [60, 61]. Perhaps future Monte Carlo simulations, coupled with experiments, will allow this supercoiling-looping relationship to be understood in vitro and in vivo.…”

Motif for controllable toggle switch in gene regulatory networks

“…Bistability is discussed with respect to all-or-none cellular processes such as differentiation, proliferation, survival and apoptosis [1][2][3][4][5][6]. However, much evidence about reversible switches has been observed in the regulatory network in biological experiments [7][8][9][10][11].…”