The structure of promoter chromatin determines the ability of transcription factors (TFs) to bind to DNA and therefore has a profound effect on the expression levels of genes. However, the role of spontaneous nucleosome movements in this process is not fully understood. Here, we developed a single-molecule optical tweezers assay capable of simultaneously characterizing the base pair-scale diffusion of a nucleosome on DNA and the binding of a TF, using the luteinizing hormone β subunit gene (Lhb) promoter and Egr-1 as a model system. Our results demonstrate that nucleosomes undergo confined diffusion, and that the incorporation of the histone variant H2A.Z serves to partially relieve this confinement, inducing a different type of nucleosome repositioning. The increase in diffusion leads to exposure of a TF's binding site and facilitates its association with the DNA, which, in turn, biases the subsequent movement of the nucleosome. Our findings suggest the use of mobile nucleosomes as a general transcriptional regulatory mechanism.nucleosomes | transcription factors | chromatin | optical tweezers P ackaging of the DNA into chromatin reduces its accessibility to regulatory proteins, such as transcription factors (TFs) and RNA polymerase (RNAP), making the structure and dynamics of nucleosomes an essential part of gene expression regulation in higher organisms (1, 2). Although atomic-resolution structures of the nucleosome reveal strong contacts between the histone core and the DNA (3), evidence has accumulated indicating that these interactions are often disrupted spontaneously, and that the crystal structure represents only a snapshot of a complex conformational dynamics. Atomic force microscopy (AFM) studies observed nucleosomal DNA looping (4), and intermediates composed of tetramers and hexamers (5), while conformational changes of the histone octamer inside the nucleosome were recently detected using cryo-electon microscopy (6). Moreover, nucleosomes have been shown to spontaneously unwrap DNA from its ends in a process termed "thermal breathing" (5,(7)(8)(9)(10)(11)(12). The momentary exposure of the DNA was shown to facilitate the invasion of regulatory proteins to nucleosomal DNA (7, 8, 10) and the elongation by RNAP (4, 13), highlighting the potential effect of these dynamics on the process of transcription.Previous studies have indicated the existence of an additional type of thermally driven dynamics: the spontaneous "mobility" or "thermal sliding" of nucleosomes by which their center of mass repositions on the DNA in an unprompted longitudinal-like movement. Initial studies using 2D gel electrophoresis (14,15) reported that nucleosomes are able to reposition over timescales of hours when incubated at 37°C, but not at 4°C. Later experiments used chemically modified H4 histones, capable of inducing a cleavage at the nucleosome's dyad (16), and found that the repositioning rates depend on the positioning sequence and the length of the DNA fragment. Others showed that sin mutations, which weaken the binding ...

Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a non-template strand concomitantly with polymerization. Here, using high-resolution optical tweezers to follow the activity of the murine leukemia Virus RT, we show that strand-displacement polymerization is frequently interrupted. Abundant pauses are modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Our results emphasize the potential regulatory role of conserved structural motifs, and may provide useful information for the development of potent and specific inhibitors.

Most functional transcription factor (TF) binding sites deviate from their "consensus" recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used singlemolecule DNA unzipping with optical tweezers to study how Egr-1, a TF harbouring 3 zinc fingers (ZF1,ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 base pairs bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.

No abstract

Most functional transcription factor (TF) binding sites deviate from their ‘consensus’ recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harboring three zinc fingers (ZF1, ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 bp bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein–DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.