Rapid quantification of miRNAs using dynamic FRET-FISH.

MicroRNAs (miRNAs) are short regulatory RNAs that control gene expression at the post-transcriptional level. Various miRNAs playing important roles in cancer development are emerging as promising diagnostic biomarkers for early cancer detection. Accurate miRNA detection, however, remains challenging because they are small and highly homologous. Recently developed miRNA detection techniques based on single-molecule imaging enabled highly specific miRNA quantification without amplification, but the time required for these techniques to detect a single miRNA was larger than 10 minutes, making rapid profiling of numerous miRNAs impractical. Here we report a rapid miRNA detection technique, dynamic FRET-FISH, in which single-molecule imaging at high probe concentrations and thus high-speed miRNA detection is possible. Dynamic FRET-FISH can detect miRNAs in 10 s at 1.2 µM probe concentration while maintaining the high-specificity of single-nucleotide discrimination. We expect dynamic FRET-FISH will be utilized for early detection of cancers by profiling hundreds of cancer biomarkers in an hour.

A Novel N-terminal Region to Chromodomain in CHD7 is Required for the Efficient Remodeling Activity.

Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed biochemical and biophysical analysis on CHD7 chromatin remodeler and uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome and contains a high conserved arginine stretch, which is reminiscent of arginine anchor. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. Furthermore, smFRET analysis shows the mutations in the N-CRD causes the defects in remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.

Yeast Chd1p Unwraps the Exit Side DNA upon ATP Binding to Facilitate the Nucleosome Translocation Occurring upon ATP Hydrolysis.

Chromodomain-helicase-DNA-binding protein 1 (CHD1) remodels chromatin by translocating nucleosomes along DNA, but its mechanism remains poorly understood. We use single-molecule fluorescence experiments to clarify the mechanism by which yeast CHD1 (Chd1p) remodels nucleosomes. We find that binding of ATP to Chd1p induces transient unwrapping of the DNA on the exit side of the nucleosome, facilitating nucleosome translocation. ATP hydrolysis is required to induce nucleosome translocation. The unwrapped DNA after translocation is then rewrapped after the release of the hydrolyzed nucleotide and phosphate, revealing that each step of the ATP hydrolysis cycle is responsible for a distinct step of nucleosome remodeling. These results show that Chd1p remodels nucleosomes via a mechanism that is unique among the other ATP-dependent chromatin remodelers.

Structural basis of recognition and destabilization of the histone H2B ubiquitinated nucleosome by the DOT1L histone H3 Lys79 methyltransferase.

DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B ubiquitination. Here, we present cryo-EM structures of DOT1L complexes with unmodified or H2B ubiquitinated nucleosomes, showing that DOT1L recognizes H2B ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L, respectively. Furthermore, the structures combined with single-molecule FRET experiments show that H2B ubiquitination enhances a noncatalytic function of the DOT1L-destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.