Genome-wide detection and quantitation of RNA distribution by ChIRC13a-seq

Eukaryotic genome are transcribed extensively, but a majority of transcripts remain functionally uncharacterized. This is most ascribed to lacking of a potent RNA-centric technology that is capable of accurately quantitating putative genomic binding sites for endogenous RNAs. We describe here a detailed protocol for Chromatin Isolation by RNA-Cas13a Complex sequencing (ChIRC13a-seq), based on recently discovered CRISPR-Cas13a from Leptotrichia wadei (LwaCas13a), for profiling of RNA associated chromatin binding cites. ChIRC13a-seq employs biotinylated, enzymatically-dead Cas13a (dCas13a) that is still capable of binding target RNA and guide RNAs (gRNAs) specific for the RNA target of interest to enrich RNA and its chromatin binding sites. This assay can be performed in standard molecular biology laboratories with 2 d taken for ChIRC13a-seq library preparation.

Context-dependent DNA Polymerization Effects Can Masquerade as DNA Modification Signals

BackgroundSingle molecule measurements of DNA polymerization kinetics provide a sensitive means to detect both secondary structures in DNA and deviations from primary chemical structure as a result of modified bases. In one approach to such analysis, deviations can be inferred by monitoring the behavior of DNA polymerase using single-molecule, real-time sequencing with zero-mode waveguide. This approach measures the time between fluorescence pulse signals from consecutive nucleosides incorporated during DNA replication, called the interpulse duration (IPD). ResultsIn this paper we present an analysis of loci with high IPDs in two genomes, a bacterial genome (E. coli) and a eukaryotic genome (C. elegans). To distinguish the potential effects of DNA modification on DNA polymerization speed, we paired an analysis of native genomic DNA with whole-genome amplified (WGA) material in which DNA modifications were effectively removed. Modification sites for E. coli are known and we observed the expected IPD shifts at these sites in the native but not WGA samples. For C. elegans, such differences were not observed. Instead, we found a number of novel sequence contexts where IPDs were raised relative to the average IPDs for each of the four nucleotides, but for which the raised IPD was present in both native and WGA samples. ConclusionThe latter results argue strongly against DNA modification as the underlying driver for high IPD segments for C. elegans, and provide a framework for separating effects of DNA modification from context-dependent DNA polymerase kinetic patterns inherent in underlying DNA sequence for a complex eukaryotic genome.

Post-Transcriptional Regulation through Long Non-Coding RNAs (lncRNAs)

The discovery of thousands of non-coding RNAs (ncRNAs) pervasively transcribed from the eukaryotic genome has revolutionized the “central dogma” of biology and shifted the attention on the role of RNAs as regulatory molecules, more than simply traditional mediators of genomic information [...]

Reduce, Retain, Recycle: Mechanisms for Promoting Histone ProteinDegradation Versus Stability and Retention

The eukaryotic genome is packaged into chromatin. The nucleosome, the basic unit of chromatin, is composed of DNA coiled around a histone octamer. Histones are among the longest-lived protein species in mammalian cells, due to their thermodynamic stability and their associations with DNA and histone chaperones. Histone metabolism plays an integral role in homeostasis. While histones are largely stable, degradation of histone proteins is necessary under specific conditions. Here we review the physiological and cellular contexts which promote histone degradation. We describe specific known mechanisms that drive histone proteolysis. Finally, we discuss the importance of histone degradation and regulation of histone supply for organismal and cellular fitness.

Combinations of histone post-translational modifications

Histones are essential proteins that package the eukaryotic genome into its physiological state of nucleosomes, chromatin, and chromosomes. Post-translational modifications (PTMs) of histones are crucial to both the dynamic and persistent regulation of the genome. Histone PTMs store and convey complex signals about the state of the genome. This is often achieved by multiple variable PTM sites, occupied or unoccupied, on the same histone molecule or nucleosome functioning in concert. These mechanisms are supported by the structures of ‘readers’ that transduce the signal from the presence or absence of PTMs in specific cellular contexts. We provide background on PTMs and their complexes, review the known combinatorial function of PTMs, and assess the value and limitations of common approaches to measure combinatorial PTMs. This review serves as both a reference and a path forward to investigate combinatorial PTM functions, discover new synergies, and gather additional evidence supporting that combinations of histone PTMs are the central currency of chromatin-mediated regulation of the genome.

Remodeler Catalyzed Nucleosome Repositioning: Influence of Structure and Stability

The packaging of the eukaryotic genome into chromatin regulates the storage of genetic information, including the access of the cell’s DNA metabolism machinery. Indeed, since the processes of DNA replication, translation, and repair require access to the underlying DNA, several mechanisms, both active and passive, have evolved by which chromatin structure can be regulated and modified. One mechanism relies upon the function of chromatin remodeling enzymes which couple the free energy obtained from the binding and hydrolysis of ATP to the mechanical work of repositioning and rearranging nucleosomes. Here, we review recent work on the nucleosome mobilization activity of this essential family of molecular machines.