Steven Henikoff | Search bioRxiv

bioRxiv2015

ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo

Chromatin endogenous cleavage (ChEC) uses fusion of a protein of interest to micrococcal nuclease (MNase) to target calcium-dependent cleavage to specific genomic loci in vivo. Here we report the combination of ChEC with high-throughput sequencing (ChEC-seq) to map budding yeast transcription factor (TF) binding. Temporal analysis of ChEC-seq data reveals two classes of sites for TFs, one displaying rapid cleavage at sites with robust consensus motifs and the second showing slow cleavage at largely unique sites with low-scoring motifs. Sites with high-scoring motifs also display asymmetric cleavage, indicating that ChEC-seq provides information on the directionality of TF-DNA interactions. Strikingly, similar DNA shape patterns are observed regardless of motif strength, indicating that the kinetics of ChEC-seq discriminates DNA recognition through sequence and/or shape. We propose that time-resolved ChEC-seq detects both high-affinity interactions of TFs with consensus motifs and sites preferentially sampled by TFs during diffusion and sliding.

Genomics↗

bioRxiv2015

Methylation-sensitive expression of a DNA demethylase gene serves as an epigenetic rheostat

Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation of the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.\n\nAuthor SummaryOrganisms must adapt to dynamic and variable internal and external environments. Maintaining homeostasis in core biological processes is crucial to minimizing the deleterious consequences of environmental fluctuations. Genomes are also dynamic and variable, and must be robust against stresses, including the invasion of genomic parasites, such as transposable elements (TEs). In this work we present the discovery of an epigenetic rheostat in plants that maintains homeostasis in levels of DNA methylation. DNA methylation typically silences transcription of TEs. Because there is positive feedback between existing and de novo DNA methylation, it is critical that methylation is not allowed to spread and potentially silence transcription of genes. To maintain homeostasis, methylation promotes the production of a demethylase enzyme that removes methylation from gene-proximal regions. The demethylation of genes is therefore always maintained in concert with the levels of methylation suppressing TEs. In addition, this DNA demethylating enzyme also represses its own production in a negative feedback loop. Together, these feedback mechanisms shed new light into how the conflict between gene expression and genome defense is maintained in homeostasis. The presence of this rheostat in multiple species suggests it is an evolutionary conserved adaptation.

Genetics↗