Controlling Chromatin with small molecules

Protein binding is at the beginning of any DNA-templated reaction including readout of the genetic code. In mammals, genomic DNA forms a tight complex with proteins called chromatin. Chromatin naturally restricts binding of regulatory proteins and thus has to be dynamically altered to facilitate changes in gene expression in response to cellular cues. Chromatin-modifying enzymes catalyze chemical modifications, which have been implicated in promoting either condensation or accessibility of DNA. However, their exact mode of action is uncertain. Addition of chemical moieties to chromatin is also thought to contribute to the maintenance of gene expression states and mechanisms for the epigenetic inheritance of transcriptional information have been proposed.

HFSP Long-Term Fellow Oliver Bell and colleagues
authored on Fri, 22 June 2012

To address these questions, we have created a mouse model allowing synthetic manipulation of the chromatin landscape using small-molecule induced proximity. Addition of small, membrane-permeable molecules results in targeting of chromatin-modifying complexes to a genetically modified Oct4 gene and permits subsequent biochemical and gene expression analysis at high temporal resolution in the context of native chromatin. In embryonic stem (ES) cells, Oct4 expression is critical for pluripotency and self-renewal. Upon differentiation, Oct4 is silenced involving the HP1 heterochromatin pathway (with the signature modification of H3K9 trimethylation = H3K9me3). We investigated the kinetics of heterochromatin formation by recruiting HP1alpha to the modified Oct4 promoter in ES cells and fibroblasts. Tethering of HP1alpha induced gene repression and formation of heterochromatic domains of up to 10kb. Measuring H3K9me3 changes after HP1alpha recruitment allowed us for the first time to describe the in vivo rates of heterochromatin spreading in two distinct cell types. In addition, after HP1alpha removal, we tested the epigenetic properties of H3K9me3 to find that heterochromatic modifications provide epigenetic memory through cell generations. Yet, we also show that memory and spreading of H3K9me3 can be antagonized by transcriptional activators indicating the high plasticity of chromatin regulation.

Figure: Design of Chromatinin vivo Assay at Oct4( CiA:Oct4) ES cell line and mouse.  The CIA: Oct4 mouse contains a modified Oct4 allele harboring two arrays of DNA binding sites (12XZFHD1 and 5XGa14) in the promoter region upstream of an in-frame EGFP reporter.  The CiA system provides temporal control to recruit any chromatin modifying activity and analyze reaction kinetics in the context of physiological chromatin at single cell resolution.

Based on the balance between antagonizing H3K9me3 activities, we proposed a mathematical model, which accurately captured our empirical observation at the Oct4 locus and moreover predicts the dynamics of heterochromatin formation and turnover at the majority of facultative H3K9me3 domains in the mammalian genome. The system presents a powerful approach for the chromatin community to study the kinetic regulation of any chromatin modifying activity in any murine cell type and derive testable quantitative models.

Reference

Dynamics and Memory of Heterochromatin in Living Cells, Hathaway NA, Bell O, Hodges C, Miller EL, Neel DS, and Crabtree GR, Cell (2012), doi:10.1016/

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