Abstract
The organization of DNA into chromatin is dynamic; nucleosomes are frequently displaced to facilitate the ability of regulatory proteins to access specific DNA elements. To gain insight into nucleosome dynamics, and to follow how dynamics change during differentiation, we used a technique called time-ChIP to quantitatively assess histone H3.3 turnover genome-wide during differentiation of mouse ESCs. We found that, without prior assumptions, high turnover could be used to identify regions involved in gene regulation. High turnover was seen at enhancers, as observed previously, with particularly high turnover at super-enhancers. In contrast, regions associated with the repressive Polycomb-Group showed low turnover in ESCs. Turnover correlated with DNA accessibility. Upon differentiation, numerous changes in H3.3 turnover rates were observed, the majority of which occurred at enhancers. Thus, time-ChIP measurement of histone turnover shows that active enhancers are unusually dynamic in ESCs and changes in highly dynamic nucleosomes predominate at enhancers during differentiation.
Highlights
The organization of the genome into chromatin acts as a mechanism for regulating access to the information encoded in DNA
We generated embryonic stem cells (ESCs) lines expressing SNAP-tagged H3.3 in single copy from a defined locus. To achieve this we used recombination-mediated cassette exchange to insert H3.3 C-terminally tagged with SNAP and HA-tags into A2lox-Cre ESCs which allow for dox inducible transgene expression from the hprt locus (Iacovino et al, 2011) (Figure 1—figure supplement 1)
The clustered enhancers that make up super-enhancer domains were more rapidly turned over than conventional ESC enhancers
Summary
The organization of the genome into chromatin acts as a mechanism for regulating access to the information encoded in DNA. The simplest repeating unit of chromatin is the nucleosome consisting of a histone octamer, made up of two copies each of H2A, H2B, H3 and H4, around which approximately 147 bp of DNA is wrapped. Variants of the histone proteins exist that have distinct incorporation dynamics and functions. These include a number of histone H2A variants such as H2A.Z, H2A. Transcription and the binding of proteins to regulatory regions are known to be dependent upon increased accessibility to nucleosomal DNA. Assessment of nucleosome turnover can provide important information concerning which genomic regions are involved in regulation and the extent to which nucleosome dynamics contribute to their function
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