Quantitative methods for profiling dynamic chromatin features

Abstract

Living systems, from entire organisms down to the single cells constituting them are dynamic entities that continuously adapt and respond to their local environment. Cells achieve this through gene expression programs derived from static information encoded in the DNA made dynamic through chemical modifications at the chromatin level, collectively termed the epigenome. Numerous epigenetic regulators have been implicated in early embryonic developmental transitions and pluripotency. Ex vivo, the different states of pluripotency can be recapitulated by embryonic stem cells (ESCs) grown in defined media conditions. Many developmental gene promoters in ESCs display co-occurrence of the activating histone H3 lysine 4 trimethylation (H3K4me3) mark and the repressive H3K27me3 mark. This distinctive ð€‚µbivalentð€‚¶ signature is considered to poise expression, allowing timely resolution to an active or inactive state depending on the signal.

The distribution of histone modifications and chromatin-associated factors across the genome can be mapped using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). However, traditional ChIP-seq methods fail to quantitatively profile the nuanced global and local epigenetic rewiring that takes place in key developmental stages. This thesis addresses this limitation through the development of a quantitative multiplexed ChIP-seq technology: MINUTE (multiplexed indexed unique molecule T7 amplification end to end sequencing) ChIP. Across the three papers included in this thesis, we reveal the underpinnings of chromatin state dynamics in early mouse and human embryonic development by employing MINUTE ChIP. In Paper I, we first show that MINUTE ChIP enables accurate quantitative comparisons over a wide linear range. By employing it to characterize mouse ESCs grown in 2i and serum conditions, we find that the 2i naïve state is characterized by high global levels of H3K27me3 and low H3K4me3. At bivalent promoters, we observe that while H3K27me3 levels are stably maintained between serum and 2i, H3K4me3 levels are higher in the serum condition. Through quantitative epigenome profiling, in Paper II we find that naïve human ESCs also have broad global gain of Polycomb repressive complex 2 (PRC2)-mediated H3K27me3 and define a previously unrecognized, naïve-specific set of bivalent promoters. Bulk and single-cell transcriptomics confirmed that naïve bivalency maintains key trophectoderm and mesoderm transcription factors in a transcriptionally poised state which is resolved to an active state upon depletion of H3K27me3. Therefore, we discovered that PRC2-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development. In paper III we show how quantitative RNA polymerase II occupancy profiles generated by MINUTE ChIP can be integrated with transient transcriptomics data to unravel genome wide transcriptional kinetics in three mESCs pluripotent states: naïve, ground and paused.

Taken together, this thesis provides compelling evidence for a broad H3K27me3 hypermethylation of the genome in both naïve mouse and human ESCs and the basis for substantially revising the model for bivalency during embryonic development.