Abstract
Although chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements. RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin dynamics. Perturbation experiments of P-TEFb clusters, which are associated with active RNAPII, had similar results. Furthermore, chromatin mobility also increased in resting G0 cells and UV-irradiated cells, which are transcriptionally less active. Our results demonstrated that chromatin is globally stabilized by loose connections through active RNAPII, which is compatible with models of classical transcription factories or liquid droplet formation of transcription-related factors. Together with our computational modeling, we propose the existence of loose chromatin domain networks for various intra-/interchromosomal contacts via active RNAPII clusters/droplets.
Highlights
Genomic DNA, which encodes genetic information, is spatially and temporally organized in the cell as chromatin (Bickmore, 2013; Cardoso et al, 2012; Dekker and Heard, 2015; Hubner et al, 2013)
I used oblique illumination microscopy, which allowed me to illuminate a thin area within a single nucleus with reduced background noise
Using the CRISPR/Cas9-based genome editing, we introduced a cassette encoding mAID and fluorescent protein, mClover at the initiation site of the endogenous RNA polymerase II (RNAPII) gene locus (POLR2A) (Figure 32) in human colon adenocarcinoma DLD-1 cells expressing the OsTIR1, which is involved in the induced degradation process (Figure 31) (Natsume et al, 2016; Yesbolatova et al, 2019)
Summary
Genomic DNA, which encodes genetic information, is spatially and temporally organized in the cell as chromatin (Bickmore, 2013; Cardoso et al, 2012; Dekker and Heard, 2015; Hubner et al, 2013). Chromosome conformation capture (3C) and related methods including Hi-C (Lieberman-Aiden et al, 2009) have enabled the production of a fine contact probability map of genomic DNA and supported the formation of numerous chromatin domains, designated as topologically associating domains (TADs) (Dekker and Heard, 2015; Dixon et al, 2012; Nagano et al, 2017; Nora et al, 2012; Sexton et al, 2012; Smallwood and Ren, 2013; Szabo et al, 2018), and more recently contact domains/loop domains (Eagen et al, 2015; Rao et al, 2014; Rao et al, 2017; Vian et al, 2018), which are considered functional units of the genome with different epigenetic features. These contact probability maps have suggested various intra-chromosomal and inter-chromosomal domain contacts for global control of gene transcription (Dekker and Heard, 2015; Dixon et al, 2012; Eagen et al, 2015; Nagano et al, 2017; Nora et al, 2012; Rao et al, 2014; Sexton et al, 2012; Smallwood and Ren, 2013), the underlying mechanism remains unclear
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