Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II and Pol III

Abstract

Background:The relationship between transcription and the 3D genome organization is one of the most important questions in molecular biology, but the roles of transcription in 3D chromatin remain controversial. Multiple groups showed that transcription affects global Cohesin binding and genome 3D structures. At the same time, several studies have indicated that transcription inhibition does not affect global chromatin interactions.Results: Here, we provide the most comprehensive evidence to date to demonstrate that transcription plays a marginal role in organizing the 3D genome in mammalian cells: 1) degraded Pol I, Pol II and Pol III proteins in mESCs, and showed their loss results in little or no changes of global 3D chromatin structures for the first time; 2) selected RNA polymerases high abundance binding sitesassociated interactions and found they still persist after the degradation; 3) generated higher resolution chromatin interaction maps and revealed that transcription inhibition mildly alters small loop domains; 4) identified Pol II bound but CTCF and Cohesin unbound loops and disclosed that they are largely resistant to transcription inhibition. Interestingly, Pol II depletion for a longer time significantly affects the chromatin accessibility and Cohesin occupancy, suggesting RNA polymerases are capable of affecting the 3D genome indirectly.So, the direct and indirect effects of transcription inhibition explain the previous confusing effects on the 3D genome. Conclusions:We conclude that Pol I, Pol II, and Pol III loss only mildly alter chromatin interactions in mammalian cells, suggesting the 3D chromatin structures are pre-established and relatively stable.

CTCF and transcription influence chromatin structure re-configuration after mitosis

“…Inhibition of transcription compromises boundary strength in Drosophila melanogaster embryos 20, 21 . Yet, neither genetic nor chemical inhibition of transcription elicited a significant impact on higher-order structures of mammalian genomes 22, 23 . A recent gain-of-function study demonstrated that ectopic insertions of TSSs can lead to the formation of new domain-like structures spanning the lengths of the de novo transcripts 24 .…”

Decoding the organization, dynamics, and function of the 4D genome

“…However, CRISPR-mediated recruitment of RNAPII was not sufficient to drive the formation of insulated boundaries, nor increase the insulation level at the target loci, suggesting that additional factors may be required to mediate local chromatin contact changes (Bonev et al, 2017). Consistent with these findings, prolonged depletion of RNAPI, RNAPII, or RNAPIII causes a modest reduction of local chromatin interactions, which is likely an indirect effect of reduced chromatin accessibility and cohesin binding (Jiang et al, 2020b). Mediator, a large complex that is involved in RNAPII activation, has been proposed to act as a bridge that tethers E-P contacts (Malik and Roeder, 2016).…”

Newly synthesized RNA Sequencing Characterizes Transcription Dynamics in Three Pluripotent States

“…RNA polymerases ChIP-seq datasets ((Jiang et al , 2020), GSE145791) were aligned to mm10 genome by bowtie2 with “--local” setting. Each TU’s Pol I, Pol II and Pol III density were divided by the sum to obtain relative occupancies, and a threshold of 90% enrichment was used for the classification indicated in the ternary plot (Fig EV1H).…”

“…G. RNA Pol I, II and III occupancy (Jiang et al , 2020) on the total intergenic TUs. Student’s t-test was performed with the common TUs against the method-specific TUs (****p < 2e-16).…”