Mol Cell+iScience team revealed RNA polymerase II subunit heterogeneity and CTCF insulation mechanism

On September 15, 2022, Ji Xiong's research group at the Center for Life Sciences and the School of Life Sciences of Peking University published a research paper
entitled "Targeted protein degradation reveals RNA Pol II heterogeneity and functional diversity" online in Molecular Cell 。 This study systematically explains the direct regulatory role and potential mechanism of RNA polymerase II subunits in eukaryotic cells on transcription and post-transcription processes, and for the first time shows that RNA polymerase II is an optimized enzyme (OPPO), and its subunits play different and even specific roles
in the transcription and post-transcription process.

RNA polymerase II, which is primarily responsible for the transcription of protein-coding genes, is a protein complex consisting of 12 subunits
.

The researchers first established a transiently degraded cell line of 12 subunits of RNA polymerase II in mouse embryonic stem cells, and the chromatin genome and proteome were quantified by chromatin interaction genomic sites and proteins
of each subunit.

Next, the researchers used the neonatal RNA sequencing experiment (PRO-Seq) to explore the effect
of transient degradation (1h) of each subunit on the production of neonatal RNA.

To further confirm the above conjecture, the researchers conducted PolyA-RNA-Seq experiments after long-term degradation (12h) to explore the effects of each subunit on the mature mRNAs of
different genes 。 The researchers found that the degradation of RPB1, RPB2, RPB5, RPB6, RPB7, and RPB8 could lead to a decline in most gene transcription, indicating that these subunits were involved in RNA polymerase intact enzyme stability, which echoed the results of the significant reduction of intranuclear enzymatic enzymes after degradation of these subunits in immunofluorescence imaging experiments, but the degradation of RPB4 and RPB12 did not show changes in multiple independent experiments, and the researchers believe that they may have the effect of protein splicing variants compensating for subunit degradation
。 Interestingly, the degradation of RPB3, RPB9, RPB10 and RPB11 only causes a decrease in transcription of some genes, and immunofluorescence imaging experiments have shown that the degradation time is extended, and some RNA polymerase II complexes are still retained in the nucleus, suggesting that these subunits play a regulatory role
in whole enzymes.

Figure 1.

PolyA-RNA-Seq experimental results also show that some subunits can lead to different degrees of expression upregulation of some genes, and the researchers analyzed that gene upregulation may be caused by the imbalance of the post-transcriptional process, such as transcript 3' end processing defects, splicing processing defects, etc
。 Using the Dogfinder software developed by predecessors, the researchers identified the gene class of transcriptional reading before and after subunit degradation, and then combined with the calculation of the processing coefficient of the end of the gene 3', it was found that RPB1, RPB3, RPB5, and RPB8 could significantly increase transcriptional clearance after degradation, which is consistent
with the results of Robert Roeder's laboratory published in Molecular Cell in 2020 to find that RPB1-CTD degradation caused gene clearance 。 At the same time, the researchers identified the genes with transcriptional splicing abnormalities through rMATs, and also found that the degradation of different subunits can cause splicing abnormalities
of different genes.

Figure 2.

In the RNA polymerase subunit work, Ji Xiong, a researcher at the School of Life Sciences of Peking University, is the corresponding author of the paper, and Dr.

On September 16, 2022, the Center for Life and Ji Xiong's research group of the School of Life Sciences of Peking University, together with the Center for Life and the Qi Zhi Group of the Center for Quantitative Biology, published a research paper entitled "CTCF DNA binding domain undergoes dynamic and selective protein–protein interactions" in iScience.

CTCF (CCCTC-binding factor) is the most important insulator-binding protein currently known in vertebrates, which can block the activation of the promoter by the enhancer, inhibit the expression of genes, and can also act as a "barrier" to prevent the spread
of heterochromatin.

The researchers used the "optoDroplet" system of light-induced protein aggregation to screen the DNA-binding domain of CTCF to aggregate; In vitro, it was verified that the DBD of CTCF could aggregate to form dynamic droplets; Next, through protein colocalization analysis combined with live cell tracking imaging, the researchers found that the aggregation of CTCF DBD can recruit insulator-related proteins CHD8 and BRD2, and drain the transcriptional activator OCT4.

Figure 3.

In the work of CTCF insulation mechanism, Ji Xiong, a researcher at the Center for Life Sciences and the School of Life Sciences of Peking University, and Qi Zhi, a researcher at the Center for Life Sciences and the Center for Quantitative Biology, are the co-corresponding authors
of this paper.

Ji Xiong's research group has long been engaged in the study
of RNA polymerase subunit function regulation and disease mechanism.

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