Ji Xiong's team in the School of Life Sciences identified the RNA polymerase II transcription factory control factor RUVBL2

RNA polymerase II (Pol II), as the transcription core machine in eukaryotic cells, is mainly responsible for transcribing mRNA encoding proteins and some non-coding RNAs
.
Pol II is not evenly distributed in cells, but rather forms a large number of clustered clusters in the nucleus, often referred to as transcription factories or transcriptional agglutinations
.
The transcription plant was discovered by chance in 1993 by Peter Cook and Luitzen de Jong's group during experiments imaging RNA polymerases and nascent RNA markers1,2, and further confirmed by ChIA-PET sequencing analysis of RNA polymerase in 201233,2,2012 by Yijun Ruan's group by ChIA-PET sequencing analysis of RNA polymerase3
。 In 2013, Xavier Darzacq and Ibrahim I.
Cissé discovered through ultra-high-resolution microscopy that transcription factories exhibit dynamic changes and correlate with RNA production in living ^cells4
.
Previous studies have found that transcription factories can mediate the co-expression of genes at different chromatin locus, enhance gene transcription efficiency, organize three-dimensional chromatin structure, and help with chromosomal translocation
in cancer.
One of the important problems with transcription factory-related research is that its key control factors
are not clear.
On the other hand, the mechanism of gene expression regulation usually focuses on the process of initiation, extension and termination of transcription, and it is still unknown
whether gene expression manipulation can be achieved by controlling transcription factories.

On September 28, 2022, Nature Communications published a research paper entitled "The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors" from Ji Xiong's research group at the School of Life Sciences, Peking University
。 Through multiple mass spectrometry screening and multidisciplinary techniques, the researchers confirmed that the AAA+ family ATPase molecule RUVBL2 can directly control the Pol II transcription plant
near the active promoter.
RUVBL2 is highly expressed in a variety of cancers, is associated with cancer development, and is one of
the popular targets of cancer inhibitors.
The study further identified direct regulatory genes of the RUVBL2-Pol II pathway, including c-Myc.

Related work provides novel factors for understanding the Pol II transcription factory, and also provides new ideas
for anti-tumor target applications.

Screenshot of the paper

Identification
of Pol II transcriptional agglutate regulators.
In order to identify transcriptional Pol II condensate regulatory proteins, the researchers first analyzed the chromatin proteome data of transcription machine-related factors, combined with histone modification interaction proteome data, and found that the AAA+ family ATPase molecule RUVBL2 can closely
interact with Pol II on chromatin.
RUVBL2 was found to be mainly involved in the assembly of biomacromolecular complexes in previous studies, and can play a function similar to that of chaperones, so it can be used as a potential regulatory protein
for Pol II condensations.
The researchers further captured the chromatin binding site information of RUVBL2 and found that the protein was mainly colocalized with Pol II on the active promoter
of the gene.
Pulldown experiments on specific Pol II-CTD degradation of cell lines and in vitro purified proteins demonstrated direct interaction between RUVBL2 and non-phosphorylated Pol II-CTD
.
After the rapid degradation of RUVBL2 protein in cells, the number of Pol II condensates in the nucleus decreased significantly, and the overall transcription level of the cells also decreased significantly after nascent RNA sequencing, indicating that RUVBL2 was closely related to
Pol II condensates and gene transcriptional activity.

Mechanism
by which RUVBL2 molecules regulate Pol II aggregates.
In order to confirm the regulation of transcriptional initiation agglomerates by RUVBL2, Pol II and transcription initiation factor TBP were observed by live-cell time-series photoactivation localization microscopy (tcPALM) and stimulated emission depletion (STED) microscopy before and after the degradation of RUVBL2 protein, and found that the degradation of RUVBL2 protein could significantly reduce the transient and stabilize Pol II condensation, and the cohesion ability of transcription initiation factor TBP was also significantly reduced
。 On the other hand, the recruitment of RUVBL2 protein to the Lac O promoter region can enhance the formation of Pol II condensate in the promoter region when the gene is activated.
Conversely, the recruitment of Pol II-CTD also leads to the aggregation of RUVBL2, indicating that RUVBL2 can promote the formation
of Pol II condensation in cells 。 In order to further explore its mechanism, the researchers found that the oligomerization ability and ATPase activity of RUVBL2 and its congenitent protein RUVBL1 had key contributions to the formation of Pol II condensates through intracellular targeted recruitment, in vitro droplet experiments, and functional region truncation analysis.
In vitro droplet and DNA curtain experiments confirmed that RUVBL1/2 heterohexamer in the physiological concentration range could significantly promote the phase separation and formation of Pol II-CTD and transcription factors, and in vitro transcription experiments showed that RUVBL1/2 could significantly enhance the transcription initiation of classical TATA promoter, indicating that RUVBL2 could directly promote the dynamic interaction between Pol II-CTD and transcription factors and enhance transcription initiation activity
.

Functions of
the RUVBL2-Pol II axis.
RUVBL2 is highly expressed in a variety of tumor cells and participates in many complexes, but the direct target gene and gene regulatory network of RUVBL2 are not clear
.
In order to explore the function of RUVBL2 in promoting the formation of Pol II agglomerates, the researchers applied indoleacetic acid-mediated rapid degradation of RUVBL2 protein system, and analyzed
the gene transcription and post-transcriptional effects caused by RUVBL2 degradation 。 By integrating chromatin co-immunoprecipitation sequencing (ChIP-Seq), time-series transcriptome sequencing (RNA-Seq), and neonatal transcriptome sequencing (PRO-Seq) data, 45 direct target genes that responded rapidly to RUVBL2 degradation at the transcriptional level and 41 immediate response genes
that responded rapidly to RUVBL2 degradation at the post-transcriptional level were identified 。 The researchers further demonstrated the universality of the 45 target genes regulated by RUVBL2 in different cell lines by identifying the chromatin binding sites of RUVBL2 in various tumor cell lines, and mapped the transcriptional regulatory network
with RUVBL2-Pol II as the axis 。 The comprehensive analysis of existing chromatin binding data by machine learning found that RUVBL2 co-binds to a variety of transcription factors in the promoter region of active genes more extensively, which may co-regulate gene expression, which further suggests that RUVBL2 exerts the function
of transcriptional regulation by promoting the formation of transcriptional agglomerates between Pol II and various transcription factors.
To this end, the researchers also confirmed the model
by RNA in situ hybridization combined with immunofluorescence experiments (smRNA FISH + IF) at the RUVBL2 direct target gene locus.

Ruvbl1/2 gene expression regulation model
.
As a chaperone, RUVBL1/2 is involved in many complexes, including the assembly of RNA polymerases, chromatin regulation and post-transcriptional RNA processing-related complexes, etc.
, and previous studies have shown that RUVBL1/2 mainly interacts with specific transcription factors in the nucleus to exert the function of transcriptional co-activation or inhibition (gray arrow).

。 The main findings of this study demonstrate that RUVBL1/2 promotes the formation of Pol II transcriptional agglomerates near active promoters by directly interacting with Pol II-CTD, thereby exerting the function of enhancing transcriptional activity (blue arrow), and multiple transcription factors are involved in this biological process

In conclusion, the researchers discovered the direct regulatory function of RUVBL1/2 on Pol II transcriptional aggregates, identified the RUVBL2-Pol II axis rapid response gene, and promoted a deeper understanding of the regulatory mechanism between Pol II, transcription factor interaction and transcription phase separation.
At the same time, RUVBL2 is abnormally expressed in many tumor cells, and a variety of inhibitors have good anti-tumor effects, which will provide new ideas
for the mechanism of RUVBL2 regulating gene expression in tumors and its application as a target.

Ji Xiong is the corresponding author
of this paper.
Dr.
Wang Hui and Dr.
Li Boyuan, School of Life Sciences, Peking University, are the co-first authors
of this paper.
Prof.
Deng Wulan of the Biomedical Frontier Innovation Center of Peking University, Qi Zhi of the Center for Quantitative Biology, Gao Juntao of Tsinghua University, Professor Xu Yanhui of Fudan University, Yu Yang and Liu Chaopei of the Institute of Biophysics, Chinese Academy of Sciences, etc.
provided important help
for this work.
This work was supported
by the National Key R&D Program of the Ministry of Science and Technology, the National Natural Science Foundation of China, the Peking University-Tsinghua Joint Center for Life Sciences, and the Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education.
Peking University Phoenix Project has provided strong support
for this project.

Ji Xiong's research group has long been engaged in the research of the functional regulation and disease mechanism of RNA polymerase subunits, mainly focusing on RNA polymerase-related novel functions, brain diseases and nucleic acid aptamer screening, and recent results have been published in journals such as Molecular Cell, Nature Communications, Genome Biology, Cell Discovery, CMLS and iScience.
To provide a new hypothesis
for selective gene expression regulation.

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