The human body has over 200 different cell types, and gene regulation is key to establishing and maintaining cell identity. During mitosis, chromatin condenses and long-range interactions between distal enhancers are lost [1]. As a result, scientists have long believed that transcription during mitosis is silenced, raising the question of how cells reactivate transcription to maintain cell identity. How cell identity is controlled is a fundamental biological question, and understanding this process could also provide insights for cell reprogramming and regenerative medicine.
Addressing how cells maintain identity during mitosis is not a trivial issue, given the technical challenges. Since the nuclear envelope breaks down during mitosis, one cannot isolate the nuclei to label transcripts. Previous approaches included using RNA polymerase II (RNAP2) cross-linking [2-3], but antibody-based methods are less sensitive than direct measurements of nascent transcription. Other studies have used the thymidine analog bromodeoxyuridine (BrdU) to label nascent RNAs, but labeling live cells with BrdU, which is not cell-permeable, can be challenging.
Dr. Kenneth Zaret’s lab investigates mechanisms of gene regulation involved in controlling cell identity. Dr. Kate Palozola, a recent alumna from this laboratory and a former Genetics and Epigenetics Program student, developed an assay, EU-RNA- seq, to capture nascent transcription during mitosis and mitotic exit [4]. The uridine analog 5-ethynyluridine (EU) was used to pulse-label nascent transcripts in HUH7 human hepatoma cells during nocodazole-induced mitotic arrest, mitotic exit, and in asynchronous cells. Azide-biotin was used to isolate these transcripts from total RNA,and RNA-sequencing was then performed to identify genes that are actively transcribed during mitosis and mitotic exit.
Surprisingly, Kate found that transcription still occurs during mitosis. Sequences from EU-RNA- seq were mostly primary transcripts, spanning introns and exons but not intergenic regions, suggesting that this method can robustly detect the nascent transcriptome. Since the mitotic population contained a small fraction of non-mitotic cells, several controls were used, including spike-in controls, to confirm the authenticity of the signal from mitotic cells. Kate identified 8074 transcripts (3689 genes) that were consistently expressed during mitosis. RNA fluorescence in situ hybridization (RNA FISH) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to independently assess these genes. While the mean expression level of mitotically expressed genes was approximately five-fold lower in mitotic cells compared to asynchronous cells, there were several genes with increased expression, including KLF4 and ATF3, which encode transcription factors.
Kate then sought to determine whether there was a particular order in which gene expression is reactivated upon mitotic exit. Since previous work in the Zaret lab had shown that most of transcription initiates about 80 minutes after release from mitotic arrest [2], Kate used EU-RNA- seq to monitor transcription upon mitotic exit at different intervals after release from nocodazole-induced mitotic arrest. While some transcripts appeared as early as 40 minutes, she reaffirmed that the largest burst of transcription indeed occurs at 80 minutes [2,5]. Kate found that the earliest transcripts encoded proteins involved in basic cell structure and growth. In addition, using this sensitive technique,Kate was able to identify additional waves of gene reactivation. The next wave of gene reactivation included adhesion genes (HUH7 cells are epithelial cells), followed by transcripts involved in cell cycle and DNA replication, as cells prepared for S phase. Although there were some liver-specific genes identified throughout the mitotic exit, most of the 149 liver-specific genes, including APOC3 and ASGR2, which encodeapolipoprotein C3 and asialoglycoprotein receptor 2, respectively, were reactivated much later. Kate also found that enhancer RNAs (eRNAs) were downregulated during mitosis and appeared early during mitotic exit, around the same time as their putative target genes.
Overall, these results overturned the dogma that transcription is silent during mitosis. On the contrary, RNA polymerases are still active during mitosis and may help contribute to the inheritance of a cell’s transcription pattern. Based on these findings, Palozola et al. propose a model in which mitotic expression units (MEUs) maintain a low level of transcription during mitosis that could help maintain cell identity (Fig. 1) [4]. Future work may focus on how cells regulate the order in which genes are reactivated during mitotic exit, which could provide insights for reprogramming cells and tissues for regenerative therapies.
Figure 1: Mitotic expression unit model
A low level of transcription is maintained during mitosis. During mitotic exit, gene reactivation occurs in multiple waves, with cell type-specific genes appearing in later stages.
Kate is now a post-doctoral fellow in the laboratory of Jean Bennett, where she studies gene therapy for heritable forms of blindness. As a co-founder of the CAMB student newsletter, Kate is also passionate about science communication. Her favorite CAMB memories include recruitment, orientation, the annual holiday party, and the CAMB Student Newsletter.
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2. Prasanth, K.V., Sacco-Bubulya, P.A., Prasanth, S.G., and Spector, D.L. Sequential entry of components of gene expression machinery into daughter nuclei. Mol. Biol. Cell 14(3):1043-1057, Mar 2003.
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4.Palozola, K.C., Donahue, G., Liu, H., Grant, G.R., Becker, J.S., Cote, A., Yu, H., Raj,A., and Zaret, K.S. Mitotic transcription and waves of gene reactivation during mitotic exit. Science 358(6359): 119-122, Oct 2017.
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