As early as the 19th century, Walther Flemming and other biologists knew that a complex of genetic material and proteins—what we now refer to as chromatin—condenses during cell division. In fact, it was the condensed nature of mitotic chromosomes that enabled their visualization under a standard light microscope. Fast forward to the mid 20th century during the era of molecular genetics: early biochemical assays showed a severe reduction in RNA production during mitosis. More recently, antibody-based protein localization assays have indicated that most proteins involved in gene expression, such as RNA polymerase II and transcription factors (TFs), are largely absent from mitotic chromatin. Given this apparent cessation of RNA synthesis during division, the mystery that remains is how transcriptional machinery is properly recruited and reassembled in G1 so that the cell type-specific gene expression program is reestablished, and the cell’s functional identity is maintained. This concept of transcriptional memory, of how phenotype is inherited, is the heart of epigenetics.
Several studies have shown that general transcriptional regulators, including polycomb (PRC1), trithorax (MLL), and TATA-binding protein (TBP) remain bound to a subset of their interphase binding sites, thus providing a bookmark of the cell’s interphase transcriptome. A study from the Blobel lab led by recent GGR alumnus Chris Hsiung found that the macromolecular accessibility of promoters is overall maintained during mitosis, despite the microscopic compaction of chromatin. In addition, many histone modifications, which are indicative of transcriptional state, are also maintained on mitotic chromatin. Given the role of tissue-specific TFs in establishing cell identity during development, it was hypothesized that they play an important role in this process. Several studies conducted at Penn, Stephan Kadauke (GTV alumnus in Blobel lab), Juanma Caravaca (former Zaret lab member) and Robert Lake (Fan lab member) have shown that certain tissue-specific TFs in several different cell types indeed bookmark select regions of the mitotic genome.
Despite these insightful studies, there remained a lack of comprehensive analysis of whether transcription of the genome immediately after mitosis is in any way different from later in interphase. Early in his Ph.D., Chris decided to tackle this problem through a co-mentorship by Dr. Arjun Raj and Dr. Gerd Blobel. To do so, Chris performed chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) for RNA Pol II on G1E cells (a murine erythroid cell line) arrested in mitosis and at time points after the cells were released from the arrest and entered G1. Chris found that the bulk of RNA Pol II is recruited to promoters and intergenic enhancers after 90 minutes of mitotic exit, in strong agreement with previous cytological approaches. Interestingly, RNA Pol II at promoters and intergenic enhancers was more abundant earlier in mitotic exit compared to later in interphase. Based on this observation, Chris suggests that there is a "spike" in genome-wide RNA Pol II recruitment. This spike in RNA Pol II recruitment occurs when the promoter-enhancers loops are first re-established after mitosis, though there is no concomitant spike in promoter-enhancer contacts. Chris says that his favorite part of the Pol II ChIP-Seq was when he saw the signal progressing from the 5’ to 3’ end of genes as cells transitioned from mitosis to G1. He says that this made it clear to him that they had mapped the first round of transcription.
One important technique that allows for visualization of primary transcripts is single-molecule RNA fluorescence in situ hybridization (RNA-FISH), a method developed by Chris’s co-advisor Dr. Arjun Raj. Using spectrally distinct probes for introns and exons, Chris quantified primary transcripts during mitotic exit and found that not all of the cells experience a spike in transcription. However, the cells that did display a spike in transcription produced more mRNA than those that did not. From this observation, Chris proposed a model in which the spike in transcription during mitotic exit contributes to population heterogeneity. “It is still not clear what causes this phenomenon, but we identified the localized levels of a histone modification (histone 3 lysine 27 acetylation) during mitosis as, so far, the best predictor of the mitosis-G1 transcriptional spike. We observed the mitosis-G1 transcriptional spike in two different cell types, suggesting it is a generalizable property of dividing cells.”
During mitosis, RNA Pol II is evicted from the DNA, though some histone modifications indicative of active chromatin, such as histone 3 lysine 27 acetylation (H3K27Ac), are retained at some sites more than others. Chris Hsiung found that when cells exit mitosis, the earliest rounds of transcription are unexpectedly higher in output than that later in G1. This spike in transcriptional activity can be observed at about half of the genes and intergenic enhancers, and correlates with the local level of H3K27Ac during mitosis. Chris’ results from single-molecule RNA FISH suggest that this transcriptional spike during mitotic exit contributes to population heterogeneity. Illustration by Siddharth Kishore.
As for the overall experience, Chris says “We tested many hypotheses about the potential mechanism underlying the phenomenon we observed. They were either wrong or inconclusive, and never made it in the paper despite all the efforts. Most conclusions we drew from the data are far from some of the hypotheses we started with. I enjoyed how the data led to fresh conclusions rather than being limited to addressing pre-existing hypotheses. I learned a tremendous amount about experimental and data analytical approaches from the different scientific backgrounds of my advisors and lab mates.”
Chris defended his thesis in November 2015 and is currently completing medical school as part of the MD-PhD program at Penn.