Abstract
Normal hematopoiesis involves the coordination of cell division and gene expression to produce physiologically appropriate cell numbers of various developmental stages across lineages. While studies have demonstrated intricate links between cell cycle progression and developmental gene regulation -- two cellular programs whose concomitant dysregulation is central to many malignant and non-malignant hematologic diseases -- researchers currently lack clear, general principles of how intrinsic properties of cell division could influence developmental gene regulation.
In each round of division, mitosis imposes a striking disruption to gene expression: the nucleus is disassembled, bulk RNA synthesis ceases, and the transcription machinery and most transcription factors -- including repressive complexes -- are evicted from mitotic chromatin. Since hematopoietic lineage fidelity often requires the continued presence of repressive complexes to inhibit expression of developmentally inappropriate genes, we hypothesized that such repression may be inefficient during a narrow window immediately post-mitosis, resulting in transient aberrant transcription in a probabilistic manner.
We tested for the presence of transient post-mitotic aberrant transcription at genes whose repression is known to depend on continued occupancy of repressive complexes. We used an experimentally tractable cell line, G1E cells, a rapidly dividing model of lineage-committed murine pro-erythroblasts that genetically lack the erythroid master regulator Gata1. Transduction with a Gata1-estrogen receptor fusion construct and treatment with estradiol restores Gata1 function, leading to recapitulation of early erythroid maturation events, including rapid repression of stemness-associated genes, such as Gata2 and c-Kit.
We examined in fine temporal detail the post-mitotic transcriptional behavior of Gata2, c-Kit and other genes using population-based assays facilitated by drug-mediated cell cycle synchronization. In addition, we bypassed the use of synchronization drugs and their associated potential experimental artifacts by developing novel complementary methods to study the relationship between cell cycle status and transcription in asynchronous populations: 1. We harnessed single-molecule RNA fluorescence in situ hybridization technology to quantitatively assess transcription in individual cells at various cell cycle stages, and 2. We adapted a fluorescent protein cell cycle reporter to separate, using fluorescence-activated cell sorting, subpopulations of specific cell cycle stages for epigenomic and transcriptomic analyses. Together, our results revealed a post-mitotic pulse of increased RNA polymerase II recruitment and transcript synthesis most clearly exhibited by Gata2, c-Kit, and other genes whose repression is known to depend on co-repressor complexes in these cells. Our results support the notion that the mitosis-G1 transition presents a window of transcriptional plasticity. We are beginning to explore how this property of post-mitotic transcriptional control applies to hematopoietic cell types across the developmental spectrum and could contribute to functionally important variations in gene expression, such as in stem cell lineage commitment, experimental reprogramming, and non-genetic heterogeneity in malignancy.
Disclosures:
No relevant conflicts of interest to declare.
Tracking pre-mRNA maturation across subcellular compartments identifies developmental gene regulation through intron retention and nuclear anchoring
