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Promoter-Mediated Diversification of Transcriptional Bursting Dynamics Following Gene Duplication

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Promoter-Mediated Diversification of Transcriptional Bursting Dynamics Following Gene Duplication Promoter-mediated diversification of transcriptional bursting dynamics following gene duplication Edward Tunnacliffea,b, Adam M. Corrigana,b,1, and Jonathan R. Chubba,b,2 aMedical Research Council Laboratory for Molecular Cell Biology, University College London, WC1E 6BT London, United Kingdom; and bDepartment of Cell and Developmental Biology, University College London, WC1E 6BT London, United Kingdom Edited by Joseph S. Takahashi, Howard Hughes Medical Institute and University of Texas Southwestern Medical Center, Dallas, TX, and approved July 6, 2018 (received for review January 21, 2018) During the evolution of gene families, functional diversification of is a broadly applicable evolutionary strategy, shared by species that proteins often follows gene duplication. However, many gene diverged more than 400 Mya (SI Appendix,TableS1)(7). families expand while preserving protein sequence. Why do cells Dictyostelium cells are highly motile, so may require lots of maintain multiple copies of the same gene? Here we have actin, perhaps beyond the production capacity of a single gene. addressed this question for an actin family with 17 genes encoding However, estimates of their actin content are of the same order an identical protein. The genes have divergent flanking regions as skeletal muscle, which derives its actin from only one gene and are scattered throughout the genome. Surprisingly, almost (8, 9). Divergent flanking sequences (10, 11) and different ge- nomic contexts of the act8 genes suggest different regulatory the entire family showed similar developmental expression pro- — files, with their expression also strongly coupled in single cells. Using dynamics and responses for example, during development. The expansion of the family may also buffer against gene ex- live cell imaging, we show that differences in gene expression were pression noise—it may be undesirable for the expression of an apparent over shorter timescales, with family members displaying “ ” essential protein to be unpredictable, and additional genes may different transcriptional bursting dynamics. Strong bursty behav- average out noise. iors contrasted steady, more continuous activity, indicating different Here, we evaluate the potential for different regulatory dy- regulatory inputs to individual actin genes. To determine the sources namics within the gene family. The family shows comparatively of these different dynamic behaviors, we reciprocally exchanged the similar expression profiles over development and strong coupling upstream regulatory regions of gene family members. This revealed between genes in single cells. However, the genes differ in the that dynamic transcriptional behavior is directly instructed by up- dynamics of their transcriptional bursts and show different stream sequence, rather than features specific to genomic context. bursting responses upon induction of development. Switching A residual minor contribution of genomic context modulates the promoters of actin genes demonstrates that transcriptional dy- gene OFF rate. Our data suggest promoter diversification following namics are instructed predominantly by upstream sequence, gene duplication could expand the range of stimuli that regulate the rather than genomic context. expression of essential genes. These observations contextualize the significance of transcriptional bursting. Results Developmental Dynamics of Actin Gene Expression. Having multiple transcriptional bursting | stochastic gene expression | single-cell genes encoding the same protein dispersed throughout the genome transcriptomics | Dictyostelium | gene family may have enabled diversification and refinement of actin expres- sion. Consistent with this view, there is considerable diversity of ene duplication is recognized as an important process for Ggenerating complexity in evolution (1). Following duplica- Significance tion, gene sequences are present in at least two copies in the genome. Assuming these sequences are identical and subject to Gene transcription occurs in discontinuous bursts. Although the same regulatory constraints, they will perform the same bursts are conserved in all forms of life, the causes and impli- function—they are redundant. Over time, duplicate genes typi- cations of bursting are not clear. Here we delineate a specific cally diverge in sequence and function; however, in some cases, cause of bursts and contextualize the significance of bursting, strong selection acts to maintain identical amino acid or nucle- using analysis of a gene family encoding 17 identical actin pro- otide sequences over long periods of evolution. Examples in- teins. Although the genes show similar developmental ex- clude histones, where humans have 14 genes for histone H4, pression, which is coupled in single cells, they show strong each encoding the same protein (2). Similarly, ribosomal RNA differences in bursting dynamics. These distinct bursting pat- genes are present in hundreds to thousands of copies in eu- terns indicate that different signals regulate the individual genes karyotes with extremely high sequence conservation between and suggest expansion of the family may have allowed di- family members (3). versification of actin gene regulation. By exchanging the pro- Why does an organism require so many genes encoding an moters of genes, we show that the dominant driver of bursting apparently identical end product? One explanation is that a large dynamics is the gene promoter, not the genome context. amount of gene product is required, and multiple genes allow more transcription. However, while histone genes can be under Author contributions: E.T. and J.R.C. designed research; E.T. performed research; E.T. and A.M.C. contributed new reagents/analytic tools; E.T. and J.R.C. analyzed data; and E.T. coordinate control during the cell cycle (4), they have different and J.R.C. wrote the paper. promoter elements and show varying contributions to total his- tone content in normal and cancer cells, suggesting regulatory The authors declare no conflict of interest. differences between family members. This article is a PNAS Direct Submission. To understand how differences in gene regulation have Published under the PNAS license. influenced the evolution of multigene families, we investigated 1Present address: AstraZeneca, Discovery Sciences, Cambridge Science Park, CB4 0WG the actin gene family of the amoeba Dictyostelium discoideum. Cambridge, United Kingdom. This organism has more than 30 actin genes, of which 17 (the 2To whom correspondence should be addressed. Email: [email protected]. act8 group) encode an identical amino acid sequence (5). Act8 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. family genes produce more than 95% of total actin (6) and are 1073/pnas.1800943115/-/DCSupplemental. dispersed throughout the genome (5). This actin family organization Published online July 30, 2018. 8364–8369 | PNAS | August 14, 2018 | vol. 115 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1800943115 Downloaded by guest on October 1, 2021 upstream regulatory sequence between act8 family members, Single-Cell Coupling of Actin Gene Expression. To obtain single-cell with TATA and 3′ UAS motifs conserved across most of the resolution and more accurate quantitation of actin gene ex- family, while other motifs such as a G-box are found in only some pression, we measured the relative abundance of act8 family promoters (SI Appendix, Fig. S1A). While some promoters transcripts using single-molecule RNA FISH (smFISH). To contain several elements, others, such as act8, show little com- sample genes with contrasting promoter architecture, we chose plexity, with large runs of A and T. Conserved elements were act1, act5, act6, and act8. Twenty-four MS2 stem loops (14) were also found at the 3′ end of act genes, which could enable further targeted at the 5′ of coding sequences, causing the MS2 loops to regulatory diversification, although earlier studies showed no be included in the transcribed RNA. Cells with MS2-tagged strong differences in RNA turnover within the family (12). genes were probed with a fluorescent oligonucleotide comple- To test whether actin genes are differentially regulated, we mentary to the MS2 array. Cytoplasmic particles corresponding B act5 act6 act8 used RNA-sequencing data to determine developmental profiles to single RNAs were counted (Fig. 1 ). The , , and act8 of gene expression (13). Fig. 1A shows the developmental ex- genes were all strongly expressed, with the strongest and act8 act6 the weakest, but each gene showed tens to hundreds of pression patterns of all 17 genes. All genes are induced upon act1 differentiation onset with a peak between 1 and 3 h. Most genes RNAs per cell. In contrast, expression was close to back- show decreased expression during middevelopment (6–10 h) ground (cells without MS2), indicating that in undifferentiated cells, actin expression at full capacity is not required. In line with and, by 16 h, show little expression. Promoter differences may act8 explain the subtle variations in developmental expression—genes this, we found that disruption of up to four family genes had no effect on cell-doubling times, and a six-gene mutant showed with similar promoters, such as act9, act13, and act14, show more only a weak growth defect (SI Appendix, Fig. S1 B and C). similar expression during development. An exception to the act act1 Each gene showed considerable expression variability (Fig. general pattern is , which has additional peaks
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