Periodic Reporting for period 4 - Allelic Regulation (Revealing Allele-level Regulation and Dynamics using Single-cell Gene Expression Analyses) Período documentado: 2020-01-01 hasta 2020-12-31 Resumen del contexto y de los objetivos generales del proyecto Cells of diploid organisms inherit one gene copy (allele) from each parent, and often the alleles have genetic differences that in some cases affect the function of the protein encoded and organismal phenotypes. The two gene copies of each gene can be expressed from both alleles (biallelic) or from only one allele (monoallelic). Until recently studies have required the simultaneous measuring of gene expression in hundreds of thousands of cells, and these analyses showed that most genes are expressed biallelically. However, we and other pioneered the development of single-cell analysis of gene expression, which revealed a much more variable pattern of allelic expressions. Indeed frequently we observed only RNA from one othe gene copies in each cell. This project aim to answer to what extent this variable allelic expression (with frequent monoallelic expression) is regulated in the cells, or whether they could be the consequence of stochastic transcriptional processes. To this end, we will investigate both clonally stable and dynamic random monoallelic expression across a large number of cell types, including cells from embryonic and adult stages. This research program will be accomplished with the novel single-cell RNA-seq method developed within my lab to obtain quantitative, genome-wide gene expression measurement. To distinguish between mitotically stable and dynamic patterns of allelic expression, we will analyze large numbers a clonally related cells per cell type, from both primary cultures (in vitro) and in clonally related cells in vivo. The biological significance of the research program is first an understanding of allelic transcription, including the nature and extent of random monoallelic expression across in vivo tissues and cell types. These novel insights into allelic transcription will be important for an improved understanding of how variable phenotypes (e.g. incomplete penetrance and variable expressivity) can arise in genetically identical individuals. Additionally, the single-cell transcriptome analyses of clonally related cells in vivo will provide unique insights into the clonality of gene expression per se. Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha We have demonstrated that essentially all monoallelic expression in cells (in vitro and in vivo) was due to a stochastic process (and therefore not regulated as is the case for X-chromosome inactivation or imprinting). We profiled primary fibroblasts that were clonally propagated, and compared those to fibroblasts that were not clonally related. This revealed only a few genes with consistent monoallelic expression within a clone. We expanded these analyses in vivo, by studying human T-cells for which we also obtained clonal information by inferring their T-cell receptor rearrangements. From this data, we could demonstrate that clonal random monoallelic expression for autosomal genes are scarce (in contrast to earlier papers) and that random monoallelic expression instead frequently arise as a consequence of stochastic gene expression. We went further to demonstrate that determinants for monoallelic expression resulting from stochastic gene expression is cell size, activation state and cell cycle state (This work was publised in Reinius, Mold et al. Nature Genetics 2016). To obtain deeper insights into stochastic transcription, we developed a statistical inference procedure to infer transcriptional bursting kinetics from single-cell RNA-seq data, which revealed that mammalian genes are all expressed in a bursty manner, and it identified distinct regulatory sequences that control bursting frequencies and sizes. This work was published (Larsson et al. Nature 2019). We next demonstrated that stochastic transcription, in the form of transcriptional bursting, could explain eseentially all the allelic patterns observed in single cells. Moreover, we demonstrated that the burst frequency is determining the frequency of monollelic observations at the single-cell level. This work was published (Larsson e al. PLoS Comp biol 2021). All this work together clearly demonstrated the nature of monoallelic expression in cells, the extent of transcriptional bursting, and how bursting kinetics affect allelic flucatuations. We have also investigated the clonality in gene expression, which looks drastically different than the allelic observations. Clonal structures underly heterogeneity observed at the RNA and protein levels. This work us curretly under peer-review. Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha) The research in this project is deepening our understanding for how our genetic material is regulated, and will have broad impact on biology and medicine. We demonstrated that core promoter elements control burst size of transcription, and that enhancer activities drive burst frequencies. We further show that burst frequency differences among cells are mainly responsible for allelic fluctuations and the observed levels of monoallelic expression in cells. The stochastic and regulated forms of allelic gene expression regulation could impact on how disease symptoms manifest and therefore have implications for human genetic disorders. Demonstration of scarce numbers of mitotically stable monoallelic expression for autosomal genes.
