mRNA is thought to predominantly reside in the cytoplasm, where it is translated and eventually degraded. Although nuclear retention of mRNA has a regulatory potential, it is considered extremely ...rare in mammals. Here, to explore the extent of mRNA retention in metabolic tissues, we combine deep sequencing of nuclear and cytoplasmic RNA fractions with single-molecule transcript imaging in mouse beta cells, liver, and gut. We identify a wide range of protein-coding genes for which the levels of spliced polyadenylated mRNA are higher in the nucleus than in the cytoplasm. These include genes such as the transcription factor ChREBP, Nlrp6, Glucokinase, and Glucagon receptor. We demonstrate that nuclear retention of mRNA can efficiently buffer cytoplasmic transcript levels from noise that emanates from transcriptional bursts. Our study challenges the view that transcripts predominantly reside in the cytoplasm and reveals a role of the nucleus in dampening gene expression noise.
Display omitted
•Genome-wide catalog of nuclear and cytoplasmic mRNA in mouse tissues•Spliced, polyadenylated mRNA is retained in the nucleus for many protein-coding genes•Retained genes include ChREBP and liver Nlrp6, co-localized with nuclear speckles•Nuclear retention of mRNA reduces cytoplasmic gene expression noise
Bahar Halpern et al. combine whole-transcriptome and single-molecule approaches to demonstrate that a substantial fraction of genes have higher levels of spliced, polyadenylated mRNA in the nucleus compared to the cytoplasm in mammalian tissues. This nuclear retention reduces cytoplasmic gene expression noise created by transcriptional bursts.
Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter ...referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.
Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation ...with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.
Mouse models play a major role in understanding key biochemical and physiological mechanisms. However, differences between humans and mice, such as metabolic measures, pose significant hurdles for ...translating findings from the laboratory into the clinic. One prominent example is glucose homeostasis and the determination of the glycemic setpoint. Proper physiological function requires the maintenance of blood glucose (BG) level at a narrow range. Any deviation from this range triggers an immediate response that restores it back to normal. This is achieved primarily by two pancreatic hormones: insulin and glucagon. Dysregulated insulin secretion can disrupt glucose homeostasis and cause severe metabolic disorders, including diabetes mellitus. While many tissues contribute to glucose homeostasis, pancreatic islets have been suggested to be the primary glucostat, a machinery that controls the glycemic setpoint. However, the underlying mechanisms have not been fully elucidated. I hypothesized that glucose transporters (GLUTs) are crucial for glucose sensing. The primary glucose transporter expressed in murine islets is GLUT2, which has a relatively low binding affinity for glucose, with a Km value of ~15-17mM. In contrast, the dominant glucose transporter in human islets is the high affinity GLUT1, with a Km of ~6m. These Km values correspond to the optimal extracellular glucose levels for insulin secretion from mouse and human islets, suggesting that the differences between GLUT1/2 expression could account for the species-specific glucose setpoint. However, despite this correlation, the precise requirements for GLUT1/2 in the human system remain unclear. I utilized two complementary systems in this project: human primary islets and stem cells derived β-like (SC-β) cells. Both were transplanted independently into recipient mice, which induced a decrease in murine BG to a human glycemic range. Furthermore, chemical and genetic perturbation were applied to study the roles of GLUT1/2 in human β cell functionality. Disruption of either or both transporters led to diminished β cell functionality, as evidenced by reduced glucose transport and insulin secretion. These findings demonstrate and emphasize the role of GLUTs in glucose homeostasis and their impact on determining species-specific normal glycemia.
DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, ...but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene,
, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.
The molecular program underlying infrequent replication of pancreatic β-cells remains largely inaccessible. Using transgenic mice expressing green fluorescent protein in cycling cells, we sorted ...live, replicating β-cells and determined their transcriptome. Replicating β-cells upregulate hundreds of proliferation-related genes, along with many novel putative cell cycle components. Strikingly, genes involved in β-cell functions, namely, glucose sensing and insulin secretion, were repressed. Further studies using single-molecule RNA in situ hybridization revealed that in fact, replicating β-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in β-cell function. These data suggest that the quiescence-proliferation transition involves global amplification of gene expression, except for a subset of tissue-specific genes, which are "left behind" and whose relative mRNA amount decreases. Our work provides a unique resource for the study of replicating β-cells in vivo.
Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an ...alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.
PDF
