Showing papers by "Stefan Hohmann published in 2017"

Transcription factor clusters regulate genes in eukaryotic cells

Abstract: Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

The yeast osmostress response is carbon source dependent

Abstract: Adaptation to altered osmotic conditions is a fundamental property of living cells and has been studied in detail in the yeast Saccharomyces cerevisiae. Yeast cells accumulate glycerol as compatible solute, controlled at different levels by the High Osmolarity Glycerol (HOG) response pathway. Up to now, essentially all osmostress studies in yeast have been performed with glucose as carbon and energy source, which is metabolised by glycolysis with glycerol as a by-product. Here we investigated the response of yeast to osmotic stress when yeast is respiring ethanol as carbon and energy source. Remarkably, yeast cells do not accumulate glycerol under these conditions and it appears that trehalose may partly take over the role as compatible solute. The HOG pathway is activated in very much the same way as during growth on glucose and is also required for osmotic adaptation. Slower volume recovery was observed in ethanol-grown cells as compared to glucose-grown cells. Dependence on key regulators as well as the global gene expression profile were similar in many ways to those previously observed in glucose-grown cells. However, there are indications that cells re-arrange redox-metabolism when respiration is hampered under osmostress, a feature that could not be observed in glucose-grown cells.

The yeast Mig1 transcriptional repressor is dephosphorylated by glucose-dependent and -independent mechanisms

Abstract: A yeast Saccharomyces cerevisiae Snf1 kinase, an analog of mammalian AMPK, regulates glucose derepression of genes required for utilization of alternative carbon sources through the transcriptional repressor Mig1. It has been suggested that the Glc7-Reg1 phosphatase dephosphorylates Mig1. Here we report that Mig1 is dephosphorylated by Glc7-Reg1 in an apparently glucose-dependent mechanism but also by a mechanism independent of glucose and Glc7-Reg1. In addition to serine/threonine phosphatases another process including tyrosine phosphorylation seems crucial for Mig1 regulation. Taken together, Mig1 dephosphorylation appears to be controlled in a complex manner, in line with the importance for rapid and sensitive regulation upon altered glucose concentrations in the growth medium.

Single-cell study links metabolism with nutrient signaling and reveals sources of variability

Abstract: Background: The yeast AMPK/SNF1 pathway is best known for its role in glucose de/repression. When glucose becomes limited, the Snf1 kinase is activated and phosphorylates the transcriptional repressor Mig1, which is then exported from the nucleus. The exact mechanism how the Snf1-Mig1 pathway is regulated is not entirely elucidated. Results: Glucose uptake through the low affinity transporter Hxt1 results in nuclear accumulation of Mig1 in response to all glucose concentrations upshift, however with increasing glucose concentration the nuclear localization of Mig1 is more intense. Strains expressing Hxt7 display a constant response to all glucose concentration upshifts. We show that differences in amount of hexose transporter molecules in the cell could cause cell-to-cell variability in the Mig1-Snf1 system. We further apply mathematical modelling to our data, both general deterministic and a nonlinear mixed effect model. Our model suggests a presently unrecognized regulatory step of the Snf1-Mig1 pathway at the level of Mig1 dephosphorylation. Model predictions point to parameters involved in the transport of Mig1 in and out of the nucleus as a majorsource of cell to cell variability. Conclusions: With this modelling approach we have been able to suggest steps that contribute to the cell-to-cell variability. Our data indicate a close link between the glucose uptake rate, which determines the glycolytic rate, and the activity of the Snf1/Mig1 system. This study hence establishes a close relation between metabolism and signalling.

Transcription factor clusters regulate genes in eukaryotic cells

Abstract: Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

Transcription factor clusters regulate genes in eukaryotic cells

Abstract: Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

Editor’s comment on “CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein”

Abstract: gene editing in human zygotes/embryos for establishing, for instance, correction of genetic defects carried by the parents. The embryos generated in the present study were destroyed as part of the analysis. The work by Tang et al. also points to limitations and complications. First of all, only a rather small number of embryos were investigated. Ten wild-type oocytes were inseminated with sperm from heterozygous patients. In the case of HBB (chromosome 11), this resulted in four heterozygous zygotes and in the case of G6PDH deficiency (X-chromosome) in only two female embryos heterozygous for the mutation. Of the four HBB zygotes, two were edited but just one by homology-directed recombination (HDR), which results in correct repair of the mutation. In the case of G6PDH deficiency both zygotes showed homologydirected editing and hence repair of the mutation. One of the two embryos was chosen for whole genome sequencing and no off-target genetic events were determined. Far more work with a larger number of embryos will be required to demonstrate how the technology can safely be used for gene editing in human embryos. One critical aspect concerns the repair pathway employed by target cells following the CAS9-mediated double strand-break, i.e., HDR (which results in correct editing) or non-homologous end-joining (NHEJ), which results in other changes at the repair site. Such events were observed in the case of the HBB experiments. Another issue concerns the generation of mosaic embryos, which also were observed in this study. For instance, one of the two embryos in the G6PDH experiments contained wild type, corrected and edited cells at a ratio of 2:1:1. It still appears a rather long way until gene editing in human embryos becomes feasible with high fidelity and safety. But the present and previous work implicate that The ethical aspects of genetic engineering have been discussed since the early 1970s, already then with the expectation that eventually it may be possible to perform targeted genetic changes in the human germline. Technically, however, this has not been feasible until very recently. The development of CRISPR/CAS9 technology has made it possible to perform with reasonable effort and specificity genetic changes in mammalian cells, including zygotes or embryos (Ledford 2015). In this issue of Molecular Genetics and Genomics, we publish work by Tang et al. that, to our knowledge, for the first time, demonstrates in diploid (2N) human embryos the correction of genetic defects. The defects studied in this case are a mutation in the HBB (haemoglobin subunit beta) gene and one in the gene encoding the enzyme glucose-6-phosphate dehydrogenase (G6PDH). These rather common genetic defects cause different types of anaemia, β-thalassemia and favism, respectively. Previous work has already shown that CRISPR/CAS9 technology can be employed to perform specific changes in triploid (and hence non-viable) human embryos (Kang et al. 2016; Liang et al. 2015). Hence, the findings reported by Tang et al. are not surprising. However, in vitro inseminated and genetically altered 2N human embryos can principally be implanted and develop into human beings, thereby bringing genetic manipulation of the human germline one step closer to reality. The work reported in this issue of MGG demonstrates that, as expected, it is possible to perform

Moving from Genetics to Systems Biology

Abstract: Access to competence in mathematical modelling may be a major hindrance for employing a systems biology approach in many research projects. Very commonly, the most successful systems biology studies are based on collaborations between experimentalists, contributing with the biological questions and experimental data and theoreticians, who contribute with skills in modelling, simulation and prediction. Collaboration across disciplines is not a trivial enterprise and very commonly requires skills to communicate across borders. This challenge, as well as the mere availability of matching theoreticians locally may prevent in many cases the integration of modelling and prediction into projects even if the interdisciplinary approaches offer great potential.”

The Yeast Mig1 Transcriptional Repressor Is Dephosphorylated By Glucose-Dependent And Independent Mechanisms

Abstract: Saccharomyces cerevisiae AMPK/Snf1 regulates glucose derepression of genes required for utilization of alternative carbon sources through the transcriptional repressor Mig1. It has been suggested that the Glc7-Reg1 phosphatase dephosphorylates Mig1. Here we report that Mig1 is dephosphorylated by Glc7-Reg1 in an apparently glucose-dependent mechanism but also by a mechanism independent of glucose and Glc7-Reg1. In addition to serine/threonine phosphatases another process including tyrosine phosphorylation seems crucial for Mig1 regulation. Taken together, Mig1 dephosphorylation appears to be controlled in a complex manner, in line with the importance for rapid and sensitive regulation upon altered glucose concentrations in the growth medium.