Stress causes a general down-regulation of gene expression in cells, together with the induction of a set of stress-responsive genes. How do cells know which specific genes to activate when they are silencing most of the others? The (yeast) answer is called Hog1, as shown in a recent paper published in Genome Biology by the Cell Signalling research group at the UPF.
The authors, led by Francesc Posas, used yeast as a model organism to study the response to osmostress, and they focused on Hog1, a stress-activated protein kinase which is related to p38. Using chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-Seq) they did genome-wide localization studies of RNA polymerase II (RNA Pol II) and Hog1. The results show that upon stress, RNA Pol II localization shifts toward stress-responsive genes relative to housekeeping genes, and that this relocalization required Hog1, which also localized to stress-responsive loci.
Posas and colleagues also looked at the re-organization of nucleosomes by micrococcal nuclease followed by sequencing (MNase-Seq). The analysis showed that, even though chromatin structure was not significantly altered at a genome-wide level in response to stress, there was pronounced chromatin remodeling at stress-responsive loci, which displayed Hog1 association.
The authors conclude that Hog1 serves to bypass the general down-regulation of gene expression that occurs in response to osmostress, and does so both by targeting RNA Pol II machinery and by inducing chromatin remodeling at stress-responsive loci.
Nadal-Ribelles M, Conde N, Flores O, Gonzalez-Vallinas J, Eyras E, Orozco M, de Nadal E, Posas F. Hog1 bypasses stress-mediated down-regulation of transcription by RNA polymerase II redistribution and chromatin remodeling. Genome Biol. 2012 Nov 18;13(11):R106
The English researcher Ben Lehner started as a junior group leader at the CRG in December 2006 and has been an ICREA Professor since 2009. His lab, Genetics Systems, consists of five postdoctoral fellows, four PhD students, and a technician who hail from Italy, the UK, Germany, the Netherlands, Poland, Chile, Peru, Canada and Switzerland. About half of the group members are computational biologists and the rest work primarily in the ‘wet’ lab. They all have the same aim – to understand basic questions in genetics – but they use diverse approaches and model systems.
From individual genome sequences to individual phenotypes
“In humans many mutations in genes are associated with an increased risk of particular diseases such as cancer”, the scientist explains. “But human geneticists are terrible at predicting disease risk. Most people with disease mutations never get the disease”. One aim of Lehner’s group is to better understand how the thousands of mutations in the genome of a particular individual interact to influence phenotypes such as disease risk. “What causes the same mutation to have a different outcome in different individuals? That’s one of our favourite questions”, states the researcher. This means understanding how genetics, the environment and ‘chance’ influence the outcome of particular genetic variants.
Recent research has focused on finding ways to predict the ‘normal’ outcome when genes are inhibited. In collaboration with labs from Korea, Toronto and Texas the group has created prediction models using generalisations such as the shared function of genes. “If two proteins interact physically, one can assume that they are involved in similar processes”, Lehner explains. The consequence of this hypothesis is that a mutation in either of the genes is likely to result in a similar phenotype, according to their shared function. By assuming this, one can generalise and expand the findings by using all available information on physical data, genetic interaction and co-evolution of every single gene analysed.
The next step for the group is to understand how the thousands of mutations in an individual’s genome combine to influence their characteristics. “Two humans differ by thousands of mutations, so how do we evaluate the outcome of all of this genetic variation in one go?” Beyond this they are also trying to understand why it can be impossible to predict disease phenotypes from a genome sequence. “Even genetically ‘identical’ twins are not identical when it comes to disease susceptibility. The same is true in simple organisms – if you control the genetics and you control the environment, you still cannot predict what will happen. We are working to understand why this is”, says the head of the lab.
Focus on basic problems
In their studies the group uses experimentally tractable model organisms like yeast or the transparent roundworm C. elegans, but they also use existing data from many different sources. “Rather than working with a single system or approach, we like to choose the best system to study a particular problem with. And if you look at the history of biology, ‘best’ normally means ‘simplest’”, explains the biologist. “The problem is the important thing – it doesn’t really matter how you choose to solve it. But the problem must be basic and general, and one that you can actually solve!”.
This article was published in the El·lipse publication of the PRBB.
Anna Bigas and Lluís Espinosa, of the Stem Cell and Cancer group of the IMIM are two principal investigators who have joined forces to investigate different aspects of cancer development. Together with their jointed group of 14 researchers, Bigas focuses on hematopoietic stem cells, while Espinosa concentrates on solid cancer and intestinal stem cells.
Bigas aims to understand how a pluripotent stem cell becomes a hematopoietic stem cell during embryogenesis. `It is a great challenge in the regenerative medicine field to understand where these stem cells come from and how they conserve this self- renewing capacity which enables them to maintain a tissue´, she explains. She focuses on a major signalling pathway controlling decisions in both normal and leukemic cells and which is also important for tissue maintenance: the Notch pathway.
Searching for Notch target genes
In order to specify the molecular mechanisms driving an undifferentiated cell towards the hematopoietic lineage or to the leukemic phenotype, the group’s current objective is to find Notch target genes and to describe their mechanism of function.
The researchers use techniques like such as chromatin precipitation and promoter arrays. This results in lists of possible candidate genes from which the real targets have to be isolated and validated in a series of experiments, including FACS to isolate the cells of interest and to determine whether the candidate gene is expressed. Further molecular and biochemical analysis, as well as experiments using mutant mice, help define possible interactions of the target molecules and their effect in the organism.
One target gene is GATA2, an important hematopoietic transcription factor that is not expressed in Notch mutant mice and is altered in human leukaemia. The Bigas group have characterized the GATA2 promoter and found that Notch exerts both positive and negative signals that restrict the intensity and the duration of GATA2 expression in hematopoietic cells.
In parallel, Espinosa is studying whether Notch cooperates with other signalling pathways in different contexts. He found that specific elements of the NF-κB pathway, which is involved in cancer development, directly regulate the transcription of genes which are known to be Notch-dependent.
In a common work Bigas and Espinosa identified a new role of the Notch signalling pathway in the maintenance of leukemic stem cells. Previous studies had shown that both the NF-κB and the Notch pathway are involved in T-cell acute lymphoblastic leukaemia, and future therapeutical strategies may employ both Notch and NF-kB inhibitors to fight this leukaemia.
However only the recent findings of Bigas and Espinosa describe the exact mechanism by which Notch activates the NF-kB transcription factor. These new insights published in Cancer Cell, could be translated to clinical trials and result in better pharmaceutical treatments with less side effects.
Despite all the advances in the field, it is not yet known whether Notch is also important for the leukaemia initiating cells, a question that Bigas would like to answer in the close near future. These cells are of major interest, since they are resistant to standard leukaemia treatments, remain in the organism and ultimately are the source of a recurrent outbreak.
Bigas and Espinosa maintain collaborations both inside and outside of the PRBB. `It is a great advantage to have so many scientists within a few square metreers´ Bigas states, `and we just have a great collaborative work in progress, including involving scientists from the CRG, the UPF, Hospital del Mar and others´.
This article was published in the El·lipse publication of the PRBB.