He explained there are only about 20,000 human genes, but more than 1,000,000 genome ‘switches’, short DNA regions which control which genes are expressed and at what levels. And the so-called ‘gene deserts’, areas of the genome with very few genes in them, are actually very rich in these conserved non-coding elements which act as cis-regulators of gene expression. So, as he said, perhaps more than gene deserts they should be called ‘regulatory jungles’.
Just because of the sheer number of these regulatory units, their importance should not be overlooked – if there are so many they must be doing something. And their key role in gene regulation is more obvious each day.
But in the context of evolution and the apparition of new, beneficial mutations, their role takes a new dimension I, for one, hadn’t thought much about. Let me use Bejerano’s example, the Sall1 gene, a key gene during development that is expressed in limb, brain and neural tube. There are three enhancers for this gene, one for each of these locations. If a mutation appears in the gene itself, it will cause problems in all the regions the gene is expressed and therefore that mutation might be lethal and won’t be successful (evolutionary speaking). But if the mutation affects only one of the enhancers, it would have an effect just on one of the regions where the gene is expressed. Such a mutation would have more chances to be passed on to the next generation. Therefore, beneficial mutations are more likely to appear in enhancers (highly conserved non-coding regions) than in genes. Enhancers would then play an essential role in evolution.
Report by Maruxa Martinez, Scientific Editor at the PRBB
The first keynote speaker at the XI Bioinformatics Symposium was Søren Brunak, director of the Centre for Biological Sequence Analysis (CBS) in Denmark. He gave an interesting overview on the need to integrate the very detailed molecular information we have with the phenotypic data we can get from the healthcare sector. He explained the best source of this type of data are the electronic patient records (EPR or EMR – for medical records), which are very well established in his country as well as other small European countries such as the Netherlands, but not at all in bigger countries such as France or Germany. Spain was halfway between both, but closer to the less advanced countries in terms of having standardized EMRs.
There are difficulties with these EMRs, not the least the barrier language amongst different countries. But there exists a unified medical language system (UMLS), a controlled vocabulary for the clinical disease descriptions which facilitates the comparison.
Brunak mentioned an interesting article by Elaine Mardis, “The $1,000 genome, the $100,000 analysis?”, which points out that even though we are quickly getting more sequence information at a lower price, the costs of analyzing it are still very high, and the task is difficult. Brunak believes the cost of these analyses will only go down if genotypic data is integrated with phenotypic information. So his take-home message was that we need to collect and analyse phenotypic data in a more fine-grained way, which will then make it easier to approach network biology from both the genotype and the phenotype data.
Report by Maruxa Martinez, Scientific Editor at the PRBB
More than 225 people registered to the XI Bioinformatics Symposium, co-organised by the Spanish Institute of Bioinformatics (INB) and the Portuguese Bioinformatics Network and hosted by the PRBB. From yesterday, January 23 and until tomorrow, Jan 25 they will enjoy the talks of three keynote speakers and another 31 scientists, as well as more than 100 scientific posters.
Alfonso Valencia, director of the INB – and who had recently been at the PRBB for another bioinformatics meeting – explained at the inauguration that the aim of the conference, organized jointly with the Portuguese community for the last three years, was to stimulate collaborations between both countries and to grow as a community, something that seemed to have been achieved as evidenced by the record audience.
The event hosted a student symposium in the first morning, and a discussion session in the afternoon on the challenges and limitations for bioinformatics researchers. One of the issues raised in this round table was how to deal with the large amounts of data that are being gathered, and Ana Rojas (IMPPC) mentioned that a centralized repository of all the data would be a good thing, but that funding is necessary –and nowadays very difficult to get! Another issue related to the sheer amount of data is the need for more computer power and in that sense a call was sent from the Barcelona Supercomputing Centre to all scientists to make more use of the existing supercomputers in Europe.
Challenges of another nature were also discussed. Bioinformatics is an interdisciplinary area by nature. Its three pillars are molecular biology, mathematics/statistics/physics, and computer science/engineering. The difficulty of finding researchers that are trained in all three areas is large, but one of the speakers also mentioned the difficulties that those well-trained researchers meet during their career development. A biologist doesn’t fit very well in a Computer Science department, nor an engineer in the Biology department. And the Impact Factor of the computer science journals is much lower than those of biology, so when fighting for a competitive position, computer scientists would always be in disadvantage… all in all, everyone agreed that sooner or later, every experimental group will need to have a bioinformatics expert with them – or at least someone with a basic knowledge of data analysis who can speak ‘bioinformaticians’ language.
Report by Maruxa Martinez, Scientific Editor at the PRBB
Artificial zinc-finger DNA-binding proteins (ZFPs) have been engineered for quite some time now, and can be very useful in molecular biology. Zinc fingers are small protein structural motifs that can coordinate one or more zinc ions to help stabilize their folds. These structural motifs are involved in a broad range of biological activities including DNA binding, DNA and RNA recognition, as well as coordinating protein-protein interactions. One use of ZFPs is the creation of a sequence-specific nuclease, which can be used, for example, to mutate chromosomal targets, via a double-stranded DNA break, or to integrate foreign DNA into a locus. These are called zinc finger nucleases (ZFNs).
Even though a multitude of methods exist for engineering zinc fingers, making ZFNs is not a solved problem. Mark Isalan, head of the Gene Network Engineering group at the CRG, has recently written a commentary in Nature Methods on the issues faced when engineering these nucleases. This follows the group’s recent work on making zinc finger nucleases to repair p53 – the gene most commonly mutated in human cancer.
The Zinc Finger Consortium provides convenient computational tools to help design constructs, although this can still be challenging for inexperienced users, warns Isalan.
One of the reasons that ZFNs are challenging to build is that it is not possible to target just any desired DNA sequence. Most of the zinc fingers engineered have G-rich consensus sequences, because G-rich sequences are the natural preference of zinc fingers. Assembly of fingers for other types of targets is frequently unsuccessful.
The second difficulty is that in order to engineer a nuclease one needs to put together two domains (it’s a dimer) and it is much easier to make a single designer DNA-binding domain than to join together two domains, in the appropriate orientation, with the correct spacing, etc.
Isalan comments about these and other problems, and talks about some alternative new technologies, such as meganucleases and transcription activator–like effector (TALE) nucleases.
Computer simulations with pharmacological interest
In this image by Ignasi Buch, from the Computational Biochemistry and Biophysics Laboratory of the GRIB (IMIM/UPF), we can see a simulation of the union of a drug with its target. The small hexagonal molecule represents the benzamidine drug, an inhibitor of tripsine, which is represented by the 3D grey molecule.
Tripsine is an enzyme which cuts proteins. Its binding to benzamidine makes it impossible for it to connect to other proteins that it should cut. This dynamic simulation of the inhibition shows how benzamidine first interacts with several regions of tripsine which help the drug to find its final binding site. Understanding the path that benzamidine follows in order to bind its target gives new clues to designing more efficient drugs.
His group, in collaboration with scientists in Mount Sinai in New York, has just discovered the presence of neutrophils in the marginal zone (MZ) of the spleen, a B cell area positioned at the interface between the circulation and the immune system. The presence of these cells is constant, even in the absence of infection.
The authors obtained lymphoid organs from people with no inflammation or infection. Unexpectedly, they detected many neutrophils in the perifollicular area of spleens that had no histological alterations – up to 10% of the total cells in the spleens were neutrophils. They also detected perifollicular neutrophils in spleens from healthy rhesus macaques and mice, suggesting these spleen neutrophils are probably common to all mammals.
They compared this newly found population of neutrophils with the circulating ones, and found they had a distinct phenotype. They also had a different function: they interacted with marginal zone B cells, and elicited immunoglobulin class switching, somatic hypermutation and antibody production by activating those B cells via BAFF and APRIL and the cytokine IL-21 – an immunoregulatory function that is new to neutrophils.
Being strategically located at the interface with the circulation, the major role of the marginal zone is to trap particulate antigen from the circulation and present them to the lymphocytes of the spleen. Marginal zone B cells are therefore geared to rapidly respond to blood-borne antigens. The authors believe that the discovered spleen neutrophils may generate an innate layer of antimicrobial immunoglobulin defense by undergoing B cell–helper reprogramming in the spleen. Actually, they found these ‘B cell–helper neutrophils’ to activate marginal zone B cells as effectively as splenic CD4+ T cells, and more effectively than splenic macrophages or dendritic cells (DCs).
Cerutti and colleagues conclude that an insufficiency of these ‘B cell–helper neutrophils’ could contribute to the pathogenesis of systemic infections in patients with neutrophil disorders. Conversely, harnessing these cells with specific adjuvants may enhance vaccine-induced immunoglobulin responses to poorly immunogenic antigens in healthy people.
Puga I, Cols M, Barra CM, He B, Cassis L, Gentile M, Comerma L, Chorny A, Shan M, Xu W, Magri G, Knowles DM, Tam W, Chiu A, Bussel JB, Serrano S, Lorente JA, Bellosillo B, Lloreta J, Juanpere N, Alameda F, Baró T, de Heredia CD, Torán N, Català A, Torrebadell M, Fortuny C, Cusí V, Carreras C, Diaz GA, Blander JM, Farber CM, Silvestri G, Cunningham-Rundles C, Calvillo M, Dufour C, Notarangelo LD, Lougaris V, Plebani A, Casanova JL, Ganal SC, Diefenbach A, Aróstegui JI, Juan M, Yagüe J, Mahlaoui N, Donadieu J, Chen K, & Cerutti A (2011). B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen. Nature immunology PMID: 22197976
A team of researchers from the CMRB directed by Juan Carlos Izpisúa Belmonte has discovered two novel inhibitors of the phosphorylase kinase subunit G1 (PhKG1) that has been identified for the first time to be involved in angiogenesis in vivo. Furthermore, they found that PhKG1 mRNA levels are elevated by more than two-fold in the majority of human tumors (breast, colon, kidney, lung, liver and thyroid), except in prostate cancer. The study was published in Oncogene.
Pathological angiogenesis, the growth of microvessels from existing vasculature, is associated with tumor progression and is a pre-requisite of tumor growth and metastasis. Therefore, inhibitors of angiogenesis are desirable candidates for anti-tumoral therapies.
First, the researchers screened for angiogenesis inhibitors from a compound library of putative kinases from the Dutch company Galapagos, BV, using an automatic quantitative screening assay. They used embryos of a zebrafish line in which the vascular system is visible through endothelial-specific enhanced green fluorescent protein (EGFP) expression. The assay was implemented at the high-throughput screening platform of Biobide SL.
The authors selected two new compounds that were found in the assay as inhibitors of angiogenesis and identified PhKG1 as their target through an in vitro kinase profiling. Finally they confirmed that the two compounds inhibited specifically the angiogenic process of vessel sprouting, as opposed to inhibition of general vasculogenesis, by treating embryos with either drug before the vasculogenic vessels had formed.
Effects of the PhKG1 inhibitors F10 and F11 on the processes of angiogenesis and vasculogenesis
Camus S, Quevedo C, Menéndez S, Paramonov I, Stouten PF, Janssen RA, Rueb S, He S, Snaar-Jagalska BE, Laricchia-Robbio L, Izpisua Belmonte JC. Identification of phosphorylase kinase as a novel therapeutic target through high-throughput screening for anti-angiogenesis compounds in zebrafish. Oncogene. 2011 Dec 19;
Mutations often have consequences that vary across individuals. A study published in Science by Ben Lehner, head of the Genetic Systems Group at the CRG, shows that the stimulation of a stress response can reduce mutation penetrance in Caenorhabditis elegans. Moreover, this induced mutation buffering varies across isogenic individuals because of interindividual differences in stress signaling. This variation has important consequences in wild-type animals, producing some individuals with higher stress resistance but lower reproductive fitness and other individuals with lower stress resistance and higher reproductive fitness.
In the experimental setting they used a transgene to overexpress the heat shock factor 1 (HSF-1) in C. elegans, a master regulator of the environmental stress response. Then they crossed the HSF-1 transgenic animals with strains carrying diverse mutations that affect development but with outcomes that vary across individuals. In 8 out of 11 tested cases, mutation penetrance was reduced in the transgenic animals both for embryonic and postembryonic development, especially when a mutation was chaperone-dependent.
Mutations may affect the three-dimensional structure of proteins, causing unstable and toxic products. But proteins can remain stable if there are enough chaperones around, because the chaperones generally assist the folding of the linear amino acid chain into the protein and provide stability to the structure. “The levels of chaperones induced by any level of stress vary from one individual to the next, even if individuals are genetically identical. We were very excited to find that for small and protective stress levels, the stochastic fluctuation in the dose of the chaperones can partly explain why only some individuals develop the disease,” explains Ben Lehner.
Animals received a 2-hour 35°C heat shock as L1 larvae and were sorted 1 day later into “high” (right worm) and “low” (left worm) populations, according to the induction of an hsp-16.2 chaperone promoter reporter.
Casanueva MO, Burga A, Lehner B. Fitness Trade-Offs and Environmentally Induced Mutation Buffering in Isogenic C. elegans. Science. 2011 Dec 15