Pluripotency and differentiation in embryos and stem cells - Pavia, 17-18 January 2008

Each year many scientific meetings are held on stem cells to appraise the state of knowledge on their potency, differentiation and applications. So why did we hold another meeting? Because we thought one aspect was not adequately addressed in the others. When thinking of how our body is derived from a single fertilized egg, it is self-evident that the embryo is the ‘mother’ of all stem cells. This fact is probably overlooked because it is so remote (decades back in our lives!) and because embryonic stem cells do not exist as such in the embryo. However, this also tends to be ignored on purpose in many stem cell meetings because working on (human) embryos brings up substantial ethical concerns that bear on the scientific undertaking like nothing else. The origin of stem cells has become even more of a sensitive issue since the discovery in 2006 that embryonic stem (ES) cell-like cells can be generated in a petri dish straight from somatic cells by retrovirus-mediated transfer of selected genes. These new cells have been named ‘induced pluripotent stem‘ (iPS) cells and have been obtained without any egg or embryo consumption (Takahashi and Yamanaka, 2006). This leads to the first topic of our meeting: natural and induced pluripotency..

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Identification and validation of transcription factors that regulate chromatin dynamics

Gene expression has to be tightly regulated during all cellular processes. During embryonic development differentiating cells loose their developmental potential and acquire specific functions by activating lineage-specific genes. Gene transcription programs are regulated by transcription factors (TFs) in concert with dynamic changes in local chromatin organisation of the DNA template. Both pathways are crucial for specific reprogramming of cells. However, how TFs and chromatin marks exactly contribute to regulate gene expression programs is not fully understood. For instance, the binding patterns of most mammalian TFs are still unknown as well as how binding specificity is achieved. Chromatin modifications are highly dynamic and cell-type specific. By regulating access to the DNA template they might guide TF binding. As most chromatin modifications have simply been associated with gene activity, a central remaining question is how chromatin modifications impact on gene expression and if they are a cause or consequence of the transcriptional state of a gene. Further it is still an open question how chromatin marks are targeted to specific loci and how they are dynamically regulated. Trimethylation of histone 3 at lysine 27 (H3K27me3) is set by the Polycomb group of proteins, which regulate body patterning during development. Polycomb-mediated H3K27me3 is associated with gene repression and essential for cellular differentiation. Further work shows that H3K27me3 targets are cell-type specific and highly dynamic during differentiation. It is unclear how these changes are regulated. Thus, we hypothesise that TFs, by recognising distinct DNA motifs, could contribute to the required specificity of chromatin reprogramming. In collaboration with the group of Erik van Nimwegen we applied an unbiased approach to model changes in H3K27me3 methylation during in vitro neuronal differentiation in terms of predicted transcription factor binding sites. This approach predicts many TFs to regulate H3K27me3 at specific stages of cellular differentiation. We experimentally focus on the validation of the RE-1 silencing transcription factor (REST) and the family of SNAIL TFs, which are both predicted to regulate a gain of H3K27me3 levels as stem cells differentiate to neuronal progenitor cells. We determine genome-wide binding sites of REST at these two cellular stages and show that measured binding sites of REST show a high overlap with predicted ones. Mapping H3K27me3 in stem cells and progenitor cells of wild type and REST knock out (RESTko) cells shows a specific loss of H3K27me3 at promoter-proximal REST binding sites in neuronal progenitors, validating the computational prediction. Moreover, short promoter fragments containing either REST or SNAIL binding sites are sufficient to recruit H3K27me3, whereas deletion of the respective binding sites results in a significant loss of H3K27me3. These results suggest that TFs are important contributors in the regulation of chromatin dynamics. However, further experiments are required to test if this is a general feature of TFs or a specialised role for REST and SNAIL proteins. In this context the extension of TF binding maps is crucial, as binding preferences for only 20-30% of all TFs are known at present. Extending this list, together with further perturbation experiments, will elucidate to what extent TF binding patterns can explain both changes in chromatin state as well as transcription

Metagenomic analyses reveal no differences in genes involved in cellulose degradation under different tillage treatments

Incorporation of plant litter is a frequent agricultural practice to increase nutrient availability in soil and heavily relies on the activity of cellulose degrading microorganisms. Here we address the question how different tillage treatments affect soil microbial communities and their cellulose degrading potential in a long-term agricultural experiment. To identify potential differences in microbial taxonomy and functionality, we generated six soil metagenomes of conventional (CT) and reduced (RT) tillage-treated topsoil samples, which differed in their potential extracellular cellulolytic activity as well as microbial biomass. Taxonomic analysis of metagenomic data revealed few differences between RT and CT and a dominance of Proteobacteria and Actinobacteria, whereas eukaryotic phyla were not prevalent. Prediction of cellulolytic enzymes revealed glycoside hydrolase families 1, 3, 5, 94, auxiliary activity family 8 and carbohydrate binding module 2 as the most abundant in soil. These were annotated mainly to the phyla of Proteobacteria, Actinobacteria and Bacteroidetes. These results suggest that the observed higher cellulolytic activity in RT soils can be explained by a higher microbial biomass or changed expression levels but not by shifts in the soil microbiome. Overall this study reveals stability of soil microbial communities and cellulolytic gene composition under the investigated tillage treatments

Ezh2 is required for neural crest-derived cartilage and bone formation

The emergence of craniofacial skeletal elements, and of the jaw in particular, was a crucial step in the evolution of higher vertebrates. Most facial bones and cartilage are generated during embryonic development by cranial neural crest cells, while an osteochondrogenic fate is suppressed in more posterior neural crest cells. Key players in this process are Hox genes, which suppress osteochondrogenesis in posterior neural crest derivatives. How this specific pattern of osteochondrogenic competence is achieved remains to be elucidated. Here we demonstrate that Hox gene expression and osteochondrogenesis are controlled by epigenetic mechanisms. Ezh2, which is a component of polycomb repressive complex 2 (PRC2), catalyzes trimethylation of lysine 27 in histone 3 (H3K27me3), thereby functioning as transcriptional repressor of target genes. Conditional inactivation of Ezh2 does not interfere with localization of neural crest cells to their target structures, neural development, cell cycle progression or cell survival. However, loss of Ezh2 results in massive derepression of Hox genes in neural crest cells that are usually devoid of Hox gene expression. Accordingly, craniofacial bone and cartilage formation is fully prevented in Ezh2 conditional knockout mice. Our data indicate that craniofacial skeleton formation in higher vertebrates is crucially dependent on epigenetic regulation that keeps in check inhibitors of an osteochondrogenic differentiation program

Modeling of epigenome dynamics identifies transcription factors that mediate Polycomb targeting

Although changes in chromatin are integral to transcriptional reprogramming during cellular differentiation, it is currently unclear how chromatin modifications are targeted to specific loci. To systematically identify transcription factors (TFs) that can direct chromatin changes during cell fate decisions, we model the relationship between genome-wide dynamics of chromatin marks and the local occurrence of computationally predicted TF binding sites. By applying this computational approach to a time course of Polycomb-mediated H3K27me3 marks during neuronal differentiation of murine stem cells, we identify several motifs that likely regulate the dynamics of this chromatin mark. Among these, the sites bound by REST and by the SNAIL family of TFs are predicted to transiently recruit H3K27me3 in neuronal progenitors. We validate these predictions experimentally and show that absence of REST indeed causes loss of H3K27me3 at target promoters in trans, specifically at the neuronal progenitor state. Moreover, using targeted transgenic insertion, we show that promoter fragments containing REST or SNAIL binding sites are sufficient to recruit H3K27me3 in cis, while deletion of these sites results in loss of H3K27me3. These findings illustrate that the occurrence of TF binding sites can determine chromatin dynamics. Local determination of Polycomb activity by REST and SNAIL motifs exemplifies such TF based regulation of chromatin. Furthermore, our results show that key TFs can be identified ab initio through computational modeling of epigenome data sets using a modeling approach that we make readily accessibl

Chromatin measurements reveal contributions of synthesis and decay to steady-state mRNA levels

Messenger RNA levels in eukaryotes are controlled by multiple consecutive regulatory processes, which can be classified into two layers: primary transcriptional regulation at the chromosomal level and secondary, co- and post-transcriptional regulation of the mRNA. To identify the individual contribution of these layers to steady-state RNA levels requires separate quantification. Using mouse as a model organism, we show that chromatin features are sufficient to model RNA levels but with different sensitivities in dividing versus postmitotic cells. In both cases, chromatin-derived transcription rates explain over 80% of the observed variance in measured RNA levels. Further inclusion of measurements of mRNA half-life and microRNA expression data enabled the identification of a low quantitative contribution of RNA decay by either microRNA or general differential turnover to final mRNA levels. Together, this establishes a chromatin-based quantitative model for the contribution of transcriptional and post-transcriptional processes to steady-state levels of messenger RNA