Histone deacetylase inhibition accelerates the early events of stem cell differentiation: transcriptomic and epigenetic analysis

BACKGROUND: Epigenetic mechanisms regulate gene expression patterns affecting cell function and differentiation. In this report, we examine the role of histone acetylation in gene expression regulation in mouse embryonic stem cells employing transcriptomic and epigenetic analysis. RESULTS: Embryonic stem cells treated with the histone deacetylase inhibitor Trichostatin A (TSA), undergo morphological and gene expression changes indicative of differentiation. Gene profiling utilizing Affymetrix microarrays revealed the suppression of important pluripotency factors, including Nanog, a master regulator of stem cell identity, and the activation of differentiation-related genes. Transcriptional and epigenetic changes induced after 6-12 hours of TSA treatment mimic those that appear during embryoid body differentiation. We show here that the early steps of stem cell differentiation are marked by the enhancement of bulk activatory histone modifications. At the individual gene level, we found that transcriptional reprogramming triggered by histone deacetylase inhibition correlates with rapid changes in activating K4 trimethylation and repressive K27 trimethylation of histone H3. The establishment of H3K27 trimethylation is required for stable gene suppression whereas in its absence, genes can be reactivated upon TSA removal. CONCLUSION: Our data suggest that inhibition of histone deacetylases accelerates the early events of differentiation by regulating the expression of pluripotency- and differentiation-associated genes in an opposite manner. This analysis provides information about genes that are important for embryonic stem cell function and the epigenetic mechanisms that regulate their expression

The FunGenES Database: A Genomics Resource for Mouse Embryonic Stem Cell Differentiation

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the “Functional Genomics in Embryonic Stem Cells” consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in “Expression Waves” and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells

Genetic and epigenetic analysis of gene expression during mouse embryonic stem cell differentiation

In the present study we developed a new way of mESC differentiation by treating cells with the deacetylase inhibitor, Trichostatin A (TSA). This treatment resulted in rearrangements of chromatin structure and modifications as well as changes in the expression profile. Pluripotency factors were downregulated and differentiation factors were induced. These changes are similar to those observed during differentiation via the formation of embryoid bodies (EB), but they occur within 12 hours instead of 8 days that the EB need to form. Nevertheless, the effect of TSA was not permanent since after removal of the reagent from the culture, cells were partly reversed in the undifferentiated state, restoring the initial levels of certain factors and certain histone modifications on their promoters. Combination of the above results demonstrated that genes that were transcriptionally down-regulated with simultaneous appearance of 3meK27H3 levels on their promoters, were the ones that could not restore their initial exepression levels upon TSA removal. However, even with the partial reversal, cells remained pluripotent as they were capable of inducing differentiation factors of all 3 germ layers (endoderm, mesoderm, ectoderm) during subsequent differentiation (EB). Among the genes that were severely downregulated during differentiation was sall1. Silencing of this gene resulted in downregulation of Nanog, one of the master pluripotency regulators. After in vitro and in vivo protein interaction experiments it was found that Sall1 protein could physically interact with Nanog and another master pluripotency regulator Sox2. ChIP-on-chip experiments for Nanog and Sall1 revealed that these two factors had a large number of common target genes. Finally, maintenance of Sall1 expression during differentiation prevented certain differentiation factors from being induced, showing that Sall1 plays a role in their suppression in the undifferentiated state and placing it among the factors that a have a role in pluripotency maintenance.Στην παρούσα μελέτη αναπτύχθηκε ένας νέος τρόπος διαφοροποίησης των εμβρυϊκών βλαστοκυττάρων με τη χρήση ενός αναστολέα απακετυλασών, την Τριχοστατίνη Α (TSA). Επώαση των κυττάρων με TSA προκάλεσε αναδιαμόρφωση της χρωματινικής δομής και των τροποποιήσεων των ιστονών αλλά και αλλαγές στο πρότυπο γονιδιακής έκφρασης με τους παράγοντες πλειοδυναμίας και διαφοροποίησης να καταστέλλονται και να επάγονται μεταγραφικά, αντίστοιχα. Οι αλλαγές αυτές είναι παρόμοιες με εκείνες που λαμβάνουν χώρα κατά τη διαφοροποίηση των κυττάρων μέσω του σχηματισμού των εμβρυϊκών σωματίων (ΕΒ) αλλά συμβαίνουν σε διάστημα 12 ωρών αντί 8 ημερών. Η δράση της TSA δεν ήταν μόνιμη καθώς μετά την απομάκρυνσή της από την καλλιέργεια τα κύτταρα επανήλθαν μερικώς στην αρχική κατάσταση, αποκαθιστώντας τα αρχικά επίπεδα έκφρασης ορισμένων παραγόντων και κάποιες από τις αρχικές τροποποίησεις στους αντίστοιχους υποκινητές. Συνδυασμός των παραπάνω αποτελεσμάτων έδειξε πως εκείνα τα γονίδια που κατά την καταστολή τους αποκτούσαν αυξημένα επίπεδα τριμεθυλίωσης της λυσίνης 27 στην ιστόνη 3 (3meK27H3), δεν μπορούσαν να επανέλθουν στην αρχική κατάσταση έκφρασης μετά την απομάκρυνση της TSA. Παρόλα αυτά, τα κύτταρα παρέμεναν πλειοδύναμα αφού όταν διαφοροποιήθηκαν μέσω του σχηματισμού των ΕΒ παρατηρήθηκε επαγωγή παραγόντων διαφοροποίησης και των τριών κατευθύνσεων (ενδόδερμα, μεσόδερμα, εκτόδερμα). Ανάμεσα στους παράγοντες που εμφάνισαν τη μεγαλύτερη καταστολή κατά τη διαφοροποίηση ήταν ο παράγοντας Sall1. Αποσιώπηση του γονιδίου του οδήγησε στη μεταγραφική καταστολή του nanog, ενός από τους σημαντικότερους παράγοντες πλειοδυναμίας, ενώ μετά από in vitro και in vivo πειράματα πρωτεϊνικών αλληλεπιδράσεων βρέθηκε πως αλληλεπιδρά με τους παράγοντες πλειοδυναμίας Nanog και Sox2. Μετά από ανοσοκατακρήμνιση χρωματίνης και ανάλυση μικροσυστοιχειών υποκινητών βρέθηκε πως οι παράγοντες Sall1 και Nanog εντοπίζονται στους υποκινητές κοινών γονιδίων-στόχων. Τέλος, διατήρηση της έκφρασης του Sall1 κατά τη διαφοροποίηση παρεμπόδισε την επαγωγή μεσοδερμικών και εκτοδερμικών παραγόντων διαφοροποίησης υποδηλώνοντας πως ο παράγοντας αυτός παίζει ρόλο στην καταστολή τους στην αδιαφοροποίητη κατάσταση και τοποθετώντας τον ανάμεσα στους παράγοντες που συμβάλλουν στη διατήρηση της πλειοδυναμίας των εμβρυικών βλαστοκυττάρων

Sall1 Regulates Embryonic Stem Cell Differentiation in Association with Nanog*

Sall1 is a multi-zinc finger transcription factor that regulates kidney organogenesis. It is considered to be a transcriptional repressor, preferentially localized on heterochromatin. Mutations or deletions of the human SALL1 gene are associated with the Townes-Brocks syndrome. Despite its high expression, no function was yet assigned for Sall1 in embryonic stem (ES) cells. In the present study, we show that Sall1 is expressed in a differentiation-dependent manner and physically interacts with Nanog and Sox2, two components of the core pluripotency network. Genome-wide mapping of Sall1-binding loci has identified 591 genes, 80% of which are also targeted by Nanog. A large proportion of these genes are related to self-renewal and differentiation. Sall1 positively regulates and synergizes with Nanog for gene transcriptional regulation. In addition, our data show that Sall1 suppresses the ectodermal and mesodermal differentiation. Specifically, the induction of the gastrulation markers T brachyury, Goosecoid, and Dkk1 and the neuroectodermal markers Otx2 and Hand1 was inhibited by Sall1 overexpression during embryoid body differentiation. These data demonstrate a novel role for Sall1 as a member of the transcriptional network that regulates stem cell pluripotency