In the second part of his talk, Dr He presented FTO, an Huck Hui Ng, from the Agency of Science, Technology obesity-associated protein with oxidative demethylation activity. and Research in Singapore, performed a siRNA library screen Intriguingly, FTO and homologs target the abundant mRNA to investigate the transcription regulatory network required for modification N6-methyladenosine (m6A), converting it to ade- Oct4 expression. With this screen, Dr Ng identified Prdm14.9 In nosine.5 Mammalian mRNAs carry in average three to six m6A, humans, Prdm14 associates with Ezh2 and is required for correct often in the UTR region. The discovery of RNA demethylases H3K27me3 levels. Prdm14-depleted cells were impaired in cel- shows, for the first time, the existence of reversible RNA methyla- lular reprogramming. tion in mammalian cells, thus providing a novel mode of biologi- To understand better the limitations in the ability of tran- cal regulation. scription factors to engage chromatin and reprogram cells, Michiel Vermeulen (UMC Utrecht, Netherlands) utilized a Abdenour Soufi and Greg Donahue, from the lab of Ken Zaret combination of SILAC and DNA pull-down6 to identify novel (University of Pennsylvania), investigated the initial chroma- 5-mC or 5-hmC interacting or repelled proteins. Dr Vermeulen tin binding events during the conversion of somatic fibroblast identified Klf4 as a novel methyl CG binding protein and showed cells to pluripotency with the Yamanaka factors OSKM. Dr that MeCP2 was also enriched among 5-hmC binding proteins. Soufi showed that Oct4, Sox2 and Klf4 act as pioneers, engag- Proteins of the CXXC family were repelled by 5-mC and 5-hmC, ing silent, DNase-resistant chromatin sites that lack known his- while other proteins, such as RFX, bound 5-mC but not 5-hmC. tone modifications, whereas c-Myc is dependent upon pioneer His findings suggest that a large number of proteins specifically factors for binding such sites. Distal enhancer sites are engaged interact with the newly discovered cytosine modifications. extensively prior to promoters, analogous to what has been seen in developmental fate choices.10 The Zaret lab also discovered Pluripotency and Cell Reprogramming large domains of the somatic cell genome that are resistant to pioneer factor binding due to heterochromatic features and a spe- Hendrik Marks, from Henk Stunnenberg’s lab at the NCMLS cific histone modification. Diminishing the latter facilitates the at Radboud University Nijmegen (Netherlands), investigated the binding and reprogramming by OSKM. These studies show how transcriptional and epigenomic foundations of ground state plu- a mechanistic understanding of transcription factor engagement ripotency. Mouse ESCs are traditionally cultured in media con- with chromatin reveals features by which cells stably maintain taining serum and the cytokine LIF. A few years ago, a defined their differentiated states. serum-free ESC medium was developed that contains inhibitors Tony Kouzarides, from the University of Cambridge (UK), of Mek and GSK3 kinases, known as “2i.”7 Cells cultured in 2i showed that Padi4, the enzyme mediating citrullination of argi- are reported to be more homogeneous in morphology and in the nine,11 also plays a role in pluripotency. Padi4 is expressed in ESCs. expression of various pluripotency factors. Dr Marks investi- Proteomic analysis shows that most proteins contain one to two gated the transcriptomes and epigenomes of ESCs derived and citrullinations, often at their active sites. Citrullinated proteins maintained in both types of media and tested the hypothesis that are involved in DNA repair, replication, transcription and chro- cells grown in 2i, as opposed to cells cultured in serum + LIF, matin remodeling. Overexpression of Padi4 induced activation are in a pluripotent ground state.8 ESCs cultured in 2i showed of stem cell-specific factors like Tcl1 or Nanog, and Padi4 was lower expression of ectoderm and mesoderm linked genes and shown to bind directly the promoters of these genes. Inhibition of showed a 3-fold reduction in H3K27me3 at PRC2 target sites. Padi4 reduced the reprogramming capacity of neural stem cells. Paused RNA polymerase II (RNAP II) was more prevalent in 2i This raises the question of how Padi4 is involved in reprogram- ESCs. Notably, bisulfite genome-wide sequencing showed that ming. Dr Kouzarides identified a citrullination site in the DNA DNA methylation was greatly reduced (mainly outside of CG binding domain of the repressive histone H1. Citrullination of islands) in 2i-cultured ESCs as compared with ESCs maintained H1 likely evicts H1 from the chromatin, thus facilitating chro- in serum + LIF. The differences in transcriptome and epigenome matin decondensation. Indeed, Padi4 was shown to control H1 between 2i-ESCs and ESCs grown in serum + LIF were revers- levels on chromatin, particularly in promoter regions. Moreover, ible by changing the media type. Both cell populations, however, Padi4 was induced upon the reprogramming of cells to iPS cells. showed similar differentiation dynamics suggesting both cultur- Histone H1, however, is not the only Padi4 substrate. ing conditions yield equally potent cells. This suggests that there are at least two different pluripotent ESC states, with similar CTD Tyrosine Phosphorylation developmental potential. and RNAP II Regulation Marc Timmers, from the University Medical Center Utrecht (Netherlands), showed that the canonical TFIID is a critical Patrick Cramer (Gene Center, University of Munich, Germany) component of the transcription network of pluripotent mouse showed that CTD tyrosine phosphorylation is important to cells. Selective Taf knockdown revealed that most Tafs, but not prevent the premature binding of RNAP II termination factors Taf7 and 8, are essential to maintain the ESC state. Intriguingly, to phosphorylated serine 2 (Ser2P). Although Ser2P gradually transient expression of Tafs greatly increased the reprogramming increases during transcriptional elongation, Ser2P-binding ter- efficiency of fibroblasts by Oct4, Sox2, Klf4 and c-Myc (OSKM) mination factors are not recruited until transcription approaches factors. Together, these data reveal a central role for TFIID in the 3'end of the transcribed region. This is because transcribing cellular reprogramming. RNAP II is phosphorylated at Tyr1 of the CTD, which stimulates 1212 Epigenetics Volume 7 issue 10