10th Transcription and Chromatin 2012

EMBL

Highlights

An All-star lineup of presenters, and a reputation for quality content promised to make the EMBL Transcription and Chromatin meeting one to remember. According to UC San Diego’s Sascha Duttke, who was in Germany to help us cover the event, it definitely didn’t disappoint. In fact, he had his hands full just trying to capture as much of the cutting edge research as he could. If you missed out on attending the meeting, you’ll want to give this report a read.

10th EMBL Transcription and Chromatin Conference Overview

Over 400 scientists from 38 countries gathered from the 25th – 28th of August in the marvelous city of Heidelberg, Germany. Surrounded by dark-green forest and overlooking a historic city, the charming environment of the EMBL as well as an outstanding program attracted scientists from all over the world to discuss the latest science and the future directions in the field. The main topics included: enhancer structure and function, transcriptional regulation, the basal transcription machinery, cellular differentiation and stem cell reprogramming, DNA, RNA and histone modifications as well as chromatin dynamics and the effect of chromatin on gene regulation.

In a pleasant summer climate which facilitated a stimulating and collaborative atmosphere, over 50 excellent talks and more than 200 posters were presented. Prominent investigators and students alike agreed that this was a very successful and well-organized meeting. The sheer volume of quality work made it very difficult to mention all the highlights at the meeting, and I apologize in advance to those speakers I have been unable to include.

Transcriptional and Epigenomic Foundations of Ground State Pluripotency

Hendrik Marks, NCMLS, Radboud University
Mouse embryonic stem cells (ESCs) are traditionally cultured in media containing serum + LIF. A few years ago, a defined serum-free ES cell medium has been developed that contains inhibitors of Mek and GSK3 kinases, known as “2i” (Ying et al., 2008). Cells cultured in 2i are reported be more homogeneous in morphology and in the expression of various pluripotency factors. Dr. Marks investigated the transcriptomes and epigenomes of ESCs derived and maintained in both types of media and tested the hypothesis that cells grown in 2i, as opposed to cells cultured in serum + LIF, are in a pluripotent ground state (Marks et al., 2012).

2i ESCs showed lower expression of ecto- and mesoderm linked genes and had a threefold reduction in H3K27me3 at PRC2 target sites. Paused Pol II was more prevalent in 2i. Interestingly, bisulphite genome-wide sequencing showed that DNA methylation was greatly reduced in 2i as compared to serum + LIF, mainly outside of CpG islands. The differences in transcriptome and epigenome between 2i ESCs and ESCs grown in serum + LIF were convertible by changing the media type. Both cell populations, however, showed similar differentiation dynamics suggesting both culturing condition to yield equally potent cells. This suggests that there are at least two different pluripotent ESC states, with similar developmental potential.

The Pluripotent Genome in 3D

Wouter de Laat, Hubrecht Institute
The genome of pluripotent embryonic stem cells (ESC) undergoes major structural changes during differentiation. Dr. de Laat utilized 4C, a method that combines chromosome conformation capture with next-generation sequencing to investigate all contact points of on particular sequence in the genome. The resolution of the method permitted mapping of regulatory sequences within a few kilobases and identified contact points as close as 10kb (Van de Werken, Splinter et al 2012).

Work from Dr. de Laat and others showed that inactive chromatin regions show little contacts in ESC while the genome of differentiated cells is spatially more defined. Inactive regions in neural precursor cells showed more specific contacts than ESC. Reprogramming of neural precursor cells into induced pluripotent stem cell made the genome spatially less defined. These findings are consistent with Dixon (2012) and provide further evidence for the presence of spatially unorganized inactive chromatin in ESC. Dr. de Laat also observed this phenomenon in more slowly dividing human iPS.

What causes this change in chromatin architecture? Hi-C showed preferred contacts among Nanog binding sites in ESC but not in neural precursor cells suggesting a role for pluripotency factors in shaping higher order chromatin structure.

Function of Post Translational Modification on Histones

Jürg Müller, MPI of Biochemistry
Histones are essential for viability. Nevertheless, the groups of Jürg Müller (MPI of Biochemistry, Germany)  and Alf Herzig (MPI of Biophysical Chemistry, Göttingen) took an elegant genetic and biochemical approach to further characterize the function of histones and their modifications in vivo.

Using a conditional knockout of the histone cluster and a transgene system, they were able to investigate the importance of histones, their modifications and the role of histone variants in the fruit fly Drosophila melanogaster (Gunesdogan et al., 2010). Due to its genetics, Drosophila provided an excellent model system for this study. Moreover, the developing wing disc provided a good model to analyze viability and division of cells with altered histones during development.

With this assay system, Müller, Herzig and colleagues showed that histone variants are unable to compensate for loss of the S-phase synthesized core histones. However, despite showing somewhat reduced proliferation rates, loss of the linker histone H1 permitted cells to continue to proliferate possibly because lack of H1 was compensated for by non-histone chromatin proteins. The assay system also permitted to investigate the importance of specific histone modifications. It was found that mutation of histone residues that are subject to particular modifications cause phenotypic alterations due to perturbed gene expression. Together, this study provided novel insights into the role of histones and its modifications during development.

Inducible Gene Expression by Natural and Synthetic “Histone Mimics”

Alexander Tarakhovsky, The Rockefeller University
Pathogenic or synthetic proteins with histone-like sequences (histone mimics) are potent inhibitors of inflammatory gene (Marazzi et al., 2012; Nicodeme et al., 2010). Dr. Tarakhovsky presented novel findings and a mechanism for how one such mimic, the influenza NS1 protein, can interfere with the human antiviral response via inhibiting the transcription elongation complex hPAF1C. This discovery also identified hPAF1 as a potential target for therapeutic applications.

Upon viral infection, mammalian cells recruit chromatin remodeling complexes that open up the chromatin of anti-viral gene promoters. This response specifically facilitates the expression of anti- viral genes.

Non-structural protein 1 (NS1) protein of H3N2 influenza is important for viral infection.  Like the synthetic histone mimic I-BET, the C terminal tail of NS1 contains a sequence motif that mimics the N terminus of histone H3. The histone like sequence (ARSK) can be methylated at lysine229 by histone modifying enzymes Set1C and Set7/9 or acetylated by histone acetyltransferase TIP60. Biochemical analysis showed that NS1 interacts with the hPAF1C and the hCHD1 chromatin remodeling complex. This propensity may facilitate NS1 positioning in proximity of viral induced promoters. NS1 interferes with the transcription of virus induced genes while transcription of housekeeping genes was not affected.  Repression of the virus induced transcription of anti-viral genes was hPAF1C-depenent. Moreover, depletion of the histone mimic of N1 in recombinant H3N2 influenza greatly enhanced transcription of anti-viral genes and reduced the viral infection titer. This suggests that the histone mimic in the tail of NS1 interferes with hPAF1C dependent transcriptional elongation of anti-viral response genes.

Histone Modifications, Cryptic Transcription and Expression Kinetics

Jerry Workman, The Stowers Institute for Medical Research
Jerry Workman presented his lab’s latest contributions to understanding Set2-mediated H3K36 methylation and its role in regulating histone exchange at transcribed genes. Set2 interacts with elongating Pol II and methylates H3K36  co-transcriptionally. This not only prevents histone exchange over the coding regions, but also recruits the histone deacetylase Rpd3S leading to suppression of cryptic transcripts. This suggests that co-transcriptional acetylation is a consequence of histone exchange. Probing for a mechanism to explain histone exchange suppression.

Workman’s group found the Isw1 and Chd1 chromatin remodelers to associate with H3K36me3, whereby the Isw1b complex is recruited directly by H3K36 methylation. Catalytic mutants or depletion of ISW1 and CHD1 caused widespread cryptic transcription and caused increased exchange of histones at transcribed genes. Depletion of ISW1 predominantly affected infrequently transcribed genes while CHD1 had a stronger effect on genes transcribed over ten times per hour. Depletion of both remodeling enzymes increased histone acetylation. Together, these data reveal an exciting new role for ISW1, CHD1 and chromatin structure in suppressing cryptic transcription in yeast.

 Histone Modifications, Chromatin Dynamics and Transcription

Robert Schneider, Max Planck Institute (now at IGBMC)
Modifications of histone tails are well known and were shown to play an important role in gene regulation. The role of modifications on the lateral surface of histones, however, is less known. Dr. Schneider presented a mechanism for a previously unidentified acetylation residue to function as a transcription stimulating, lateral surface chromatin mark.

This residue is located close to the pseudo-dyad of the nucleosome and its modifications have the potential to directly impact on chromatin dynamics and structure. Schneider showed that acetylation of this residue correlates with gene expression, active chromatin marks and histone variants H3.3 and H2A.Z. It also co-localizes with H3K4me and marked active and tissue specific enhancers.

Transcriptional activity of chromatin templates was enhanced by acetylation of this single residue in vitro, which was rapidly acetylated by p300 upon gene induction in vivo. Mechanistic studies showed that acetylation on this core residue decreased nucleosome stability and increased the rate of DNA dissociation, thus likely stimulating transcription via histone eviction.

Dr. Schneider’s studies show that the list of discovered histone modifications is far from complete. He also highlighted the possibility of histones to directly alter nucleosome structure through modifications of the nucleosomal core.

DNA and RNA Demethylation

Chuan He, University of Chicago
Chemical modifications of proteins and nucleic acids can have significant effects on gene expression. Dr. He presented a technique to investigate the presence of the modified base 5-hydroxymethylcytosine (5-hmC) on genome wide scale.  Tet methylcytosine dioxygenase (TET) oxidizes 5-methylcytosine (5-meC) to 5-hmC and further to 5-formylcytosine and 5-carboxylcytosine (He et al., 2011; Ito et al., 2011; Tahiliani et al., 2009). Yet both 5-meC and 5-hmC are protected from C to U conversion upon bisulfite treatment making it impossible to distinguish both DNA modifications with traditional bisulfite sequencing. This limitation can be overcome by TET-assisted bisulfite sequencing (TAB-Seq) (Yu et al., 2012).

Method: The bacteriophage T4 β-glucosyltransferase selectively glycosylates the hydroxyl group of 5-hmC.  Utilization of glucose with an azide group (N3) allowed further modification such as addition of a biotin molecule (by simple click chemistry) which facilitates enrichment. Glycosylated, and thus masked 5-hmC is, unlike 5-meC, protected from TET-mediated oxidation. Hence, 5-hmC is read as C after bisulfite treatment while C and 5-meC are read as T.

TET-Seq provided single base resolution mapping of 5-hmC sites in the mammalian genome. 5-hmC displayed an asymmetric genome wide distribution and 99% of all sites were at CG nucleotides.  It was further enriched in distal regulatory elements including p300 and CTCF binding sites as well as DNAseI hyper-sensitive sites. Dr. He also provided evidence for 5-hmC to be an active demethylation intermediate. It will thus be exciting to look forward to the functional roles of 5-hmC. TAB-Seq is commercially available from Wisegene, USA.

In the second part of his talk, Dr. He presented FTO, an obesity-associated protein with oxidative demethylation activity. Intriguingly, FTO and homologues target the abundant mRNA modification N6-methyladenosine (m6A), converting it to adenosine (Jia et al., 2011). Mammalian mRNAs carry in average three to six m6A, often in the UTR region. The discovery of RNA demethylases shows the presence of reversible RNA methylation in mammalian cells for the first time, thus providing a novel mode of biological regulation.

**EpiGenie thanks Sascha Duttke from Jim Kadonaga’s lab at University of California, San Diego for providing this conference coverage. An expanded version of this report is also available in the Landes Bioscience journal Epigenetics, October 2012.