By holding the Chromatin:Structure and Function meeting in Aruba during the winter, Abcam made anyone holding a registration pass the envy of their labmates. Attendees came back with more than just a tan though, as some serious chromatin knowledge was exchanged as well. See what you missed in this report from Harvard’s Manching Ku, who escaped the Boston cold for a few days to cover this year’s event for us:
Abcam Chromatin: Structure and Function
After traveling 2000 miles and gaining about 50 degrees of warmth, I arrived in a tropical paradise, Aruba. I shared a short taxi ride from the airport with one of the speakers and we’re both excited about the cutting-edge scientific advances in chromatin research to come for the next few days, not to mention interacting with world renowned scientists under the sunshine and ocean breeze.
Linking RNA to Human Health and Disease
John Rinn, Broad Institute, Harvard University
John Rinn spearheaded the 4-day conference with a 50 year-history of non-coding RNA, which is one of the most discussed topics during the meeting. RNA was long believed to act solely as the transcript of DNA to be translated into protein, but large amounts of non-coding RNA have now been discovered because of noisy transcription levels in cells. In order to infer the functions of several novel lincRNAs (large intergenic noncoding RNAs), Rinn’s lab has developed a ‘guilt by association’ approach to predict potential targets of lincRNAs that are located closely in the genome. LincRNAs were found to closely regulate several pathways including cancer and adipogenesis.
In one study, the Rinn group profiled the transcriptome of different adipocyte populations, such as precursor, primary and cultured. During adipogenesis, they found that over four hundred lincRNAs are regulated specifically. Adipogenic master regulators PPARg and CEBPa are found to occupy promoters of many adipose-specific lincRNAs and induce their expression during adipogenesis. According to these data, they also carry out an RNAi-screen to validate the ability of these lincRNAs to shift cellular states and phenotypes between pre-adipocytes and mature adipocytes.
lincRNAs Function in Pluripotency and Differentiation in Embryonic Stem Cells
Mitchell Guttman, Broad Institute
Mitchell Guttman, a graduate student from Eric Lander’s lab presented on lincRNA regulation in mouse embryonic stem (ES) cells and during differentiation. He undertook the non-biased approach to knock down all ES specific lincRNAs, their effects were evaluated by the loss of Oct4-GFP reporter expression and ES-specific phenotypes. He found that knocking down majority of the ES-specific lincRNAs both up- and down-regulate global gene expression of protein coding genes, validated by multiple shRNAs. Contradictory to some reports observed by studying only a handful of lincRNAs, Guttman found that most lincRNAs regulate gene expression in trans.
Additionally, ES-specific lincRNAs regulate cell fates both by repressing other differentiation pathways and sustaining ES cell self renewal. His group also performed RIP-seq (RNA-immunoprecipitation coupled with sequencing) on ES-specific core transcription factors and chromatin associating proteins, including chromatin writers (PRC2, Eset, Setd8, Suv39h1, tip60/p400), readers (PRC1, Yy1) and erasers (Jarid1b, Jarid1c, Hdac1). Guttman proposed a model that lincRNAs can form flexible protein-interacting scaffold with specific protein complexes depending on the context of a particular cell state.
Understanding Nuclear Structure and Epigenetics at a Molecular Level
Danny Reinberg, New York University
Dr. Reinberg’s group aimed to understand the molecular mechanisms of how epigenetic changes in nuclear structure is inherited. Furthermore, this knowledge may shed light on the potential link between the aberrancies of nuclear architecture and cancer. First, the Reinberg lab is also interested in studying ncRNAs (non-coding RNAs) and their associating proteins. Polycomb group proteins (PcG) were shown to interact with several ncRNAs. They study particularly two proteins in the PcG complex, Jarid2 and Scml2. Using in vitro assays, the Reinberg group mapped the RNA-binding domains (RBDs) of Jarid2 and Scml2 to a protein domain that spans about forty amino acid residues. They generated mutants for both Jarid2 and Scml2 that lack RBDs, and found that RBD is important for the chromatin occupancy of Scml2, but not Jarid2.
In order to study specific RNAs that interact with Scml2 and Jarid2, they performed RIP-seq on these proteins and used the RBD-mutant as negative controls. They are in the process of validating potential functions of Jarid2 specific ncRNAs in ES cells self-renewal and lineage specification. Secondly, Scml2 isoforms are functionally and spatially distinct. Scml2A associates with chromatin, while Scml2B localizes in nucleoplasm. Scml2A regulates transcription by interacting with PRC1 proteins, Usp7 and directly occupancy to promoters. Scml2B regulates cell cycle by interacting with cdk/cyclin/p27 complexes.
Tet Proteins and Hydroxymethylcytosine in Transcription and DNA Methylation Fidelity
Kristine Williams, University of Copenhagen
Dr. Williams, a postdoc fellow in Dr. Kristian Helin’s group in Copenhagen presented interesting data on Tet proteins. DNA methylation has been a popular topic in epigenetic research. It has been a long mystery to search for the enzyme(s) that catalyze the demethylation of methylated DNA. Recently, there were several reports on Tet proteins and their cellular effects, because of its novel function in converting methyl-cytosine (mC) into hydroxymethylcytosine (hmC). Thus, Tet proteins serve as attractive candidate(s) for this potentially complicated, multi-step process. Tet1 was found to localize at transcription start sites (TSSs), while hmC enriched at gene regions. Opposed to the localization of mC, hmC enriches at CpG islands, suggesting that mC was specifically converted to hmC at these sites.
Another member of the Tet family of proteins is Tet2. Interestingly Tet2 lacks CpG-binding domain, which was found in Tet1, but Tet2 still associates CpG-rich promoters in mouse ES cells. Tet1 and Tet2 co-occupy many target genes and potentially regulate DNA methylation at CpG islands cooperatively.
TET-mediated Hydroxymethylation in Transcriptional Regulation
Francois Fuks, Free University of Brussels
To continue the second most popular subject during the conference on Tet proteins, Dr. Fuks was interested in finding protein interacting partners for Tet proteins, which may shed light on their functions. Using the halo-tag technology, they found that OGT glycosylase interacts strongly with Tet2 and Tet3. OGT is an enzyme that can add O-GlcNAc residue to serine and threonine amino acid residues of several target proteins. The Fuks group was interested in deciphering the epigenetic control by OGT action in cells.
Knocking down of Tet2 and Tet3 decrease O-linked glycosylation both on target proteins and globally, in an OGT dependent manner. Tet proteins and OGT also co-localize to many target genes by ChIP-Seq analyses. Dr. Fuks and his group are currently trying to understand the relationship of Tet proteins and OGT on their chromatin occupancy. Their data suggest that OGT-mediated GlcNAcylation may serve as direct mechanistic downstream for the hydroxymethylation pathways.
**EpiGenie would like to give a big “Thank you!” to Manching Ku, PhD at Massachusetts General Hospital and Broad Institute of Harvard and MIT in Boston for providing this conference coverage.