Epigenetics and Stem Cells 2012

Abcam

Highlights

The latest Abcam Epigenetics and Stem Cells conference of took place in Robinson College in Cambridge, UK on October 16-17th, 2012. Anne Rochtus from KU Leuven in Belgium was the eyes and ears for EpiGenie at this event and sent back a report on everything that went on. Check it out the full write up below:

Epigenetics and Stem Cells Summary

The different presentations covered a broad aspect of epigenetics and stem cells; from non-coding RNA regulators to epigenetic regulation in lineage decisions, the epigenetic control of development and the regulation of reprogramming. All of the speakers gave very interesting talks. Here you can find some summaries to let you share in the intriguing evolutions on the field that were discussed.

Large ncRNAs Controlling Pluripotency and Differentiation in ES Cells

Mitchell Guttman, Massachusetts Institute of Technology

Although the mammalian genome encodes many thousands of large non-coding transcripts including a class of large intergenic non-coding RNAs (lincRNAs), few have been functionally characterized, leading to debate about their biological role. Dr. Guttman and his team performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem cells (ESCs) and characterized the effects on gene expression.

They found out that the majority of lincRNA had functional consequences on overall gene expression of comparable magnitude to the known transcriptional regulators in ESCs. To determine whether lincRNA had a role in the maintenance of the pluripotency program, their effect on the expression of pluripotency markers was studied. Dozens of lincRNAs were identified that upon-loss-of-function caused an exit from the pluripotent state and dozens of lincRNAs acted to repress lineage-specific gene expression programs in ESCs. LincRNAs were integrated into the molecular circuitry of ESCs and showed that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts themselves bind to multiple chromatin regulatory proteins to affect shared gene expression programs.

The results demonstrate that lincRNAs have key roles in the circuitry controlling ESC state.

PRC2 Function in Hematopoietic Stem Cells

Marnie Blewitt, Walter and Eliza Hall Institute of Medical Research

Dr. Blewitt focused on Polycomb Repressive Comlex 2. In addition to the core PRC2 members Ezh2, Suz12 and Eed, accessory factors have been described that appear to modulate PRC2 activity and direct its binding to specific targets in ES cells. Using retroviral shRNA-mediated knockdown they examined the function of known PRC2 accessory factors in normal blood cell development by analyzing the capacity of transduced murine fetal liver cells to reconstitute irradiated recipients. They found that fetal liver cells depleted of the enzymatically inactive histone methyltransferase Jarid2 show enhanced contribution to all mature blood cell lineages compared to cells containing non-silencing control constructs, similar to the phenotype observed upon Ezh2 or Suz12 depletion. These data suggest that the enhanced activity of Jarid2 depleted cells is due to an acute increase in hematopoietic stem and progenitor cell number post Jarid2 knockdown.

TET Proteins and 5-methylcytosine Oxidation

Anjana Rao, Sanford Consortium for Regenerative Medicine

Dr. Rao talked about TET proteins. They constitute a new family of 2-oxoglytarate- and Fe(II)-dependent dioxygenases that alter the methylation status of DNA by catalyzing the successive oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) in DNA. The oxidized methylcytosines and the TET enzymes that produce them have major roles in embryonic development, stem cell properties, cellular differentiation, oncogenic transformation and neurol function.

Guo-Liang Xu, Institute of Biohemistry and Cell Biology, Shanghai

Dr. Xu found out that TET-mediated oxidation is important for DNA methylation and gene activation in the early embryo following natural fertilization, as well for the reprogramming of somatic cell nuclei during animal cloning.

Alexey Ruzov, University of Nottingham

Dr. Ruzov examined the distribution of 5-hydroxymethylcytosine (5-hmC) during embryonic brain development. Various brain cell populations start to differ in their 5-hmC content between 12.5 and 13.5 days post coitum (dpc) and this correlates with the appearance of detectable 5-carboxylcytosine, which is not found in the brain at later developmental stages. Moreover 5-caC and 5-hmC exhibit partially overlapping but not identical patterns of nuclear distribution in most of the 13.5 dpc brain cells. These data suggest that 5-hmC content is dynamically regulated in post-implantation development and its oxidation into 5-carboxylcytosine at certain genomic regions is involved in epigenetic reprogramming taking place during lineage specification in embryonic brain.

Epigenetic boundaries define Embryonic Stem Cells

Myriam Hemberger, Babraham Institute

Dr. Hemberger and her team studied the different methylation profiles of all four stem cell types of the early mouse embryo. Embryonic (ES) and epiblast (EpiSC) cells originate from the epiblast, throphoblast stem (TS) cells from the trophectoderm layer of the blastocyst and extraembryonic endoderm (XEN) stem cells from the primitive endoderm. ES and EpiSC are committed to an embryonic lineage fate; conversely TS and XEN contribute to tissues of placenta and yolk sac.

Each of these four stem cell types is defined by a unique DNA methylation profile. Despite their distinct origin, TS and XEN cells share an extraembryonic lineage signature, i.e. most notably robust DNA methylation of embryo-specific developmental regulators as well as a subordinate role of 5-hydroxymethylation, that is markedly different from that of ES en EpiSCs. Embryonic lineage-specific de novo methylation is robust and maintained in ES and EpiSC cell culture. TS and XEN acquire additional methylation in a lineage-specific pattern early during the stem cell derivation, which is stably maintained thereafter. The two extraembryonic lineages are characterized by globally similar promoter and CGI methylation profiles, satellite repeat hypomethylation and enforcement of embryo-specific gene silencing by multiple epigenetic repressive mechanisms. This shared regulation is developmentally intriguing as trophectoderm cells are the first to be set apart in the morula-to-blastocyst transition, whereas primitive endoderm cells are the last to delaminate from a common pool of cells within the inner cell mass.

Taken together Hemberger showed that global DNA methylation patterns are a defining feature of each stem cell type that underpin lineage commitment and differentiative potency of early embryo-derived stem cells.

Characterization of Mouse Haploid ES Cells

Anton Wutz & Martin Leeb, Wellcome Trust Centre for Stem Cell Research (University of Cambridge)

Most animals are diploid but haploid-only and male haploid species have been described. Diploid genomes of complex organisms limit genetic approaches in biomedical model species. To overcome this problem experimental induction of haploidy has been used in fish. In contrast to fish, haploidy is not compatible with development in mammals. Anton Wutz and Martin Leeb recently derived haploid embryonic stem cells (ESCs) from mouse embryos. Genome-wide expression analysis showed a high correlation between haploid ESCs and control diploid male ESCs. Thus, haploid ESCs largely maintain a mouse ESC transcription profile. To investigate the developmental potential of haploid ESCs, Wutz et al introduced a piggyBag transposon vector for expressing green fluorescent protein (GFP) into HAP-2 ESCs. GFP marked haploid ESCs contributed substantially to chimeric embryos when injected into C57BL/6 blastocysts. The great majority of GFP positive cells extracted from chimeric embryos had a diploid DNA content indicating that haploid ESCs contributed extensively to development after diploidization. They obtained 2 male and 2 female live born chimeras with a substantial contribution from haploid ESCs, these mice developed normally.

Taken together haploid ESCs maintain a wide differentiation potential and have an interesting developmental potential. It is interesting to speculate whether differentiated haploid lineages can be generated perhaps through suppression of X inactivation or whether a screen can be developed for detecting factors for Xist transcription.

**EpiGenie sends out a great big “Thanks” to Anne Rochtus, who is a PhD student in the Center for Molecular and Vascular Biology at KU Leuven in Belgium, for providing this conference coverage.