After attending this year’s International Society for Stem Cell Research (ISSCR) meeting, Rutgers grad student and EpiGenie reporter Kristina Hernandez sent us such thorough coverage that it took us a while to sort through it all. Now that we have it all posted, check out Kristina’s coverage and see what you may have missed at the conference.
A short one hour flight across the U.S.-Canadian border landed me in one of the cleanest cities I have ever come across. Toronto is located on the northwestern shore of Lake Ontario and the conference was held at the Metro Toronto Convention Centre, situated a short walking distance from the shoreline. Considering Toronto’s wettest season is during the summer months we were lucky to have nice weather for the entire five day stay.
As I walked into the large convention center I was ready to immerse myself in the international cutting-edge research. A truly international vibe was readily felt as I observed many different greeting customs among the top scientists in stem cell biology. The meeting featured many of the latest findings in stem cell research and highlighted how rapidly progressing this field is today.
MicroRNAs to Pathways in Stem Cell Fate Decisions
Robert Blelloch, UC San Francisco
Robert Blelloch’s research goal is to use microRNAs to uncover the pathways in cellular processes underlying reprogramming to pluripotency. His previous research has shown that DGCR8-deficient mouse ES cells cannot silence pluripotency genes. He further demonstrated that DGCR8 knock-out cells accumulate in the G1 phase of the cell cycle.
A shortened G1 phase is thought to be required for the rapid proliferation of stem cells. The embryonic stem cell–specific cell cycle–regulating (ESCC) family of microRNAs suppresses the G1/S checkpoint in the CDK-cyclin E pathway. Whereas the ESCC family of microRNAs functions in self-renewal, the Let-7 family of microRNAs functions in differentiation. To prevent the silencing of self-renewal, ESSC microRNAs function as general inhibitors of microRNA-induced differentiation.
One target of inhibition by the ESSC microRNAs is the Rb family of proteins. However, an Rb family triple knockout did not block microRNA-induced differentiation. ESCC microRNAs must have additional targets besides the RB family. In fact, hundreds of transcripts are targeted by the ESCC microRNAs, which promote induced pluripotency in human cells. Currently, they have followed thirty-four likely targets of the ESCC microRNAs during reprogramming. Many of these targets are also down-regulated during reprogramming. In addition, knockout of individual miRNA targets partially-mimic miRNA effects on reprogramming.
The functional classification of these targets includes cell-cycle regulators, epithelial to mesenchymal transition regulators, epigenetic enzymes, which gives insight into how these processes are altered during reprogramming.
Direct Reprogramming of Somatic Cell Nuclei to Egg or Oocyte Patterns of Gene Expression
John Gurdon, University of Cambridge
John Gurdon uses somatic cell nuclear transfer to eggs and oocytes to investigate the mechanisms responsible for nuclear reprogramming and gene activation. In particular, he is using this system to identify the components of oocytes that can elicit or repress the reprogramming of somatic cell nuclei.
Stem cell genes (SOX2, OCT4, Nanog) are rapidly activated when mammalian somatic nuclei are transplanted to the germinal vesicle of Xenopus oocytes. Transplanted nuclei undergo frequent transcriptional reinitiation in oocytes and John Gurdon’s interest lie in understanding the mechanisms of this transcriptional reprogramming. Within a few hours after transplantation, the somatic linker histone H1o is replaced by an oocyte or embryo-specific linker histone B4 H1foo. The B4 histone is required for gene activation in oocytes. Using B4 dominant negatives or antibodies specific for B4, they show that SOX2 transcription is knocked out.
Another hallmark for the reversal of differentiation is DNA methylation, and in particular H3K4 histone methylation. Histone H3.3 is a transcription-related histone and is 100 times more abundant in oocytes than in somatic cells. Histone H3.3 is incorporated into transplanted nuclei 6 to 10 hours after transplantation. The exchange of H3.3 is required for reprogramming of the somatic nucleus. SOX2 transcription was measured after using an antibody for H3.3 and they observed a decrease in SOX2 expression. Oocytes are exposed to very high concentrations of oocyte-specific proteins. These proteins function in decondensing nuclear chromatin, allowing once repressed genes access to reprogramming factors.
Ubiquitin Proteasome System Regulates Self Renewal and Differentiation of Mouse ES Cells
Shannon Buckley, NYU School of Medicine
Among the many mechanisms that regulate stem cells, Dr. Buckley is interested in investigating how the ubiquitin proteasome pathway is involved in this regulation. Proteasome function is essential for mouse embryonic stem (ES) cell maintenance. Mass spectrometry identified differentially ubiquitinated proteins in self-renewing and differentiated cells. When an inhibitor of the proteasome, MG132, is used there is an increase in K48 ubiquitin chains and a decrease in OCT4 expression. In addition, multi-ubiquitinated Nanog is seen after treatment with MG132 for 6 hours. She next wanted to identify members of the ubiquitin proteasome system (UPS) that regulate stem cell fate decisions.
Her results demonstrated that members of the UPS are required for ES cell maintenance. Knockdown of UPS genes leads to morphology changes and decreased OCT4 expression. Transcriptional profiling was conducted, which revealed that members of the UPS inhibit ES cell differentiation. One of these members is Psmd14, a deubiquitinating enzyme and also a subunit of the 19S proteasome lid. During differentiation Psmd14 is down-regulated and the knockdown of Psmd14 results in differentiation and increased apoptosis.
To gain a better understanding of how Psmd14 was inhibiting ES differentiation its interacting partners were identified using tandem affinity purification coupled with mass spectrometry. Some of the interacting partners of Psmd14 identified include a number of subunits of the proteasome. Her results demonstrate how strongly involved the UPS and the function of the proteasome are in the maintenance of ES cell pluripotency and self-renewal.
The Role of DNA Methylation in Regulating Transcriptome in Mouse ES Cells
Kevin Huang, UCLA
Dr. Huang suggests that DNA methylation has other roles besides its primary role in gene repression. To study the effect DNA methylation has on the RNA transcriptome, DNA methyltransferase (DNMT) triple knock-out (TKO) mouse embryonic stem (ES) cells were generated. The genomes of TKO ES cells lack any DNA methylation and this deficiency in methylation results in global deregulation. DNA methylation acts in concert with other epigenetic mechanisms, such as OCT4/Nanog expression and histone modifications. Thirty percent of genes are not associated with histone modifications and they hypothesized that these genes are directly controlled by DNA methylation.
RNA-seq was used in control and TKO ES cells to identify genes that are differentially expressed. Genes found to be up-regulated in TKO ES cells are involved in development, specifically in neurogenesis and ectoderm development. Genes found to be down-regulated, likely due to indirect mechanisms, are linked to the immune response and metabolism. However, a number of deregulated genes found are indeed direct targets of DNA methylation. Interestingly, the pluripotency network of genes remained intact in the TKO ES cells. In addition, the microRNA landscape is globally perturbed in TKO ES cells, for example miR-290 was found to be down-regulated. His results were also able to identify genes with differential isoform expression in TKO ES cells.
Kevin Huang used the power of RNA-seq to find genes that are differentially expressed in DNA methylation mutant ES cells, and in fact this tool was able to identify more genes than microarray analysis. His results show that limiting the role of DNA methylation in regulating the RNA transcriptome of mouse ES cells to simply repression excludes the effects it has on other processes such as RNA splicing and microRNA regulation.
Tet Proteins in DNA Methylation and ES Cell Self-Renewal
Yi Zhang, University of North Carolina at Chapel Hill
The enzymes responsible for DNA methylation have been studied extensively; however, the enzymes required for active DNA demethylation have not been as strongly characterized. Yi Zhang’s talk focuses on the role of the Tet family proteins in the DNA demethylation process, and in particular how Tet1 is uniquely linked to this process. The Tet oncogene family has three members with two conserved motifs. All three Tet family members are able to hydrolyze 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), a potential step toward DNA demethylation. However, the over-expression of Tet1 and Tet2, but not Tet3 reduces 5mC levels. Only Tet1 and Tet2 have been linked to leukemia.
Dr. Zhang used shRNA against Tet1 and found Tet1 to be required for mouse embryonic stem (ES) cell maintenance. Tet1 knock-outs cause self-renewal defects, preferential differentiation into trophectoderm lineages, and a decrease in Nanog expression. To be noted, the over-expression of Nanog alone was not sufficient to totally rescue the phenotype. Tet1 is found to be enriched in protein coding regions. In addition, Tet1 is highly enriched in CpG-rich promoters and Tet1 binding is inversely correlated to 5mC levels in mouse ES cells. Using microarray analysis, he found that Tet1 both positively and negatively regulates its targets.
Tet1 activates pluripotency genes and silences genes that are important for differentiation. Knowing this, he next wanted to investigate the relationship between Tet1 and the Polycomb Repressive Complex 2 (PRC2). Amazingly, 90% of PRC2-bound genes are also bound by Tet1. Using an in vitro chromatin binding assay, he found that Tet1 facilitates PRC2 recruitment indirectly through maintaining a DNA hypomethylated state. He finished his talk by discussing the role Tet1 plays in mouse embyrogenesis. Tet1 knock-out mice were made, but not all of these mice were able to survive. He also showed data that Tet1 is specifically expressed in primordial germ cells (PGCs) during their migration and a Tet1 deficiency impairs PGC development.
**EpiGenie would like to thank Kristina Hernandez, who is a grad student in the Firestein lab, Cell Biology and Neuroscience department at Rutgers University for providing coverage of this conference.