The impact of TE-derived sequences on human pluripotency
Dr. Jichang Wang
Most of TEs and TE-derived sequences are transcriptionally inactive in somatic cells. However, due to the DNA de-methylation, some families of TEs can be transcriptionally reactivated during early development. Recent studies show that a family of retrotransposon, ERVL is uniquely reactivated in 2-cell embryo stage in mice, and might be associated with the totipotency. These studies indicate that some families of retroelements, such as HERVH might have roles in the acquisition and maintenance of pluripotency. Using the human pluripotent stem cells as models, our main interest is to decipher the potential biological functions of TEs in human pluripotency ().
Uncover the role of TFCP2 in human pluripotency using transposon based inducible Cas9 system
Our previous study has proved LBP9 contributed to pluripotency by driving human specific Human endogenous retrovirus subfamily H (HERVH) transcription. Belonging to the same transcription family as TFCP2L1, TFCP2 share the same structure and conserved sequences with LBP9. Both CP2 family and the evolutionally closed GRHL family involved in development. In my study, I explored the role of TFCP2 in human pluripotency. Transcription factors that regulate pluripotency may have distinct functions in different status of hESCs or hESCs-derived cells. To study the genes in a temporal manner, it is useful to generate conditional knockout model in hESCs. Piggybac and Sleeping Beauty are widely used non-viral transposon systems in mammalian cells. Sleeping Beauty has been used in clinical research because it supports stable and high efficient gene transfer in many cell lines. Importantly, Sleeping beauty transposon generated a close to random insertion pattern thus proved to be a safe gene transfer tool compared to viral vector and Piggybac. By controlling the amount of transposon and transposase, low copy integrants can be obtained using Piggybac transposon. The integrants with genomic safe harbor locus can be screened out by Splinkerette PCR. In my study, I generated doxycycline-inducible Cas9 expressing cell line in commonly used H1 and H9 hESCs cell line. Importantly, we identified low copy integrants at genomic safe harbor loci, yet supporting robust Cas9 expression without leaky effect. A GFP-tagged gRNA containing vector allows enrichment for gRNA expressing cells thus further increased the knockout efficiency.
Cross-talk of ERVs and host factors in re-wiring transcriptional networks in primates
Transcriptional expression of repetitive elements originating from endogenous retroviruses (ERVs) in human cell-types is tightly controlled by host factors. Despite systematically expressed, they are differentially expressed during different stages of early embryogenesis. Host genome and transcriptome has been modified by multiple waves of retroviral insertions. We investigate the cross-talk between host-factors and ERVs during primate evolution (collaboration with L. D. Hurst, University of Bath, UK) and in various human cell lines. Analysis of our data and recently published datasets enables us to look into the complexity of regulatory networks and the possible involvement of spatio-temporal expression and repression of retroelements in biological processes. A second major area of our interest involves chromatin regulation in embryonic stem cells (ESCs), molecular basis of naive and primed pluripotency and role of transposed elements in cell fate decisions.
Decipering the function of domesticated TE-derived sequences in the human genome
Dr. Tamás Raskó and Dr. Attila Szvetnik
Summary description of the piggyBac projects
Although transposable elements (TEs) are considered as selfish creatures, inactivated TEs are frequently used as building boxes to generate novel cellular functions for the benefit of the host-organism. In the human genome, five domesticated genes (piggyBac transposable element derived 1-5, PGBD1-5) exist, originated from the piggyBac transposon. While the function of PGBD3 and PGBD5 are partially characterized, only little is known about the other three PGBD genes. PGBD1 has been identified in two independent GWAS studies, as a susceptibility locus for schizophrenia. Our preliminary studies indicate that PGBD1 is associated with cellular processes including, protein folding, stress response, oxidative stress, cell survival and differentiation. Remarkably, altogether five protein interacting partners of PGBD1 are also indicated in schizophrenia susceptibility. We also try to uncover the molecular function of PGBD4, one of the youngest human DNA element. Based on our preliminary results PGBD4 seems to be involved in the regulation of RNA splicing.
In these projects, we aim at characterizing the cellular function of domesticated PGBD1 and PGBD4, and deciphering their role in the manifestation of neurological diseases both in vitro and in vivo, by combining genetic and systems biological approaches.
Elucidating the role of transposable element chimeric genes in human pluripotency
Dr. Christine Römer
The modern human genome is in 8% comprised of endogenous retroviruses. These repetitive sequences regulate gene expression and give rise to novel chimeric genes. One of these chimeric genes is the human embryonic stem cell related gene (ESRG), which has become essential for human pluripotency acquisition and maintenance. We aim at elucidating the protein-coding ability of the ESRG gene and the mechanisms through which it regulates pluripotency. Transposable element chimeric genes give also rise to alternative RNA forms, such as circular RNAs. In collaboration with Prof. Irmtraud Margret Meyer we will identify transposable element derived circular RNAs in human pluripotent stem cells and investigate their functional relevance.
Deciphering the function of an evolutionary conserved medium reiterated frequency repeat (MER)
PhD student Vaishnavi Raghunathan
TEs make the most significant components of both prokaryotic and eukaryotic genomes, and have been widely regarded as molecular parasites owing to their potential to give rise to a variety of genetic alterations. These alterations might interrupt gene function. Alternatively, TEs could make themselves useful to the host genome (domestication). Due to their mobile nature, TEs have likely played a key role in distributing non-coding, regulatory elements in the vertebrate genome. Medium reiterated frequency repeats-6 (MER6), are a class of DNA transposons belonging to Tc1-Mariner-Tigger family of interspersed repeats. We propose to identify the potential impact of the evolutionary conserved MER6.