Funktionelle Untersuchung der aus Transposons entstandenen, wirbeltierspezifischen Gene Harbi1 und Naif1
Biochemie
Zusammenfassung der Projektergebnisse
Spatio-temporal coordination of eukaryotic gene expression relies on transcriptional and posttranscriptional regulatory networks. Although some of the components and function of these networks have been elucidated, the mechanisms by which such intricate circuits originate and evolve remain poorly understood. Significant fractions of eukaryotic genomes are composed of transposable elements (TEs) that are able to move and replicate in the genome. TEs are best viewed as genomic parasites with no apparent adaptive value to the host cell in which they reside. Thus, in the absence of selective pressure to maintain mobility, the vast majority of TEs has become transpositionally inactivated over evolutionary time, and has been viewed as “junk DNA” without any apparent cellular function. However, it is being increasingly realized that the spread of TEs in the genome likely played a key role in the evolution of novel gene functions. For example, through an evolutionary process termed “domestication” TE-derived transposase proteins have been recurrently recruited into cellular pathways as regulatory elements. Harbi1 and Naif1 are highly conserved genes in vertebrates that have been derived from an ancient PIF/Harbinger transposon in a common ancestor of jawed vertebrates some 500 million years ago. Conservation of these genes implies that they have been under selection for important cellular functions, and their phylogenetic relationship suggests that both are involved in the same molecular pathway. Naif1 has been implicated in apoptotic functions. Our preliminary data suggest that Naif1 is a DNA-binding protein that promotes nuclear import of Harbi1, where it may recruit Harbi1 to genomic sites through protein-protein interactions. Sequence conservation of the catalytic domain of Harbi1 suggests that it might have retained nuclease activity. However, the role(s) of these two genes in cellular homeostasis and organismal development have been enigmatic. In this project we leveraged genome-wide ChIP and transcriptional profiling in cultured cells ex vivo and phenotyping of knockout (KO) animals in the zebrafish model in vivo to understand and functionally annotate these genes and the genetic networks they regulate. By applying ChIP-Seq analysis in human cells transiently overexpressing Naif1 in the presence and absence of Harbi1 we mapped hundreds of chromosomal sites where the Harbi1/Naif1 complex interacts with chromatin. The vast majority of these sites map to the 5’ regulatory regions of genes, within 1 kb of their promoters, and show a symmetrical distribution centered around transcriptional start sites. Consistent with a transcriptional regulatory function, RNA-Seq analysis revealed genes that are differentially expressed (up- or down-regulated) in response to overexpression of Naif1. Bioinformatic analyses based on an overlay of the ChIP-Seq and RNA-Seq data established a group of genes that are bound and transcriptionally regulated by Naif1. Gene onthology analyses revealed that the overwhelming majority of these differentially regulated genes play a role in cancer and cellular signalling, including TGF-beta and Wnt signalling pathways. In situ hybridization and qRT-PCR assays in zebrafish embryos as well as in tissues of adult fish indicates maternal expression in the early embryo, followed by a predominant expression in the brain. Knockout fish generated by CRISPR/Cas9 technology that lacked either Harbi1 or Naif1 were viable and did not produce any overt phenotype, indicating auxiliary pathways compensating for the lack of these genes in animals. Overexpression of Harbi1/Naif in transgenic human cells induces DNA damage in the form of double strand DNA breaks (DSBs) as assessed by antibody stainings and apoptosis assays, consistent with the hypothesis that the domesticated, transposase-derived Harbi1 protein retains nuclease activity in human cells. As the myb-like protein is required to complement the Harbinger transposase by providing a DNA-binding function during the transposition process, so is Naif1 required for introducing DSBs by Harbi1. Our current efforts are focusing on an unbiased, genome-wide mapping of Harbi1/Naif1 cleavage sites in the human genome. Collectively, our data indicate the cellular and organismal recruitment of DNA-binding, transcriptional and nuclease functions over the evolution of Harbinger transposons in vertebrate animals.
