Mechanisms targeting epigenetic states in mammals

Our group is interested in mammalian development and in particular in how genes are regulated. We focus on epigenetic mechanisms, which are about how our genes are packaged along the chromosome, to make them able to be turned on or off. In human, almost half of the genome is composed of bits of DNA that are repeated over and over again. They do not resemble the protein coding genes that are important for our cells. However, these repetitive elements have the ability to "jump" and integrate themselves all over the genome with the potential to disrupt the proper regulation of our genes. A large family of proteins, the KRAB zinc finger proteins (KZFP), have evolved to be able to silence these mobile repetitive elements by targeting repressive epigenetic states to these repeats. Thus, KZFPs are thought to have evolved in parallel with mobile repetitive elements to repress their activity.

Interestingly, one KZFP that we have been studying has been shown to target unique regions of the genome, and not just repeat elements. This has completely changed our understanding of this family, as it suggests that these proteins evolved to have more specialised functions regulating protein-coding genes. In a preliminary study, we have identified another KZFP that also targets unique regions. It is an ancient protein highly conserved in mammals, meaning that it must have been preserved during evolution and so is very likely to have an essential role in mammals. It is also expressed throughout mouse development and in most human tissues. We identified its target genes in mouse cells, and found that this is a very unusual KZFP. It seems to act in a different way than most others, and does not seem to be able to recruit the same co-factor as the others. It appears that this is not a silencing KZFP but one that does the opposite and contributes to gene activation.

Because this KZFP is so unusual and so highly conserved in mammals, we propose to investigate its functions further using mice models. We have generated mice that are mutant for the gene encoding this KZFP and hence are depleted of the protein. We will follow their development and assess what is wrong with them and impact of the mutation on their life-course. This will tell us what this protein is doing and why it has evolved its unusual properties. We will aim at understanding the action of this protein by identifying its molecular partners and the role of different parts of it.

Overall, this project will not only help us to characterize an important protein in mammals but also it will help us to gain greater insight into the evolution and function of this large family of KZFP. We believe we will learn more about how these proteins behave on the molecular level and will better understand how epigenetic mechanisms are targeted to specific unique regions of the genome.

Half of the human genome is composed of repeats including elements with the ability to retrotranspose. Retrotransposons are silenced by KRAB zinc finger proteins (KZFPs) and their co-factor KAP1, by recruiting repressive epigenetic states. KZFPs represent one of the largest families of DNA binding factors, with over 400 proteins in mouse and human. We have shown that one KZFP (ZFP57) can target not only repetitive elements but also unique regions of the genome; in particular, the differentially DNA methylated regions that regulate genomic imprints. This suggests that KZFPs acquired more diverse functions at unique portions of the genome.

We asked if other KZFPs have functions at unique regions of the genome. Our preliminary results identified a highly conserved SCAN domain-containing KZFP. Although 71 SCAN proteins have been identified in human, this domain is not well understood. This KZFP targets genes in embryonic stem cells interacting with a specific DNA binding motif. Its targets have a range of expression levels and are involved in diverse biological processes. Interestingly, target sites do not co-localise with KAP1 peaks, and most are associated with active histone marks, in particular H3K122 acetylation, which has recently been identified as novel mark at active enhancers.

We propose to characterise this unusual KZFP. We will identify its functions in vivo and in ES cells using knock-out models, and investigate cellular phenotypes, development, ageing, transcription and epigenetic states in these mutants. Biochemical analyses will be conducted to identify protein partners. Finally, we aim to determine the role of the SCAN domain and its contribution to this KZFP properties. This work will shed light on our understanding of the evolution and function of this remarkable family of proteins contributing to our knowledge of the mechanisms that target particular epigenetic modifications to unique and repetitive portions of the genome.

1. Educational impact
The group has a long-term track-record in training undergraduates and graduate students, from within and outside the UK. For this project, we will create an environment where students can be trained on different aspects of the work: mouse phenotypic analysis, cell culture experiments and bioinformatics analysis. Every year during the course of the project, we will offer a 3-month project for three undergraduate Genetics students. Not only will we teach them cutting-edge laboratory techniques, but we will also tutor them more broadly to become honest, independent, critical students. This way we believe we are preparing our future scientists for a high quality future in science. In addition, we accept summer trainees, work experience students and Erasmus and other students from abroad. On average, the lab trains 9 visitors/students per year - mostly junior trainees. During the course of this project, such individuals will become familiar with research using animal models, biochemical approaches to functional genomics and genome-wide datasets. We know from past experience that the opportunities we provide these young beneficiaries, has an impact on the science they conduct in the future.
2. Therapeutics and economics impact
Our project aims at understanding the targeting of epigenetics states to specific regions of the genome. We will make new genomics and proteomics data available to the scientific community, both for academic and industry researchers and provide novel insights into the formation of complexes that may write, read or erase the epigenetic code. We will better understand how this family of DNA-binding proteins specifically targets the genome, what epigenetic marks it recruits, the implications of this for the silencing and activation of particular regions of the genome and in gene regulation and the networks it associates with. Understanding the pathways and mechanisms by which epigenetic states are targeted is crucial, since scientific evidence clearly indicates that health across adult lifespan is programmed by genetic and epigenetic mechanisms. Thus, the project has a potential impact on improving human health by understanding the pathways affecting epigenetic sensitivity. Our work has the potential to identify novel biomarkers of particular functional chromatin states leading to the development of intervention tools against epigenetic mis-programming, or as therapeutics targets that might be modulated to control epigenetic modifications in a targeted fashion.
3. Societal impact - Public dissemination
Members of the group and particularly Anne Ferguson-Smith is regularly asked to provide information to the press and media about their work and to comment on the wider implications of epigenetics and its relation to society and the public at large. Public dissemination talks and career talks will be given at least once a year. These meetings have the potential to inform a wide audience about the state of scientific research and contribute to public awareness and science education. The PI and the postdocs have a track record in communicating the importance of epigenetics with considerable emphasis being placed on the relationship between genome and epigenome. This is a topic that really engages the public. We will continue with our public outreach activities during the course of this project.