KRAB zinc finger protein-mediated control of transposon-embedded regulatory sequences from human germline to early embryos

Alexandra Iouranova
EPFL, 2021
EPFL thesis

Abstract

Transposable elements (TEs), also called jumping genes, are genetic elements capable of changing their position within the genome of their host. They make up large fractions of genomes, including 45% of human DNA content, according to current estimates.

Some of these elements are of retroviral origin and are known as endogenous retroviruses (ERVs). Long terminal repeats (LTRs) of ERVs, like other TEs, influence host gene expression in various ways. They can for example create new promoters, enhancers or imprinted regions upon integration. As ERVs derive from a germline infection in the common ancestor of a species, they are often restricted to a particular evolutionary clade.

While rewiring by TEs can harm a host genome, some of the features of TE invasions can increase host fitness and appear to be universally exploited by various organisms. For instance, TE insertions happen faster than protein-coding genes evolve. They can therefore be coopted for a species-specific evolutionary remodeling of transcriptional networks. Specifically, the LTR12/ERV9 TE family has colonized primate genomes over the past 30 million years and has been widely reported to contribute regulatory elements to the human genome.

In this work, we set out to establish the features that made this family successful, and to assess the prevalence of LTR12-derived regulatory elements in early development. We observe the hominoid-restricted LTR12C subfamily to be a driver of transcription in both gametogenesis and early embryogenesis. LTR12C loci have rewired genes involved in ciliogenesis and flagellogenesis that are critical for male fertility. Relatedly, they also present unusual behavior during the epigenetic reprogramming characteristic of these periods by resisting genome-wide demethylation in primordial germ cell (PGC) development.

To identify the determinants of the epigenetic status of LTR12C elements that made them attractive for cooption, we turn to KRAB zinc finger proteins (KZFPs). TEs appear to be prominent targets of this large family of epigenetic repressors.

Through functional studies, we determine ZNF676 is a regulator of LTR12C, with its targets resisting genome-wide demethylation. This resistance possibly pre-marks them for zygotic rather than maternal expression. We show that ZNF676 levels influence the expression of some cilium-related genes and are able to disrupt ciliogenesis.

We conclude this KZFP likely coevolved with its target ERVs, which carried its binding sites into the vicinity of multiple genes, including those important for male fertility. KZFPs can therefore become regulators of genes with a common function along with the transposable elements they target.

More generally, in our study of TEs expressed in human embryonic development, we find the expression of some TE families to be a hallmark of a particular development stage, an indicator of the current chromatin state. These markers provide a criterion that can be used to assess the similarity of embryonic development models to real embryos. Transcription of some marker TEs anticorrelates with that of their putative KZFP regulators, implying proper KZFP expression to be important to properly recapitulate embryonic development.

Our results provide insight into the TE- and KZFP-mediated rewiring of transcriptional networks related to the germline and early development, and suggest that the improper expression of a KZFP can have consequences ranging up to infertility.

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Alexandra Iouranova
EPFL, 2021
EPFL thesis

Abstract

Official source

https://infoscience.epfl.ch/record/286266?ln=en

About this result

Related concepts (32)

Related concepts

Related publications (114)

Related publications

KRAB zinc-finger proteins contribute to the evolution of gene regulatory networks

Pierre-Yves Joseph Laurent Helleboid, Michaël Imbeault, Didier Trono

The human genome encodes some 350 Kruppel-associated box (KRAB) domain-containing zinc-finger proteins (KZFPs), the products of a rapidly evolving gene family that has been traced back to early tetrapods(1,2). The function of most KZFPs is unknown, but a few have been demonstrated to repress transposable elements in embryonic stem (ES) cells by recruiting the transcriptional regulator TRIM28 and associated mediators of histone H3 Lys9 trimethylation (H3K9me3)-dependent heterochromatin formation and DNA methylation(3-9). Depletion of TRIM28 in human or mouse ES cells triggers the upregulation of a broad range of transposable elements(4,10,11), and recent data based on a few specific examples have pointed to an arms race between hosts and transposable elements as an important driver of KZFP gene selection(5). Here, to obtain a global view of this phenomenon, we combined phylogenetic and genomic studies to investigate the evolutionary emergence of KZFP genes in vertebrates and to identify their targets in the human genome. First, we unexpectedly reassigned the root of the family to a common ancestor of coelacanths and tetrapods. Second, although we confirmed that the majority of KZFPs bind transposable elements and pinpoint cases of ongoing co-evolution, we found that most of their transposable element targets have lost all transposition potential. Third, by examining the interplay between human KZFPs and other transcriptional modulators, we obtained evidence that KZFPs exploit evolutionarily conserved fragments of transposable elements as regulatory platforms long after the arms race against these genetic invaders has ended. Together, our results demonstrate that KZFPs partner with transposable elements to build a largely species-restricted layer of epigenetic regulation.

Nature Publishing Group2017

There are 377 Krüppel-associated box (KRAB) domain-containing zinc finger proteins (KZFPs) in the human genome, making them the largest family of transcription factors. KZFPs are defined by a N-terminal KRAB domain and several zinc-finger domains arranged in an array at the C-terminus of the proteins. The zinc-finger domains each form sequence specific interactions with double stranded DNA, allowing the zinc-finger array to target specific genomic sequences. The KRAB domain, through its interaction with the protein TRIM28 (also known as KAP1) allows for the stable silencing of transcription in a genomic region. Together these two domains allow KZFPs to bind specific regions of the genome and generally lead to the formation of heterochromatin and silencing of transcription allthough other functions for some KZFPs have been recently reported. KZFPs usually target transposable elements (TEs), mobile genomic elements that can move through the genome either by cut-and-paste or copy-paste mechanisms and make up almost half of the human genome. Different KZFPs bind to specific regions of TEs, limiting their expression and thus mitigating some of the threat they pose to human development, allowing both the TE and its host to survive. In recent years it became apparent that both certain TEs and KZFPs have roles beyond the previously described threat and mitigation of that threat. TEs harbor gene regulatory regions allowing, through their spread, for a dissemination of those regions through the genome and for new gene regulatory networks to form. KZFPs can affect the transcription of genes located in proximity to their binding sites leading to changes in gene regulation as well. A multitude of regulatory roles involving KZFPs and TEs have been described in recent years making them an exciting field of study. A major hurdle in understanding both KZFPs and TEs is to identify the binding sites of KZFPs, as they reveal both the targets of the KZFP and how, if at all, a TE is regulated by KZFPs. To do so, we expanded on previous efforts and aimed to perform chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on every member of the human KZFP family. Here we present ChIP-seq experiments for 110 new KZFPs, which together with previously published data, allow us to study the binding of almost all human KZFPs (95%). The entirety of the data was analysed together to generate a coherent dataset and results for each KZFP are made available to the public on our web portal (https://tronoapps.epfl.ch/web/krabopedia/). The identified binding sites allowed us to witness the adaptation of the KZFP family to new TEs and showed how targeted sequences shift after segmental gene duplication events. Furthermore, we could corroborate that several KZFPs target the same TE subfamilies in a seemingly redundant fashion and show that unexpectedly these KZFPs arose independently in different genomic locations. These results represent a valuable tool for anyone studying KZFPs and should aid in the pursuit of both understanding KZFPs and TEs.

EPFL2022

The Interplay between Cytidine Deaminases, HIV and Domesticated Genetic Parasites

"There is more virus in us than us in us". John Coffin's famous sentence illustrates that particular nucleic acid sequences related to exogenous viruses, called retrotransposons, constitute almost half of the human genome and largely exceeds the amount of protein-coding DNA (section 1.4). At the beginning of the past century, biologists realized the size of the genomes of various eukaryotes did not correlate with the level of complexity of these species. In extreme cases, certain species such as the fish L.paradoxa had 30-times more genomic DNA than do primates. In 1980, Francis Crick and Carmen Sapienza independently postulated that part of this once-called "junk DNA" must stem from self-replicating units, categorized as "selfish DNA" to distinguish them from more "altruistic" cellular genes. Indeed, while genes amplify in a population because of the selective advantage they confer to their host, the selfish DNA amplifies without making any contribution to the host phenotype and survives rather as a not-too-deleterious parasite. In early 2000's, the accomplishment of the human genome sequencing projects gave formal credence to Crick's and Sapienza's initial hypothesis. Retrotransposons replicate in the genome of their host by a copy-and-paste mechanism. Accordingly, any new retrotransposition event causes a genetic alteration potentially detrimental to the host. On an evolutionary perspective however, accumulation of retrotransposon-derived sequences can be beneficial to the species after these once "selfish" elements become "domesticated" to deserve more physiological roles. In human or mouse for instance, retrotransposons are known to cover a large spectrum of cellular functions, from gene regulation to antiviral defense (section 1.6). In order to maintain under tight control the balance between the good and adverse effects of retroelements, eukaryotes have evolved cellular defenses aimed at inhibiting the replication of such elements (section 1.7). Gene silencing by DNA methylation is a mechanism widely used in plants, fungi or vertebrates to control the expression of self-replicating elements. For instance in mammals, specific KRAB-zinc finger proteins can target retroviral-derived sequences and mediate their transcriptional silencing (section 3). The first aim of this study was to characterize the sensitivity of retroviruses to KRAB-mediated epigenetic silencing in varied chromosomal contexts (section 5). Since some retroviruses like HIV favor integration within transcriptionally active regions, these viruses may escape blockade by integrating chromosomal area refractory to epigenetic silencing. We found that instead, KRAB-mediated repression mechanism was not dependent upon integration site of a retroviral vector, hence the emergence of viruses escaping KRAB-mediated repression would be unlikely. Mammalian cells are also able to control the replication of retrotransposons at a post-transcriptional level via the APOBEC3 family of cytidine deaminases (section 2). Since certain retrotransposons are related to exogenous retroviruses, it is perhaps not surprising that APOBEC3 proteins can also inhibit a range of such elements including HIV. While there is only one APOBEC3 gene in the mouse genome, the family considerably expanded in other species such as primates were it comprises seven members (section 2.1). Although it is tempting to postulate that the expansion of the family in primates was driven by the strong selective pressure imposed by various genetic threats growing in number and complexity, it is not understood how only a few homologous proteins can target such a disparate group of replicating elements. The second aim of this study was to understand the mechanism by which different APOBEC3 proteins can target and inactivate particular self-perpetuating elements (section 4). We launched an integrative study focused on two human APOBEC3 homologues with distinct antiviral functions. APOBEC3A, a single-domain cytidine deaminase, can block efficiently the parvovirus adeno-associated virus type 2 (AAV-2) or the retrotransposon LINE-1 but cannot inhibit HIV replication. APOBEC3G is formed of two independent cytidine deaminase domains, only the C-terminal of which is catalytically active. APOBEC3G is endowed with restriction activity against HIV, but is largely ineffective against AAV-2 or LINE-1. We combined functional studies with structural modeling and phylogenetic analyses and found that restriction in each case involved similar regions at the surface of the protein and nearby the catalytic center, yet with distinct shapes and nucleic-acid interacting properties between the homologous proteins. While on APOBEC3G, the amino-acids in this region mediate intersubunit contacts between monomers and the predicted dimer interface shapes a groove that may facilitate binding to single-stranded RNA, on APOBEC3A however, it is corresponding residues on the monomer that form a DNA-binding groove important for editing. In summary, this study describes the nucleic-acid-interacting domains of APOBEC3A and APOBEC3G essential for their intrinsic restriction activities, and further suggests that structural variations in this domain may contribute to define the sets of targets inhibited by each APOBEC3 protein. APOBEC3 proteins may be considered as modular units, whose restriction function can evolve with emerging selective pressures by specific variations in their nucleic-acid binding domain, alternatively by fusion of two APOBEC3 proteins endowed with different functionalities. APOBEC3 proteins would efficiently protect the genome integrity of their host against highly variable genetic threats or "selfish DNA", in combination with other restriction factors such as the KRAB-zinc finger proteins. Contrary to epigenetic silencing however, post-transcriptional restriction as provided by APOBEC3 proteins may prohibit the deleterious effect of retrotransposition, without altering other retroelement functions putatively important for the physiology of the cell.

EPFL2010

EPFL2022

The Interplay between Cytidine Deaminases, HIV and Domesticated Genetic Parasites

EPFL2010