Beyond silence : a functional and evolutionary study of KRAB zinc finger proteins

Transposable elements (TEs) are genetic units capable of spreading within the genomes of their host. TEs contribute a readily recognizable 45% of the human DNA, reflecting in part their co-option for some as source of protein-coding sequences, for others as regulators of genome architecture and expression. Nevertheless, new TE integrants can occasionally cause diseases. The KZFP/KAP1 (KRAB-Zinc Finger Protein /KRAB-associated Protein 1) system plays a major role in the transcriptional control of TEs. Encoded in the hundreds by the genomes of most higher vertebrates, KZFPs are endowed with a N-terminal KRAB (KruÌppel- associated box) domain, some of which can recruit KAP1, and a C-terminal array of zinc fingers (ZNF) with polynucleotide binding potential. When we started this work, KZFP genes were thought to be restricted to tetrapods, and a few of their products had been found to tether KAP1 and associated heterochromatin-inducing factors to TE-derived sequences during mouse or human early embryogenesis, resulting in the deposition at these loci of H3K9me3 (histone 3 trimethylated on lysine 9) and DNA methylation, two repressive marks. Additionally, the KZFP/KAP1 system had been implicated in events as diverse as imprinting, cell differentiation and metabolic homeostasis. In order to gain insight into the biology of human KZFPs, we first performed a large-scale identification of their genomic targets. We found that the majority was enriched at TEs, where they bound to regions also recognized by other TFs. Accordingly, many KZFP-targeted TE loci displayed differential chromatin patterns in human tissues, consistent notably with their putative role of cell-specific enhancers. This strongly suggests that KZFPs participate in orchestrating TE-modulated regulatory networks. Finally, we could trace KZFP genes to a common ancestor of the coelacanth, lungfishes and tetrapods, and determined that their evolutionary emergence was most often contemporaneous to that of their TE targets, with signs supporting both an arms race and a domestication model. To pursue this exploration, we analyzed the protein partners of more than a hundred human KZFPs. We could observe that the interactome of evolutionarily recent KZFPs mainly comprised KAP1 and associated effectors, whereas that of more ancient family members, conserved from marsupials to sauropsids, tended not to associate with KAP1 and were likely to engage in interactions with more original factors, including proteins related to RNA processing or genome architecture. While these results suggested that KAP1 binding might have been a late emerging property of KZFPs, we could demonstrate that KRAB domains derived from Latimeria chalumnae associated with the cognate KAP1 protein, albeit not with its human orthologue. These results support a model whereby KZFPs first emerged as TE- controlling repressors, were continuously renewed by turnover of their hostsâ TE loads, and occasionally produced derivatives that escaped this evolutionary flushing by development and exaptation of novel functions. In sum, this work provides important insights into the evolutionary history and biological function of KRAB zinc finger proteins, the largest family of transcriptional regulators encoded by human and other higher vertebrates.

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

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

KRAB'n'KAP, DNA methylation and epigenetic memory

Andrea Coluccio

The KZFP/KAP1 (Krüppel associated box zinc finger proteins/KRAB-associated protein 1) complex is a transcriptional repressor that triggers the deposition of heterochromatin (H3K9me3) and DNA methylation. The main targets of the KZFP/KAP1 complex are transposable elements (TEs), which are generally maintained transcriptionally inactive to avoid retrotransposition and preserve genomic stability, and imprinting control regions (ICRs), where DNA methylation is protected to ensure proper parent-of-origin specific expression of crucial developmental genes. The role of the KZFP/KAP1 system in forming a heterochromatic environment is of particular relevance during the wave of genome-wide epigenetic reprogramming that takes place in the early embryo between fertilization and implantation. During these stages, TEs and ICRs are amongst the few sequences that retain the epigenetic information inherited from the gametes. While the importance of KZFP/KAP1 in maintenance of imprinting and TE silencing is well established, the mechanisms by which KAP1 counteracts demethylation and interacts with this epigenetically unstable environment to regulate TEs are still largely unknown. In this work, we uncover the complex interplay between KAP1, DNA methylation and active demethylation in maintaining imprinting and regulating TE expression in naïve embryonic stem cells. We first confirmed that KAP1 was able to bind and repress TEs in absence of DNA methylation. We then demonstrated that the KZFP/KAP1 complex was required to maintain heterochromatin and DNA methylation at ICRs and TEs, and was able to protect these sequences from active demethylation. We comprehensively characterized the responsiveness of TEs to removal of KAP1 in absence of DNA methylation and active demethylation, which unveiled a complex, multi-layered and integrant-specific regulation for these elements. ZFP57 is known to recruit KAP1 on ICRs, but here we identified ZNF445 as a second KZFP that specifically binds ICRs both in human and mouse. We found that ZNF445 regulates imprinting differentially in human versus murine embryonic stem cells. Our findings suggest for the first time that two different KZFPs could have evolved with species-specific roles in imprinting regulation. The high degree of conservation of ZNF445 in the human population argues in favour of a previously unsuspected critical role for this protein in development. This work provides useful insights in the role of KAP1 and its KZFPs partners both in preserving epigenetic memory and in transposon silencing during early development.

EPFL2017

The Interplay between Cytidine Deaminases, HIV and Domesticated Genetic Parasites

EPFL2010

KRAB'n'KAP, DNA methylation and epigenetic memory

Andrea Coluccio

EPFL2017