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.

Genomic targets and molecular partners of the chromatin regulator KAP1

Annamaria Kauzlaric

KAP1 is an enigmatic regulatory protein, first described some twenty years ago, shown to be involved in multiple and diverse cellular functions. Specifically, it mediates tasks critical to cell growth and differentiation, pluripotency, apoptosis, gene silencing, DNA repair and genomic integrity maintenance. In line with these findings, Kap1 deletion results in embryonic lethality, and its conditional ablation impairs crucial processes such as erythropoiesis, hepatic metabolism homeostasis and stress response. Initially named also TIF1ðœ, transcriptional intermediary factor 1ðœ, KAP1 was thought to mediate mostly chromatin-related functions. Accordingly, global maps of genomic recruitment of KAP1, generated with the advent of high-throughput technologies, display binding of this protein to thousands of loci in a multiplicity of cell types. Coupling such maps with data of transcriptional perturbations detected upon Kap1 depletion, and with the protein complexes associated with KAP1, proved to be a highly efficient approach to elucidate the molecular functions of KAP1. In particular, such studies unveiled the mechanisms of KAP1-mediated repression of different classes of transposable elements (TEs), and led to important evolutionary considerations on the strategies evolved by higher organisms to restrict the activity of these entities. Through its multiple functional domains, KAP1 interacts with a broad range of proteins, and our first goal was to establish the complete set of such potential cofactors. We found marked enrichment of KAP1 in the chromatin fraction of the nucleus, and accordingly, among its associated proteins, a strong preponderance of nucleic-acid contacting proteins. We revealed that KAP1 is part of a variety of relatively low molecular weight complexes, co-existing at comparable abundances. Study of the molecular functions of the hits suggested that KAP1 could partake in processes thus far unexplored in this context, such as DNA replication and active transcription. We then pursued with the thorough characterization of genome-wide KAP1 binding sites, and found significant fractions of RNA polymerase II (PolII)-dependent promoters and RNA polymerase III (PolIII)-transcribed elements, for the majority enriched with euchromatic marks, to be targeted by KAP1. Using high-throughput techniques, we uncovered that KAP1 variably affected the PolII pausing index of defined sets of genes, and downregulated transcription of tRNA genes. Our analyses of KAP1 genomic recruitments further led us to the discovery of novel genetic entities in the mouse genome, highly related to KRAB zinc finger protein (KZFP) genes. We described possible mechanisms underlying the evolution and amplification of these sequences and more broadly of the KZFP genes family. Furthermore, our data supported the existence of a KAP1-dependent regulatory circuit taming TE-contained enhancers located in the vicinity of these genetic units, thereby regulating their expression. We explored the proposed models of KAP1 functions in euchromatic regions of the genome, and with our results we provided the most complete analysis of the interactome of KAP1 thus far reported. Moreover, we annotated new potentially protein-coding transcripts belonging to the biggest single family of transcription factors encoded in the murine and human genomes. In sum, this work brings novel pieces of information that complement the current knowledge in the field, opening the way to further molecular studies clarifying the physiological roles of KAP1 in higher vertebrates.

EPFL2017

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

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

Pierre-Yves Joseph Laurent Helleboid

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.