Genomic targets and molecular partners of the chromatin regulator KAP1

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.

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

Phase specific transcriptional regulation of circadian clock and metabolism in mouse liver

Jonathan Aryeh Sobel

The molecular clock has been conserved from cyanobacteria to mammals and is believed to align behavioral and biochemical processes with the diurnal cycle. This cellular mechanism has been an advantage to increase the fitness of organisms through the ability to anticipate food availability or predator presence. Accumulating evidence has revealed that the circadian clock is intimately interconnected with the metabolic cycle. But, the nature of these interconnections is yet not clear at the transcriptional level, and the contribution of cis-regulatory modules has not been elucidated. Accessible chromatin regions of the genome are implicated in diverse processes, such as gene regulation through enhancers and promoters, insulation of genomic domains or alternative splicing. They allow cell-type specific programs that are controlled by tissue-specific transcription factors and chromatin modifiers, although tissue-specific regulation of the circadian clock remains unclear. Therefore, we compared genome-wide BMAL1 binding and chromatin accessibility in the liver and in NIH3T3 fibroblasts. Moreover, for the first time, our study explores the dynamics of accessible regions, histone 3 lysine 27 acetylation (H3K27ac) and RNA polymerase II (Pol II) every 4 hours over one day in mouse liver. In this study, we show that a substantial fraction of these accessible sites are oscillating during a diurnal cycle with a circadian period. We observed that these accessible regions are enriched in the proximity of actively transcribed genes, and that they are dynamically affected by the binding of transcription factors such as BMAL1. We investigated wild-type (WT) and Bmal1-/- genotypes, in night restricted feeding regimen and in Light-Dark (LD) cycle, to study the circadian clock regulatory network underlying diurnal transcription. Therefore, we applied a penalized generalized linear model to infer the activity of transcription factor binding motifs in oscillating accessible sites using Pol II loadings at the transcription start sites of nearby genes. We were able to recapitulate the known regulatory elements of the circadian clock, notably E-box, D-Box, and ROR-responsive elements (RRE) in the WT genotype. On the other hand, we found that Forkhead box (FOX), glucocorticoids responsive elements (GRE), and C-AMP Response Element (CRE) were the main contributors of the regulation of oscillating genes in Bmal1-/-. Finally, using a mixture model to detect footprints with a base pair resolution, we studied the dynamics of the accessibility overlapping E-box. Our last analysis suggested that BMAL1/CLOCK is binding on double E-boxes with a spacer of 6 or 7 bp in a hetero-tetramer configuration. A 3D structure model further supported this binding mode. In Summary, we used DNase I-seq and ChIP-seq of Pol II and H3K27ac to study the circadian chromatin landscape in a 4h time-resolved experimental design. We uncovered the underlying circadian transcriptional regulatory network and, we dissected the chromatin accessibility around BMAL1 binding sites at a base pair resolution, which led to an unappreciated mode of binding of BMAL1/CLOCK in a hetero-tetramer conformation on double E-boxes. Lastly, we found tissue-specific factors that might contribute to tissue-specific binding of BMAL1/CLOCK.

EPFL2016

Rhythmic and clock-dependent chromatin loops regulate circadian gene expression

Jérôme Mermet

In mammals the circadian clock drives daily behavioural and physiological changes that resonate with environmental cues, which can be observed, for example, in the intricate timing of rest during the night and activity during the day in humans. The circadian clock consists of a network of hierarchical clocks in which the central pacemaker is located in the suprachiasmatic nucleus, which is sensitive to external light and propagates time to the peripheral organ-based clocks. Within organs, clocks tick in each individual cell and are molecularly encoded in the genome as a network of dynamic feedback loops. By mediating the expression of the appropriate gene products at the correct moment of the day, the molecular clocks op-timize physiological functions of virtually every cell in our body, allowing us to synchronize with daily fluc-tuations in the environment. In mammalian cells the chromatin organization in the nucleus consists of a topological network, in which chromatin interactions between distal regulatory elements such as enhancers and target genes are essen-tial to regulate gene expression. Important questions remain: is the complex chromatin folding dynamic and if so on which genomic and temporal scales? In fact, dynamic rearrangements of the chromatin have been reported on multiple genomic and time scales, from the entire genome to enhancer-promoter con-tacts, across developmental transitions, cellular differentiation, and transcription cycles. With this per-spective in mind, the circadian oscillator provides a unique model to study the dynamics of genomic inter-actions in the context of fluctuating transcription. Here, we explored chromatin contacts of core-clock and clock-controlled gene promoters in the mouse. We aimed at evaluating the putative dynamics of chromatin contacts, at estimating their conservation across tissues, and at exploring the contribution of the circadian clock to this interaction network. To achieve this goal, we performed 4C-sequencing assays at two circadian time points in the liver and kidney of WT and clock-deficient animals. Overall, we observe that chromatin contacts are largely conserved across circadian time points and genetic backgrounds, and are tissue-specific. Our experiments reveal that the promoters of core-clock and clock-controlled genes contact distal genomic regions containing en-hancer chromatin signatures, suggesting a functional role of these distal sites in target gene regulation. Our data also suggest a clock-driven module of rhythmic genes that are colocalized in the nucleus. However, we also observe dynamic contacts between oscillating gene promoter and enhancer-like ge-nomic regions. In this thesis, we discuss in detail two examples of dynamic chromatin looping that are ob-served at the Cry1 and Gys2 loci. Our data reveal not only a dynamic coupling between the chromatin loop and the transcriptional state but also that the dynamic of chromatin looping depends on a functional clock. Furthermore, genome editing experiments using CRISPR-Cas9 tools reveal that the Cry1 distal site is essential for the transcriptional regulation of Cry1 and contributes to the circadian clock robustness both in-vivo and in cultured cells. These results demonstrate how local chromatin rearrangements take place in a 24-hour time-scale and set chromatin looping between gene promoters and distal regions as a new reg-ulatory layer for circadian gene expression.

EPFL2017