A Plasmodium falciparum transcriptional CDK-related kinase with a crucial role in parasite proliferation associates with histone deacetylase activity

Cyclin-dependent protein kinases (CDKs) are key regulators of the eukaryotic cell cycle and of the eukaryotic transcription machinery. Here, we report the characterisation of Pfcrk-3 (P. falciparum CDK-related kinase 3, PlasmoDB identifier PFD0740w), an unusually large CDK-related protein whose kinase domain displays maximal homology to those CDKs which are, in other eukaryotes, involved in the control of transcription. The closest enzyme in yeast is BUR1 ("Bypass Upstream activating sequence Requirement"), known to control gene expression through interaction with chromatin modification enzymes. Consistent with this, immunofluorescence data show that Pfcrk-3 colocalises with histones. We show that recombinant Pfcrk-3 associates with histone H1 kinase activity in parasite extracts, and that this association is detectable even if the catalytic domain of Pfcrk-3 is rendered inactive by site-directed mutagenesis, indicating that Pfcrk-3 is part of a complex that includes other protein kinases. Immunoprecipitates obtained from extracts of transgenic parasites expressing HA-tagged Pfcrk-3 using a hemagglutinin (HA) antibody displayed both protein kinase and histone deacetylase activities. Reverse genetics data show that the pfcrk-3 locus can be targeted only if the genetic modification does not cause loss-of-function. Taken together, our data strongly suggests that Pfcrk-3 fulfils a crucial role in the intraerythrocytic development of P. falciparum, presumably through chromatin modification-dependent regulation of gene expression.

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

The role of OCT4 and SOX2 in regulating chromatin accessibility and cell fate across the cell cycle in embryonic stem cells

Torgil Elias Friman

Precise spatiotemporal regulation of gene expression is essential for development and homeostasis of complex organisms. This is achieved in large part by sequence-specific transcription factors (TF) that bind to genomic regulatory elements to activate or repress transcription. DNA in eukaryotic nuclei is wrapped around histones into nucleosomes, which are inaccessible to most proteins. However, certain TFs can access their binding sites in the presence of histones and promote nucleosome eviction to increase chromatin accessibility, allowing for binding of other protein. These TFs, called pioneer factors, include OCT4 and SOX2, which are master regulators of embryonic stem (ES) cells. Dividing cells, including stem cells, must achieve proper gene regulation despite the progression of the cell cycle, which imposes several constraints. During S phase, the replication fork passes through the genome to make a copy of each chromosome, which leads to a loss of chromatin accessibility. Furthermore, the substantial chromatin compaction that occurs during mitosis to prepare for chromosome segregation is associated with eviction of most DNA-binding proteins and reduced accessibility at distal cis-regulatory elements including enhancers. How pioneer factors operate in the context of cell cycle progression is unclear. In this thesis, I will present work studying different features of OCT4 and SOX2, including how they interplay to regulate chromatin accessibility in ES cells, the impact of small endogenous fluctuations of their protein levels on cell fate and chromatin accessibility, and how their roles in regulating cell fate and chromatin accessibility is related to different stages of the cell cycle. Using live-cell imaging of fluorescently labeled proteins, we discovered that OCT4 and SOX2 were associated to mitotic chromatin. Using cell-cycle specific degradation strategies, we could show that the presence of SOX2 and OCT4 at the mitosis-G1 (M-G1) transition contributes to maintain the pluripotency of ES cells. We further showed that ES cells with high levels of OCT4 in G1, but not in S phase, are more prone to differentiate towards neuroectoderm and mesendoderm cell fates. Cells with high levels of OCT4 displayed increased chromatin accessibility at enhancers of differentiation genes, suggesting accessibility fluctuates with OCT4 levels and can prime cells for differentiation. To explore potential cell cycle-dependent regulation of chromatin accessibility, we degraded OCT4 at the M-G1 transition, which led to a reduction in accessibility at many OCT4-bound regions. When OCT4 returned later in the cell cycle, chromatin accessibility was recovered, although at many loci this recovery was incomplete, suggesting that OCT4 is required at the M-G1 transition to maintain these regions open. We then rapidly degraded OCT4 at other phases of the cell cycle. Surprisingly, loss of chromatin accessibility was highly similar at all cell cycle phases, showing that OCT4 is constantly required to maintain regulatory elements accessible. To explore the dynamic regulation of accessibility by OCT4, we performed time-course measurements of accessibility upon its degradation. This revealed that loss of accessibility was temporally correlated to the degradation of OCT4. These results suggest that chromatin accessibility is highly dynamic and continuously dependent on the presence of OCT4 across the cell cycle in ES cells.

EPFL2019