Search

Genome-wide rhythms in RNA polymerase II loading and dynamic chromatin remodeling underlie periodic gene expression during diurnal cycles in the mouse liver., Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II) as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver., Author Summary In mammalian organs such as the liver, many metabolic and physiological processes occur preferentially at specific times during the 24-hour daily cycle. The timing of these rhythmic functions depends on a complex interplay between the endogenous circadian clock and environmental timing cues relayed through the master circadian clock in the suprachiasmatic nucleus, or via feeding rhythms. These rhythms can be implemented on several regulatory levels, and here we aimed at a better understanding of the transcriptional and epigenetic changes that regulate diurnal rhythms. We performed genome-wide analysis of the locations of RNA polymerase II (Pol II) and the epigenetic histone modifications H3K4me3 and H3K36me3 at specific times of day, relating these data to mRNA expression levels. Our analyses show that Pol II transcriptional rhythms are biphasic in mouse liver, having predominant peak activities in the morning and evening. Moreover, dynamic changes in histone marks lag transcription rhythms genome-wide, indicating that the epigenetic landscape can be remodeled during the 24-hour cycle. Finally, a quantitative analysis of temporal Pol II and mRNA accumulation profiles indicates that posttranscriptional regulation significantly contributes to the amplitude and phase of mRNA accumulation profiles. While many studies have analyzed how transcription and chromatin states are modified during irreversible cell differentiation processes, our work highlights how these states can evolve reversibly in a system exhibiting periodicity in time.

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species. ISSN:1544-9173 ISSN:1545-7885

The TGF-β homolog Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. We then, using novel genetic tools, experimentally monitored the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution., Author Summary Morphogens are signaling molecules that trigger specific responses in cells in a concentration-dependent manner. The formation of morphogen gradients is essential for the patterning of tissues and organs. Decapentaplegic (Dpp) is the Drosophila homolog of the bone morphogenic proteins in vertebrates and forms a morphogen gradient along the anterior-posterior axis of the Drosophila wing imaginal disc, a single-cell layered epithelium. Dpp determines the growth and final size of the wing disc and serves as an ideal model system to study gradient formation. Despite extensive studies the mechanism by which morphogen gradients are established remains controversial. In the case of Dpp two mechanisms have been postulated, namely extracellular diffusion and receptor-mediated transcytosis. In the first model Dpp is suggested to move by diffusion through the extracellular matrix of a tissue, whereas in the latter model Dpp is transported through the cells by receptor-mediated uptake and re-secretion. In this work we combined novel genetic tools with mathematical modeling to discriminate between the two models. Our results suggest that the Dpp gradient forms following the extracellular diffusion mechanism. Moreover, our data suggest that the majority of the extracellular Dpp is free and not bound to its receptor, a property likely to play a role for the long-range gradient formation.