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Author

Marc Boehning

Other affiliations: University of Göttingen
Bio: Marc Boehning is an academic researcher from Max Planck Society. The author has contributed to research in topics: RNA polymerase II & Stress granule. The author has an hindex of 6, co-authored 7 publications receiving 567 citations. Previous affiliations of Marc Boehning include University of Göttingen.

Papers
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Journal ArticleDOI
TL;DR: It is reported that human and yeast CTDs undergo cooperative liquid phase separation, with the shorter yeast CTD forming less-stable droplets and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.
Abstract: The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation, with the shorter yeast CTD forming less-stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association, whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters or hubs at active genes through interactions between CTDs and with activators and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

422 citations

Journal ArticleDOI
22 Aug 2018-Nature
TL;DR: In this article, the authors showed that the formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6.
Abstract: Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Homo sapiens DSIF, PAF and SPT6 was determined at 3.1 A resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminal-domain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.

251 citations

Journal ArticleDOI
TL;DR: Two active mechanisms that can explain how cells regulate condensate formation and size are proposed and can guide studies into the many emerging roles of biomolecular condensates.

115 citations

Journal ArticleDOI
TL;DR: In this paper, the negative elongation factor (NELF) is recruited to gene promoters to impair RNA polymerase II elongation, which is required for cellular viability under stressful conditions.

68 citations

Journal ArticleDOI
29 Jan 2019-Mbio
TL;DR: A previously unreported phytase activity of the alkaline phosphatase and sulfatase superfamily and of purple acid phosphatases from nonvegetal origin is revealed, suggesting that the classical concept comprising four classes of phytases should be modified and indicates high performance of the screening method for retrieving novel types of phosphatased/phytases hidden in metagenomes of complex environments.
Abstract: Phosphatases, including phytases, play a major role in cell metabolism, phosphorus cycle, biotechnology, and pathogenic processes. Nevertheless, their discovery by functional metagenomics is challenging. Here, soil metagenomic libraries were successfully screened for genes encoding phosphatase activity. In this context, we report the largest number and diversity of phosphatase genes derived from functional metagenome analysis. Two of the detected gene products carry domains which have never been associated with phosphatase activity before. One of these domains, the SNARE-associated domain DedA, harbors a so-far-overlooked motif present in numerous bacterial SNARE-associated proteins. Our analysis revealed a previously unreported phytase activity of the alkaline phosphatase and sulfatase superfamily (cl23718) and of purple acid phosphatases from nonvegetal origin. This suggests that the classical concept comprising four classes of phytases should be modified and indicates high performance of our screening method for retrieving novel types of phosphatases/phytases hidden in metagenomes of complex environments.IMPORTANCE Phosphorus (P) is a key element involved in numerous cellular processes and essential to meet global food demand. Phosphatases play a major role in cell metabolism and contribute to control the release of P from phosphorylated organic compounds, including phytate. Apart from the relationship with pathogenesis and the enormous economic relevance, phosphatases/phytases are also important for reduction of phosphorus pollution. Almost all known functional phosphatases/phytases are derived from cultured individual microorganisms. We demonstrate here for the first time the potential of functional metagenomics to exploit the phosphatase/phytase pools hidden in environmental soil samples. The recovered diversity of phosphatases/phytases comprises new types and proteins exhibiting largely unknown characteristics, demonstrating the potential of the screening method for retrieving novel target enzymes. The insights gained into the unknown diversity of genes involved in the P cycle highlight the power of function-based metagenomic screening strategies to study Earth's phosphatase pools.

19 citations


Cited by
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Journal ArticleDOI
TL;DR: A review of the mechanisms of lncRNA biogenesis, localization and functions in transcriptional, post-transcriptional and other modes of gene regulation, and their potential therapeutic applications is presented in this article.
Abstract: Evidence accumulated over the past decade shows that long non-coding RNAs (lncRNAs) are widely expressed and have key roles in gene regulation. Recent studies have begun to unravel how the biogenesis of lncRNAs is distinct from that of mRNAs and is linked with their specific subcellular localizations and functions. Depending on their localization and their specific interactions with DNA, RNA and proteins, lncRNAs can modulate chromatin function, regulate the assembly and function of membraneless nuclear bodies, alter the stability and translation of cytoplasmic mRNAs and interfere with signalling pathways. Many of these functions ultimately affect gene expression in diverse biological and physiopathological contexts, such as in neuronal disorders, immune responses and cancer. Tissue-specific and condition-specific expression patterns suggest that lncRNAs are potential biomarkers and provide a rationale to target them clinically. In this Review, we discuss the mechanisms of lncRNA biogenesis, localization and functions in transcriptional, post-transcriptional and other modes of gene regulation, and their potential therapeutic applications.

1,630 citations

Journal ArticleDOI
27 Jul 2018-Science
TL;DR: Live-cell single-molecule imaging revealed that TF LCDs interact to form local high-concentration hubs at both synthetic DNA arrays and endogenous genomic loci, suggesting that under physiological conditions, rapid, reversible, and selective multivalent LCD-LCD interactions occur between TFs and the RNA Pol II machinery to activate transcription.
Abstract: Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.

710 citations

Journal ArticleDOI
01 Aug 2019-Nature
TL;DR: In this paper, the authors investigated whether the phosphorylation of the C-terminal domain of the RNA polymerase II (PolII) C-interaction subunit regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing.
Abstract: The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex1–4. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus5,6. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain7–12. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers7,8, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites9–12. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference. RNA polymerase II with a hypophosphorylated C-terminal domain preferentially incorporates into mediator condensates, and with a hyperphosphorylated C-terminal domain into splicing-factor condensates, revealing phosphorylation as a regulatory mechanism in condensate preference.

404 citations

Journal ArticleDOI
TL;DR: It is found that the evidence for in vivo LLPS is often phenomenological and inadequate to discriminate between phase separation and other possible mechanisms, and the causal relationship and functional consequences of LLPS in vivo are even more elusive.
Abstract: The idea that liquid-liquid phase separation (LLPS) may be a general mechanism by which molecules in the complex cellular milieu may self-organize has generated much excitement and fervor in the cell biology community. While this concept is not new, its rise to preeminence has resulted in renewed interest in the mechanisms that shape and drive diverse cellular self-assembly processes from gene expression to cell division to stress responses. In vitro biochemical data have been instrumental in deriving some of the fundamental principles and molecular grammar by which biological molecules may phase separate, and the molecular basis of these interactions. Definitive evidence is lacking as to whether the same principles apply in the physiological environment inside living cells. In this Perspective, we analyze the evidence supporting phase separation in vivo across multiple cellular processes. We find that the evidence for in vivo LLPS is often phenomenological and inadequate to discriminate between phase separation and other possible mechanisms. Moreover, the causal relationship and functional consequences of LLPS in vivo are even more elusive. We underscore the importance of performing quantitative measurements on proteins in their endogenous state and physiological abundance, as well as make recommendations for experiments that may yield more conclusive results.

382 citations

Journal ArticleDOI
28 Aug 2019-Nature
TL;DR: Structural and microscopy studies of gene transcription underpin a model in which phosphorylation controls the shuttling of RNA polymerase II between promoter and gene-body condensates to regulate transcription initiation and elongation.
Abstract: The regulated transcription of genes determines cell identity and function. Recent structural studies have elucidated mechanisms that govern the regulation of transcription by RNA polymerases during the initiation and elongation phases. Microscopy studies have revealed that transcription involves the condensation of factors in the cell nucleus. A model is emerging for the transcription of protein-coding genes in which distinct transient condensates form at gene promoters and in gene bodies to concentrate the factors required for transcription initiation and elongation, respectively. The transcribing enzyme RNA polymerase II may shuttle between these condensates in a phosphorylation-dependent manner. Molecular principles are being defined that rationalize transcriptional organization and regulation, and that will guide future investigations. Structural and microscopy studies of gene transcription underpin a model in which phosphorylation controls the shuttling of RNA polymerase II between promoter and gene-body condensates to regulate transcription initiation and elongation.

356 citations