Project (Patrick Cramer - Michael Lidschreiber)
Genome transcription and regulation

We are uncovering the molecular mechanisms and systemic principles of genome transcription and regulation using a combination of structural biology with functional genomics and bioinformatics in human and yeast cells. We offer several exciting projects in these areas and will recruit highly motivated and talented PhD students with experimental or computational backgrounds. As part of the IMPRS-GS, our laboratory can offer PhD projects in (A) functional genomics (Project leader: Kristina Zumer), (B) computational biology (Project leader: Michael Lidschreiber), and (C) genome-wide in vitro chromatin reconstitution (Project leader: Elisa Oberbeckmann). Below please find more information regarding our recent and ongoing work relating to the PhD projects.

A, B: We have developed the experimental and computational tools for transient transcriptome sequencing, or TT-seq, a method that enables measuring of RNA synthesis rates (1) and mapping of enhancer landscapes in a dynamic way in vivo (1, 2). We have used TT-seq to follow cell differentiation events and cellular responses to hormones and other signals (3, 4), and to study transcriptional misregulation in cancer cells (5). Combining these data with other genome-wide data sets for "multi-omics" approaches, which integrated with theoretical modeling, enables us to extract additional kinetic parameters of transcription. We can estimate several essential mechanisms of transcriptional regulation in human cells. Specifically, how long RNA polymerase II pauses at the beginning of genes (6, 7); what is the RNA polymerase II elongation velocity across entire genes (8) and how pre-mRNA splicing and transcription are coupled (9). Additionally, we utilize CRISPR/Cas9 to engineer cells in which we can rapidly and specifically degrade or inhibit proteins regulating transcription.
We continue to develop and use functional genomics methods and computational approaches to study the mechanisms of enhancer function, differentiation, and RNA metabolism, including transcription-coupled RNA processing. Work on these projects requires experimentalists (life sciences background) and computational biologists (background in mathematics, informatics etc.) with a strong interest in gene regulation. Possible projects will be developed together with candidates interested in specific areas.

C: Chromatin architecture can regulate transcriptional activity. Therefore, we aim to understand which proteins influence chromatin architecture to which extent. The most important players are ATP-dependent chromatin remodelers, certain transcription factors and cohesin. In vivo, many of these factors are either redundant or essential. Thus, it is inherently difficult to disentangle their individual contributions and direct activity in vivo. To solve this problem, we set up a bottom-up approach in which each protein can be tested individually or in combination (10). We reconstitute chromatin from a plasmid library containing the entire yeast genome and purified histone octamers. After incubation of chromatin with the protein of interest, the protein activity is measured genome-wide with MNase-seq and MicroC (in collaboration with the Oudelaar lab). The project involves genome engineering by CRISPR/Cas9, endogenous and recombinant protein purification from human or insect cells, Next-Generation Sequencing techniques, bioinformatics and potentially cryo-electron tomography. Thus, a strong background in protein biochemistry or NGS is of advantage.




Homepage Research Group
http://www.mpinat.mpg.de/cramer



Publications:

(1) Schwalb B, Michel M, Zacher B, Frühauf K, Demel C, Tresch A, Gagneur J, Cramer P. TT-seq maps the human transient genome. Science. 2016 Jun 3; 352(6290):1225-8.

(2) Michel M, Demel C, Zacher B, Schwalb B, Krebs S, Blum H, Gagneur J, Cramer P. TT-seq captures enhancer landscapes immediately after T-cell stimulation. Molecular Systems Biology. 2017 Mar 7;13(3):920.

(3) Choi J, Lysakovskaia K, Stik G, Demel C, Söding J, Tian TV, Graf T, Cramer P. Evidence for additive and synergistic action of mammalian enhancers during cell fate determination. eLife. 2021 10, e65381.

(4) Sawicka A, Villamil G, Lidschreiber M, Darzacq X, Dugast‐Darzacq C, Schwalb B, Cramer P. Transcription activation depends on the length of the RNA polymerase II C‐terminal domain. EMBO Journal. 2021 40 (9), e107015.

(5) Lidschreiber K, Jung LA, von der Emde H, Dave K, Taipale J, Cramer P, Lidschreiber M. Transcriptionally active enhancers in human cancer cells. Molecular Systems Biology. 2021 17 (1), e9873.

(6) Gressel S, Schwalb B, Cramer P. The pause-initiation limit restricts transcription activation in human cells. Nature Communications. 2019 10 (1), 3603.

(7) Gressel S, Schwalb B, Decker TM, Qin W, Leonhardt H, Eick D, Cramer P. CDK9-dependent RNA polymerase II pausing controls transcription initiation. eLife. 2017 6, e29736.

(8) Zumer K, Maier KC, Farnung L, Jaeger MG, Rus P, Winter G, Cramer P. Two distinct mechanisms of RNA polymerase II elongation stimulation in vivo. Molecular Cell. 2021 81 (15), pp. 3096 - 3109.e8.

(9) Caizzi L, Monteiro-Martins S, Schwalb B, Lysakovskaya K, Schmitzova J, Sawicka A, Chen Y, Lidschreiber M, Cramer P. Efficient RNA polymerase II pause release requires U2 snRNP function. Molecular Cell. 2021 May 6;81(9):1920-1934.e9.

(10) Oberbeckmann E, Niebauer V, Watanabe S, Farnung L, Moldt M, Schmid A, Cramer P, Peterson C, Eustermann S, Hopfner KP, Korber P. Ruler elements in chromatin remodelers set nucleosome array spacing and phasing. Nature Communications. 2021 12: 3232.