| dc.contributor.advisor | Cramer, Patrick Prof. Dr. | |
| dc.contributor.author | Gressel, Saskia | |
| dc.date.accessioned | 2019-08-01T08:19:14Z | |
| dc.date.available | 2020-05-04T22:50:03Z | |
| dc.date.issued | 2019-08-01 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0003-C183-E | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7586 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | Multi-omics analysis of transcription kinetics in human cells | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Cramer, Patrick Prof. Dr. | |
| dc.date.examination | 2019-05-06 | |
| dc.description.abstracteng | Transcription of the eukaryotic genome is a highly regulated process which is accomplished in a number of steps, known as the transcription cycle. One of these steps is promoter-proximal pausing, a regulatory halt of the RNA polymerase II (Pol II) shortly after transcription initiation that is released by the P-TEFb kinase CDK9. A kinetic model of transcription predicted that pause duration delimits the initiation frequency and suggested that paused Pol II sterically interferes with initiation. The relationship between promoter-proximal pausing and initiation could thus far not be tested, as no method was available to measure initiation frequencies in vivo. Other kinetic parameters such as pause duration and elongation velocity could likewise not be described genome-wide. Here we show that in human hematopoietic cells the ‘pause-initiation limit’ restricts transcription in steady state, upon perturbation of the CDK9 kinase, and upon heat shock. To elucidate this relationship, we develop a novel multi-omics approach that quantifies transcription kinetic parameters by combining two state-of-the-art sequencing methods with novel kinetic modeling. Specifically, mammalian native elongating transcript sequencing (mNET-seq) and transient transcriptome sequencing (TT-seq). mNET-seq maps the position and amount of Pol II in high-resolution across each strand of the entire human genome. TT-seq allows to distinguish newly synthesized from pre-existing RNA, and thus, measures immediate transcription activity as transcribed nucleotides per time. Combining measurements of TT-seq and mNET-seq enables us to derive productive initiation frequency, pause duration and elongation velocity genome-wide. For highly specific and fast inhibition of the pause release kinase CDK9, we engineer an analog-sensitive human cell line using CRISPR/Cas9. Upon CDK9 kinase inhibition, pause duration increases and productive initiation frequency decreases genome-wide. This shows that CDK9 activity stimulates the release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus increases the amount of RNA synthesized per time. We find that highly CDK9 responsive genes are associated with long-range chromatin interactions. We show that human pause sites are located ~50 bp downstream of the transcription start site and show an enrichment for G/C-C/G dinucleotides. Furthermore, transcribed RNA of genes with longer pause durations shows higher secondary structure propensity upstream of the pause site. We next explore whether the pause-initiation limit applies to transcription activation upon heat shock. To this end, we annotate protein-coding RNAs, and six major long noncoding transcript classes in human hematopoietic cells. Using our multi-omics approach, we observe a reciprocal behavior of productive initiation frequency and pause duration in steady state for all gene classes except enhancers. We show that upon heat shock the pause-initiation limit indeed restricts transcription activation at most genes. Surprisingly, enhancer elements are not limited by pausing and depend less on CDK9 activity than protein-coding genes. Together these results suggest a model in which transcription might be activated by an increase of the productive initiation frequency at enhancer elements, accompanied by a decrease in pause duration at the target gene. This allows for an increase of productive initiation events at the target gene. Our multi-omics approach can now be used to further dissect the effect of other known Pol II pause and release factors in a quantitative and genome-wide manner, ultimately revealing the nature of gene regulation in human cells. This will pave the way for novel treatments of diseases with transcriptional malignancies such as cancers, HIV infection or cardiac hypertrophy. | de |
| dc.contributor.coReferee | Bohnsack, Markus Prof. Dr. | |
| dc.subject.eng | Multiomics | de |
| dc.subject.eng | Transient Transcriptome sequencing (TT-seq) | de |
| dc.subject.eng | mammalian Native Elongating Transcript sequencing (mNET-seq) | de |
| dc.subject.eng | pause-initiation limit | de |
| dc.subject.eng | productive initiation frequency | de |
| dc.subject.eng | pause duration | de |
| dc.subject.eng | 4sU metabolic labeling | de |
| dc.subject.eng | promoter-proximal pausing | de |
| dc.subject.eng | Pol II pause sites | de |
| dc.subject.eng | P-TEFb | de |
| dc.subject.eng | analog sensitive CDK9 kinase (CDK9as) | de |
| dc.subject.eng | GenoSTAN | de |
| dc.subject.eng | transcription regulation | de |
| dc.subject.eng | transcription kinetics | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0003-C183-E-8 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2020-05-04 | |
| dc.identifier.ppn | 1672307147 | |