Directly to content
  1. Publishing |
  2. Search |
  3. Browse |
  4. Recent items rss |
  5. Open Access |
  6. Jur. Issues |
  7. DeutschClear Cookie - decide language by browser settings

Understanding pluripotency by global and targeted quantification of chromatin-associated proteins

Rafiee, Mahmoud-Reza

[img]
Preview
PDF, English
Download (58MB) | Terms of use

[img] PDF, English (plus Excel-Tabellen in zip-File)
Download (66MB) | Terms of use

Citation of documents: Please do not cite the URL that is displayed in your browser location input, instead use the DOI, URN or the persistent URL below, as we can guarantee their long-time accessibility.

Abstract

The maintenance of pluripotency in embryonic stem cells (ESC) is regulated by a network of chromatin-associated proteins coordinated by three master transcription factors Oct4, Sox2 and Nanog. To understand how different states of pluripotency are established, I developed three methods for studying chromatin-associated proteins globally, protein-targeted and locus-targeted in mouse ESC. Firstly, to study chromatin protein composition in a global manner, for the first time I developed in-vitro enzymatic labeling of chromatin by biotinylated nucleotides using Terminal deoxynucleotidyltransferase (TdT). As a result, more than 5000 proteins were significantly enriched in mouse ESCs in comparison to the negative control omitting the biotinylation step. In addition to the canonical chromatin-binding proteins, SICAP suggests chromatin association of some unexpected proteins such as Fgf4, which is a growth factor. This observation was further verified by immuno-staining. Secondly, I combined SICAP with chromatin immuno-precipitation (ChIP-SICAP) to identify proteins that interact with a target protein specifically on chromatin. Using endogenous Oct4, Sox2 and Nanog (OSN) as the targets of ChIP-SICAP, I identified about 400 proteins, as the overlap of the three assays. These 400 proteins include a large number of established interaction partners of the target proteins known to participate in the core pluripotency network (e.g. Rex1, Prdm14, Tcf3, Sall4, Esrrb, Tbx3, Stat3 etc). To reveal the co-localization sites with OSN, I selected Trim24. Interestingly, using ChIP-seq it turned out that Trim24 co-localizes with OSN on many super-enhancers of pluripotency. Thirdly, I developed a method to identify proteins bound to the Nanog promoter using biotinylated oligonucleotides. The specificity of the method, called targeted isolation of genomic regions (TIGR), was validated using qPCR and high-throughput sequencing. Hence, several proteins have been identified that are known to bind to, and regulate transcriptional activity of Nanog. Comparing the meta-stable and the ground-state of pluripotency, TIGR identified several nucleoporins that associate with the Nanog promoter preferentially in the ground-state of pluripotency. Using ChIP-qPCR I could validate the association of Nup98 to the Nanog promoter. Taken together, the data generated by the aforementioned methods expand the circuitry of pluripotency, and shed a new light on the differences between the ground-state and meta-stable state of pluripotency. Additionally, the newly developed methods are highly generalizable and independent of cell culture or genetic engineering so that they can be used for studying diverse biological systems.

Item Type: Dissertation
Supervisor: Knop, Prof. Dr. Michael
Place of Publication: Heidelberg, EMBL
Date of thesis defense: 25 November 2015
Date Deposited: 17 Dec 2015 12:43
Date: 2015
Faculties / Institutes: The Faculty of Bio Sciences > Institute of Pharmacy and Molecular Biotechnology
Subjects: 500 Natural sciences and mathematics
About | FAQ | Contact | Imprint |
OA-LogoDINI certificate 2013Logo der Open-Archives-Initiative