Construction of a nucleosome array for analysis of pioneer transcription factor interactions
Year: 2023
Type: dissertation
Abstract: The large genome of eukaryotes is compacted into the complex structure of chromatin fibers and stored in the tiny nucleus. Transcription factors (TFs) play the role of deciphering the DNA code bundled in chromatin, however, the efficient packaging of nucleosomal arrays into chromatin fibers poses a barrier to regular TFs. Pioneer transcription factors (pTFs) can recognize their target binding sequence in the tightly packed chromatin, and initiate transcription by recruiting the rest of the transcriptional machinery to the target gene´s promoter. This master regulator function of pTFs has been previously used in cell fate reprogramming, such as the reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs). Purine-rich sequence DNA-binding protein 1 (PU.1) is one such pTF, which acts as a master regulator for hematopoietic stem cell renewal as well as lymphoid and myeloid differentiation. Although the interactions of PU.1 with chromatin have been explored in previous research, there is still a vast pool of the unknown surrounding it. One of the first steps in understanding these interactions is successfully replicating the chromatin environment in-vitro. In this project, an attempt was made to construct a thirteen-nucleosome array containing PU.1´s target binding sequence, which can serve as chromatin-like structure for in-vitro experiments such as single molecule Förster resonance energy transfer (sm-FRET). The plasmid containing the Cx3cr1 DNA sequence was successfully isolated from bacterial cultures, the strand separated by restriction enzyme digestion, and purified using gel extraction purification and anion exchange chromatography (AIEX). A nucleosome array reconstitution reaction was successfully set up with a variety of different ratios, and an EcoRI digest of the reconstitution reactions showed the pattern of formation of the array.
Source: Skemman
Author: Mariya Petrova Monova
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7018055616