School of Science Division of Life Science 18 Investigating the Effects of MEF2D on Transcription Dynamics at a Single-Molecule Level Supervisor: ISHIBASHI Toyotaka / LIFS Student: LEONG Kin Nam / BCB Course: UROP2100, Spring MEF2D (Myocyte-specific enhancer factor 2D) is present in nucleus and mitochondria and enhances RNA polymerases transcription activity. The nucleus transcription enhancement effects by MEF2D are relatively well studied, whereas the transcription enhancing mechanism by MEF2D in mitochondria remains unknown. This project aims to investigate the effects of MEF2D on the dynamics of mitochondrial RNA polymerase (RNAPMT) transcription and further examine the effects quantitatively using quantitative sequencing techniques, in vitro transcription elongation assay, and singlemolecule optical tweezers transcription elongation assay. Comparing the WT and CRISPR-Cas9 MEF2D knockout 293T cells, we found that RNAPMT has multiple pausing sites throughout the human mitochondrial genome, and MEF2D can modulate some pausing sites. These results are confirmed by in vitro biochemical transcription elongation assay. Furthermore, to quantitatively measure its effects on RNAPMT, we have established high-spatial and temporal resolution optical tweezers transcription elongation assay. This study will allow us to quantify the effects of MEF2D on mitochondrial transcription. Investigating the Effects of MEF2D on Transcription Dynamics at a Single-Molecule Level Supervisor: ISHIBASHI Toyotaka / LIFS Student: MALLORCA Paolo Miguel Magallanes / BISC Course: UROP1000, Summer Transcription is a step that RNA polymerase uses DNA as a template to synthesize RNA. In Eukaryotic cells, genomic DNA is formed chromatin structure. It is known that condensed heterochromatin is associated to transcription repression whereas the loosen euchromatin is linked with transcription activation. The fundamental basic unit to form chromatin is histone proteins. There are core histones (H2A, H2B, H3 and H4) and linker histone (H1). Both core and linker histones have several types of histones and histone variants. It is known that some histone variants change their chromatin stability, thus RNA polymerase is much easier to transcribe through some histone variants containing chromatin. I focused to study histone variants mFM, TH2B, TH2A, and H2AL2, and how they change for the effect of transcription regulation. They are known to be expressed specifically during spermatogenesis. Their functions are not yet fully understood but are believed to play a role in histone-to-protamine transition as spermatogenic cell differentiation takes place. The aim of this project is to discover histone chaperones involved in the giving rise to these histone variants. In this UROP1000 course, we focused on creating and cloning the full recombinant DNA template construct containing a histidine tag, a sumo tag, two histone variants, and a flag tag fused together.