טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSergei Rudnizky
SubjectSingle-Molecule Studies of Transcription Regulation:
Insights from the LH Genes
DepartmentDepartment of Biology
Supervisors Professor Kaplan Ariel
Professor Melamed Philippa
Full Thesis textFull thesis text - English Version


Abstract

The structure and dynamics of promoter chromatin have a profound effect on the expression levels of genes. However, the contribution of DNA sequence, histone variant usage and other factors to shaping the architecture of chromatin, and the mechanisms by which this architecture modulates expression of specific genes, are not yet completely understood. Here, we combined chromatin mapping in cells with single-molecule optical tweezers experiments to study the roles that DNA sequence and the histone variant H2A.Z play in shaping the structure and dynamics of the chromatin at the promoters of two model genes, Cga and Lhb, which encode, respectively, the alpha and beta subunits of luteinizing hormone (LH). We developed a single-molecule assay that allows measurement of nucleosome mobility at bp precision and seconds to minutes timescales. We discovered that although the nucleosomes reposition spontaneously, their movements are constrained to ~10 bp. This restriction is overcome by the incorporation of H2A.Z, leading to the production of larger DNA excursions. The distinct localization of this histone variant relative to the transcription start site (TSS) at the promoter regions of Cga and Lhb suggest a possible role for the mobility of nucleosomes in the regulation of transcription. In the case of the highly-expressed Cga, the nucleosome located at the TSS is evicted, as opposed to the nucleosome positioned downstream () which is remodeled with H2A.Z, likely to facilitate elongation of RNA polymerase. For the more tightly regulated Lhb, the TSS nucleosome is not evicted, but remodeled with H2A.Z. This incorporation leads to enhanced nucleosome diffusion, which results in an increase in the exposure of the transcription factor Egr1 binding site, leading to its association with DNA. This binding is also modulated by the identity of the nucleotides at the core of the binding site, and the flanking sequence context, which govern the symmetry of local TF-DNA interactions. Our study highlights how the fundamental physical properties of promoter chromatin are modulated by the usage of local DNA sequence, alternative histone variants and remodeling machinery to modulate synergistically gene expression at the initiation and elongation phases.