Regulation of transcription in pathogens, yeast and people
- Author(s): Nelson, Christopher Steven
- Advisor(s): DeRisi, Joseph L
- et al.
In the post genomic and high throughput era we have a wealth of sequence and expression data, yet we are still learning to understand the punctuation and syntax of the genome. This thesis presents case studies in gene regulation for a range of organisms, addressing whole transcriptome pattern changes in Plasmodium falciparum and Bartonella quintana, and then more focused descriptions of the binding sites of transcription factors from Mycobacterium tuberculosis, yeast, chimps, and humans. Early on in my thesis, I studied the immunology of Plasmodium infections. Later I profiled the transcriptomes of Plasmodium parasites after exposure to artemisinin, currently the front-line standard of care, and uncovered evidence for a developmental stall. The parasite was not previously thought to undergo any cell cycle arrest, and this may explain observed clinical recrudescence of infection after artemisinin monotherapy treatment and could represent a means for the parasite to adapt to drug pressure and select for resistance. After these studies I turned to a technique, called MITOMI2.0 (for mechanical trapping of molecular interactions), a means of measuring the specificity and energetic affinities of DNA binding proteins. The primary data produced with a lab-on-a-chip is the amount of DNA binding to a given protein, over a library of DNA sequences. We improved made the technique more robust and were able to apply it to several systems. We found that the yeast unfolded protein response factor Hac1 bound two DNA sites with distinct sequences. For another yeast stress regulator, Msn2, we measured its absolute affinity for regulated sites in target promoters and confirmed that it was a low affinity DNA binder, capable of a linear induction of its targets, as opposed to a frequently observed more binary induction response. ChIP-seq and MITOMI analysis revealed that FOXP2 the best-studied example of a protein involved in the development of human language, has had a conserved binding site preference, yet the complement of available binding sites has changed in humans. We confirmed the sequence specificity of a Mycobacterium tuberculosis factor that controls virulence in macrophage infection, and studied the way the protein interacts with DNA both as a dimer and a monomer.