UC Santa Cruz

The key proteins that function during eukaryotic transcription elongation are likely to have all been identified, but in vitro reconstitution of this process remains incomplete. Nucleosomes inhibit transcription elongation in vitro and in vivo. However, actively transcribed genes in vivo remain nucleosomal. Thus, there must be some mechanism(s) that overcomes the nucleosomal barrier to transcription elongation and subsequently restores nucleosomes. Spt5 is a universally conserved transcription elongation factor thought to play a role in this process. However, the in vivo relevance of Spt5 for transcription through nucleosomes remains uncertain. Here we present evidence that Spt5 facilitates transcription through nucleosomes in vivo. Chapter 1 summarizes the literature and current gaps in our knowledge of transcription elongation through chromatin. Chapter 2 presents a novel of class of histone H3 mutations that suppress spt5-242, a transcription elongation defective mutation that genetically interacts with chromatin regulators. These H3 mutations disrupt transcribed chromatin, and result in reduced nucleosome density at the 3’ ends of genes. Single-molecule analysis of one of these mutations revealed that nucleosomes predicted to be stable are selectively lost from the body of PHO5 when it is actively transcribed. The selective loss of stable nucleosomes is also observed genome wide. Furthermore, we describe spt5 mutations with chromatin disruption phenotypes that cluster in a poorly conserved region of Spt5 that we name the NGN-Proximal Region (NPR). AlphaFold predicts that the Spt5 NPR has structure in many organisms. We propose that the NPR functions as a hinge to dynamically facilitate transcription through nucleosomes. Chapter 3 describes genetic suppressors of one of the h3 mutations described in chapter 2. Loss of sgf73/hfi1 SAGA, ubp10, chd1, or spt5 function all suppress the temperature sensitive growth defect of this histone mutation. We establish an epistatic relationship between the h3 genetic suppressors and key enzymes that function during transcription elongation: Rad6 and Gcn5. These results suggest that RSC chromatin remodeler and/or Spt16 function work in collaboration with Spt5 to transcribe through nucleosomal DNA. The concluding Chapter 4 summarizes the main findings throughout this work and its relation to the knowledge gaps identified in Chapter 1.