The genetic material of eukaryotic cells is packaged into chromatin. DNA is wrapped around histone proteins to form nucleosomes, the basic repeating unit of chromatin. The histone N-termini protrude from the nucleosomal core and are extensively modified during transcription. Many transcription factors harbor domains that will interact with the modified histone N-termini. Additionally, modification of the N-termini can alter the charge of the basic residues resulting in the unfolding of higher order chromatin structures. In S. cerevisiae, the histone N-termini are nonessential and this has allowed for investigations into how N-termini mutations affect the regulation of transcription. Initial characterizations of histone N-termini mutants revealed transcription defects; however, the mechanism of these defects has not been examined. In this study, we explore roles for the histone N-termini in transcription initiation, elongation, termination, and mRNA processing.
Using the DNA damage-inducible RNR3 gene, we explore how deletions and lysine to glutamine substitutions would affect transcription induction. We establish that the H2A, H3, and H4 N-termini are important for RNR3 activation. Interestingly, we observe that these transcription defects are the results of different mechanisms. For example, we demonstrate that the H4 N-terminus, specifically, is required for the release of the Crt1-Ssn6-Tup1 corepressor upon DNA damage. Additionally, deletion of the H4 N-terminus results in constitutive SWI/SNF recruitment in a mechanism that likely involves the loss of Isw2 function. Deletion of the H2A N-terminus results in a significant reduction of H3 lysine 4 tri-methylation. The methylation loss in this mutant is suggestive of another form of trans-tail regulation of histone modifications. Our characterization of the histone N-termini in transcription initiation revealed possible roles for the N-termini in transcription elongation. We demonstrate that deletion of the H3 N-terminus or substitution of the H4 lysine to glutamines result in a shift of RNA polymerase density that is indicative of a transcription elongation defect.
In the second part of this study, we explore how mutations to the H4 N-terminus affect transcription elongation. Substituting all of the H4 N-terminal lysine residues for glutamines results in genetic interactions with elongation factors and suggests that the H4 lysines have roles in elongation. Interestingly, mutation of only three lysines did not result in growth defects. This implies redundancy among the acetylatable lysines. We also demonstrate that the H4 N-terminus is important for the repression of intragenic transcription at FLO8. This repression mechanism is separate from the Set2-Rpd3 pathway and likely involves the elongation factors Spt6 and Spt16. Further examination revealed novel roles for the H4 N-termini in transcription termination at snoRNAs as well as mRNA splicing. These results suggest that the H4 N-terminus is an important regulator throughout transcription.
The final section of this study involves the characterization of novel intra-tail regulation of Set2 by the H3 N-terminus. Deletion of the H3 N-terminus, or substitution of the lysine residues within the tail for glutamine, results in significant defects in H3 lysine 36 tri-methylation. We demonstrate that the H3 N-terminus is not required for the recruitment of Set2, but does stimulate Set2 activity. Furthermore, we demonstrate that the charge of the H3 N-terminus is important for Set2 activity. We propose a mechanism by which Set2 activity is regulated by novel intra-tail interactions within H3, which can be regulated by modifications to the tail.
It has been the goal of this study to better understand the mechanisms by which the histone N-termini regulate transcription. Initially, we expected the histone N-termini to regulate transcription initiation predominantly. However, our characterization of initiation led us to the hypothesis that the histone N-termini regulate multiple facets of transcription. Experiments to test this hypothesis revealed novel roles for the H4 N-terminus in elongation, termination, and mRNA splicing. Additionally, we identified novel intra-tail regulation of Set2 activity and characterized the mechanism.