Investigation of cryptic transcription suppressing factors using a FLO8:sfGFP reporter

Chromatin disruption caused by polymerase passage during transcription or DNA replication has the potential to lead to cryptic transcription originating within aberrant initiation sites across the genome. We developed a sfGFP fluorescence reporter to track cryptic transcription initiating from an intragenic cryptic promoter within the FLO8 coding region and confirmed reporter utility in a spt16-197 mutant strain. Using this reporter, our results showed that the proteins, Spt10, Spt21, Cac1, and Asf1 are required for chromatin stability during DNA replication, while Spt2 plays a replication-independent role in maintaining chromatin. We also showed that blocking DNA replication only partially suppresses cryptic transcription in Spt5, Spt6, or Spt16-depleted cells, indicative of these factors functioning in a process independent of replication. Taken together, our results demonstrate that restoration of nucleosome stability following disruption is facilitated by a number of collaborating factors functioning in various pathways.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat