| This dissertation provides an initial characterization of two nuclear amine oxidase (NAO) proteins, saf110+ /kdm1a+ and saf140 +/kdm1b+, from the fission yeast S. pombe. We show that Kdm1a and Kdm1b associate with one another in a four-component complex that we have named SAPHIRE. Genetic analyses demonstrate that SAPHIRE is essential, with distinct enzymatic versus nonenzymatic functions. Both in vitro and in vivo experiments reveal that SAPHIRE binds chromatin, and we map binding sites for the two NAO subunits across the genome.;Next, we focus upon a distinct class of SAPHIRE-occupied loci: the 3'-UTR region of genes bearing specialized noncoding RNAs of the small nucleolar family (snoRNAs). Here, we find that SAPHIRE facilitates gene-internal transcriptional activation, enabling target snoRNAs to be transcribed internally, independent of upstream promoter elements. Further investigation of the chromatin structure at these SAPHIRE targets reveals important differences between these loci and typical gene-internal regions. These findings define the first sites of natural gene-internal transcription initiation, and suggest that SAPHIRE activity creates a promoter-like environment within genes to facilitate noncoding RNA production at these specialized loci.;The final SAPHIRE targets discussed in this dissertation are kinetochores, which are specialized chromatin domains required for chromosome segregation. SAPHIRE occupancy at these loci is phased, and displays an inverse relationship relative to Cnp1, a conserved centromere-specific histone H3 variant. Importantly, we find that specific regions within kinetochore domains are transcribed by Pol II. We further show that kinetochore transcription is enhanced by SAPHIRE, and correlate changes in transcription with alterations in kinetochore structure and function. These findings provide the first insights into NAO function within kinetochore domains, and suggest that Pol II transcription may regulate kinetochore chromatin.;The most populated SAPHIRE target class is Pol II promoters, and genome-wide analyses reveal that the complex activates transcription at target promoters. Detailed analysis of promoter binding reveals that SAPHIRE localizes to the transcription start site. Importantly, SAPHIRE is a dynamic complex, as newly activated genes rapidly acquire SAPHIRE within their promoter regions. SAPHIRE catalytic mutants show increased levels of histone H3K4-dimethylation (H3K4me2) within target promoters; suggesting a possible link between transcriptional activation and H3K4-specific demethylation. |
