The genome of metazoan cells is organized into topologically associating domains 
(TADs) that have similar histone modifications, transcription level, and DNA 
replication timing. Although similar structures appear to be conserved in 
fission yeast, computational modeling and analysis of high-throughput chromosome 
conformation capture (Hi-C) data have been used to argue that the small, highly 
constrained budding yeast chromosomes could not have these structures. In 
contrast, herein we analyze Hi-C data for budding yeast and identify 200-kb 
scale TADs, whose boundaries are enriched for transcriptional activity. 
Furthermore, these boundaries separate regions of similarly timed replication 
origins connecting the long-known effect of genomic context on replication 
timing to genome architecture. To investigate the molecular basis of TAD 
formation, we performed Hi-C experiments on cells depleted for the Forkhead 
transcription factors, Fkh1 and Fkh2, previously associated with replication 
timing. Forkhead factors do not regulate TAD formation, but do promote 
longer-range genomic interactions and control interactions between origins near 
the centromere. Thus, our work defines spatial organization within the budding 
yeast nucleus, demonstrates the conserved role of genome architecture in 
regulating DNA replication, and identifies a molecular mechanism specifically 
regulating interactions between pericentric origins.