Identification of Fkh1 and Fkh2 binding site variants associated with dynamically bound DNA elements including replication origins

Abstract

Forkhead Box (Fox) DNA binding proteins control multiple genome activities, including transcription, replication, and repair. These activities are organized spatially and temporally in the nucleus, and Fox proteins Fkh1 and Fkh2 have emerged as regulators of long-range chromosomal interactions involved with these activities, such as the clustering of replication origins programmed for early initiation. Fkh1 and Fkh2 bind a subset of replication origins and are thought to dimerize to mediate long-range chromosomal contacts between these origins. The binding of Fkh1 and/or Fkh2 (Fkh1/2) to replication origins and the recombination enhancer (RE), which is involved in DNA repair required for mating-type switching, is cell cycle-regulated and thus appears to be more dynamic than Fkh1/2 binding at regulated target genes. Here we report the identification of Fkh1/2 binding sequence variants at replication origins and the RE compared with Fkh1/2 binding sequences found at target genes of the CLB2 group. These different binding sequences have previously been characterized as weak and strong, respectively, suggesting that the presence of weak sites contributes to more dynamic interactions at replication origins and RE, possibly facilitated by Fkh1/2 dimerization and cooperative interactions with accessory proteins. We discuss the wealth of regulatory potential imbued in these features of the DNA and its binding proteins.

Keywords: DNA motif; DNA replication foci; Fox proteins; chromatin binding; genome architecture.

Figure 1.

Density of Fkh1/2 binding motif variants correlates with differential regulation of Fkh1/2-chromatin binding. (A) For actual results, genomic sequences of 500 bp centered on the indicated features were queried for the presence of AYMAAYA or GYMAAYA motifs. Significance was calculated by comparison with simulation results, entailing searches (10,000 iterations per feature-binding motif combination) of n random 500 bp genomic regions; n is indicated for each feature class. For the Recombination Enhancer, n = 2 because two non-overlapping, 500 bp regions on each side of the center point were used to calculate mean motifs per window. For each feature class, search results for both motifs were recorded from the same simulation. Fkh-activated and –repressed origins were defined in [2], and CLB2 cluster genes were defined in. (B) Model depicting the possible effect of Fkh1/2 dimerization and presence of DNA motif variants on binding site hierarchies. Fkh1-dsm binds CLB2 cluster promoters and Fkh-activated origins similarly to Fkh1, but fails to cluster early origins in G1, potentially leaving Fkh-activated origins with a lower density of Fkh1/2 binding motifs unbound. In G2/M phase (right panel), origins are unbound while CLB2 cluster promoters (enriched for GYMAAYA motifs) remain bound by either Fkh1 or Fkh1-dsm. Intra-chromosomal origin clustering is likely to be much more frequent than inter-chromosomal origin cluster, which is depicted for emphasis. Cell cycle phase and Fkh1 genotype are depicted above.