An additional level of regulation for CENP-A assembly at centromeres occurs at the level of gene expression. CENP-A expression was limited to S-phase by placing CENP-A under the control of a cloned, S-phase dependent histone H3 promoter and 3'-UTR. This abolished the specific centromeric targeting of CENP-A, resulting in accumulation of protein throughout the nucleus. Analysis of the timing of CENP-A expression during the cell cycle revealed that it is expressed later than histone H3, with CENP-A mRNA beginning to accumulate in late S phase and reaching peak levels in G2. Analysis of the 5' proximal genomic DNA sequence revealed a pair of DNA sequence motifs that mediate cell cycle dependent transcriptional regulation of genes such as cdc25C and cdc2.

These data suggest that transcriptional regulation of the CENP-A gene normally couples CENP-A expression to centromere assembly during the cell cycle.

Placing CENP-A expression under the S-phase regulatory elements of a histone H3 gene abolishes centromeric targeting.	CENP-A mRNA (black in C) accumulates later in the cell cycle than histone H3 (grey). CENP-A mRNA rapidly drops after mitosis.	Analysis of the proximal 5' region of genomic DNA revealed DNA sequence motifs in that mediate cell cycle-dependent gene expression