The Isw2 Chromatin-Remodeling ATPase Cooperates with the Fkh2 Transcription Factor To Repress Transcription of the B-Type Cyclin Gene CLB2

Abstract

Forkhead (Fkh) transcription factors influence cell death, proliferation, and differentiation and the cell cycle. In Saccharomyces cerevisiae, Fkh2 both activates and represses transcription of CLB2, encoding a B-type cyclin. CLB2 is expressed during G 2 /M phase and repressed during G 1 . Fkh2 recruits the coactivator Ndd1, an interaction which is promoted by Clb2/Cdk1-dependent phosphorylation of Ndd1, suggesting that CLB2 is autoregulated. Ndd1 is proposed to function by antagonizing Fkh2-mediated repression, but nothing is known about the mechanism. Here we ask how Fkh2 represses CLB2. We show that Fkh2 controls a repressive chromatin structure that initiates in the early coding region of CLB2 and spreads up the promoter during the M and G 1 phases. The Isw2 chromatin-remodeling ATPase cooperates with Fkh2 to remodel the chromatin and repress CLB2 expression throughout the cell cycle. In addition, the related factors Isw1 and Fkh1 configure the chromatin at the early coding region and negatively regulate CLB2 expression but only during G 2 /M phase. Thus, the cooperative actions of two forkhead transcription factors and two chromatin-remodeling ATPases combine to regulate CLB2. We propose that chromatin-mediated repression by Isw1 and Isw2 may serve to limit activation of CLB2 expression by the Clb2/Cdk1 kinase during G 2 /M and to fully repress expression during G 1 .The forkhead (Fkh) family of transcription factors is highly conserved in eukaryotes, with roles in cell cycle control, cell death, proliferative responses, and differentiation (1,3,4,27). All four forkhead factors have been characterized in Saccharomyces cerevisiae. Fhl1 and its coregulator Ihf1 regulate cell proliferation primarily through control of ribosomal protein gene expression (5,18,21,35,50,52,59). Different phases of the cell cycle are regulated by two forkhead factors, Hcm1 and Fkh2. Hcm1 regulates chromosome segregation genes and controls the S-phase transition (46). Fkh2 and its coactivator Ndd1 influence the expression of a wide range of genes (56), including the genes of the CLB2 cluster that control the G 2 /M and M/G 1 phases of the cell cycle (15,23,62) which are the best-characterized targets of Fkh2/Ndd1 regulation. Genes belonging to the CLB2 cluster, including CLB2 itself, contain one or more SFF binding sites at which Fkh2, Ndd1, and the MADS box protein Mcm1 bind. Both Fkh2 and Ndd1 are subject to extensive phosphorylation during the cell cycle. The Clb5/Cdc28 complex phosphorylates residues on the C-terminal region of Fkh2 between the S phase and G 2 (44). The interaction between Fkh2 and Ndd1 is optimal when both proteins are phosphorylated. Ndd1 is a substrate for the Clb2/ Cdc28 G 2 /M kinase, and phosphorylation promotes the interaction between Ndd1 and the FHA domain of Fkh2 (11,49). Thus, CLB2 expression is likely to be subjected to a positivefeedback loop (11,23,44,49) and may be regulated differently from other genes in the CLB2 cluster. Very little is understood about how this feedback loop is broken to repres...

Coupling Phosphate Homeostasis to Cell Cycle-Specific Transcription: Mitotic Activation of Saccharomyces cerevisiae PHO5 by Mcm1 and Forkhead Proteins

“…10). It is possible that Rpd3 histone deacetylase activity helps establish and/or maintain the repressive chromatin configuration that silences PHO5 and CLB2 transcription in G 1 phase of cycling cells (74,77). Rpd3 is released from CLB2 by Cln-kinase activity as cells progress through START (87), much like the temporal binding profile for Mcm1-Fkh2 that we observed at PHO5.…”

“…A straightforward explanation for positive binding to CLB2 in these studies is localization to an extended nuclease-hypersensitive site of 4 to 5 Mcm1 sites and the single known Fkh site at this gene (1,38,46,50,77). Cooperative binding to this region would account for the high occupancy of Mcm1 and forkheads in both late G 1 (␣-factor arrest) and M phase (Noc arrest) (1,34).…”

Nucleosome Remodeling and Transcriptional Repression Are Distinct Functions of Isw1 in Saccharomyces cerevisiae

“…There are strong correlations between the nucleosome remodeling activity of Isw1 and transcriptional repression (12,45,53), and current models link these two functions although direct evidence is lacking. Moreover, information concerning the structural and biochemical aspects of the SANT and SLIDE domains supports their involvement in nucleosome mobilization and reorganization (13,21).…”

Deregulation of DSE1 Gene Expression Results in Aberrant Budding within the Birth Scar and Cell Wall Integrity Pathway Activation in Saccharomyces cerevisiae

“…The Isw2 chromatin remodeling complex is involved in the transcriptional regulation of a broad spectrum of genes by influencing the accessibility of chromatin to the transcriptional machinery (32). It was found to participate, e.g., in the repression of the mitotic cyclin-encoding gene CLB2 (24) and MATa-specific genes in MAT␣ cells (20,31). The absence of either Isw2 or Itc1 in MAT␣ cells results in an inappropriate a-factor production and an autocrine activation of the pheromone response pathway.…”