The genome-wide program of gene expression during the cell division cycle in a human cancer cell line (HeLa) was characterized using cDNA microarrays. Transcripts of >850 genes showed periodic variation during the cell cycle. Hierarchical clustering of the expression patterns revealed coexpressed groups of previously well-characterized genes involved in essential cell cycle processes such as DNA replication, chromosome segregation, and cell adhesion along with genes of uncharacterized function. Most of the genes whose expression had previously been reported to correlate with the proliferative state of tumors were found herein also to be periodically expressed during the HeLa cell cycle. However, some of the genes periodically expressed in the HeLa cell cycle do not have a consistent correlation with tumor proliferation. Cell cycle-regulated transcripts of genes involved in fundamental processes such as DNA replication and chromosome segregation seem to be more highly expressed in proliferative tumors simply because they contain more cycling cells. The data in this report provide a comprehensive catalog of cell cycle regulated genes that can serve as a starting point for functional discovery. The full dataset is available at http://genome-www.stanford.edu/Human-CellCycle/HeLa/.

The expression of the replication-dependent histone mRNAs is tightly regulated during the cell cycle. As cells progress from G 1 to S phase, histone mRNA levels increase 35-fold, and they decrease again during G 2 phase. Replication-dependent histone mRNAs are the only metazoan mRNAs that lack polyadenylated tails, ending instead in a conserved stem-loop. Much of the cell cycle regulation is posttranscriptional and is mediated by the 3 stem-loop. A 31-kDa stem-loop binding protein (SLBP) binds the 3 end of histone mRNA. The SLBP is necessary for pre-mRNA processing and accompanies the histone mRNA to the cytoplasm, where it is a component of the histone messenger RNP. We used synchronous CHO cells selected by mitotic shakeoff and HeLa cells synchronized at the G 1 /S or the M/G 1 boundary to study the regulation of SLBP during the cell cycle. In each system the amount of SLBP is regulated during the cell cycle, increasing 10-to 20-fold in the late G 1 and then decreasing in the S/G 2 border. SLBP mRNA levels are constant during the cell cycle. SLBP is regulated at the level of translation as cells progress from G 1 to S phase, and the protein is rapidly degraded as they progress into G 2 . Regulation of SLBP may account for the posttranscriptional component of the cell cycle regulation of histone mRNA.The replication-dependent histone mRNAs are tightly regulated during the cell cycle, increasing 35-fold as cells progress from G 1 to S phase (18). There is only a three-to five-fold increase in the rate of transcription of the histone genes (7,19), indicating that much of the regulation is posttranscriptional. The posttranscriptional component of cell cycle regulation is mediated by the 3Ј end of the histone mRNA (32, 35), which is a highly conserved stem-loop (34). The only processing step necessary for formation of the mature histone mRNA is an endonucleolytic cleavage to form the 3Ј end (14). Cleavage is directed by two cis-acting elements, the stem-loop which binds to the hairpin binding factor (37, 38), and a purine-rich sequence about 10 nucleotides (nt) 3Ј of the cleavage site that binds the 5Ј end of U7 snRNA (3,39,55). Hairpin binding factor is composed of a single 31-kDa protein, the stem-loop binding protein (SLBP) (10, 60) or hairpin binding protein (33), which is required for processing in vivo (44) and which remains with the mature mRNA as a component of the cytoplasmic messenger RNP (mRNP) (9, 17). At least one additional factor, a heat-labile factor, is required for processing, but this factor has not been well defined biochemically (15).Two posttranscriptional regulatory steps contribute to the cell cycle regulation of histone mRNA concentrations. Processing is regulated as cells progress from G 1 to S phase, and the half-life of histone mRNA is reduced to about 10 min at the end of S phase (18), resulting in the destruction of histone mRNA prior to mitosis. We (60) and others (33) recently cloned the cDNA for the SLBP from several species using the yeast three-hybrid system (50). Here we show tha...

SummaryThe aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts with both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3), thereby recruiting chaperone-associated protein cargos to dynein motors for their transport to aggresomes. This molecular cascade entails functional dimerization of 14-3-3, which we show to be crucial for the formation of aggresomes in both yeast and mammalian cells. These results suggest that 14-3-3 functions as a molecular adaptor to promote aggresomal targeting of misfolded protein aggregates and may link such complexes to inclusion bodies observed in various neurodegenerative diseases.

Yin-Yang 1 (YY1) is a ubiquitously expressed zinc finger transcription factor. It regulates a vast array of genes playing critical roles in development, differentiation, and cell cycle. Very little is known about the mechanisms that regulate the functions of YY1. It has long been proposed that YY1 is a phosphoprotein; however, a direct link between phosphorylation and the function of YY1 has never been proven. Investigation of the localization of YY1 during mitosis shows that it is distributed to the cytoplasm during prophase and remains excluded from DNA until early telophase. Immunostaining studies show that YY1 is distributed equally between daughter cells and rapidly associates with decondensing chromosomes in telophase, suggesting a role for YY1 in early marking of active and repressed genes. The exclusion of YY1 from DNA in prometaphase HeLa cells correlated with an increase in the phosphorylation of YY1 and loss of DNA-binding activity that can be reversed by dephosphorylation. We have mapped three phosphorylation sites on YY1 during mitosis and show that phosphorylation of two of these sites can abolish the DNA-binding activity of YY1. These results demonstrate a novel mechanism for the inactivation of YY1 through phosphorylation of its DNA-binding domain. INTRODUCTIONYin-Yang 1 (YY1) is a ubiquitously expressed transcription factor that has been implicated in the regulation of a large number of genes critical for basic processes of development, cell growth, differentiation, cell cycle, and even apoptosis. The essential role of YY1 is underscored by the fact that its deletion resulted in peri-implantation lethality in mice, and disruption of only one allele caused severe developmental abnormalities (Donohoe et al., 1999). A substantial amount of information has been compiled over the past decade about the wide variety of target genes regulated by YY1; however, the regulation of the various functions of YY1 remains enigmatic.The expression of the human yy1 gene is under the control of a promoter that contains three Sp1-binding sites but neither a TATA box nor a CCAAT box, thus classifying YY1 among the constitutively expressed house keeping genes (Yao et al., 1998). Although higher YY1 expression levels have been detected in some types of cancers (Erkeland et al., 2003;Seligson et al., 2005;de Nigris et al., 2006), this type of regulation is not commonly observed for YY1. Other modes of regulation have been proposed including localization, cleavage, interaction with other proteins, and posttranslational modification. Regulation of YY1 through changes in subcellular localization of YY1 into the cytoplasm has been reported during early stages of development in Xenopus (Ficzycz et al., 2001), muscle cell differentiation (Delehouzee et al., 2005), G1/S transition of the mammalian cell cycle (Palko et al., 2004), and apoptosis (Krippner-Heidenreich et al., 2005). YY1 has also been shown to be cleaved under certain conditions of muscle cell differentiation (Walowitz et al., 1998) and apoptosis (Krippner-Heidenr...

Cessation of transcriptional activity is a hallmark of cell division. Many biochemical pathways have been shown and proposed over the past few decades to explain the silence of this phase. In particular, many individual transcription factors have been shown to be inactivated by phosphorylation. In this report, we show the simultaneous phosphorylation and mitotic redistribution of a whole class of modified transcription factors. C(2)H(2) zinc finger proteins (ZFPs) represent the largest group of gene expression regulators in the human genome. Despite their diversity, C(2)H(2) ZFPs display striking conservation of small linker peptides joining their adjacent zinc finger modules. These linkers are critical for DNA binding activity. It has been proposed that conserved phosphorylation of these linker peptides could be a common mechanism for the inactivation of the DNA binding activity of C(2)H(2) ZFPs, during mitosis. Using a novel antibody, raised against the phosphorylated form of the most conserved linker peptide sequence, we are able to visualize the massive and simultaneous mitotic phosphorylation of hundreds of these proteins. We show that this wave of phosphorylation is tightly synchronized, starting in mid-prophase right after DNA condensation and before the breakdown of the nuclear envelope. This global phosphorylation is completely reversed in telophase. In addition, the exclusion of the phospho-linker signal from condensed DNA clearly demonstrates a common mechanism for the mitotic inactivation of C(2)H(2) ZFPs.