The E2F transcription factor family is known to play a key role in the timely expression of genes required for cell cycle progression and proliferation, but only a few E2F target genes have been identified. We explored the possibility that E2F regulators play a broader role by identifying additional genes bound by E2F in living human cells. A protocol was developed to identify genomic binding sites for DNA-binding factors in mammalian cells that combines immunoprecipitation of cross-linked protein-DNA complexes with DNA microarray analysis. Among ∼1200 genes expressed during cell cycle entry, we found that the promoters of 127 were bound by the E2F4 transcription factor in primary fibroblasts. A subset of these targets was also bound by E2F1. Most previously identified target genes known to have roles in DNA replication and cell cycle control and represented on the microarray were confirmed by this analysis. We also identified a remarkable cadre of genes with no previous connection to E2F regulation, including genes that encode components of the DNA damage checkpoint and repair pathways, as well as factors involved in chromatin assembly/ condensation, chromosome segregation, and the mitotic spindle checkpoint. Our data indicate that E2F directly links cell cycle progression with the coordinate regulation of genes essential for both the synthesis of DNA as well as its surveillance. The E2F transcription factor family plays a crucial and well-established role in cell cycle progression (Dyson 1998). This family comprises six different polypeptides (E2F1-E2F6) that pair with a heterodimeric partner (DP1 or DP2). E2F is thought to function by activating a panel of genes involved in progression through the G 1 phase as well as DNA replication. An important function of E2F is the recruitment of the retinoblastoma (pRB) tumor suppressor family of proteins ("pocket" proteins); this family includes the related proteins p107 and p130 (Harbour and Dean 2000). We and others have shown that E2F-mediated recruitment of pocket proteins in quiescent cells and the early G 1 phase of the cell cycle results in repression of genes that are subsequently activated at the G 1 /S phase transition. The pRB family both inhibits transcriptional activation of E2F and globally represses promoters by recruiting histone deacetylases (HDACs) (Brehm et al. 1998;Luo et al. 1998;Magnaghi-Jaulin et al. 1998;Ross et al. 1999Ross et al. , 2001.The E2F family can be functionally subdivided into repressors (E2F4 and E2F5) and activators (E2F1, E2F2, and E2F3) based on a number of findings. First, E2F target gene expression is reduced in E2F3-deficient mouse embryonic fibroblasts, and these cells show significant proliferative defects (Humbert et al. 2000). Chromatin immunoprecipitation (ChIP) experiments have shown that E2F1, E2F2, and E2F3 associate with the promoters of previously described human and mouse E2F-responsive genes coincident with their activation at the G 1 /S phase boundary (Takahashi et al. 2000; J. Rayman, Y. Takahashi, and B. Dynlach...

The behavior of As in paddy fields is of great interest considering high As contents of groundwater in several Asian countries where rice is the main staple. We determined the concentrations of Fe, Mn, and As in soil, soil water, and groundwater samples collected at different depths down to 2 m in an experimental paddy field in Japan during the cycle of flooded and non-flooded periods. In addition, we measured the oxidation states of Fe, Mn, and As in situ in soil samples using X-ray absorption near-edge structure (XANES) and conducted sequential extraction of the soil samples. The results show that Fe (hydr)oxide hosts As in soil. Arsenic in irrigation waters is incorporated in Fe (hydr)oxide in soil during the non-flooded period, and the As is quickly released from soil to water during the flooded period because of reductive dissolution of the Fe (hydr)oxide phase and reduction of As from As(V) to As(III). The enhancement of As dissolution by the reduction of As is supported by high As/Fe ratios of soil water during the flooded period and our laboratory experiments where As(III) concentrations and As(III)/As(V) ratios in submerged soil were monitored. Our work, primarily based on data from an actual paddy field, suggests that rice plants are enriched in As because the rice grows in flooded paddy fields when mobile As(III) is released to soil water.

Despite biochemical and genetic data suggesting that E2F and pRB (pocket protein) families regulate transcription via chromatin-modifying factors, the precise mechanisms underlying gene regulation by these protein families have not yet been defined in a physiological setting. In this study, we have investigated promoter occupancy in wild-type and pocket protein-deficient primary cells. We show that corepressor complexes consisting of histone deacetylase (HDAC1) and mSin3B were specifically recruited to endogenous E2F-regulated promoters in quiescent cells. These complexes dissociated from promoters once cells reached late G 1 , coincident with gene activation. Interestingly, recruitment of HDAC1 complexes to promoters depended absolutely on p107 and p130, and required an intact E2F-binding site. In contrast, mSin3B recruitment to certain promoters did not require p107 or p130, suggesting that recruitment of this corepressor can occur via E2F-dependent and -independent mechanisms. Remarkably, loss of pRB had no effect on HDAC1 or mSin3B recruitment. p107/p130 deficiency triggered a dramatic loss of E2F4 nuclear localization as well as transcriptional derepression, which is suggested by nucleosome mapping studies to be the result of localized hyperacetylation of nucleosomes proximal to E2F-binding sites. Taken together, these findings show that p130 escorts E2F4 into the nucleus and, together with corepressor complexes that contain mSin3B and/or HDAC1, directly represses transcription from target genes as cells withdraw from the cell cycle.