The retinoblastoma protein (Rb) silences specific genes that are active in the S phase of the cell cycle and which are regulated by E2F transcription factors. Rb binds to the activation domain of E2F and then actively represses the promoter by a mechanism that is poorly understood. Here we show that Rb associates with a histone deacetylase, HDAC1, through the Rb 'pocket' domain. Association with the deacetylase is reduced by naturally occurring mutations in the pocket and by binding of the human papilloma virus oncoprotein E7. We find that Rb can recruit histone deacetylase to E2F and that Rb cooperates with HDAC1 to repress the E2F-regulated promoter of the gene encoding the cell-cycle protein cyclin E. Inhibition of histone deacetylase activity by trichostatin A (TSA) inhibits Rb-mediated repression of a chromosomally integrated E2F-regulated promoter. Our results indicate that histone deacetylases are important for regulating the cell cycle and that active transcriptional repression by Rb may involve the modification of chromatin structure.

The DNA methyltransferase Dnmt1 is responsible for cytosine methylation in mammals and has a role in gene silencing. DNA methylation represses genes partly by recruitment of the methyl-CpG-binding protein MeCP2, which in turn recruits a histone deacetylase activity. Here we show that Dnmt1 is itself associated with histone deacetylase activity in vivo. Consistent with this association, we find that one of the known histone deacetylases, HDAC1, has the ability to bind Dnmt1 and can purify methyltransferase activity from nuclear extracts. We have identified a transcriptional repression domain in Dnmt1 that functions, at least partly, by recruiting histone deacetylase activity and shows homology to the repressor domain of the trithorax-related protein HRX (also known as MLL and ALL-1). Our data show a more direct connection between DNA methylation and histone deacetylation than was previously considered. We suggest that the process of DNA methylation, mediated by Dnmt1, may depend on or generate an altered chromatin state via histone deacetylase activity.

Negative regulators include the tumour supressor protein Søren J.Nielsen, Alexander Brehm and p53 (O'Connor et al., 1995) and the most important Tony Kouzarides 1 regulator, the retinoblastoma protein RB (Flemington et al., Wellcome/CRC Institute and Department of Pathology, University of 1993; Hagemeier et al., 1993; Luo Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK et al., 1998;Magnaghi-Jaulin et al., 1998 (Weintraub et al., 1995;Zhang et al., 1999) and by During the G 1 phase of the cell cycle, an E2F-RB recruiting a histone deacetylase complex (Brehm et al., complex represses transcription, via the recruitment 1998; Luo et al., 1998;Magnaghi-Jaulin et al., 1998). of histone deacetylase activity. Phosphorylation of RB at the G 1 /S boundary generates a pool of 'free' E2F, During G 1 phase, RB is phosphorylated, which blocks the which then stimulates transcription of S-phase genes.binding of E2F-RB and releases transcriptionally active Given that E2F1 activity is stimulated by p300/CBP E2F at the G 1 /S transition (reviewed by Mittnacht, 1998). acetylase and repressed by an RB-associatedOther levels of control that influence E2F activity include deacetylase, we asked if E2F1 was subject to modificaits own phosphorylation (Dynlacht et al., 1994(Dynlacht et al., , 1997 , 1997). and 3. Acetylation by P/CAF has three functional There are five E2F family members that have highly consequences on E2F1 activity: increased DNA-binding conserved DNA-binding and activation domains (Helin ability, activation potential and protein half-life. Kaelin et al., 1992; reviewed by Dyson, results suggest that acetylation stimulates the functions 1998; Helin, 1998) whereas a sixth protein, EMA, is only of the non-RB bound 'free' form of E2F1. Consistent conserved in the DNA-binding domain. Three of these, with this, we find that the RB-associated histone E2F1, 2 and 3, have the ability to induce S phase (Johnson deacetylase can deacetylate E2F1. These results identify et al., 1993;DeGregori et al., 1995;Lukas et al., 1996). acetylation as a novel regulatory modification that However, all E2F members are able to bind to a similar stimulates E2F1's activation functions.consensus sequence when heterodimerized with a member

#Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.