In eukaryotic cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1 (ref. 2), creating a binding site for the chromo domain of HP1 (refs 3, 4). Here we show that SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter, and in fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins.

MicroRNAs (miRNAs) constitute a class of small cellular RNAs (typically 21-23nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, although they are relatively few in number (less than 2000 human miRNAs). The high relative stability of miRNA in common clinical tissues and biofluids (e.g. plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. Furthermore miRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology. To facilitate discovery and clinical development of miRNA-based biomarkers, we developed a genome-wide Locked Nucleic Acid (LNA™)-based miRNA qPCR platform with unparalleled sensitivity and robustness. The platform allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Using this system, we have profiled thousands of biofluid samples including blood derived plasma and serum. An extensive quality control (QC) system has been implemented in order to secure technical excellence and reveal any unwanted bias coming from pre-analytical or analytical variables. We present our approaches to sample and RNA QC as well as data QC and normalization. Specifically we have developed normal reference ranges for circulating miRNAs in serum and plasma as well as a hemolysis indicator based on microRNA expression.

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

The acetylation state of histones can influence transcription. Acetylation, carried out by acetyltransferases such as CBP/p300 and P/CAF, is commonly associated with transcriptional stimulation, whereas deacetylation, mediated by the three known human deacetylases HDAC1, 2 and 3, causes transcriptional repression. The known human deacetylases represent a single family and are homologues of the yeast RPD3 deacetylase. Here we identify and characterize HDAC4, a representative of a new human histone deacetylase family, which is homologous to the yeast HDA1 deacetylase. We show that HDAC4, unlike other deacetylases, shuttles between the nucleus and the cytoplasm in a process involving active nuclear export. In the nucleus, HDAC4 associates with the myocyte enhancer factor MEF2A. Binding of HDAC4 to MEF2A results in the repression of MEF2A transcriptional activation, a function that requires the deacetylase domain of HDAC4. These results identify MEF2A as a nuclear target for HDAC4-mediated repression and suggests that compartmentalization may be a novel mechanism for controlling the nuclear activity of this new family of deacetylases.