The Rb/E2F complex represses S-phase genes both in cycling cells and in cells that have permanently exited from the cell cycle and entered a terminal differentiation pathway. Here we show that S-phase gene repression, which involves histone-modifying enzymes, occurs through distinct mechanisms in these two situations. We used chromatin immunoprecipitation to show that methylation of histone H3 lysine 9 (H3K9) occurs at several Rb/E2F target promoters in differentiating cells but not in cycling cells. Furthermore, phenotypic knock-down experiments using siRNAs showed that the histone methyltransferase Suv39h is required for histone H3K9 methylation and subsequent repression of S-phase gene promoters in differentiating cells, but not in cycling cells. These results indicate that the E2F target gene permanent silencing mechanism that is triggered upon terminal differentiation is distinct from the transient repression mechanism in cycling cells. Finally, Suv39h-depleted myoblasts were unable to express early or late muscle differentiation markers. Thus, appropriately timed H3K9 methylation by Suv39h seems to be part of the control switch for exiting the cell cycle and entering differentiation.

Short interfering RNAs (siRNAs) are short (21-23 nt) doublestranded RNAs that direct the sequence-specific degradation of corresponding mRNAs, resulting in suppression of gene activity. siRNAs are powerful tools for gene functional analysis in mammals. Chemically synthesized siRNAs permit transient gene repression but preclude inhibition of stable gene products as well as long-term phenotypic analyses. Permanent gene suppression can be achieved by transcribing siRNAs as stem-loop precursors from Pol III promoters. This approach, however, has a major limitation: inhibition cannot be controlled in a time-or tissuespecific manner. Thus, the approach cannot be applied to genes essential for cell survival or cell proliferation. To overcome these limitations, we have designed a CRE-lox-based strategy that allows one to repress gene activity in a time-dependent manner in cells, and in a time-or tissue-dependent manner in animals. Our approach promises to improve dramatically the procedures for functional genetics in mammals.

Bloom's syndrome is a rare human autosomal recessive disorder that combines a marked genetic instability and an increased risk of developing all types of cancers and which results from mutations in both copies of the BLM gene encoding a RecQ 3 -5 DNA helicase. We recently showed that BLM is phosphorylated and excluded from the nuclear matrix during mitosis. We now show that the phosphorylated mitotic BLM protein is associated with a 3 -5 DNA helicase activity and interacts with topoisomerase III␣. We demonstrate that in mitosis-arrested cells, ionizing radiation and roscovitine treatment both result in the reversion of BLM phosphorylation, suggesting that BLM could be dephosphorylated through the inhibition of cdc2 kinase. This was supported further by our data showing that cdc2 kinase activity is inhibited in ␥-irradiated mitotic cells. Finally we show that after ionizing radiation, BLM is not involved in the establishment of the mitotic DNA damage checkpoint but is subjected to a subcellular compartment change. These findings lead us to propose that BLM may be phosphorylated during mitosis, probably through the cdc2 pathway, to form a pool of rapidly available active protein. Inhibition of cdc2 kinase after ionizing radiation would lead to BLM dephosphorylation and possibly to BLM recruitment to some specific sites for repair.Mutations in both copies of the BLM gene give rise to Bloom's syndrome (BS), 1 a rare disorder characterized by marked genetic instability combined with a greatly increased predisposition to a wide range of cancers commonly affecting the general population. The BLM gene is located on chromosome 15 at 15q26.1 and encodes the BLM protein, which belongs to the DExH box-containing RecQ helicase subfamily (1) and displays ATP-and Mg 2ϩ -dependent 3Ј-5Ј-DNA helicase activity (2). The major cellular consequences of a BLM defect are an increase in homologous recombination and in the rate of widespread mutations. Indeed, BS cells display spontaneous hypermutability and several cytogenetic abnormalities including an increase in chromosome breaks, symmetric quadriradial chromatid interchanges between homologous chromosomes, and sister chromatid exchanges (for review, see Ref.3). Until recently, very little information was available about the physiological function of BLM. Now, several lines of evidences strongly support the involvement of BLM during DNA replication and in the cellular response to DNA damage. Recently, BLM protein has been shown to accumulate during the S phase of the cell cycle (4), to interact selectively in vitro with Holliday junctions (5), and to coimmunoprecipitate with hRAD51 from cells synchronized in early S phase (6). BLM has also been shown to participate in the BRCA1-associated genome surveillance complex (7), to be phosphorylated and to accumulate through an ATM-dependent pathway in response to ionizing radiation (8), to assemble with promyelocytic leukemia protein at sites of single-stranded DNA after ␥-irradiation (9), and to be cleaved early during apoptosis (10).We s...

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