http://repub.eur.nl/res/aut/26415/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/32495/ 2012-01-25T00:00:00Z Cellular compartments are believed to serve functional separation within the cell and allow it to perform multiple metabolic processes at the same time. Processes are divided between compartments with respect to the physiological requirements for a certain process. Thus, regulatory processes involving chromatin organization and control of gene activity are performed in the nucleus while metabolic processes are believed to be performed in the aqueous cytoplasm and cytoplasmic organelles. Numerous reports demonstrate that transcriptional modulation has a strong impact on metabolism, as expression levels of metabolic enzymes are limited by signaling pathways and the transcriptional machinery (Blanchet et al., 2011; Desvergne et al., 2006; Dufour et al., 2011). On the other hand, there should be mechanisms allowing feedback control from metabolism to gene expression that would tune the transcriptional program of the cell in accordance with its physiological needs (McKnight, 2010; Ray, 2010). Essentially, metabolic flux, including metabolic enzymes, small metabolites, nutrients and redox state, should impose reciprocal control upon nuclear events. However, there is little knowledge on mechanisms that allow such regulation. In recent years, several groups addressed this question and discovered that concentrations of intermediate metabolites indeed affect gene control (Shi, 2004). In all cases, metabolic compounds such as NADH, Acetyl-CoA and poly-ADP ribose bind to chromatin and change gene activity. Potentially many more metabolites should touch upon the gene expression machinery. These “metabolic” regulatory complexes might, in turn, affect expression of the transcriptional regulators of metabolism and close the feedback loop. http://repub.eur.nl/res/pub/21317/ 2010-11-01T00:00:00Z ATP-dependent chromatin-remodeling complexes (remodelers) are essential regulators of chromatin structure and gene transcription. How remodelers can act in a gene-selective manner has remained enigmatic. A yeast two-hybrid screen for proteins binding the Drosophila transcription factor Tramtrack69 (TTK69) identified MEP1. Proteomic characterization revealed that MEP1 is a tightly associated subunit of the NuRD remodeler, harboring the Mi2 enzymatic core ATPase. In addition, we identified the fly homolog of human Deleted in oral cancer 1 (DOC1), also known as CDK2-associated protein 1 (CDK2AP1), as a bona fide NuRD subunit. Biochemical and genetic assays supported the functional association between MEP1, Mi2, and TTK69. Genomewide expression analysis established that TTK69, MEP1, and Mi2 cooperate closely to control transcription. The TTK69 transcriptome profile correlates poorly with remodelers other than NuRD, emphasizing the selectivity of remodeler action. On the genes examined, TTK69 is able to bind chromatin in the absence of NuRD, but targeting of NuRD is dependent on TTK69. Thus, there appears to be a hierarchical relationship in which transcription factor binding precedes remodeler recruitment. http://repub.eur.nl/res/pub/27846/ 2010-02-01T00:00:00Z Drosophila GMP synthetase binds ubiquitin-specific protease 7 (USP7) and is required for its ability to deubiquitylate histone H2B. Previously, we showed that the GMPS/USP7 complex cooperates with the Polycomb silencing system through removal of the active ubiquitin mark from histone H2B (H2Bub). Here, we explored the interplay between GMPS and USP7 further and assessed their role in hormone-regulated gene expression. Genetic analysis established a strong cooperation between GMPS and USP7, which is counteracted by the histone H2B ubiquitin ligase BRE1. Loss of either GMPS or USP7 led to increased levels of histone H2Bub in mutant animals. These in vivo analyses complement our earlier biochemical results, establishing that GMPS/USP7 mediates histone H2B deubiquitylation. We found that GMPS/ USP7 binds ecdysone-regulated loci and that mutants display severe misregulation of ecdysone target genes. Ecdysone receptor (EcR) interacts biochemically and genetically with GMPS/USP7. Genetic and gene expression analyses suggested that GMPS/USP7 acts as a transcriptional corepressor. These results revealed the cooperation between a biosynthetic enzyme and a ubiquitin protease in developmental gene control by hormone receptors. Copyright http://repub.eur.nl/res/pub/29251/ 2008-05-15T00:00:00Z SUUR (Suppressor of Under-Replication) protein is responsible for late replication and, as a consequence, for DNA underreplication of intercalary and pericentric heterochromatin in Drosophila melanogaster polytene chromosomes. However, the mechanism by which SUUR slows down the replication process is not clear. To identify possible partners for SUUR we performed a yeast two-hybrid screen using full-length SUUR as bait. This identified HP1, the well-studied heterochromatin protein, as a strong SUUR interactor. Furthermore, we have determined that the central region of SUUR is necessary and sufficient for interaction with the C-terminal part of HP1, which contains the hinge and chromoshadow domains. In addition, recruitment of SUUR to ectopic HP1 sites on chromosomes provides evidence for their association in vivo. Indeed, we found that the distributions of SUUR and HP1 on polytene chromosomes are interdependent: both absence and overexpression of HP1 prevent SUUR from chromosomal binding, whereas SUUR overexpression causes redistribution of HP1 to numerous sites occupied by SUUR. Finally, HP1 binds to intercalary heterochromatin when histone methyltransferase activity of SU(VAR)3-9 is increased. We propose that interaction with HP1 is crucial for the association of SUUR with chromatin.