Karim Bouazoune

The dMi-2 chromodomains are DNA binding modules important for ATP-dependent nucleosome mobilization

Drosophila Mi‐2 (dMi‐2) is the ATPase subunit of a complex combining ATP‐dependent nucleosome remodelling and histone deacetylase activities. dMi‐2 contains an HMG box‐like region, two PHD fingers, two chromodomains and a SNF2‐type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. We have tested a panel of dMi‐2 deletion mutants in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi‐2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome‐stimulated ATPase activity. In contrast to dHP1, dMi‐2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi‐2 chromodomains display DNA binding activity that is not shared by other chromodomains. Our results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein–DNA interactions. Furthermore, we identify DNA binding as a novel chromodomain‐associated activity.

ATP-dependent chromatin remodeling complexes in Drosophila

The regulation of chromatin structure is of fundamental importance for many DNA-based processes in eukaryotes. Activation or repression of gene transcription or DNA replication depends on enzymes which can generate the appropriate chromatin environment. Several of these enzymes utilize the energy of ATP hydrolysis to alter nucleosome structure. In recent years our understanding of the multisubunit complexes within which they function, their mechanisms of action, their regulation and their in-vivo roles has increased. Much of what we have learned has been gleaned from studies in Drosophila melanogaster. Here we will review what we know about the main classes of ATP-dependent chromatin remodelers in Drosophila.

dMi-2 Chromatin Binding and Remodeling Activities Are Regulated by dCK2 Phosphorylation

A plethora of ATP-dependent chromatin-remodeling enzymes have been identified during the last decade. Many have been shown to play pivotal roles in the organization and expression of eukaryotic genomes. It is clear that their activities need to be tightly regulated to ensure their coordinated action. However, little is known about how ATP-dependent remodelers are regulated at the molecular level. Here, we have investigated the ATP-dependent chromatin remodeling enzyme Mi-2 of Drosophila melanogaster. Radioactive labeling of S2 cells reveals that dMi-2 is a phosphoprotein in vivo. dMi-2 phosphorylation is constitutive, and we identify dCK2 as a major dMi-2 kinase in cell extracts. dCK2 binds to and phosphorylates a dMi-2 N-terminal region. Dephosphorylation of recombinant dMi-2 increases its affinity for the nucleosome substrate, nucleosome-stimulated ATPase, and ATP-dependent nucleosome mobilization activities. Our results reveal a potential mechanism for regulation of the dMi-2 enzyme and point toward CK2 phosphorylation as a common feature of CHD family ATPases.

Analysis of individual remodeled nucleosomes reveals decreased histone–DNA contacts created by hSWI/SNF

Chromatin remodeling enzymes use the energy of ATP hydrolysis to alter histone–DNA contacts and regulate DNA-based processes in eukaryotes. Whether different subfamilies of remodeling complexes generate distinct products remains uncertain. We have developed a protocol to analyze nucleosome remodeling on individual products formed in vitro. We used a DNA methyltransferase to examine DNA accessibility throughout nucleosomes that had been remodeled by the ISWI and SWI/SNF families of enzymes. We confirmed that ISWI-family enzymes mainly created patterns of accessibility consistent with canonical nucleosomes. In contrast, SWI/SNF-family enzymes generated widespread DNA accessibility. The protection patterns created by these enzymes were usually located at the extreme ends of the DNA and showed no evidence for stable loop formation on individual molecules. Instead, SWI/SNF family proteins created extensive accessibility by generating heterogeneous products that had fewer histone–DNA contacts than a canonical nucleosome, consistent with models in which a canonical histone octamer has been ‘pushed’ off of the end of the DNA.

Methylation‐Sensitive Single‐Molecule Analysis of Chromatin Structure

Methylation‐sensitive single‐molecule analysis of chromatin structure is a high‐resolution method for studying nucleosome positioning. As described in this unit, this method allows for the analysis of the chromatin structure of unmethylated CpG islands or in vitro–remodeled nucleosomes by treatment with the CpG‐specific DNA methyltransferase SssI (M.SssI), followed by bisulfite sequencing of individual progeny DNA molecules. Unlike nuclease‐based approaches, this method allows each molecule to be viewed as an individual entity instead of an average population. Curr. Protoc. Mol. Biol. 89:21.17.1‐21.17.16.

EcR recruits dMi-2 and increases efficiency of dMi-2-mediated remodelling to constrain transcription of hormone-regulated genes

Gene regulation by steroid hormones plays important roles in health and disease. In Drosophila, the hormone ecdysone governs transitions between key developmental stages. Ecdysone-regulated genes are bound by a heterodimer of ecdysone receptor (EcR) and Ultraspiracle. According to the bimodal switch model, steroid hormone receptors recruit corepressors in the absence of hormone and coactivators in its presence. Here we show that the nucleosome remodeller dMi-2 is recruited to ecdysone-regulated genes to limit transcription. Contrary to the prevalent model, recruitment of the dMi-2 corepressor increases upon hormone addition to constrain gene activation through chromatin remodelling. Furthermore, EcR and dMi-2 form a complex that is devoid of Ultraspiracle. Unexpectedly, EcR contacts the dMi-2 ATPase domain and increases the efficiency of dMi-2-mediated nucleosome remodelling. This study identifies a non-canonical EcR-corepressor complex with the potential for a direct regulation of ATP-dependent nucleosome remodelling by a nuclear hormone receptor.

The dosage-compensation complex in flies and humans

A report on the 6th EMBL Transcription Meeting, Heidelberg, Germany, 28 August-1 September 2004.

Chromatin: Assembly, remodelled

Biochemical assays reveal that nucleosome maturation and chromatin remodelling by the motor protein Chd1 are distinct, separable enzymatic activities.

Chromatin remodeling by the CHD7 protein is impaired by mutations that cause human developmental disorders

Background Mutations in the CHD7 gene cause CHARGE, a developmental syndrome which affect most organs. In addition, CHD7 mutations also cause puberty and reproductive organ formation disorders such as Idiopathic Hypogonadotropic Hypogonadism and Kallmann Syndrome. Genetic studies in model organisms have further established CHD7 as a central regulator of vertebrate development. To understand how the CHD7 proteins achieve its function and how mutation of CHD7 leads to developmental disorders, it is critical to characterize WT and mutant CHD7 proteins biochemically. However to date, CHD7 has not been characterized for activity, as it is extremely large and has resisted purification.