Alexander Brehm

Retinoblastoma protein recruits histone deacetylase to repress transcription

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.

DNA methyltransferase Dnmt1 associates with histone deacetylase activity

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.

Regulation of E2F1 activity by acetylation.

During the G1 phase of the cell cycle, an E2F–RB complex represses transcription, via the recruitment of histone deacetylase activity. Phosphorylation of RB at the G1/S boundary generates a pool of ‘free’ E2F, which then stimulates transcription of S‐phase genes. Given that E2F1 activity is stimulated by p300/CBP acetylase and repressed by an RB‐associated deacetylase, we asked if E2F1 was subject to modification by acetylation. We show that the p300/CBP‐associated factor P/CAF, and to a lesser extent p300/CBP itself, can acetylate E2F1 in vitro and that intracellular E2F1 is acetylated. The acetylation sites lie adjacent to the E2F1 DNA‐binding domain and involve lysine residues highly conserved in E2F1, 2 and 3. Acetylation by P/CAF has three functional consequences on E2F1 activity: increased DNA‐binding ability, activation potential and protein half‐life. These results suggest that acetylation stimulates the functions of the non‐RB bound ‘free’ form of E2F1. Consistent with this, we find that the RB‐associated histone deacetylase can deacetylate E2F1. These results identify acetylation as a novel regulatory modification that stimulates E2F1s activation functions.

The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth

E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2β, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2β and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways.

Native E2F/RBF Complexes Contain Myb-Interacting Proteins and Repress Transcription of Developmentally Controlled E2F Target Genes

The retinoblastoma tumor suppressor protein (pRb) regulates gene transcription by binding E2F transcription factors. pRb can recruit several repressor complexes to E2F bound promoters; however, native pRb repressor complexes have not been isolated. We have purified E2F/RBF repressor complexes from Drosophila embryo extracts and characterized their roles in E2F regulation. These complexes contain RBF, E2F, and Myb-interacting proteins that have previously been shown to control developmentally regulated patterns of DNA replication in follicle cells. The complexes localize to transcriptionally silent sites on polytene chromosomes and mediate stable repression of a specific set of E2F targets that have sex- and differentiation-specific expression patterns. Strikingly, seven of eight complex subunits are structurally and functionally related to C. elegans synMuv class B genes, which cooperate to control vulval differentiation in the worm. These results reveal an extensive evolutionary conservation of specific pRb repressor complexes that physically combine subunits with established roles in the regulation of transcription, DNA replication, and chromatin structure.

dMi‐2 and ISWI chromatin remodelling factors have distinct nucleosome binding and mobilization properties

Mi‐2 and ISWI, two members of the Snf2 superfamily of ATPases, reside in separate ATP‐dependent chromatin remodelling complexes. These complexes differ in their biochemical properties and are believed to perform distinct functions in the cell. We have compared the remodelling activity of recombinant Drosophila Mi‐2 (dMi‐2) with that of recombinant ISWI. Both proteins are nucleosome‐stimulated ATPases and promote nucleosome mobilization. However, dMi‐2 and ISWI differ in their interaction with nucleosome core particles, in their substrate requirements and in the direction of nucleosome mobilization. We have used antibodies to immobilize a complex containing dMi‐2 and the dRPD3 histone deacetylase from Drosophila embryo extracts. This complex shares the nucleosome‐stimulated ATPase and nucleosome mobilization properties of recombinant dMi‐2, demonstrating that these activities are maintained in a physiological context. Its functional properties distinguish dMi‐2 from both SWI2/SNF2 and ISWI, defining a new family of ATP‐dependent remodelling machines.

Physical and functional association of SU(VAR)3‐9 and HDAC1 in Drosophila

Modification of histones can have a dramatic impact on chromatin structure and function. Acetylation of lysines within the N‐terminal tail of the histone octamer marks transcriptionally active regions of the genome whereas deacetylation seems to play a role in transcriptional silencing. Recently, the methylation of the histone tails has also been shown to be important for transcriptional regulation and chromosome structure. Here we show by immunoaffinity purification that two activities important for chromatin‐mediated gene silencing, the histone methyltransferase SU(VAR)3‐9 and the histone deacetylase HDAC1, associate in vivo. The two activities cooperate to methylate pre‐acetylated histones. Both enzymes are modifiers of position effect variegation and interact genetically in flies. We suggest a model in which the concerted histone deacetylation and methylation by a SU(VAR)3‐9/HDAC1‐containing complex leads to a permanent silencing of transcription in particular areas of the genome.

Retinoblastoma protein meets chromatin

The retinoblastoma (RB) protein exerts its tumour-suppressor function by repressing the transcription of cellular genes required for DNA replication and cell division. Recent investigations into the mechanism of RB repression have revealed that RB can regulate transcription by effecting changes in chromatin structure. These findings point towards a link between chromatin regulation and cancer.

Temporal Recruitment of the mSin3A-Histone Deacetylase Corepressor Complex to the ETS Domain Transcription Factor Elk-1

ABSTRACT The transcriptional status of eukaryotic genes is determined by a balance between activation and repression mechanisms. The nuclear hormone receptors represent classical examples of transcription factors that can regulate this balance by recruiting corepressor and coactivator complexes in a ligand-dependent manner. Here, we demonstrate that the equilibrium between activation and repression via a single transcription factor, Elk-1, is altered following activation of the Erk mitogen-activated protein kinase cascade. In addition to its C-terminal transcriptional activation domain, Elk-1 contains an N-terminal transcriptional repression domain that can recruit the mSin3A-histone deacetylase 1 corepressor complex. Recruitment of this corepressor is enhanced in response to activation of the Erk pathway in vivo, and this recruitment correlates kinetically with the shutoff of one of its target promoters, c-fos. Elk-1 therefore undergoes temporal activator-repressor switching and contributes to both the activation and repression of target genes following growth factor stimulation.

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.