Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

An LNA-based loss-of-function assay for micro-RNAs.

MicroRNAs (miRNAs) have recently emerged as being essential for development and for the control of cell proliferation/differentiation in various organisms. However, little is known about miRNA function and mode of action at the cellular level. We have designed a miRNA loss-of-function assay, based on chemically modified locked nucleic acids (LNA) antisense oligonucleotides and usable in tissue culture cells. We show that LNA/DNA mixed oligonucleotides form highly stable duplexes with miRNAs in vitro. Ex vivo, the target miRNA becomes undetectable in cells transfected with the antisense oligonucleotide. The effect is dose-dependent, long-lasting, and specific. Moreover, using a reporter assay, we show that antisense LNA/DNA oligonucleotides inhibit short non-coding RNAs at the functional level. Thus LNA/DNA mixmers represent powerful tools for functional analysis of miRNAs.

CBP/p300 and muscle differentiation: no HAT, no muscle.

Terminal differentiation of muscle cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both muscle-specific and ubiquitous genes. Two distinct families of transcriptional co-activators, GCN5/PCAF and CREB-binding protein (CBP)/p300, are crucial to this process. While both possess histone acetyl-transferase (HAT) activity, previous studies have failed to identify a requirement for CBP/p300 HAT function in myogenic differentiation. We have addressed this issue directly using a chemical inhibitor of CBP/p300 in addition to a negative transdominant mutant. Our results clearly demonstrate that CBP/p300 HAT activity is critical for myogenic terminal differentiation. Furthermore, this requirement is restricted to a subset of events in the differentiation program: cell fusion and specific gene expression. These data help to define the requirements for enzymatic function of distinct coactivators at different stages of the muscle cell differentiation program.

Transcriptional repression by the retinoblastoma protein through the recruitment of a histone methyltransferase.

The E2F transcription factor controls the cell cycle-dependent expression of many S-phase-specific genes. Transcriptional repression of these genes in G(0) and at the beginning of G(1) by the retinoblasma protein Rb is crucial for the proper control of cell proliferation. Rb has been proposed to function, at least in part, through the recruitment of histone deacetylases. However, recent results indicate that other chromatin-modifying enzymes are likely to be involved. Here, we show that Rb also interacts with a histone methyltransferase, which specifically methylates K9 of histone H3. The results of coimmunoprecipitation experiments of endogenous or transfected proteins indicate that this histone methyltransferase is the recently described heterochromatin-associated protein Suv39H1. Interestingly, phosphorylation of Rb in vitro as well as in vivo abolished the Rb-Suv39H1 interaction. We also found that Suv39H1 and Rb cooperate to repress E2F activity and that Suv39H1 could be recruited to E2F1 through its interaction with Rb. Taken together, these data indicate that Suv39H1 is involved in transcriptional repression by Rb and suggest an unexpected link between E2F regulation and heterochromatin.

Cell cycle-dependent recruitment of HDAC-1 correlates with deacetylation of histone H4 on an Rb-E2F target promoter.

The transcription factor E2F, which is a key element in the control of cell proliferation, is repressed by Rb and other pocket proteins in growth-arrested differentiating cells, as well as in proliferating cells when they progress through early G1. It is not known whether similar mechanisms are operative in the two situations. A body of data suggests that E2F repression by pocket proteins involves class I histone deacetylases (HDACs). It has been hypothesized that these enzymes are recruited to E2F target promoters where they deacetylate histones. Here we have tested this hypothesis directly by using formaldehyde cross-linked chromatin immunoprecipitation (XChIP) assays to evaluate HDAC association in living cells. Our data show that a histone deacetylase, HDAC-1, is stably bound to an E2F target promoter during early G1 in proliferating cells and released at the G1-S transition. In addition, our results reveal an inverse correlation between HDAC-1 recruitment and histone H4 acetylation on specific lysines.

The histone deacetylase HDAC3 targets RbAp48 to the retinoblastoma protein.

The product of the retinoblastoma susceptibility gene, the Rb protein, functions partly through transcriptional repression of E2F-regulated genes. Repression by Rb is mediated, at least in part, by a histone deacetylase complex, whose enzymatic activity relies on HDAC1, HDAC2 or HDAC3. Recently, we have shown that the Rb-associated histone deacetylase complex contains RbAp48 protein, which interacts with HDAC1 and HDAC2. RbAp48 could favour the deacetylation of histones since it binds directly to histone H4. In agreement with that, we show that transcriptional repression of E2F activity requires the presence of RbAp48. HDAC3 was thought not to interact with RbAp48. However, we found that it shared with HDAC1 the ability to favour the recruitment of RbAp48 to Rb. This latter effect was unlikely to be due to activation of Rb function, since HDAC3 did not increase Rb-E2F1 interaction. Rather, we found, surprisingly, that HDAC3 could physically interact with RbAp48 both in vitro and in living cells. Taken together, our data suggest a model in which Rb mediates the recruitment to E2F-regulating promoters of a repressive complex containing either HDAC1, HDAC2 or HDAC3 and the histone-binding protein RbAp48.

The Rb/chromatin connection and epigenetic control: opinion.

The balance between cell differentiation and proliferation is regulated at the transcriptional level. In the cell cycle, the transition from G1 to S phase (G1/S transition) is of paramount importance in this regard. Indeed, it is only before this point that cells can be oriented toward the differentiation pathway: beyond, cells progress into the cycle in an autonomous manner. The G1/S transition is orchestrated by the transcription factor E2F. E2F controls the expression of a group of checkpoint genes whose products are required either for the G1-to-S transition itself or for DNA replication (e.g. DNA polymerase alpha). E2F activity is repressed in growth-arrested cells and in early G1, and is activated at mid-to-late G1. E2F is controlled by the retinoblastoma tumor suppressor protein Rb. Rb represses E2F mainly by recruiting chromatin remodeling factors (histone deacetylases and SWI/SNF complexes), the DNA methyltransferase DNMT1, and a histone methyltransferase. This review will focus on the molecular mechanisms of E2F repression by Rb during the cell cycle and during cell-cycle exit by differentiating cells. A model in which Rb irreversibly represses E2F-regulated genes in differentiated cells by an epigenetic mechanism linked to heterochromatin, and involving histone H3 and promoter DNA methylation, is discussed.

Programming the transcriptional state of replicating methylated dna.

CpG methylation is maintained in daughter chromatids by the action of DNA methyltransferase at the replication fork. An opportunity exists for transcription factors at replication forks to bind their cognate sequences and thereby prevent remethylation by DNA methyltransferase. To test this hypothesis, we injected a linearized, methylated, and partially single-stranded reporter plasmid into the nuclei of Xenopus oocytes and followed changes in the transcriptional activity after DNA replication. We find that dependent on Gal4-VP16, the action of DNA methyltransferase, and replication-coupled chromatin assembly DNA replication provides a window of time in which regulatory factors can activate or repress gene activity. Demethylation in the promoter region near the GAL4 binding sites of the newly synthesized DNA did not occur even though the Gal4 binding sites were occupied and transcription was activated. We conclude that "passive" demethylation at the replication fork is not simply dependent on the presence of DNA binding transcriptional activators.

DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters.

Methylation of CpG islands is associated with transcriptional silencing and the formation of nuclease-resistant chromatin structures enriched in hypoacetylated histones. Methyl-CpG-binding proteins, such as MeCP2, provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylase, but the mechanisms establishing the methylation patterns themselves are unknown. Whether DNA methylation is always causal for the assembly of repressive chromatin or whether features of transcriptionally silent chromatin might target methyltransferase remains unresolved. Mammalian DNA methyltransferases show little sequence specificity in vitro, yet methylation can be targeted in vivo within chromosomes to repetitive elements, centromeres and imprinted loci. This targeting is frequently disrupted in tumour cells, resulting in the improper silencing of tumour-suppressor genes associated with CpG islands. Here we show that the predominant mammalian DNA methyltransferase, DNMT1, co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1 and that DNMT1 cooperates with Rb to repress transcription from promoters containing E2F-binding sites. These results establish a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.

HATs off: selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF.

Histone acetyltransferases (HATs) play important roles in the regulation of gene expression. In this report, we describe the design, synthesis, and application of peptide CoA conjugates as selective HAT inhibitors for the transcriptional coactivators p300 and PCAF. Two inhibitors (Lys-CoA for p300 and H3-CoA-20 for PCAF) were found to be potent (IC(50) approximately = 0.5 microM) and selective (approximately 200-fold) in blocking p300 and PCAF HAT activities. These inhibitors were used to probe enzymatic and transcriptional features of HAT function in several assay systems. These compounds should be broadly useful as biological tools for evaluating the roles of HATs in transcriptional studies and may serve as lead agents for the development of novel antineoplastic therapeutics.

CBP/p300 histone acetyl-transferase activity is important for the G1/S transition.

Transforming viral proteins such as E1A which force quiescent cells into S phase have two essential cellular target proteins, Rb and CBP/p300. Rb regulates the G1/S transition by controlling the transcription factor E2F. CBP/p300 is a transcriptional co-activator with intrinsic histone acetyl-transferase activity. This activity is regulated in a cell cycle dependent manner and shows a peak at the G1/S transition, suggesting a function for CBP/p300 in this crucial step of the cell cycle. Here, we have artificially modulated CBP/p300 levels in individual cells through microinjection of specific antibodies and expression vectors. We show that CBP/p300 is required for cell proliferation and has an essential function during the G1/S transition. Using the same microinjection system and GFP-reporter vectors, we demonstrate that CBP/p300 is essential for the activity of E2F, a transcription factor that controls the G1/S transition. In addition, our results suggest that CBP HAT activity is required both for the G1/S transition and for E2F activity. Thus CBP/p300 seems to be a versatile protein involved in opposing cellular processes, which raises the question of how its multiple activities are regulated.

Unambiguous demonstration of triple-helix-directed gene modification.

Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies. However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated. To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region. The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified. Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.

Histone acetylation and the control of the cell cycle.

The critical steps of the cell cycle are generally controlled through the transcriptional regulation of specific subsets of genes. Transcriptional regulation has been recently linked to acetylation or deacetylation of core histone tails: acetylated histone tails are generally associated with active chromatin, whereas deacetylated histone tails are associated with silent parts of the genome. A number of transcriptional co-regulators are histone acetyl-transferases or histone deacetylases. Here, we discuss some of the critical cell cycle steps in which these enzymes are involved.

Autonomously binding protein detected on ets box of c-fos serum response element in proliferating cells.

The serum response element (SRE) in the c-fos promoter contains an ets box whose integrity is required for full activation of this proto-oncogene by nerve growth factor (NGF) in PC12 rat pheochromocytoma cells. Electrophoretic mobility shift assays (EMSA) detect a protein in nuclear extracts that binds to the wild-type SRE, but not to an SRE containing a mutated ets box. Competition studies using unlabeled probes, and supershift experiments using antibodies and in vitro translated core serum response factor (SRF) indicate that the protein in question is not YY1, SAP-1, nor Elk-1 and that it does not exhibit ternary complex factor (TCF) activity, so that it may correspond to an autonomously binding Ets family protein. The complete disappearance of this "Ets-like autonomous binding factor" upon terminal differentiation of both L6alpha2 myoblastic and PC12 pheochromocytoma cells points to a possible role in the proliferation/differentiation process.

Gene activation by triplex-forming oligonucleotide coupled to the activating domain of protein VP16.

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a chimera peptide-TFO able to activate transcription from a target gene. The designed hybrid molecule contains a triplex-forming sequence, linked through a phosphoroamidate bond to several minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). We show here that this TFO-peptide chimera (TFO-P) can specifically recognise its DNA target at physiological salt and pH conditions. Bound to the double-stranded target DNA in a promoter region, the TFO-P is able to activate gene expression. Our results suggest that this type of molecule may prove useful in the design of new tools for artificial modulation of gene expression.

Histone acetylation in signal transduction by growth regulatory signals.

Cell fate is determined by extracellular signals which are transmitted to the nucleus and result in the transcriptional regulation of specific subsets of genes. Transcriptional regulation has been recently linked to enzymatic activities which are able to acetylate or deacetylate core histone tails. A number of transcriptional co-regulators are histone acetyl-transferases or histone deacetylases. Here, we discuss the involvement of these enzymes in critical steps of cell proliferation or cell differentiation control

Phosphorylation by p44 MAP Kinase/ERK1 stimulates CBP histone acetyl transferase activity in vitro.

The transcriptional coactivator CBP displays an intrinsic histone acetyl transferase (HAT) activity which seems to participate in transcriptional activation through the destabilization of nucleosome structure. CBP is involved in the activity of several transcription factors that are nuclear endpoints of intracellular signal transduction pathways. In some instances, the transcription factors are phosphorylated upon cell activation, which induces their interaction with CBP. CBP itself is a phosphoprotein and can be phosphorylated by cycle-dependent kinases or by MAP kinases. Here we show that CBP phosphorylation by p44 MAP kinase/ERK1 results in the stimulation of its HAT enzymatic activity. The p44 MAP kinase/ERK1 phosphorylation sites are located in the C-terminal part of the protein, outside of the HAT domain. These sites are required for enzymatic stimulation, suggesting that phosphorylation by p44 MAP kinase/ERK1 induces a conformational change of the CBP molecule. Our data suggest that, in some instances, CBP itself might be a target for signal transduction pathways.

The Wilms' tumor gene product represses the transcription of thrombospondin 1 in response to overexpression of c-Jun.

Thrombospondin 1 (TSP1) is known for its significant anti-angiogenic properties. In a previous study, we have shown that transient or stable overexpression of the transcription factor c-Jun, in rat fibroblasts, leads to repression of TSP1. We now demonstrate that the c-Jun-induced repression of TSP1 does not occur directly and does not require binding of c-Jun to the TSP1 promoter. Instead, repression involves a factor secreted by c-Jun-overexpressing cells. This secreted factor triggers a signal transduction pathway from the membrane to the nucleus, and these signals lead to the binding of the product of the Wilms' tumor suppressor gene, WT1, to the -210 region of the TSP1 promoter. This region binds WT1 and SP1, but not EGR1, although its sequence fits the consensus binding site for this transcription factor. WT1 overexpression in transfected cells inhibits endogenous TSP1 gene expression and TSP1 transcription in experiments using TSP1 promoter-reporter constructs. The WT1 - KTS isoform is more active in repressing TSP1 transcription than WT1 + KTS, while EGR1 is inactive. Enhancement of WT1 binding to DNA in response to c-Jun does not require de novo protein synthesis. The above mechanism for TSP1 repression could apply to other genes, thus coordinating their regulation in the vicinity of a c-Jun-overexpressing cell. We conclude that WT1, which was discovered as a result of its tumor suppressor properties, may also possess oncogenic characteristics in the c-Jun transformation process, and thus repress the anti-angiogenic protein, TSP1.

Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A.

Transforming viral proteins such as E1A force cells through the restriction point of the cell cycle into S phase by forming complexes with two cellular proteins: the retinoblastoma protein (Rb), a transcriptional co-repressor, and CBP/p300, a transcriptional co-activator. These two proteins locally influence chromatin structure: Rb recruits a histone deacetylase, whereas CBP is a histone acetyltransferase. Progression through the restriction point is triggered by phosphorylation of Rb, leading to disruption of Rb-associated repressive complexes and allowing the activation of S-phase genes. Here we show that CBP, like Rb, is controlled by phosphorylation at the G1/S boundary, increasing its histone acetyltransferase activity. This enzymatic activation is mimicked by E1A.

A rapid and sensitive assay for histone acetyl-transferase activity.

Histone acetyl-transferases (HATs) seem to be key elements in the regulation of transcription. We have designed an enzymatic assay to quantify HAT enzymatic activity. In this assay, the substrate is a peptide corresponding to the 24 first amino acids of histone H4 which is coupled to biotin. After acetylation using [14C]acetyl-CoA, the peptide is purified on streptavidin beads and the associated radioactivity is measured. This assay is sensitive, rapid and convenient.