Functional characterization of ATAD2 as a new cancer/testis factor and a predictor of poor prognosis in breast and lung cancers.

Cancer cells frequently express genes normally active in male germ cells. ATAD2 is one of them encoding a conserved factor harbouring an AAA type ATPase domain and a bromodomain. We show here that ATAD2 is highly expressed in testis as well as in many cancers of different origins and that its high expression is a strong predictor of rapid mortality in lung and breast cancers. These observations suggest that ATAD2 acts on upstream and basic cellular processes to enhance oncogenesis in a variety of unrelated cell types. Accordingly, our functional studies show that ATAD2 controls chromatin dynamics, genome transcriptional activities and apoptotic cell response. We could also highlight some of the important intrinsic properties of its two regulatory domains, including a functional cross-talk between the AAA ATPase domain and the bromodomain. Altogether, these data indicate that ATAD2 overexpression in somatic cells, by acting on basic properties of chromatin, may contribute to malignant transformation.

[Spermiogenesis: histone acetylation triggers male genome reprogramming]. / Spermiogenèse: l'acétylation des histones déclenche la reprogrammation du génome mâle.

During their post-meiotic maturation, male germ cells undergo an extensive reorganization of their genome, during which histones become globally hyperacetylated, are then removed and progressively replaced by transition proteins and finally by protamines. The latter are known to tightly associate with DNA in the mature sperm cell. Although this is a highly conserved and fundamental biological process, which is a necessary prerequisite for the transmission of the male genome to the next generation, its molecular basis remains mostly unknown. We have identified several key factors involved in this process, and their detailed functional study has enabled us to propose the first model describing molecular mechanisms involved in post-meiotic male genome reprogramming. One of them, Bromodomain Testis Specific (BRDT), has been the focus of particular attention since it possesses the unique ability to specifically induce a dramatic compaction of acetylated chromatin. Interestingly, a mutation was found homozygous in infertile men which, according to our structural and functional studies, disrupts the function of the protein. A combination of molecular structural and genetic approaches has led to a comprehensive understanding of new major actors involved in the male genome reprogramming and transmission.

Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways.

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

New arylpiperazine derivatives as antagonists of the human cloned 5-HT(4) receptor isoforms.

New derivatives of arylpiperazine 9 were designed from ML 10302, a potent 5-HT(4) receptor agonist in the gastrointestinal system. Compounds were synthesized by condensation of a number of available arylpiperazines or heteroarylpiperazines with 2-bromoethyl 4-amino-5-chloro-2-methoxybenzoate. They were evaluated in binding assays on the recently cloned human 5-HT(4(e)) isoform stably expressed in C6 glial cells with [(3)H]GR 113808 as the radioligand. The affinity values (K(i)) depended upon the substituent on the aromatic ring. A chlorine atom produced a marked drop in activity (K(i) > 100 nM), while a m-methoxy group gave a compound with nanomolar affinity (K(i) = 3 nM). The most potent compounds were the heterocyclic derivatives with pyrimidine, pyrazine, pyridazine, or pyridine moieties (compounds 9r, 9t, 9u, 9x, respectively). K(i) values for 9a and 9r were determined for the 5-HT(4(a)), 5-HT(4(b)), 5-HT(4(c)), and 5-HT(4(d)) receptor isoforms transiently expressed in COS cells. The results indicated that the compounds were not selective. They produced an inhibition of the 5-HT-stimulated cyclic AMP synthesis in the C6 glial cells stably expressing the 5-HT(4(e)) receptor and shifted the 5-HT concentration-effect curve on adenylyl cyclase activity with pK(D) values of 7.44 and 8.47, respectively. In isolated human atrial myocytes, 9r antagonized the stimulatory effect of 5-HT on the L-type calcium current (I(Ca)) with a K(D) value of 0.7 nM.

Synthesis of a small library of phenylalkylamide derivatives as melatoninergic ligands for human mt1 and MT2 receptors.

Focused small libraries of melatonin receptor ligands from arylalkylamine derivatives were synthesised by combinatorial chemistry using the mix and split method in the solid phase. A library of 108 compounds was then synthesised from 12 arylalkyl amines and nine carboxylic acids. The compound mixtures were evaluated on chicken brain melatonin and recombinant human mt1 and MT2 receptors. Deconvolution of the most potent mixture demonstrated the superiority of 3-methoxy and 2,5-dimethoxy substitution on the phenyl ring with isopropyl, propyl and ethyl amido chains. Several compounds with nanomolar affinity for human melatonin receptors were obtained.

Active maintenance of mHDA2/mHDAC6 histone-deacetylase in the cytoplasm.

The intracellular localization, and thereby the function, of a number of key regulator proteins tagged with a short leucine-rich motif (the nuclear export signal or NES) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus [1]. A common characteristic of these regulators is their transient action in the nucleus during either a specific phase of the cell cycle or in response to specific signals [1]. Here, we show that a particular member of the class II histone-deacetylases mHDA2/mHDAC6 [2] belongs to this family of cellular regulators that are present predominantly in the cytoplasm, but are also capable of shuttling between the nucleus and the cytoplasm. A very potent NES present at the amino terminus of mHDAC6 was found to play an essential role in this shuttling process. The sub-cellular localization of mHDAC6 appeared to be controlled by specific signals, since the arrest of cell proliferation was found to be associated with the translocation of a fraction of the protein into the nucleus. Data presented here suggest that mHDAC6 might be the first member of a functionally distinct class of deacetylases, responsible for activities not shared by other known histone deacetylases.

mHDA1/HDAC5 histone deacetylase interacts with and represses MEF2A transcriptional activity.

Recently we identified a new family of histone deacetylases in higher eukaryotes related to yeast HDA1 and showed their differentiation-dependent expression. Data presented here indicate that HDAC5 (previously named mHDA1), one member of this family, might be a potent regulator of cell differentiation by interacting specifically with determinant transcription factors. We found that HDAC5 was able to interact in vivo and in vitro with MEF2A, a MADS box transcription factor, and to strongly inhibit its transcriptional activity. Surprisingly, this repression was independent of HDAC5 deacetylase domain. The N-terminal non-deacetylase domain of HDAC5 was able to ensure an efficient repression of MEF2A-dependent transcription. We then mapped protein domains involved in the HDAC5-MEF2A interaction and showed that MADS box/MEF2-domain region of MEF2A interacts specifically with a limited region in the N-terminal part of HDAC5 which also possesses a distinct repressor domain. These data show that two independent class II histone deacetylases HDAC4 and HDAC5 are able to interact with members of the MEF2 transcription factor family and regulate their transcriptional activity, thus suggesting a critical role for these deacetylases in the control of cell proliferation/differentiation.

Differential recognition of histone H10 by monoclonal antibodies during cell differentiation and the arrest of cell proliferation.

Individual anti-H1(0) monoclonal antibodies were screened in an immunolocalization assay to isolate clones able to recognize H1(0) in a differentiation-dependent manner using a murine erythroleukemia cell line. Two clones were selected, one recognizing H1(0) only in differentiating cells (clone 27 antibody), and the other recognizing the protein constitutively (clone 34 antibody). Both antibodies recognized a restricted region of the protein located at the N-terminal part of the globular domain. Amino acids 24-30, essential for the recognition of the protein by the clone 27 antibody, are extremely conserved in all known H1(0)-like proteins from sea urchin to human. Within these residues, proline 26, responsible for a bend in this region, plays a particularly important role in the epitope recognition. The region involved in the protein recognition by clone 34 antibody is larger and encompasses amino acids 20-30. However, proline 26 does not play an essential role in the structure of this epitope. Detailed analysis of the differential recognition of H1(0) in chromatin during cell differentiation and proliferation suggests that the modification of chromatin structure as well as that of H1(0) conformation can account for this effect. Indeed, in vitro study of H1(0)-four-way junction DNA interaction showed that the N-terminal tail domain of the protein can influence the recognition of H1(0) by these antibodies when the protein interacts with DNA. The two monoclonal antibodies described here therefore seem to be valuable tools for investigating fine modulations in chromatin structure and the concomitant changes occurring in the conformation of the protein.