A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability.

FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.

Neural restrictive silencer factor recruits mSin3 and histone deacetylase complex to repress neuron-specific target genes

Accumulative evidence suggests that more than 20 neuron-specific genes are regulated by a transcriptional cis-regulatory element known as the neural restrictive silencer (NRS). A trans-acting repressor that binds the NRS, NRSF [also designated RE1-silencing transcription factor (REST)] has been cloned, but the mechanism by which it represses transcription is unknown. Here we show evidence that NRSF represses transcription of its target genes by recruiting mSin3 and histone deacetylase. Transfection experiments using a series of NRSF deletion constructs revealed the presence of two repression domains, RD-1 and RD-2, within the N- and C-terminal regions, respectively. A yeast two-hybrid screen using the RD-1 region as a bait identified a short form of mSin3B. In vitro pull-down assays and in vivo immunoprecipitation-Western analyses revealed a specific interaction between NRSF-RD1 and mSin3 PAH1-PAH2 domains. Furthermore, NRSF and mSin3 formed a complex with histone deacetylase 1, suggesting that NRSF-mediated repression involves histone deacetylation. When the deacetylation of histones was inhibited by tricostatin A in non-neuronal cells, mRNAs encoding several neuronal-specific genes such as SCG10, NMDAR1, and choline acetyltransferase became detectable. These results indicate that NRSF recruits mSin3 and histone deacetylase 1 to silence neural-specific genes and suggest further that repression of histone deacetylation is crucial for transcriptional activation of neural-specific genes during neuronal terminal differentiation.

Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development

Amphibian metamorphosis is marked by dramatic, thyroid hormone (TH)-induced changes involving gene regulation by TH receptor (TR). It has been postulated that TR-mediated gene regulation involves chromatin remodeling. In the absence of ligand, TR can repress gene expression by recruiting a histone deacetylase complex, whereas liganded TR recruits a histone acetylase complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving histone deacetylation. During metamorphosis, endogenous TH allows TR to activate gene expression through histone acetylation. Here using chromatin immunoprecipitation assay, we directly demonstrate TR binding to TH response genes constitutively in vivo in premetamorphic tadpoles. We further show that TH treatment leads to histone deacetylase release from TH response gene promoters. Interestingly, in whole animals, changes in histone acetylation show little correlation with the expression of TH response genes. On the other hand, in the intestine and tail, where TH response genes are known to be up-regulated more dramatically by TH than in most other organs, we demonstrate that TH treatment induces gene activation and histone H4 acetylation. These data argue for a role of histone acetylation in transcriptional regulation by TRs during amphibian development in some tissues, whereas in others changes in histone acetylation levels may play no or only a minor role, supporting the existence of important alternative mechanisms in gene regulation by TR.

CoREST is an integral component of the CoREST- human histone deacetylase complex

Here we describe the components of a histone deacetylase (HDAC) complex that we term the CoREST-HDAC complex. CoREST-HDAC is composed of polypeptides distinct from previously characterized HDAC1/2-containing complexes such as the mSin3 and nucleosome remodeling and deacetylating (NRD, also named NURD, NuRD) complex. Interestingly, we do not observe RbAp46 and RbAp48 in this complex, although these proteins have been observed in all previously identified complexes and are thought to be part of an HDAC1/2 core. We identify the transcriptional corepressor CoREST and a protein with homology to polyamine oxidases as components of CoREST-HDAC. The HDAC1/2-interacting region of CoREST is mapped to a 179-aa region containing a SANT domain, a domain found in other HDAC1/2-interacting proteins such as NCoR, MTA1, and MTA2. Furthermore, we demonstrate that the corepressor function of CoREST depends on this region. Although CoREST initially was cloned as a corepressor to REST (RE1 silencing transcription factor/neural restrictive silencing factor), we find no evidence for the existence of the eight-zinc finger REST transcription factor as an interacting partner in this complex; however, we do find evidence for association of the putative oncogene ZNF 217 that contains eight zinc fingers.

Menin associates with a trithorax family histone methyltransferase complex and with the Hoxc8 locus

The cellular function of the menin tumor suppressor protein, product of the MEN1 gene mutated in familial multiple endocrine neoplasia type 1, has not been defined. We now show that menin is associated with a histone methyltransferase complex containing two trithorax family proteins, MLL2 and Ash2L, and other homologs of the yeast Set1 assembly. This menin-associated complex methylates histone H3 on lysine 4. A subset of tumor-derived menin mutants lacks the associated histone methyltransferase activity. In addition, menin is associated with RNA polymerase II whose large subunit carboxyl-terminal domain is phosphorylated on Ser5. Men1 knockout embryos and cells show decreased expression of the homeobox genes Hoxc6 and Hoxc8. Chromatin immunoprecipitation experiments reveal that menin is bound to the Hoxc8 locus. These results suggest that menin activates the transcription of differentiation-regulating genes by covalent histone modification, and that this activity is related to tumor suppression by MEN1.

ATM, a central controller of cellular responses to DNA damage

Mutations in the ATM gene lead to the genetic disorder ataxia-telangiectasia. ATM encodes a protein kinase that is mainly distributed in the nucleus of proliferating cells. Recent studies reveal that ATM regulates multiple cell cycle checkpoints by phosphorylating different targets at different stages of the cell cycle. ATM also functions in the regulation of DNA repair and apoptosis, suggesting that it is a central regulator of responses to DNA double-strand breaks.

Actively transcribed GAL genes can be physically linked to the nuclear pore by the SAGA chromatin modifying complex

Recent work has demonstrated that some actively transcribed genes closely associate with nuclear pore complexes (NPC) at the nuclear periphery. The Saccharomyces cerevisiae Mlp1 and Mlp2 proteins are components of the inner nuclear basket of the nuclear pore that mediate interactions with these active genes. To investigate the physical link between the NPC and active loci, we identified proteins that interact with the carboxyl-terminal globular domain of Mlp1 by tandem affinity purification coupled with mass spectrometry. This analysis led to the identification of several components of the Spt-Ada-Gcn5-acetyltransferase ( SAGA) histone acetyltransferase complex, Gcn5, Ada2, and Spt7. We utilized co-immunoprecipitation and in vitro binding assays to confirm the interaction between the Mlp proteins and SAGA components. Chromatin immunoprecipitation experiments revealed that Mlp1 and SAGA components associate with the same region of the GAL promoters. Critically, this Mlp-promoter interaction depends on the integrity of the SAGA complex. These results identify a physical association between SAGA and the NPC, and support previous results that relied upon visualization of GAL loci at the nuclear periphery by microscopy ( Cabal, G. G. Genovesio, A., Rodriguez-Navarro, S., Zimmer, C., Gadal, O., Lesne, A., Buc, H., Feuerbach- Fournier, F., Olivo-Marin, J.-C., Hurt, E. C., and Nehrbass, U. ( 2006) Nature 441, 770-773). We propose that a physical interaction between nuclear pore components and the SAGA complex can link the actively transcribed GAL genes to the nuclear pore.

Myc-mediated control of gene transcription in cancer cells

The Myc oncoproteins belong to a family of transcription factors composed by Myc, N-Myc and L-Myc. The most studied components of this family are Myc and N-Myc because their expressions are frequently deregulated in a wide range of cancers. These oncoproteins can act both as activators or repressors of gene transcription. As activators, they heterodimerize with Max (Myc associated X-factor) and the heterodimer recognizes and binds a specific sequence elements (E-Box) onto gene promoters recruiting histone acetylase and inducing transcriptional activation. Myc-mediated transcriptional repression is a quite debated issue. One of the first mechanisms defined for the Myc-mediated transcriptional repression consisted in the interaction of Myc-Max complex Sp1 and/or Miz1 transcription factors already bound to gene promoters. This interaction may interfere with their activation functions by recruiting co-repressors such as Dnmt3 or HDACs. Moreover, in the absence of , Myc may interfere with the Sp1 activation function by direct interaction and subsequent recruitment of HDACs. More recently the Myc/Max complex was also shown to mediate transcriptional repression by direct binding to peculiar E-box. In this study we analyzed the role of Myc overexpression in Osteosarcoma and Neuroblastoma oncogenesis and the mechanisms underling to Myc function. Myc overexpression is known to correlate with chemoresistance in Osteosarcoma cells. We extended this study by demonstrating that c-Myc induces transcription of a panel of ABC drug transporter genes. ABCs are a large family trans-membrane transporter deeply involved in multi drug resistance. Furthermore expression levels of Myc, ABCC1, ABCC4 and ABCF1 were proved to be important prognostic tool to predict conventional therapy failure. N-Myc amplification/overexpression is the most important prognostic factor for Neuroblastoma. Cyclin G2 and Clusterin are two genes often down regulated in neuroblastoma cells. Cyclin G2 is an atypical member of Cyclin family and its expression is associated with terminal differentiation and apoptosis. Moreover it blocks cell cycle progression and induces cell growth arrest. Instead, CLU is a multifunctional protein involved in many physiological and pathological processes. Several lines of evidences support the view that CLU may act as a tumour suppressor in Neuroblastoma. In this thesis I showed that N-Myc represses CCNG2 and CLU transcription by different mechanisms. • N-Myc represses CCNG2 transcription by directly interacting with Sp1 bound in CCNG2 promoter and recruiting HDAC2. Importantly, reactivation of CCNG2 expression through epigenetic drugs partially reduces N-Myc and HDAC2 mediated cell proliferation. • N-Myc/Max complex represses CLU expression by direct binding to a peculiar E-box element on CLU promoter and by recruitment of HDACs and Polycomb Complexes, to the CLU promoter. Overall our findings strongly support the model in which Myc overexpression/amplification may contribute to some aspects of oncogenesis by a dual action: i) transcription activation of genes that confer a multidrug resistant phenotype to cancer cells; ii), transcription repression of genes involved in cell cycle inhibition and cellular differentiation.

A histone deacetylase inhibitor potentiates retinoid receptor action in embryonal carcinoma cells

Histone acetylation is thought to have a role in transcription. To gain insight into the role of histone acetylation in retinoid-dependent transcription, we studied the effects of trichostatin A (TSA), a specific inhibitor of histone deacetylase, on P19 embryonal carcinoma cells. We show that coaddition of TSA and retinoic acid (RA) markedly enhances neuronal differentiation in these cells, although TSA alone does not induce differentiation but causes extensive apoptosis. Consistent with the cooperative effect of TSA and RA, coaddition of the two agents synergistically enhanced transcription from stably integrated RA-responsive promoters. The transcriptional synergy by TSA and RA required the RA-responsive element and a functional retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimer, both obligatory for RA-dependent transcription. Furthermore, TSA led to promoter activation by an RXR-selective ligand that was otherwise inactive in transcription. In addition, TSA enhanced transcription from a minimum basal promoter, independently of the RA-responsive element. Finally, we show that TSA alone or in combination with RA increases in vivo endonuclease sensitivity within the RA-responsive promoter, suggesting that TSA treatment might alter a local chromatin environment to enhance RXR/RAR heterodimer action. Thus, these results indicate that histone acetylation influences activity of the heterodimer, which is in line with the observed interaction between the RXR/RAR heterodimer and a histone acetylase presented elsewhere.

Analyse des modifications de la chromatine impliquées dans l'effet barrière de CTF1 aux télomères humains

Eukaryotic genomes are compartmentalized in different structural domains that can affect positively or negatively gene expression. These regions of euchromatin and heterochromatin are characterized by distinct histones marks which can facilitate or repress gene transcription. The chromatin environment represents thus one of the main problems to control gene expression in biotechnological applications or gene therapy, since its expression is affected by the chromatin neighboring its locus of insertion. Some chromatin regions like telomeres are composed of constitutive heterochromatin which leads to the telomeric position effect (TPE) that silences genes adjacent to the telomere. TPE is known to spread by the selfrecruitment of the SIR histone deacetylase complex from the telomere in S.cerevisiae, but the histone marks that are associated to telomeric chromatin in mammalian cells remain mostly unknown. The transcription factor CTF1 has shown antisilencing properties in mammalian cells and also a boundary activity against TPE in yeast cells when fused to the yeast Gal4 DNA binding domain. In the work presented here, we describe a dual-reporter system to assess the boundary activity of proteins such as CTF1 at human telomeres. When located between the two reporter genes, CTF1 shields the telomere distal gene from TPE, while the telomereproximal gene remains silenced by telomeric heterochromatin. The boundary activity of CTF1 is shown to act regardless its function of transcriptional activator, by opposition to the transcriptional activator VP16 which activates indifferently both transgenes. Moreover, this study shows that CTF1 boundary activity is linked to its H3 binding function, as expected from a chromatin remodeler. ChIP experiments showed that histone deacetylation is the main histone modification involved in gene silencing at mammalian cell telomeres. Distinctly to yeast cells, the histone deacetylation signal in human cells extented over a short range along the chromosome. CTF1 may help to block this propagation and therefore to restore histones acetylation level on telomere protected locus. Surprisingly, other histone marks such as trimethyl-H3K9 or trimethyl-H4K20 were found on telomere protected locus, while in another clone, unsilencing of telomere distal transgene was associated with recruitment of the histone variant H2A.Z. Thus, I conclude that CTF1 displays a chromatin boundary function which is independent of its transcriptional activity and therefore exhibit features required for use as chromatin insulator in biotechnological applications. RESUME Les génomes eucaryotes sont compartementalisés en domaines structurels qui peuvent affecter positivement ou négativement l'expression des gènes avoisinants. Ces régions dites d'euchromatine ou d'hétérochromatine sont caractérisées par des modifications posttraductionnelles des histones qui peuvent faciliter ou au contraire inhiber la transcription des gènes qui s'y trouvent. Ainsi, isoler un gène de son environnement chromatinien est problème fréquent lorsqu'il s'agit de contrôler son expression dans le cadre d'applications en biotechnologie ou encore en thérapie génique. Certaines régions de chromatine telles que les télomères sont composées d'hétérochromatine constitutive qui mène au silençage des gènes avoisinants. Cet effet de position télomérique (TPE) est connu dans la levure S.cerevisiae comme se propageant par auto-recrutement du complexe de déacétylation d'histone SIR, alors que peu de modifications de chromatine ont pu être associées à ce phénomène dans les cellules de mammifères. Le facteur de transcription CTF1 a montré des propriétés d'anti-silençage dans les cellules de mammifères, ainsi qu'une activité barrière contre le silençage télomérique dans les cellules de levures lorsqu'il est fusionné au domaine de liaison à l'ADN de la protéine de levure Gal4. Dans le travail présenté ci-après est décrit un système à deux gènes rapporteurs permettant de mesurer l'activité barrière de protéines telles que CTF1 aux télomères humains, et les modifications de chromatine qui y sont associées. Lorsque CTF1 est placé entre les deux gènes rapporteurs, le gène distant du télomère est protégé du silençage qui lui est associé, alors que le gène proche du télomère reste soumis à ce silençage induit par l'hétérochromatine télomérique. L'activité barrière de CTF1 est montrée ici comme agissant indépendamment de son activité transcriptionnelle, par opposition à l'activateur transcriptionnel VP16 qui active indifféremment les deux transgènes. En outre, cette étude appuie l'hypothèse stipulant que CTF1 agisse comme remodeleur chromatinien puisqu'elle démontre que son activité barrière est directement dépendante de son activité de liaison avec l'histone H3. De plus, des expériences d'immuno-précipitation de la chromatine démontrent que la déacétylation des histones est le majeur phénomène intervenant dans le silençage télomérique. Par opposition à la levure, ce signal de déacétylation ne se propage dans les cellules humaines que sur une courte distance le long du chromosome. CTF1 agit ainsi en bloquant cette propagation et en restaurant le niveau d'acétylation des histones sur le locus protégé du télomère. De manière surprenante et inattendue, d'autres modifications d'histones telles que 4 les H3K9 et H4K20 triméthylées sont aussi observées à ce locus, tandis le recrutement du variant H2A.Z peut aussi être suffisant à restaurer l'expression du gène distant du télomère. En terme de cette analyse, CTF1 exhibe ainsi une fonction de barrière chromatinienne qui exclue une activité transcriptionnelle non désirée - propriété qui est requise dans l'établissement des isolateurs visant à permettre le contrôle d'un transgène dans le cadre d'applications en biotechnologies.

Crystal structure of the BTB domain from PLZF

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein–protein interaction motif found at the N terminus of 5–10% of C2H2-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism of linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 Å resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and we expect the full-length PLZF protein to exist as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins.

Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells

BACKGROUND: Histone deacetylase inhibitors (HDACi) are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB) is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. RESULTS: We show here that the HDACi Sodium Butyrate (NaB), suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA) strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. CONCLUSION: HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.

Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component, Lsm11, in histone RNA processing.

A set of seven Sm proteins assemble on the Sm-binding site of spliceosomal U snRNAs to form the ring-shaped Sm core. The U7 snRNP involved in histone RNA 3' processing contains a structurally similar but biochemically unique Sm core in which two of these proteins, Sm D1 and D2, are replaced by Lsm10 and by another as yet unknown component. Here we characterize this factor, termed Lsm11, as a novel Sm-like protein with apparently two distinct functions. In vitro studies suggest that its long N-terminal part mediates an important step in histone mRNA 3'-end cleavage, most likely by recruiting a zinc finger protein previously identified as a processing factor. In contrast, the C-terminal part, which comprises two Sm motifs interrupted by an unusually long spacer, is sufficient to assemble with U7, but not U1, snRNA. Assembly of this U7-specific Sm core depends on the noncanonical Sm-binding site of U7 snRNA. Moreover, it is facilitated by a specialized SMN complex that contains Lsm10 and Lsm11 but lacks Sm D1/D2. Thus, the U7-specific Lsm11 protein not only specifies the assembly of the U7 Sm core but also fulfills an important role in U7 snRNP-mediated histone mRNA processing.

Biochemical demonstration of complex formation of histone pre-mRNA with U7 small nuclear ribonucleoprotein and hairpin binding factors.

Histone RNA 3' end formation occurs through a specific cleavage reaction that requires, among other things, base-pairing interactions between a conserved spacer element in the pre-mRNA and the minor U7 snRNA present as U7 snRNP. An oligonucleotide complementary to the first 16 nucleotides of U7 RNA can be used to characterize U7 snRNPs from nuclear extracts by native gel electrophoresis. Using similar native gel techniques, we present direct biochemical evidence for a stable association between histone pre-mRNA and U7 snRNPs. Other complexes formed in the nuclear extract are dependent on the 5' cap structure and on the conserved hairpin element of histone pre-mRNA, respectively. However, in contrast to the U7-specific complex, their formation is not required for processing. Comparison of several authentic and mutant histone pre-mRNAs with different spacer sequences demonstrates that the formation and stability of the U7-specific complex closely follows the predicted stability of the potential RNA-RNA hybrid. However, this does not exclude a stabilization of the complex by U7 snRNP structural proteins.

Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma

We used high-resolution SNP genotyping to identify regions of genomic gain and loss in the genomes of 212 medulloblastomas, malignant pediatric brain tumors. We found focal amplifications of 15 known oncogenes and focal deletions of 20 known tumor suppressor genes (TSG), most not previously implicated in medulloblastoma. Notably, we identified previously unknown amplifications and homozygous deletions, including recurrent, mutually exclusive, highly focal genetic events in genes targeting histone lysine methylation, particularly that of histone 3, lysine 9 (H3K9). Post-translational modification of histone proteins is critical for regulation of gene expression, can participate in determination of stem cell fates and has been implicated in carcinogenesis. Consistent with our genetic data, restoration of expression of genes controlling H3K9 methylation greatly diminishes proliferation of medulloblastoma in vitro. Copy number aberrations of genes with critical roles in writing, reading, removing and blocking the state of histone lysine methylation, particularly at H3K9, suggest that defective control of the histone code contributes to the pathogenesis of medulloblastoma.