Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation

Histone deacetylases (HDACs) catalyze the removal of acetyl groups on the amino-terminal lysine residues of core nucleosomal histones. This activity is associated generally with transcriptional repression. We have reported previously that inhibition of HDAC activity by hydroxamic acid-based hybrid polar compounds, such as suberoylanilide hydroxamic acid (SAHA), induces differentiation and/or apoptosis of transformed cells in vitro and inhibits tumor growth in vivo. SAHA is a potentially new therapeutic approach to cancer treatment and is in Phase I clinical trials. In several tumor cell lines examined, HDAC inhibitors alter the expression of less than 1% of expressed genes, including the cell cycle kinase inhibitor p21WAF1. In T24 bladder carcinoma cells, SAHA induces up to a 9-fold increase in p21WAF1 mRNA and protein, which is, at least in part, because of an increase in the rate of transcription of the gene. SAHA causes an accumulation of acetylated histones H3 and H4 in total cellular chromatin by 2 h, which is maintained through 24 h of culture. An increase in the accumulation of acetylated H3 and H4 was detected throughout the p21WAF1 promoter and the structural gene after culture with SAHA. The level of histone acetylation did not change in chromatin associated with the actin and p27 genes, and their mRNA expression was not altered during culture of T24 cells with SAHA. Thus, the present findings indicate that the induction of p21WAF1 by SAHA is regulated, at least in part, by the degree of acetylation of the gene-associated histones and that this induced increase in acetylation is gene selective.

The three members of the pocket proteins family share the ability to repress E2F activity through recruitment of a histone deacetylase

The transcription factor E2F plays a major role in cell cycle control in mammalian cells. E2F binding sites, which are present in the promoters of a variety of genes required for S phase, shift from a negative to a positive role in transcription at the commitment point, a crucial point in G1 that precedes the G1/S transition. Before the commitment point, E2F activity is repressed by members of the pocket proteins family. This repression is believed to be crucial for the proper control of cell growth. We have previously shown that Rb, the founding member of the pocket proteins family, represses E2F1 activity by recruiting the histone deacetylase HDAC1. Here, we show that the two other members of the pocket proteins family, p107 and p130, also are able to interact physically with HDAC1 in live cells. HDAC1 interacts with p107 and Rb through an “LXCXE”-like motif, similar to that used by viral transforming proteins to bind and inactivate pocket proteins. Indeed, we find that the viral transforming protein E1A competes with HDAC1 for p107 interaction. We also demonstrate that p107 is able to interact simultaneously with HDAC1 and E2F4, suggesting a model in which p107 recruits HDAC1 to repress E2F sites. Indeed, we demonstrate that histone deacetylase activity is involved in the p107- or p130-induced repression of E2F4. Taken together, our data suggest that all members of the E2F family are regulated in early G1 by similar complexes, containing a pocket protein and the histone deacetylase HDAC1.

Coiled Bodies Preferentially Associate with U4, U11, and U12 Small Nuclear RNA Genes in Interphase HeLa Cells but Not with U6 and U7 Genes

Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle–regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for χ2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.

Transcriptional adaptor and histone acetyltransferase proteins in Arabidopsis and their interactions with CBF1, a transcriptional activator involved in cold-regulated gene expression

The Arabidopsis CBF transcriptional activators bind to the CRT/DRE regulatory element present in the promoters of many cold-regulated genes and stimulate their transcription. Expression of the CBF1 proteins in yeast activates reporter genes carrying a minimal promoter with the CRT/DRE as an upstream regulatory element. Here we report that this ability of CBF1 is dependent upon the activities of three key components of the yeast Ada and SAGA complexes, namely the histone acetyltransferase (HAT) Gcn5 and the transcriptional adaptor proteins Ada2 and Ada3. This result suggested that CBF1 might function through the action of similar complexes in Arabidopsis. In support of this hypothesis we found that Arabidopsis has a homolog of the GCN5 gene and two homologs of ADA2, the first report of multiple ADA2 genes in an organism. The Arabidopsis GCN5 protein has intrinsic HAT activity and can physically interact in vitro with both the Arabidopsis ADA2a and ADA2b proteins. In addition, the CBF1 transcriptional activator can interact with the Arabidopsis GCN5 and ADA2 proteins. We conclude that Arabidopsis encodes HAT-containing adaptor complexes that are related to the Ada and SAGA complexes of yeast and propose that the CBF1 transcriptional activator functions through the action of one or more of these complexes.

Differential Expression of Genes for Cyclin-Dependent Protein Kinases in Rice Plants1

Cyclin-dependent protein kinases (CDKs) play key roles in regulating the eukaryotic cell cycle. We have analyzed the expression of four rice (Oryza sativa) CDK genes, cdc2Os1, cdc2Os2, cdc2Os3, and R2, by in situ hybridization of sections of root apices. Transcripts of cdc2Os1, cdc2Os2, and R2 were detected uniformly in the dividing region of the root apex. cdc2Os1 and cdc2Os2 were also expressed in differentiated cells such as those in the sclerenchyma, pericycle, and parenchyma of the central cylinder. By contrast, signals corresponding to transcripts of cdc2Os3 were distributed only in patches in the dividing region. Counterstaining of sections with 4′,6-diamidino-2-phenylindole and double-target in situ hybridization with a probe for histone H4 transcripts revealed that cdc2Os3 transcripts were abundant from the G2 to the M phase, but were less abundant or absent during the S phase. The levels of the Cdc2Os3 protein and its associated histone H1-kinase activity were reduced by treatment of cultured cells with hydroxyurea, which blocks cycling cells at the onset of the S phase. Our results suggest that domains other than the conserved amino acid sequence (the PSTAIRE motif) have important roles in the function of non-PSTAIRE CDKs in distinct cell-cycle phases.

Histone deacetylase-dependent transcriptional repression by pRB in yeast occurs independently of interaction through the LXCXE binding cleft

We have developed a yeast model system to address transcriptional repression by the retinoblastoma protein (pRB). When fused to the DNA-binding domain of Gal4p (DB-pRB), pRB can repress transcription of reporter genes containing Gal4p binding sites; the histone deacetylase activity encoded by yeast RPD3 is required for DB-pRB repression. Mutation of the LXCXE binding cleft in pRB, a region reported to be required for histone deacetylase recruitment, does not interfere with pRB-mediated repression. From these findings based on yeast experiments, we surmise that the small pocket region of pRB must contain an additional domain that confers histone deacetylase-dependent transcriptional repression. This hypothesis was verified by experiments examining pRB-dependent histone deacetylase association in mammalian cells. In addition to RPD3, repression by pRB in yeast requires MSI1, an ortholog of RbAp48, but not SIN3 or SAP30. By comparing the genetic requirements of DB-pRB repression in yeast to those of other DB-repressor fusions, we can suggest a mechanism by which pRB recruits histone deacetylase activity.

Histone H1 overexpressed to high level in tobacco affects certain developmental programs but has limited effect on basal cellular functions.

Histone H1, a major structural component of chromatin fiber, is believed to act as a general repressor of transcription. To investigate in vivo the role of this protein in transcription regulation during development of a multicellular organism, we made transgenic tobacco plants that overexpress the gene for Arabidopsis histone H1. In all plants that overexpressed H1 the total H1-to-DNA ratio in chromatin increased 2.3-2.8 times compared with the physiological level. This was accompanied by 50-100% decrease of native tobacco H1. The phenotypic changes in H1-overexpressing plants ranged from mild to severe perturbations in morphological appearance and flowering. No correlation was observed between the extent of phenotypic change and the variation in the amount of overexpressed H1 or the presence or absence of the native tobacco H1. However, the severe phenotypic changes were correlated with early occurrence during plant growth of cells with abnormally heterochromatinized nuclei. Such cells occurred considerably later in plants with milder changes. Surprisingly, the ability of cells with highly heterochromatinized nuclei to fulfill basic physiological functions, including differentiation, was not markedly hampered. The results support the suggestion that chromatin structural changes dependent on H1 stoichiometry and on the profile of major H1 variants have limited regulatory effect on the activity of genes that control basal cellular functions. However, the H1-mediated chromatin changes can be of much greater importance for the regulation of genes involved in control of specific developmental programs.

Yeast histone H3 and H4 N termini function through different GAL1 regulatory elements to repress and activate transcription.

Previous work has shown that N-terminal deletions of yeast histone H3 cause a 2- to 4-fold increase in the induction of GAL1 and a number of other genes involved in galactose metabolism. In contrast, deletions at the H4 N terminus cause a 10- to 20-fold decrease in the induction of these same GAL genes. However, H3 and H4 N-terminal deletions each decrease PHO5 induction only 2- to 4-fold. To define the GAL1 gene regulatory elements through which the histone N termini activate or repress transcription, fusions were made between GAL1 and PHO5 promoter elements attached to a beta-galactosidase reporter gene. We show here that GAL1 hyperactivation caused by the H3 N-terminal deletion delta 4-15 is linked to the upstream activation sequence. Conversely, the relative decrease in GAL1 induction caused by the H4N-terminal deletion delta 4-28 is linked to the downstream promoter which contains the TATA element. These data indicate that the H3 N terminus is required for the repression of the GAL1 upstream element, whereas the H4N terminus is required for the activation of the GAL1 downstream promoter element.

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.

Histone-deacetylase inhibitors for the treatment of cancer

Histone deacetylase inhibitors (HDACi) are a promising new class of chemotherapeutic drug currently in early phase clinical trials. A large number of structurally diverse HDACi have been purified or synthesised that mostly inhibit the activity of all eleven class I and II HDACs. While these agents demonstrate many features required for anti-cancer activity such as low toxicity against normal cells and an ability to inhibit tumor cell growth and survival at nanomolar concentrations, their mechanisms of action are largely unknown. Initially, a model was proposed whereby HDACi-mediated transactivation of a specific gene or set of genes was responsible for the inhibition of cell cycle progression or induction of apoptosis. Given that HDACs can regulate the activity of a number of nonhistone proteins and that histone acetylation is important for events such as DNA replication and mitosis that do not directly involve gene transcription, it appears that the initial mechanistic model for HDACi may have been too simple. Herein, we provide an update on the transcription-dependent and - independent events that may be important for the anti-tumor activities of HDACi and discuss the use of these compounds in combination with other chemotherapeutic drugs.

LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression

Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow-derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac-4, 5, 7) in BMM, whereas Hdac-1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS-induced expression of the Cox-2, Cxcl2, and Ifit2 genes. In the case of Cox-2, this effect was also apparent at the promoter level. Overexpression of Hdac-8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox-2 mRNA. Another class of LPS-inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.

Análise de expressão de genes da família SETD em leucemia linfocítica crônica

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2015.

Proscillaridin A effects on histone acetylation and C-MYC degradation in acute lymphoblastic leukemia

La leucémie lymphoblastique aiguë (LLA) représente environ 25% des cancers pédiatriques diagnostiqués chaque année. Dans 80 % des cas, une rémission complète est observée. Cependant, les patients résistants aux traitements ainsi que les patients en rechute présentent un mauvais pronostique. Les altérations épigénétiques sont des facteurs essentiels dans le développement et la progression de la maladie, ainsi qu’à la résistance aux traitements. Lors d’un criblage de médicaments approuvés par la FDA, nous avons découvert des molécules ayant des caractéristiques anticancéreux et épigénétiques. Pour évaluer l’activité de ces molécules, nous avons procédé à un criblage secondaire sur plusieurs lignées cellulaires leucémiques. Nous avons découvert qu’une de ces molécules, un glucoside cardiotonique appelé la proscillaridine A, avait une activité anticancéreuse spécifique pour des cellules leucémiques. Nous faisons donc l’hypothèse que la proscillaridine A pourrait avoir des effets épigénétiques et anticancéreux dans des modèles précliniques de LLA. Pour tester cette hypothèse, nous avons traité deux lignées cellulaires de LLA Nalm-6 (LLA pre-B) et Molt-4 (T-LLA) in vitro pendant 2 à 96 heures à des doses pertinentes sur le plan clinique. Nous avons alors pu observer une inhibition de croissance qui était dépendante de la dose administrée dans les deux lignées cellulaires, avec des valeurs de 50% d’inhibition de croissance (CI50) de 3.0 nM pour les Nalm-6 et de et 2.3 nM pour les Molt-4. De plus, nos études sur le cycle cellulaire par BrdU démontrent un arrêt en phase G2/M. Nous avons également détecté par immunobuvardage de type western des baisses significatives de l’acétylation de résidus de l’histone 3. Les niveaux d’expression des enzymes responsables de cette acétylation, les histones acétyltransférases CBP, P300 et TIP60 ainsi que de l’oncogène C-MYC étaient également diminuées. Par des analyses de séquençage de l’ARN, nous avons observé une augmentation de l’expression des gènes impliquées dans les processus d’apoptose et de différentiation cellulaire, ainsi qu’une diminution des gènes impliqués dans la prolifération cellulaire comme en particulier les gènes cibles de C-MYC. Ces résultats prometteurs suggèrent le potentiel prometteur de la proscillaridine A comme nouvelle thérapie pour les patients atteints de LLA.

Proscillaridin A effects on histone acetylation and C-MYC degradation in acute lymphoblastic leukemia

La leucémie lymphoblastique aiguë (LLA) représente environ 25% des cancers pédiatriques diagnostiqués chaque année. Dans 80 % des cas, une rémission complète est observée. Cependant, les patients résistants aux traitements ainsi que les patients en rechute présentent un mauvais pronostique. Les altérations épigénétiques sont des facteurs essentiels dans le développement et la progression de la maladie, ainsi qu’à la résistance aux traitements. Lors d’un criblage de médicaments approuvés par la FDA, nous avons découvert des molécules ayant des caractéristiques anticancéreux et épigénétiques. Pour évaluer l’activité de ces molécules, nous avons procédé à un criblage secondaire sur plusieurs lignées cellulaires leucémiques. Nous avons découvert qu’une de ces molécules, un glucoside cardiotonique appelé la proscillaridine A, avait une activité anticancéreuse spécifique pour des cellules leucémiques. Nous faisons donc l’hypothèse que la proscillaridine A pourrait avoir des effets épigénétiques et anticancéreux dans des modèles précliniques de LLA. Pour tester cette hypothèse, nous avons traité deux lignées cellulaires de LLA Nalm-6 (LLA pre-B) et Molt-4 (T-LLA) in vitro pendant 2 à 96 heures à des doses pertinentes sur le plan clinique. Nous avons alors pu observer une inhibition de croissance qui était dépendante de la dose administrée dans les deux lignées cellulaires, avec des valeurs de 50% d’inhibition de croissance (CI50) de 3.0 nM pour les Nalm-6 et de et 2.3 nM pour les Molt-4. De plus, nos études sur le cycle cellulaire par BrdU démontrent un arrêt en phase G2/M. Nous avons également détecté par immunobuvardage de type western des baisses significatives de l’acétylation de résidus de l’histone 3. Les niveaux d’expression des enzymes responsables de cette acétylation, les histones acétyltransférases CBP, P300 et TIP60 ainsi que de l’oncogène C-MYC étaient également diminuées. Par des analyses de séquençage de l’ARN, nous avons observé une augmentation de l’expression des gènes impliquées dans les processus d’apoptose et de différentiation cellulaire, ainsi qu’une diminution des gènes impliqués dans la prolifération cellulaire comme en particulier les gènes cibles de C-MYC. Ces résultats prometteurs suggèrent le potentiel prometteur de la proscillaridine A comme nouvelle thérapie pour les patients atteints de LLA.

Diet Carotenoid Lutein Modulates The Expression Of Genes Related To Oxygen Transporters And Decreases Dna Damage And Oxidative Stress In Mice.

Lutein (LT) is a carotenoid obtained by diet and despite its antioxidant activity had been biochemically reported, few studies are available concerning its influence on the expression of antioxidant genes. The expression of 84 genes implicated in antioxidant defense was quantified using quantitative reverse transcription polymerase chain reaction array. DNA damage was measured by comet assay and glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were quantified as biochemical parameters of oxidative stress in mouse kidney and liver. cDDP treatment reduced concentration of GSH and increased TBARS, parameters that were ameliorated in treatment associated with LT. cDDP altered the expression of 32 genes, increasing the expression of GPx2, APC, Nqo1 and CCs. LT changed the expression of 37 genes with an induction of 13 mainly oxygen transporters. In treatments associating cDDP and LT, 30 genes had their expression changed with a increase of the same genes of the cDDP treatment alone. These results suggest that LT might act scavenging reactive species and also inducing the expression of genes related to a better antioxidant response, highlighting the improvement of oxygen transport. This improved redox state of the cell through LT treatment could be related to the antigenotoxic and antioxidant effects observed.