Involvement of histone H3 methylation in Tup1-Ssn6 repression

The Tup1-Ssn6 complex regulates the expression of diverse classes of genes in Saccharomyces cerevisiae including those regulated by mating type, DNA damage, glucose, and anaerobic stress. The complex is recruited to target genes by sequence-specific repressor proteins. Once recruited to particular promoters, it is not completely clear how it functions to block transcription. Repression probably occurs through interactions with both the basal transcriptional machinery and components of chromatin. Tup1 interactions with chromatin are strongly influenced by acetylation of histories H3 and H4. Tup1 binds to underacetylated histone tails and requires multiple histone deacetylases (HDACs) for its repressive functions. Like acetylation, histone methylation is involved in regulation of gene expression. The possible role of histone methylation in Tup1 repression is not known. Here we examined possible roles of histone methyltransferases in Tup1-Ssn6 functions. We found that like other genes, Tup1-Ssn6 target genes exhibit increases in the levels of histone H3 lysine 4 methylation upon activation. However, deletion of individual or multiple histone methyltransferases (HMTs) and other SET-domain containing genes has no apparent effect on Tup1-Ssn6 mediated repression of a number of well-defined targets. Interestingly, we discovered that Ssn6 interacts with Set2. Since deletion of SET2 does not affect Tup1-Ssn6 repression, Ssn6 may utilize Set2 in other contexts to regulate gene repression. In order examine if the two components of the Tup1-Ssn6 complex have independent functions in the cell, we identified genes differentially expressed in tup1Δ and ssn6Δ mutants using DNA microarrays. Our data indicate that ∼4% of genes in the cell are regulated by Ssn6 independently of Tup1. In addition, expression of genes regulated by Tup1-Ssn6 seems to be differently affected by deletion of Ssn6 and deletion of Tup1, which indicates that these components might have separate functions. Our data shed new light on the classical view of Tup1-Ssn6 functions, and indicate that Ssn6 might have repressive functions as well. ^

Requirement of GCN5 histone acetyltransferase in mouse neural tube closure and skeletal patterning

Histone acetylation plays an essential role in many DNA-related processes such as transcriptional regulation via modulation of chromatin structure. Many histone acetytransferases have been discovered and studied in the past few years, but the roles of different histone acetyltransferases (HAT) during mammalian development are not well defined at present. Gcn5 histone acetyltransferase is highly expressed until E16.5 during development. Previous studies in our lab using a constitutive null allele demonstrated that Gcn5 knock out mice are embryonic lethal, precluding the study of Gcn5 functions at later developmental stages. The creation of a conditional Gcn5 null allele, Gcn5flox allele, bypasses the early lethality. Mice homozygous for this allele are viable and appear healthy. In contrast, mice homozygous for a Gcn5 Δex3-18 allele created by Cre-loxP mediated deletion display a phenotype identical to our original Gcn5 null mice. Strikingly, a Gcn5flox(neo) allele, which contain a neomycin cassette in the second intron of Gcn5 is only partially functional and gives rise to a hypomorphic phenotype. Initiation of cranial neural tube closure at forebrain/midbrain boundary fails, resulting in an exencephaly in some Gcn5flox(neo)/flox(neo) embryos. These defects were found at an even greater penetrance in Gcn5flox(neo)/Δ embryos and become completely penetrant in the 129Sv genetic background, suggesting that Gcn5 controls mouse neural tube closure in a dose dependent manner. Furthermore, both Gcn5flox(neo)/flox(neo) and Gcn5 flox(neo)/Δ embryos exhibit anterior homeotic transformations in lower thoracic and lumbar vertebrae. These defects are accompanied by decreased expression levels and a shift in anterior expression boundary of Hoxc8 and Hoxc9. This study provides the first evidence that Gcn5 regulates Hox gene expression and is required for normal axial skeletal patterning in mice. ^

OXIDATIVE STRESS BASED STRATEGIES FOR ENHANCING THE EFFICACY OF HISTONE DEACETYLASE INHIBITORS (HDACi)

Histone deacetylase inhibitors (HDACi) are anti-cancer drugs that primarily act upon acetylation of histones, however they also increase levels of intracellular reactive oxygen species (ROS). We hypothesized that agents that cause oxidative stress might enhance the efficacy of HDACi. To test this hypothesis, we treated acute lymphocytic leukemia cells (ALL) with HDACi and adaphostin (ROS generating agent). The combination of two different HDACi (vorinostat or entinostat) with adaphostin synergistically induced apoptosis in ALL. This synergistic effect was blocked when cells were pre-treated with the caspase-9 inhibitor, LEHD. In addition, we showed that loss of the mitochondrial membrane potential is the earliest event observed starting at 12 h. Following this event, we observed increased levels of superoxide at 16 h, and ultimately caspase-3 activation. Pre-treatment with the antioxidant N-acetylcysteine (NAC) blocked ROS generation and reversed the loss of mitochondrial membrane potential for both combinations. Interestingly, DNA fragmentation and caspase-3 activity was only blocked by NAC in cells treated with vorinostat-adaphostin; but not with entinostat-adaphostin. These results suggest that different redox mechanisms are involved in the induction of ROS-mediated apoptosis. To further understand these events, we studied the role of the antioxidants glutathione (GSH) and thioredoxin (Trx). We found that the combination of entinostat-adaphostin induced acetylation of the antioxidant thioredoxin (Trx) and decreased intracellular levels of GSH. However, no effect on Trx activity was observed in either combination. In addition, pre-treatment with GSH ethyl ester, a soluble form of GSH, did not block DNA fragmentation. Together these results suggested that GSH and Trx are not major players in the induction of oxidative stress. Array data examining the expression of genes involved in oxidative stress demonstrated a differential regulation between cells treated with vorinostat-adaphostin and entinostat-adaphostin. Some of the genes differentially expressed between the combinations include aldehyde oxidase 1, glutathione peroxidase-5, -6, peroxiredoxin 6 and myeloperoxidase. Taken together, these experimental results indicate that the synergistic activity of two different HDACi with adaphostin is mediated by distinct redox mechanisms in ALL cells. Understanding the mechanism involved in these combinations will advance scientific knowledge of how the action of HDACi could be augmented in leukemia models. Moreover, this information could be used for the development of effective clinical trials combining HDACi with other anticancer agents.

Targeting Histone deacetylases (HDAC) for the treatment of soft tissue sarcoma

Targeting Histone deacetylases (HDAC) for the treatment of genetically complex soft tissue sarcoma Histone deactylase inhibitors (HDACi) are a new class of anticancer therapeutics; however, little is known about HDACi or the individual contribution of HDAC isoform activity in soft tissue sarcoma (STS). We investigated the potential efficacy of HDACi as monotherapy and in combination with chemotherapy in a panel of genetically complex STS. We found that HDACi combined with chemotherapy significantly induced anti-STS effects in vitro and in vivo. We then focused our study of HDACi in malignant peripheral nerve sheath tumor (MPNST), a subtype of highly aggressive, therapeutically resistant, and commonly fatal malignancies that occur in patients with neurofibromatosis type-1 (NF1) or sporadically. The therapeutic efficacy of HDACi was investigated in a panel of NF1-associated and sporadic MPNST cell lines. Our results demonstrate the NF1-assocaited cohort to be highly sensitive to HDACi while sporadic cell lines exhibited resistance. HDACi-induced productive autophagy was found to be a mode of resistance and inhibiting HDACi-induced autophagy significantly induced pro-apoptotic effects of HDACi in vitro and in vivo. HDACs are not a single enzyme consisting of 11 currently known isoforms. HDACis used in these studies inhibit a variety of these isoforms, namely class I HDACs which include HDAC1, 2, 3, and 8. Recently, HDAC8-specific inhibitors (HDAC8i) have been created and tested in various cancer cell lines. Lastly, the potential therapeutic efficacy of HDAC8i was investigated in human (NF1-associated and sporadic) and NF1-associated murine-derived MPNST. HDAC8i abrogated cell growth in human and murine-derived MPNST cells. Similar to the pattern noticed with pan-HDACis NF1-associated cells, especially murine-derived, were more sensitive to HDAC8i compared to human sporadic MPNST cell lines. S-phase arrest was observed in human and murine MPNST cells, independent of p53 mutational and NF1 status. HDAC8i induced apoptosis is all cell lines tested, with a more pronounced effects in human and murine-derived NF1-associated cells. Most importantly, HDAC8i abrogated murine-derived MPNST xenograft growth in vivo. Taken together, these findings support the evaluation of pan-HDACi and isoform-specific inhibitors as a novel therapy to treat MPNST, including in combination with autophagy blocking combination regimens in particular for patients with sporadic MPNST.

Deposition-related sites K5/K12 in histone H4 are not required for nucleosome deposition in yeast

Histone H4 can be acetylated at N-terminal lysines K5, K8, K12, and K16, but newly synthesized H4 is diacetylated at K5/K12 in diverse organisms. This pattern is widely thought to be important for histone deposition onto replicating DNA. To investigate the importance of K5/K12 we have mutagenized these lysines in yeast and assayed for nucleosome assembly. Assaying was done in the absence of the histone H3 N terminus, which has functions redundant with those of H4 in histone deposition. Nucleosome assembly was assayed by three methods. Because nucleosome depletion may be lethal, we examined cell viability. We also analyzed nucleosome assembly in vivo and in vitro by examining plasmid superhelicity density in whole cells and supercoiling in yeast cell extracts. All three approaches demonstrate that mutagenizing K5 and K12 together does not prevent cell growth and histone deposition in vivo or in vitro. Therefore, K5/K12 cannot be required for nucleosome assembly in yeast. It is only when the first three sites of acetylation—K5, K8, and K12—are mutagenized simultaneously that lethality occurs and assembly is most strongly decreased both in vivo and in vitro. These data argue for the redundancy of sites K5, K8, and K12 in the deposition of yeast histone H4.

Phosphorylation of histone H3 at serine 10 is correlated with chromosome condensation during mitosis and meiosis in Tetrahymena

H3 phosphorylation has been correlated with mitosis temporally in mammalian cells and spatially in ciliated protozoa. In logarithmically growing Tetrahymena thermophila cells, for example, H3 phosphorylation can be detected in germline micronuclei that divide mitotically but not in somatic macronuclei that divide amitotically. Here, we demonstrate that micronuclear H3 phosphorylation occurs at a single site (Ser-10) in the amino-terminal domain of histone H3, the same site phosphorylated during mitosis in mammalian cells. Using an antibody specific for Ser-10 phosphorylated H3, we show that, in Tetrahymena, this modification is correlated with mitotic and meiotic divisions of micronuclei in a fashion that closely coincides with chromosome condensation. Our data suggest that H3 phosphorylation at Ser-10 is a highly conserved event among eukaryotes and is likely involved in both mitotic and meiotic chromosome condensation.

Enhanced transcription factor access to arrays of histone H3/H4 tetramer⋅DNA complexes in vitro: Implications for replication and transcription

Defined model systems consisting of physiologically spaced arrays of H3/H4 tetramer⋅5S rDNA complexes have been assembled in vitro from pure components. Analytical hydrodynamic and electrophoretic studies have revealed that the structural features of H3/H4 tetramer arrays closely resemble those of naked DNA. The reptation in agarose gels of H3/H4 tetramer arrays is essentially indistinguishable from naked DNA, the gel-free mobility of H3/H4 tetramer arrays relative to naked DNA is reduced by only 6% compared with 20% for nucleosomal arrays, and H3/H4 tetramer arrays are incapable of folding under ionic conditions where nucleosomal arrays are extensively folded. We further show that the cognate binding sites for transcription factor TFIIIA are significantly more accessible when the rDNA is complexed with H3/H4 tetramers than with histone octamers. These results suggest that the processes of DNA replication and transcription have evolved to exploit the unique structural properties of H3/H4 tetramer arrays.

Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity

Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.

The N-terminal tail of histone H2A binds to two distinct sites within the nucleosome core

Each of the core histone proteins within the nucleosome has a central “structured” domain that comprises the spool onto which the DNA superhelix is wrapped and an N-terminal “tail” domain in which the structure and molecular interactions have not been rigorously defined. Recent studies have shown that the N-terminal domains of core histones probably contact both DNA and proteins within the nucleus and that these interactions play key roles in the regulation of nuclear processes (such as transcription and replication) and are critical in the formation of the chromatin fiber. An understanding of these complex mechanisms awaits identification of the DNA or protein sites within chromatin contacted by the tail domains. To this end, we have developed a site-specific histone protein–DNA photocross-linking method to identify the DNA binding sites of the N-terminal domains within chromatin complexes. With this approach, we demonstrate that the N-terminal tail of H2A binds DNA at two defined locations within isolated nucleosome cores centered around a position ≈40 bp from the nucleosomal dyad and that this tail probably adopts a defined structure when bound to DNA.

Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation

The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by 1H NMR as a 4-methyl-24,25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.

MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with a t(11;16)(q23;p13.3)

The recurring translocation t(11;16)(q23;p13.3) has been documented only in cases of acute leukemia or myelodysplasia secondary to therapy with drugs targeting DNA topoisomerase II. We show that the MLL gene is fused to the gene that codes for CBP (CREB-binding protein), the protein that binds specifically to the DNA-binding protein CREB (cAMP response element-binding protein) in this translocation. MLL is fused in-frame to a different exon of CBP in two patients producing chimeric proteins containing the AT-hooks, methyltransferase homology domain, and transcriptional repression domain of MLL fused to the CREB binding domain or to the bromodomain of CBP. Both fusion products retain the histone acetyltransferase domain of CBP and may lead to leukemia by promoting histone acetylation of genomic regions targeted by the MLL AT-hooks, leading to transcriptional deregulation via aberrant chromatin organization. CBP is the first partner gene of MLL containing well defined structural and functional motifs that provide unique insights into the potential mechanisms by which these translocations contribute to leukemogenesis.

Histone underacetylation is an ancient component of mammalian X chromosome inactivation

Underacetylation of histone H4 is thought to be involved in the molecular mechanism of mammalian X chromosome inactivation, which is an important model system for large-scale genetic control in eukaryotes. However, it has not been established whether histone underacetylation plays a critical role in the multistep inactivation pathway. Here we demonstrate differential histone H4 acetylation between the X chromosomes of a female marsupial, Macropus eugenii. Histone underacetylation is the only molecular aspect of X inactivation known to be shared by marsupial and eutherian mammals. Its strong evolutionary conservation implies that, unlike DNA methylation, histone underacetylation was a feature of dosage compensation in a common mammalian ancestor, and is therefore likely to play a central role in X chromosome inactivation in all mammals.

The mouse histone H2a gene contains a small element that facilitates cytoplasmic accumulation of intronless gene transcripts and of unspliced HIV-1-related mRNAs

Histone mRNAs are naturally intronless and accumulate efficiently in the cytoplasm. To learn whether there are cis-acting sequences within histone genes that allow efficient cytoplasmic accumulation of RNAs, we made recombinant constructs in which sequences from the mouse H2a gene were cloned into a human β-globin cDNA. By using transient transfection and RNase protection analysis, we demonstrate here that a 100-bp sequence within the H2a coding region permits efficient cytoplasmic accumulation of the globin cDNA transcripts. We also show that this sequence appears to suppress splicing and can functionally replace Rev and the Rev-responsive element in the cytoplasmic accumulation of unspliced HIV-1-related mRNAs. Like the Rev-responsive element, this sequence acts in an orientation-dependent manner. We thus propose that the sequence identified here may be a member of the cis-acting elements that facilitate the cytoplasmic accumulation of naturally intronless gene transcripts.

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.

Purified histone acetyltransferase complexes stimulate HIV-1 transcription from preassembled nucleosomal arrays

Protein acetylation has been implicated in the regulation of HIV-1 gene transcription. Here, we have exploited the activities of four native histone acetyltransferase (HAT) complexes from yeast to directly test whether acetylation regulates HIV-1 transcription in vitro. HAT activities acetylating either histone H3 (SAGA, Ada, and NuA3) or H4 (NuA4) stimulate HIV-1 transcription from preassembled nucleosomal templates in an acetyl CoA-dependent manner. HIV-1 transcription from histone-free DNA is not affected by the HATs, indicating that these activities function in a chromatin-specific fashion. For Ada and NuA4, we demonstrate that acetylation of only histone proteins mediates enhanced transcription, suggesting that these complexes facilitate transcription at least in part by modifying histones. To address a potential mechanism by which HAT complexes stimulate transcription, we performed a restriction enzyme accessibility analysis. Each of the HATs increases the cutting efficiencies of restriction endonucleases targeting the HIV-1 chromatin templates in a manner not requiring transcription, suggesting that histone acetylation leads to nucleosome remodeling.