E2F1's role in ultraviolet-induced DNA damage response

The E2F1 transcription factor is a well-known regulator of cell proliferation and apoptosis, but its role in the DNA damage response is less clear. It has been shown that E2F1 becomes stabilized in response to DNA double strand breaks (DSBs) and accumulates at sites of DSBs. This process requires ATM kinase and serine 31 phosphorylation, which provides a binding site for TopBp1. However, the role of E2F1 at sites of DNA damage is not clear. We expanded the study of E2F1's role in the DNA damage response by exploring its functions in ultraviolet (UV) induced DNA damage, and identified that E2F1 promotes DNA repair and cell survival. To further investigate the mechanisms underlying our findings, we examined the possibility for direct involvement of E2F1 in DNA repair. We found that E2F1 localizes to sites of UV irradiation-induced DNA damage dependent on the ATR kinase and serine 31 of E2F1. E2F1 also associates with the GCN5 histone acetyltransferase in response to UV irradiation and recruits GCN5 to sites of DNA damage. This correlates with an increase in histone H3 lysine 9 (H3K9) acetylation and chromatin relaxation. In the absence of E2F1 or GCN5, nucleotide excision repair (NER) proteins do not efficiently localize to sites of UV damage and DNA repair is impaired. E2F1 mutants unable to bind DNA or activate transcription retain the ability to stimulate NER. These findings demonstrate a non-transcriptional role for E2F1 in DNA repair involving GCN5-mediated H3K9 acetylation and increased accessibility to the NER machinery. ^

TRIM24-REGULATED ESTROGEN RESPONSE IS DEPENDENT ON SPECIFIC HISTONE MODIFICATIONS IN BREAST CANCER CELLS

In this dissertation, I discovered that function of TRIM24 as a co-activator of ERα-mediated transcriptional activation is dependent on specific histone modifications in tumorigenic human breast cancer-derived MCF7 cells. In the first part, I proved that TRIM24-PHD finger domain, which recognizes unmethylated histone H3 lysine K4 (H3K4me0), is critical for ERα-regulated transcription. Therefore, when LSD1-mediated demethylation of H3K4 is inhibited, activation of TRIM24-regulated ERα target genes is greatly impaired. Importantly, I demonstrated that TRIM24 and LSD1 are cyclically recruited to estrogen responsive elements (EREs) in a time-dependent manner upon estrogen induction, and depletion of their expression exert corresponding time-dependent effect on target gene activation. I also identified that phosphorylation of histone H3 threonine T6 disrupts TRIM24 from binding to the chromatin and from activating ERα-regulated targets. In the second part, I revealed that TRIM24 depletion has additive effect to LSD1 inhibitor- and Tamoxifen-mediated reduction in survival and proliferation in breast cancer cells.

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.

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.

Functional domains for assembly of histones H3 and H4 into the chromatin of Xenopus embryos

Histones H3 and H4 have a well defined structural role in the nucleosome and an established role in the regulation of transcription. We have made use of a microinjection strategy using Xenopus embryos to define the minimal structural components of H3 and H4 necessary for nucleosome assembly into metazoan chromosomes in vivo. We find that both the N-terminal tail of H4, including all sites of acetylation, and the C-terminal α-helix of the H4 histone fold domain are dispensable for chromatin assembly. The N-terminal tail and an N-terminal α-helix of H3 are also dispensable for chromatin assembly. However, the remainder of the H3 and H4 histone folds are essential for incorporation of these proteins into chromatin. We suggest that elements of the histone fold domain maintain both nucleosomal integrity and have distinct functions essential for cell viability.

Apicidin: A novel antiprotozoal agent that inhibits parasite histone deacetylase

A novel fungal metabolite, apicidin [cyclo(N-O-methyl-l-tryptophanyl-l-isoleucinyl-d-pipecolinyl-l-2-amino-8-oxodecanoyl)], that exhibits potent, broad spectrum antiprotozoal activity in vitro against Apicomplexan parasites has been identified. It is also orally and parenterally active in vivo against Plasmodium berghei malaria in mice. Many Apicomplexan parasites cause serious, life-threatening human and animal diseases, such as malaria, cryptosporidiosis, toxoplasmosis, and coccidiosis, and new therapeutic agents are urgently needed. Apicidin’s antiparasitic activity appears to be due to low nanomolar inhibition of Apicomplexan histone deacetylase (HDA), which induces hyperacetylation of histones in treated parasites. The acetylation–deacetylation of histones is a thought to play a central role in transcriptional control in eukaryotic cells. Other known HDA inhibitors were also evaluated and found to possess antiparasitic activity, suggesting that HDA is an attractive target for the development of novel antiparasitic agents.

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.

Promoter-specific hypoacetylation of X-inactivated genes

The histone H4 acetylation status of the active X (Xa) and inactive X (Xi) chromosomes was investigated at the level of individual genes. A moderate level of acetylation was observed along the lengths of genes on both the Xi and Xa, regardless of their X inactivation status. However, this moderate level of acetylation was modified specifically in promoter regions. Transcriptionally active genes showed elevated levels of acetylation at their promoters on both the Xi and Xa. In contrast, promoters of X-inactivated genes were markedly hypoacetylated, which coincided with the methylation of adjacent CG dinucleotides. This promoter-specific hypoacetylation may be a key component of an X inactivation machinery that operates at the level of individual genes.

Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes ≈1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed ≈8 nm from the nucleosome center and remain apposed for 3–5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

Long-distance transcriptional enhancement by the histone acetyltransferase PCAF

Enhancers are defined by their ability to stimulate gene activity from remote sites and their requirement for promoter-proximal upstream activators to activate transcription. Here we demonstrate that recruitment of the p300/CBP-associated factor PCAF to a reporter gene is sufficient to stimulate promoter activity. The PCAF-mediated stimulation of transcription from either a distant or promoter-proximal position depends on the presence of an upstream activator (Sp1). These data suggest that acetyltransferase activity may be a primary component of enhancer function, and that recruitment of polymerase and enhancement of transcription are separable. Transcriptional activation by PCAF requires both its acetyltransferase activity and an additional activity within its N terminus. We also show that the simian virus 40 enhancer and PCAF itself are sufficient to counteract Mad-mediated repression. These results are compatible with recent models in which gene activity is regulated by the competition between deacetylase-mediated repression and enhancer-mediated recruitment of acetyltransferases.