Differential effects of garcinol and curcumin on histone and p53 modifications in tumour cells.

BACKGROUND: Post-translational modifications (PTMs) of histones and other proteins are perturbed in tumours. For example, reduced levels of acetylated H4K16 and trimethylated H4K20 are associated with high tumour grade and poor survival in breast cancer. Drug-like molecules that can reprogram selected histone PTMs in tumour cells are therefore of interest as potential cancer chemopreventive agents. In this study we assessed the effects of the phytocompounds garcinol and curcumin on histone and p53 modification in cancer cells, focussing on the breast tumour cell line MCF7. METHODS: Cell viability/proliferation assays, cell cycle analysis by flow cytometry, immunodetection of specific histone and p53 acetylation marks, western blotting, siRNA and RT-qPCR. RESULTS: Although treatment with curcumin, garcinol or the garcinol derivative LTK-14 hampered MCF7 cell proliferation, differential effects of these compounds on histone modifications were observed. Garcinol treatment resulted in a strong reduction in H3K18 acetylation, which is required for S phase progression. Similar effects of garcinol on H3K18 acetylation were observed in the osteosarcoma cells lines U2OS and SaOS2. In contrast, global levels of acetylated H4K16 and trimethylated H4K20 in MCF7 cells were elevated after garcinol treatment. This was accompanied by upregulation of DNA damage signalling markers such as γH2A.X, H3K56Ac, p53 and TIP60. In contrast, exposure of MCF7 cells to curcumin resulted in increased global levels of acetylated H3K18 and H4K16, and was less effective in inducing DNA damage markers. In addition to its effects on histone modifications, garcinol was found to block CBP/p300-mediated acetylation of the C-terminal activation domain of p53, but resulted in enhanced acetylation of p53K120, and accumulation of p53 in the cytoplasmic compartment. Finally, we show that the elevation of H4K20Me3 levels by garcinol correlated with increased expression of SUV420H2, and was prevented by siRNA targeting of SUV420H2. CONCLUSION: In summary, although garcinol and curcumin can both inhibit histone acetyltransferase activities, our results show that these compounds have differential effects on cancer cells in culture. Garcinol treatment alters expression of chromatin modifying enzymes in MCF7 cells, resulting in reprogramming of key histone and p53 PTMs and growth arrest, underscoring its potential as a cancer chemopreventive agent.

C-terminal tensin-like gene functions as an oncogene and promotes cell motility in pancreatic cancer.

OBJECTIVES: C-terminal tensin-like gene (CTEN, also known as TNS4) localizes to focal adhesions and is reported to function as an oncogene in colonic, breast, lung, and gastric cancers. Its role in pancreatic cancer is unknown and was thus investigated in this study. METHODS: C-terminal tensinlike gene expression was evaluated by immunohistochemistry in a series of pancreatic cancers. Functional activity of the CTEN was tested by manipulating cellular CTEN levels using a dual approach of gene knockdown/forced expression. RESULTS: The CTEN is overexpressed in 31 (70.45%) of 44 pancreatic cancers. Functionally, changes in CTEN level did not alter cellular proliferation, but CTEN levels were positively associated with enhanced colony-forming efficiency in both Panc-1 and PSN-1 cell lines. Forced CTEN expression in Panc-1 cells stimulated cell motility, whereas knockdown of CTEN in PSN-1 inhibited cell motility in both transwell migration and wound-healing assays. Evaluation of downstream targets demonstrated that alterations in CTEN levels induced changes in focal adhesion kinase and E-cadherin, whereas integrin-linked kinase (ILK) remained unchanged. CONCLUSIONS: These are the first data showing an oncogenic role for CTEN in pancreatic cancer through promotion of colony formation and cell motility. The latter may be mediated by signaling through focal adhesion kinase and inhibiting E-cadherin.

Cten is targeted by Kras signalling to regulate cell motility in the colon and pancreas.

CTEN/TNS4 is an oncogene in colorectal cancer (CRC) which enhances cell motility although the mechanism of Cten regulation is unknown. We found an association between high Cten expression and KRAS/BRAF mutation in a series of CRC cell lines (p = 0.03) and hypothesised that Kras may regulate Cten. To test this, Kras was knocked-down (using small interfering (si)RNA) in CRC cell lines SW620 and DLD1 (high Cten expressors and mutant for KRAS). In each cell line, Kras knockdown was mirrored by down-regulation of Cten Since Kras signals through Braf, we tested the effect of Kras knockdown in CRC cell line Colo205 (which shows high Cten expression and is mutant for BRAF but wild type for KRAS). Cten levels were unaffected by Kras knockdown whilst Braf knockdown resulted in reduced Cten expression suggesting that Kras signals via Braf to regulate Cten. Quantification of Cten mRNA and protein analysis following proteasome inhibition suggested that regulation was of Cten transcription. Kras knockdown inhibited cell motility. To test whether this could be mediated through Cten, SW620 cells were co-transfected with Kras specific siRNAs and a Cten expression vector. Restoring Cten expression was able to restore cell motility despite Kras knockdown (transwell migration and wounding assay, p<0.001 for both). Since KRAS is mutated in many cancers, we investigated whether this relationship could be demonstrated in other tumour models. The experiments were repeated in the pancreatic cancer cell lines Colo357 & PSN-1(both high Cten expressors and mutant for KRAS). In both cell lines, Kras was shown to regulate Cten and forced expression of Cten was able to rescue loss of cell motility following Kras knockdown in PSN-1 (transwell migration assay, p<0.001). We conclude that, in the colon and pancreas, Cten is a downstream target of Kras and may be a mechanism through which Kras regulates of cell motility.

Interleukin-4-inducing principle from Schistosoma mansoni eggs contains a functional C-terminal nuclear localization signal necessary for nuclear translocation in mammalian cells but not for its uptake.

Interleukin-4-inducing principle from schistosome eggs (IPSE/alpha-1) is a protein produced exclusively by the eggs of the trematode Schistosoma mansoni. IPSE/alpha-1 is a secretory glycoprotein which activates human basophils via an IgE-dependent but non-antigen-specific mechanism. Sequence analyses revealed a potential nuclear localization signal (NLS) at the C terminus of IPSE/alpha-1. Here we show that this sequence (125-PKRRRTY-131) is both necessary and sufficient for nuclear localization of IPSE or IPSE-enhanced green fluorescent protein (EGFP) fusions. While transiently expressed EGFP-IPSE/alpha-1 was exclusively nuclear in the Huh7 and U-2 OS cell lines, a mutant lacking amino acids 125 to 134 showed both nuclear and cytoplasmic staining. Moreover, insertion of the IPSE/alpha-1 NLS into a tetra-EGFP construct rendered the protein nuclear. Alanine scanning mutagenesis revealed a requirement for the KRRR residues. Fluorescence microscopy depicted, and Western blotting further confirmed, that recombinant IPSE/alpha-1 protein added exogenously is rapidly internalized by CHO cells and accumulates in nuclei in an NLS-dependent manner. A mutant protein in which the NLS motif was disrupted by triple mutation (RRR to AAA) was able to penetrate CHO cells but did not translocate to the nucleus. Furthermore, the uptake of native glycosylated IPSE/alpha-1 was confirmed in human primary monocyte-derived dendritic cells and was found to be a calcium- and temperature-dependent process. Live-cell imaging showed that IPSE/alpha-1 is not targeted to lysosomes. In contrast, peripheral blood basophils do not take up IPSE/alpha-1 and do not require the presence of an intact NLS for activation. Taken together, our results suggest that IPSE/alpha-1 may have additional nuclear functions in host cells.

SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells.

The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBPDelta998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.

ZNF366 is an estrogen receptor corepressor that acts through CtBP and histone deacetylases.

The regulation of gene expression by estrogen receptor-alpha (ERalpha) requires the coordinated and temporal recruitment of diverse sets of transcriptional co-regulator complexes, which mediate nucleosome remodelling and histone modification. Using ERalpha as bait in a yeast two-hybrid screen, we have identified a novel ERalpha-interacting protein, ZNF366, which is a potent corepressor of ERalpha activity. The interaction between ZNF366 and ERalpha has been confirmed in vitro and in vivo, and is mediated by the zinc finger domains of the two proteins. Further, we show that ZNF366 acts as a corepressor by interacting with other known ERalpha corepressors, namely RIP140 and CtBP, to inhibit expression of estrogen-responsive genes in vivo. Together, our results indicate that ZNF366 may play an important role in regulating the expression of genes in response to estrogen.

Functional interaction of CREB binding protein (CBP) with nuclear transport proteins and modulation by HDAC inhibitors.

Nuclear transport proteins such as CSE1, NUP93 and Importin-alpha have recently been shown to be chromatin-associated proteins in yeast, which have unexpected functions in gene regulation. Here we report interactions between the mammalian histone acetyltransferase CBP with nuclear transport proteins CAS (a CSE1 homologue) and Importin-alpha (Impalpha) and NUP93. CAS was found to bind the SRC1 interaction domain (SID) of CBP via a leucine-rich motif in the N-terminus of the protein, that is conserved in other SID-binding proteins. Coimmunoprecipitation experiments also revealed that CBP and Impalpha proteins form a complex. As Impalpha is a known acetylation target of CBP/p300, and is recycled to the cytoplasm via the exportin CAS, we investigated whether HDAC inhibitors would alter the subcellular localization of these proteins. Treatment of COS-1 cells with the HDAC inhibitors trichostatin A or sodium butyrate resulted in sequestration of Impalpha in the nuclear envelope, accumulation of CAS in nuclear aggregates, and an increased number of CBP-containing PML bodies per cell. In addition, HDACi treatment appeared to enhance the association of Impalpha and CBP in coimmunoprecipitation experiments. Our results provide evidence for novel functional interactions between the chromatin modification enzyme CBP and nuclear transport proteins in mammalian cells.

Effects of glucocorticoids on STAT4 activation in human T cells are stimulus-dependent.

Glucocorticoids affect the immune system by a number of mechanisms, including modulation of cytokine production in lymphocytes. Glucocorticoids suppress T helper cell type 1 immune responses by decreasing the ability of T cells to respond to interleukin (IL)-12, a major inducer of interferon (IFN)-gamma. IFN-beta increases the expression of the anti-inflammatory cytokine IL-10 and suppresses IL-12. Signaling pathways through IFN-beta and the IL-12 receptor (IL-12R) involve activation by phosphorylation of signal transducer and activator of transcription 4 (STAT4). Our aim was to investigate the effects of dexamethasone on STAT4 activation by IFN-beta and IL-12 in human T cell blasts. We report that dexamethasone decreases IL-12-induced STAT4 phosphorylation and IFN-gamma production and enhances IFN-beta-induced STAT4 activation and IL-10 production. These effects are associated with a down-regulation of IL-12Rbeta1 expression but an up-regulation of IFN-betaR. These results indicate that the effect of glucocorticoids on the STAT4 signaling pathway depends on the stimulus activating that pathway.

MOZ-TIF2 alters cofactor recruitment and histone modification at the RARbeta2 promoter: differential effects of MOZ fusion proteins on CBP- and MOZ-dependent activators.

MOZ-TIF2 and MOZ-CBP are leukemogenic fusion proteins associated with therapy-induced acute myeloid leukemia. These proteins are thought to subvert normal gene expression in differentiating hematopoietic progenitor cells. We have previously shown that MOZ-TIF2 inhibits transcription by CREB-binding protein (CBP)/p300-dependent activators such as nuclear receptors and p53. Here we have shown that MOZ-TIF2 associates with the RARbeta2 promoter in vivo, resulting in altered recruitment of CBP/p300, aberrant histone modification, and down-regulation of the RARbeta2 gene. In contrast, MOZ-TIF2 up-regulated transcription mediated by the MOZ/MYST3-dependent activator AML1/RUNX1. Both wild type MOZ and MOZ-TIF2 were found to colocalize with AML1, and MOZ-TIF2 was recruited to an AML1 target promoter. A MOZ-CBP fusion protein showed similar functions to MOZ-TIF2 in that it inhibited retinoic acid receptor-mediated transcription but enhanced AML1 reporter activation. Although it contains almost the entire CBP sequence, MOZ-CBP does not appear to associate with PML bodies. In summary, our results indicate that leukemogenic MOZ fusion proteins have differential effects on the activities of CBP-dependent and MOZ-dependent activators because of their ability to alter cofactor recruitment and chromatin modification at target promoters.

MOZ fusion proteins in acute myeloid leukaemia.

MOZ (monocytic leukaemia zinc finger protein; also known as ZNF220 or MYST3) is a member of the MYST family of protein acetyltransferases. Chromosomal translocations involving the MOZ gene are associated with AML (acute myeloid leukaemia), suggesting that it has a role in haematopoiesis. Recurrent reciprocal translocations fuse the MOZ gene [or the gene encoding MORF (MOZ-related factor); also known as MYST4] to genes encoding the nuclear receptor co-activators CBP [CREB (cAMP response element-binding protein)-binding protein], p300 or the p160 protein TIF2 (transcription intermediary factor 2). The resulting fusion proteins can transform haematopoietic progenitors in vitro, and induce myeloproliferative disease in mice. Recent insights into the molecular mechanisms underlying these effects indicate that MOZ fusion proteins interfere with the activities of transcription factors such as nuclear receptors, p53 and Runx proteins. Our studies suggest that subverting the function of cellular CBP and p300 proteins may play a key role in this process. Here we review the recent progress in understanding the role of MOZ fusion proteins in the aetiology of AML.

T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif.

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-alpha. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein-protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexes.

MOZ-TIF2 inhibits transcription by nuclear receptors and p53 by impairment of CBP function.

Chromosomal rearrangements associated with acute myeloid leukemia (AML) include fusions of the genes encoding the acetyltransferase MOZ or MORF with genes encoding the nuclear receptor coactivator TIF2, p300, or CBP. Here we show that MOZ-TIF2 acts as a dominant inhibitor of the transcriptional activities of CBP-dependent activators such as nuclear receptors and p53. The dominant negative property of MOZ-TIF2 requires the CBP-binding domain (activation domain 1 [AD1]), and coimmunoprecipitation and fluorescent resonance energy transfer experiments show that MOZ-TIF2 interacts with CBP directly in vivo. The CBP-binding domain is also required for the ability of MOZ-TIF2 to extend the proliferative potential of murine bone marrow lineage-negative cells in vitro. We show that MOZ-TIF2 displays an aberrant nuclear distribution and that cells expressing this protein have reduced levels of cellular CBP, leading to depletion of CBP from PML bodies. In summary, our results indicate that disruption of the normal function of CBP and CBP-dependent activators is an important feature of MOZ-TIF2 action in AML.

A Conserved alpha-helical motif mediates the binding of diverse nuclear proteins to the SRC1 interaction domain of CBP.

CREB-binding protein (CBP) and p300 contain modular domains that mediate protein-protein interactions with a wide variety of nuclear factors. A C-terminal domain of CBP (referred to as the SID) is responsible for interaction with the alpha-helical AD1 domain of p160 coactivators such as the steroid receptor coactivator (SRC1), and also other transcriptional regulators such as E1A, Ets-2, IRF3, and p53. Here we show that the pointed (PNT) domain of Ets-2 mediates its interaction with the CBP SID, and describe the effects of mutations in the SID on binding of Ets-2, E1A, and SRC1. In vitro binding studies indicate that SRC1, Ets-2 and E1A display mutually exclusive binding to the CBP SID. Consistent with this, we observed negative cross-talk between ERalpha/SRC1, Ets-2, and E1A proteins in reporter assays in transiently transfected cells. Transcriptional inhibition of Ets-2 or GAL4-AD1 activity by E1A was rescued by co-transfection with a CBP expression plasmid, consistent with the hypothesis that the observed inhibition was due to competition for CBP in vivo. Sequence comparisons revealed that SID-binding proteins contain a leucine-rich motif similar to the alpha-helix Aalpha1 of the SRC1 AD1 domain. Deletion mutants of E1A and Ets-2 lacking the conserved motif were unable to bind the CBP SID. Moreover, a peptide corresponding to this sequence competed the binding of full-length SRC1, Ets-2, and E1A proteins to the CBP SID. Thus, a leucine-rich amphipathic alpha-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP.