David J. Meyers

The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2

The kinase mTOR has emerged as an important regulator of the differentiation of helper T cells. Here we demonstrate that differentiation into the TH1 and TH17 subsets of helper T cells was selectively regulated by signaling from mTOR complex 1 (mTORC1) that was dependent on the small GTPase Rheb. Rheb-deficient T cells failed to generate TH1 and TH17 responses in vitro and in vivo and did not induce classical experimental autoimmune encephalomyelitis (EAE). However, they retained their ability to become TH2 cells. Alternatively, when mTORC2 signaling was deleted from T cells, they failed to generate TH2 cells in vitro and in vivo but preserved their ability to become TH1 and TH17 cells. Our data identify mechanisms by which two distinct signaling pathways downstream of mTOR regulate helper cell fate in different ways. These findings define a previously unknown paradigm that links T cell differentiation with selective metabolic signaling pathways.

A Fasting Inducible Switch Modulates Gluconeogenesis Via Activator-Coactivator Exchange

During early fasting, increases in skeletal muscle proteolysis liberate free amino acids for hepatic gluconeogenesis in response to pancreatic glucagon. Hepatic glucose output diminishes during the late protein-sparing phase of fasting, when ketone body production by the liver supplies compensatory fuel for glucose-dependent tissues. Glucagon stimulates the gluconeogenic program by triggering the dephosphorylation and nuclear translocation of the CREB regulated transcription coactivator 2 (CRTC2; also known as TORC2), while parallel decreases in insulin signalling augment gluconeogenic gene expression through the dephosphorylation and nuclear shuttling of forkhead box O1 (FOXO1). Here we show that a fasting-inducible switch, consisting of the histone acetyltransferase p300 and the nutrient-sensing deacetylase sirtuin 1 (SIRT1), maintains energy balance in mice through the sequential induction of CRTC2 and FOXO1. After glucagon induction, CRTC2 stimulated gluconeogenic gene expression by an association with p300, which we show here is also activated by dephosphorylation at Ser 89 during fasting. In turn, p300 increased hepatic CRTC2 activity by acetylating it at Lys 628, a site that also targets CRTC2 for degradation after its ubiquitination by the E3 ligase constitutive photomorphogenic protein (COP1). Glucagon effects were attenuated during late fasting, when CRTC2 was downregulated owing to SIRT1-mediated deacetylation and when FOXO1 supported expression of the gluconeogenic program. Disrupting SIRT1 activity, by liver-specific knockout of the Sirt1 gene or by administration of a SIRT1 antagonist, increased CRTC2 activity and glucose output, whereas exposure to SIRT1 agonists reduced them. In view of the reciprocal activation of FOXO1 and its coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α, encoded by Ppargc1a) by SIRT1 activators, our results illustrate how the exchange of two gluconeogenic regulators during fasting maintains energy balance.

Virtual Ligand Screening of the p300/CBP Histone Acetyltransferase: Identification of a Selective Small Molecule Inhibitor

The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. Although p300 inhibitors have been reported, a potent, selective, and readily available active-site-directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646. C646 is a competitive p300 inhibitor with a K(i) of 400 nM and is selective versus other acetyltransferases. Studies on site-directed p300 HAT mutants and synthetic modifications of C646 confirm the importance of predicted interactions in conferring potency. Inhibition of histone acetylation and cell growth by C646 in cells validate its utility as a pharmacologic probe and suggest that p300/CBP HAT is a worthy anticancer target.

Butyrate Greatly Enhances Derivation of Human Induced Pluripotent Stem Cells by Promoting Epigenetic Remodeling and the Expression of Pluripotency-Associated Genes

We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15‐ to 51‐fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100‐ to 200‐fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome‐wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency‐associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming. STEM CELLS 2010;28:713–72028:713–720

Chromatin “Prepattern” and Histone Modifiers in a Fate Choice for Liver and Pancreas

Screening histone modifications reveals distinctive patterns of chromatin marks for liver and pancreas development. Transcriptionally silent genes can be marked by histone modifications and regulatory proteins that indicate the genes’ potential to be activated. Such marks have been identified in pluripotent cells, but it is unknown how such marks occur in descendant, multipotent embryonic cells that have restricted cell fate choices. We isolated mouse embryonic endoderm cells and assessed histone modifications at regulatory elements of silent genes that are activated upon liver or pancreas fate choices. We found that the liver and pancreas elements have distinct chromatin patterns. Furthermore, the histone acetyltransferase P300, recruited via bone morphogenetic protein signaling, and the histone methyltransferase Ezh2 have modulatory roles in the fate choice. These studies reveal a functional “prepattern” of chromatin states within multipotent progenitors and potential targets to modulate cell fate induction.

Inhibition of the acetyltransferases p300 and CBP reveals a targetable function for p300 in the survival and invasion pathways of prostate cancer cell lines

Inhibitors of histone deacetylases have been approved for clinical application in cancer treatment. On the other hand, histone acetyltransferase (HAT) inhibitors have been less extensively investigated for their potential use in cancer therapy. In prostate cancer, the HATs and coactivators p300 and CBP are upregulated and may induce transcription of androgen receptor (AR)-responsive genes, even in the absence or presence of low levels of AR. To discover a potential anticancer effect of p300/CBP inhibition, we used two different approaches: (i) downregulation of p300 and CBP by specific short interfering RNA (siRNA) and (ii) chemical inhibition of the acetyltransferase activity by a newly developed small molecule, C646. Knockdown of p300 by specific siRNA, but surprisingly not of CBP, led to an increase of caspase-dependent apoptosis involving both extrinsic and intrinsic cell death pathways in androgen-dependent and castration-resistant prostate cancer cells. Induction of apoptosis was mediated by several pathways including inhibition of AR function and decrease of the nuclear factor kappa B (NF-κB) subunit p65. Furthermore, cell invasion was decreased upon p300, but not CBP, depletion and was accompanied by lower matrix metalloproteinase (MMP)-2 and MMP-9 transcriptions. Thus, p300 and CBP have differential roles in the processes of survival and invasion of prostate cancer cells. Induction of apoptosis in prostate cancer cells was confirmed by the use of C646. This was substantiated by a decrease of AR function and downregulation of p65 impairing several NF-κB target genes. Taken together, these results suggest that p300 inhibition may be a promising approach for the development of new anticancer therapies. Mol Cancer Ther; 10(9); 1644–55. ©2011 AACR.

Oncogenesis by sequestration of CBP/p300 in transcriptionally inactive hyperacetylated chromatin domains

In a subset of poorly differentiated and highly aggressive carcinoma, a chromosomal translocation, t(15;19)(q13;p13), results in an in‐frame fusion of the double bromodomain protein, BRD4, with a testis‐specific protein of unknown function, NUT (nuclear protein in testis). In this study, we show that, after binding to acetylated chromatin through BRD4 bromodomains, the NUT moiety of the fusion protein strongly interacts with and recruits p300, stimulates its catalytic activity, initiating cycles of BRD4–NUT/p300 recruitment and creating transcriptionally inactive hyperacetylated chromatin domains. Using a patient‐derived cell line, we show that p300 sequestration into the BRD4–NUT foci is the principal oncogenic mechanism leading to p53 inactivation. Knockdown of BRD4–NUT released p300 and restored p53‐dependent regulatory mechanisms leading to cell differentiation and apoptosis. This study demonstrates how the off‐context activity of a testis‐specific factor could markedly alter vital cellular functions and significantly contribute to malignant cell transformation.

MicroRNA-224 is up-regulated in hepatocellular carcinoma through epigenetic mechanisms

MicroRNA‐224 (miR‐224) is one of the most commonly up‐regulated microRNAs in hepatocellular carcinoma (HCC), which affects crucial cellular processes such as apoptosis and cell proliferation. In this study, we aim to elucidate the molecular mechanism that leads to the overexpression of miR‐224 in HCC. We examined the transcript expression of miR‐224 and neighboring miR‐452 and genes on chromosome Xq28 in tumor and paired adjacent nontumorous tissues from 100 patients with HCC and found that miR‐224 is coordinately up‐regulated with its neighboring microRNA (miRNA) and genes. This coordinated up‐regulation of miRNAs and genes at the Xq28 locus can be mimicked in nontransformed immortalized human liver cells by the introduction of histone deacetylase (HDAC) inhibitors, which resulted in a corresponding increase in histone H3 acetylation in this region. This miR‐224‐residing locus in Xq28 is reciprocally regulated by HDAC1, HDAC3, and histone acetylase protein, E1A binding protein p300 (EP300). Notably, in HCC tumors that significantly overexpress microRNA‐224, EP300 is also overexpressed and displays increased binding to the Xq28 locus. In transformed HCC cells, high miR‐224 expression can be attenuated through the inhibition of EP300, using either siRNA or the specific drug C646. In summary, overexpression of EP300 may account, in part, for the up‐regulation of miR‐224 expression in patients with HCC.—Wang, Y., Toh, H. C., Chow, P., Chung, A. Y. F., Meyers, D. J., Cole, P. A., Ooi, L. L. P. J., Lee, C. G. L. MicroRNA‐224 is up‐regulated in hepatocellular carcinoma through epigenetic mechanisms. FASEB J. 26, 3032–3041 (2012). www.fasebj.org

Rapid and orthogonal logic gating with a gibberellin-induced dimerization system

Using a newly synthesized gibberellin analog containing an acetoxymethyl group (GA(3)-AM) and its binding proteins, we developed an efficient chemically inducible dimerization (CID) system that is completely orthogonal to existing rapamycin-mediated protein dimerization. Combining the two systems should allow applications that have been difficult or impossible with only one CID system. By using both chemical inputs (rapamycin and GA(3)-AM), we designed and synthesized Boolean logic gates in living mammalian cells. These gates produced output signals such as fluorescence and membrane ruffling on a timescale of seconds, substantially faster than earlier intracellular logic gates. The use of two orthogonal dimerization systems in the same cell also allows for finer modulation of protein perturbations than is possible with a single dimerizer.

p300/CBP-associated factor selectively regulates the extinction of conditioned fear

It is well established that the activity of chromatin-modifying enzymes is crucial for regulating gene expression associated with hippocampal-dependent memories. However, very little is known about how these epigenetic mechanisms influence the formation of cortically dependent memory, particularly when there is competition between opposing memory traces, such as that which occurs during the acquisition and extinction of conditioned fear. Here we demonstrate, in C57BL/6 mice, that the activity of p300/CBP-associated factor (PCAF) within the infralimbic prefrontal cortex is required for long-term potentiation and is necessary for the formation of memory associated with fear extinction, but not for fear acquisition. Further, systemic administration of the PCAF activator SPV106 enhances memory for fear extinction and prevents fear renewal. The selective influence of PCAF on fear extinction is mediated, in part, by a transient recruitment of the repressive transcription factor ATF4 to the promoter of the immediate early gene zif268, which competitively inhibits its expression. Thus, within the context of fear extinction, PCAF functions as a transcriptional coactivator, which may facilitate the formation of memory for fear extinction by interfering with reconsolidation of the original memory trace.