Vikas Rishi

CpG methylation of half-CRE sequences creates C/EBPα binding sites that activate some tissue-specific genes

DNA methylation of the cytosine in the CpG dinucleotide is typically associated with gene silencing. Genomic analyses have identified low CpG promoters that are both methylated and transcriptionally active, but the mechanism underlying the activation of these methylated promoters remains unclear. Here we show that CpG methylation of the CRE sequence (TGACGTCA) enhances the DNA binding of the C/EBPα transcription factor, a protein critical for activation of differentiation in various cell types. Transfection assays also show that C/EBPα activates the CRE sequence only when it is methylated. The biological significance of this observation was seen in differentiating primary keratinocyte cultures from newborn mice where certain methylated promoters are both bound by C/EBPα and activated upon differentiation. Experimental demethylation by either 5-azacytidine treatment or DNMT1 depletion diminished both C/EBPα binding and activation of the same methylated promoters upon differentiation suggesting that CpG methylation can localize C/EBPα. Transfection studies in cell cultures using methylated tissue-specific proximal promoters identified half-CRE (CGTCA) and half-C/EBP (CGCAA) sequences that need to be methylated for C/EBPα mediated activation. In primary dermal fibroblasts, C/EBPα activates a different set of methylated tissue-specific promoters upon differentiation into adipocytes. These data identify a new function for methyl CpGs: producing DNA binding sites at half-CRE and half-C/EBP sequences for C/EBPα that are needed to activate tissue-specific genes.

Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy

Measurement of live-cell binding interactions is vital for understanding the biochemical reactions that drive cellular processes. Here, we develop, characterize, and apply a new procedure to extract information about binding to an immobile substrate from fluorescence correlation spectroscopy (FCS) autocorrelation data. We show that existing methods for analyzing such data by two-component diffusion fits can produce inaccurate estimates of diffusion constants and bound fractions, or even fail altogether to fit FCS binding data. By analyzing live-cell FCS measurements, we show that our new model can satisfactorily account for the binding interactions introduced by attaching a DNA binding domain to the dimerization domain derived from a site-specific transcription factor (the vitellogenin binding protein (VBP)). We find that our FCS estimates are quantitatively consistent with our fluorescence recovery after photobleaching (FRAP) measurements on the same VBP domains. However, due to the fast binding interactions introduced by the DNA binding domain, FCS generates independent estimates for the diffusion constant (6.7 +/- 2.4 microm2/s) and the association (2 +/- 1.2 s(-1)) and dissociation (19 +/- 7 s(-1)) rates, whereas FRAP produces only a single, but a consistent, estimate, the effective-diffusion constant (4.4 +/- 1.4 microm2/s), which depends on all three parameters. We apply this new FCS method to evaluate the efficacy of a potential anticancer drug that inhibits DNA binding of VBP in vitro and find that in vivo the drug inhibits DNA binding in only a subset of cells. In sum, we provide a straightforward approach to directly measure binding rates from FCS data.

Activator Protein-1 Activity Regulates Epithelial Tumor Cell Identity

To examine the consequences of inhibiting activator protein-1 (AP-1) transcription factors in skin, transgenic mice were generated, which use the tetracycline system to conditionally express A-FOS, a dominant negative that inhibits AP-1 DNA binding. Older mice develop mild alopecia and hyperplasia of sebaceous glands, particularly around the eyes. When A-FOS was expressed during chemical-induced skin carcinogenesis, mice do not develop characteristic benign and malignant squamous lesions but instead develop benign sebaceous adenomas containing a signature mutation in the H-ras proto-oncogene. Inhibiting AP-1 activity after tumor formation caused squamous tumors to transdifferentiate into sebaceous tumors. Furthermore, reactivating AP-1 in sebaceous tumors results in a reciprocal transdifferentiation into squamous tumors. In both cases of transdifferentiation, individual cells express molecular markers for both cell types, indicating individual tumor cells have the capacity to express multiple lineages. Molecular characterization of cultured keratinocytes and tumor material indicates that AP-1 regulates the balance between the wnt/beta-catenin and hedgehog signaling pathways that determine squamous and sebaceous lineages, respectively. Chromatin immunoprecipitation analysis indicates that c-Jun binds several wnt promoters, which are misregulated by A-FOS expression, suggesting that members of the wnt pathway can be a primary targets of AP-1 transcriptional regulation. Thus, AP-1 activity regulates tumor cell lineage and is essential to maintain the squamous tumor cell identity.

All and only CpG containing sequences are enriched in promoters abundantly bound by RNA polymerase II in multiple tissues

BackgroundThe promoters of housekeeping genes are well-bound by RNA polymerase II (RNAP) in different tissues. Although the promoters of these genes are known to contain CpG islands, the specific DNA sequences that are associated with high RNAP binding to housekeeping promoters has not been described.ResultsChIP-chip experiments from three mouse tissues, liver, heart ventricles, and primary keratinocytes, indicate that 94% of promoters have similar RNAP binding, ranging from well-bound to poorly-bound in all tissues. Using all 8-base pair long sequences as a test set, we have identified the DNA sequences that are enriched in promoters of housekeeping genes, focusing on those DNA sequences which are preferentially localized in the proximal promoter. We observe a bimodal distribution. Virtually all sequences enriched in promoters with high RNAP binding values contain a CpG dinucleotide. These results suggest that only transcription factor binding sites (TFBS) that contain the CpG dinucleotide are involved in RNAP binding to housekeeping promoters while TFBS that do not contain a CpG are involved in regulated promoter activity. Abundant 8-mers that are preferentially localized in the proximal promoters and exhibit the best enrichment in RNAP bound promoters are all variants of six known CpG-containing TFBS: ETS, NRF-1, BoxA, SP1, CRE, and E-Box. The frequency of these six DNA motifs can predict housekeeping promoters as accurately as the presence of a CpG island, suggesting that they are the structural elements critical for CpG island function. Experimental EMSA results demonstrate that methylation of the CpG in the ETS, NRF-1, and SP1 motifs prevent DNA binding in nuclear extracts in both keratinocytes and liver.ConclusionIn general, TFBS that do not contain a CpG are involved in regulated gene expression while TFBS that contain a CpG are involved in constitutive gene expression with some CpG containing sequences also involved in inducible and tissue specific gene regulation. These TFBS are not bound when the CpG is methylated. Unmethylated CpG dinucleotides in the TFBS in CpG islands allow the transcription factors to find their binding sites which occur only in promoters, in turn localizing RNAP to promoters.

Inhibition of CCAAT/Enhancer Binding Protein Family DNA Binding in Mouse Epidermis Prevents and Regresses Papillomas

The CCAAT/enhancer binding proteins (C/EBP) are a family of B-ZIP DNA binding proteins that act as transcription factors to regulate growth and differentiation of many cell types, including keratinocytes. To examine the consequences of inhibiting the C/EBP family of transcription factors in skin, we generated transgenic mice that use the tetracycline system to conditionally express A-C/EBP, a dominant negative that inhibits the DNA binding of C/EBP family members. We expressed A-C/EBP in the basal layer of the skin epidermis during a two-step skin carcinogenesis protocol. A-C/EBP expression caused hyperplasia of the basal epidermis and increased apoptosis in the suprabasal epidermis. The mice developed fewer papillomas and had systemic hair loss. A-C/EBP expression caused C/EBPbeta protein to disappear whereas C/EBPalpha, p53, Bax, and caspase-3 protein levels were dramatically up-regulated in the suprabasal layer. Primary keratinocytes recapitulate the A-C/EBP induction of cell growth and increase in p53 protein. A-C/EBP expression after papilloma development caused the papillomas to regress with an associated increase in apoptosis and up-regulation of p53 protein. Furthermore, A-C/EBP-expressing mice heterozygous for p53 were more susceptible to papilloma formation, suggesting that the suppression of papilloma formation has a p53-dependent mechanism. These results implicate DNA binding of C/EBP family members as a potential molecular therapeutic target.

Glucose-induced lipogenesis in pancreatic β-cells is dependent on SREBP-1

Abstract High concentrations of glucose induce de novo fatty acid synthesis in pancreatic β-cells and chronic exposure of elevated glucose and fatty acids synergize to induce accumulation of triglycerides, a phenomenon termed glucolipotoxicity. Here we investigate the role of sterol-regulatory element binding proteins in glucose-induced lipogenesis in the pancreatic β-cell line INS-1E. We show that glucose induces SREBP-1c expression and SREBP-1 activity independent of insulin secretion and signaling. Using adenoviral expression of SREBP-1c and a SREBP-mutant we show that lipogenic gene expression, de novo fatty acid synthesis and lipid accumulation are induced primarily through sterol-regulatory elements (SREs) and not E-Boxes. Adenoviral expression of a dominant negative SREBP compromises glucose induction of some lipogenic genes and significantly reduces glucose-induction of de novo fatty acid synthesis. Thus, we demonstrate for the first time that SREBP activity is necessary for full glucose induction of de novo fatty acid synthesis in pancreatic β-cells.

Inhibition of CREB Function in Mouse Epidermis Reduces Papilloma Formation

We used a double transgenic tetracycline system to conditionally express A-CREB, a dominant negative protein that prevents the DNA binding and function of cAMP-responsive element binding protein (CREB) family members, in mouse basal epidermis using the keratin 5 promoter. There was no phenotype in the adult. However, following a 7,12-dimethylbenz(a)anthracene (DMBA)/phorbol-12-myristate-13-acetate two-stage skin carcinogenesis experiment, A-CREB–expressing epidermis develop 5-fold fewer papillomas than wild-type controls. However, A-CREB expression one month after DMBA treatment does not prevent papilloma formation, suggesting that CREB functions at an early stage of papilloma formation. Oncogenic H-Ras genes with A→T mutations in codon 61 were found in wild-type skin but not in A-CREB–expressing skin 2 days after DMBA treatment, suggesting that A-CREB either prevents DMBA mutagenesis or kills oncogenic H-Ras cells. In primary keratinocyte cultures, A-CREB expression induced apoptosis of v-RasHa–infected cells and suppressed the expression of cell cycle proteins cyclin B1 and cyclin D1. These results suggest that inhibiting CREB function is a valuable cancer prevention strategy.(Mol Cancer Res 2009;7(5):654–64)

The arylstibonic acid compound NSC13746 disrupts B-ZIP binding to DNA in living cells.

The inhibition of DNA binding of basic leucine zipper (B-ZIP) transcription factors is a clinically relevant molecular target. Our laboratory has previously reported two methods of inhibiting B-ZIP DNA binding in solution: 1) an arylstibonic acid compound that binds to the basic region, stabilizes the B-ZIP dimer, and prevents B-ZIP DNA binding and 2) dominant negative proteins, termed A-ZIPs, that heterodimerize with B-ZIP domains in a leucine zipper-dependent manner. To determine if these two agents also inhibit DNA binding in live cells, GFP-tagged B-ZIP domains and mCherry-tagged A-ZIP domains were transfected into NIH3T3 cells to assess protein localization and Fluorescence Recovery After nuclear Photobleaching (FRAP). FRAP, showed that all six GFP-B-ZIP domains examined recovered faster in the nucleus in the presence of drug that we interpret represents an inhibition of DNA binding. Faster recovery in the presence of the A-ZIP was leucine zipper dependent. The arylstibonic also induced a cytoplasmic localization of all B-ZIP domains while the A-ZIPs induced a leucine zipper-dependent cytoplasmic localization. Thus, the change in cellular localization of B-ZIP domains could be used as a high-throughput assay for inhibitors of B-ZIP DNA binding. Additionally, the arylstibonic acid compound was cytostatic in clear cell sarcoma cells, which express a chimera between the B-ZIP domain of ATF-1 and N-terminal activation domain of EWS but not in K562 cells that express a non-B-ZIP containing chimeric protein BCR-ABL. These studies suggest that arylstibonic acid compounds or other small molecules capable of inhibiting B-ZIP DNA binding could be valuable anticancer agents.

12 Arylstibonic acids that inhibit the DNA binding of five B-ZIP dimers.

Previously, we identified an arylstibonic acid, NSC13778 that specifically binds to the basic region of the C/EBPalpha B-ZIP domain and disrupts DNA binding. We now examine a panel of 14 additional arylstibonic acid derivatives of NSC13778 for their ability to inhibit the DNA binding of five B-ZIP dimers (c-Fos|JunD, VBP, C/EBPalpha, C/EBPbeta, and CREB). They show various specificities at inhibiting the DNA binding of five B-ZIP domains. NSC13746 inhibits the DNA binding of C/EBPbeta and CREB at 100nM and promiscuously inhibiting the DNA binding of all five proteins in the 1muM range. Dialysis experiments indicate that NSC 13746 binding to the B-ZIP domain is reversible. Thermal denaturation studies indicate that NSC13746 binds the B-ZIP domain. Some compounds specifically inhibit DNA binding, with VBP and c-Fos|JunD being most easily disrupted. These compounds inhibit, with similar specificities to the pure B-ZIP domains, the DNA binding of nuclear extract to the AP1 DNA sequence but no inhibition is observed to SP1 containing oligonucleotide. Transient transfection assays indicate that NSC13746 can inhibit the TPA induced activation of two B-ZIP dependent reporters. These experiments suggest that arylstibonic acids are promising leads for inhibiting the DNA binding of a group of B-ZIP proteins in cells.

P6981, an arylstibonic acid, is a novel low nanomolar inhibitor of cAMP response element-binding protein binding to DNA.

Several basic leucine zipper (B-ZIP) transcription factors have been implicated in cancer, substance abuse, and other pathological conditions. We previously identified arylstibonic acids that bind to B-ZIP proteins and inhibit their interaction with DNA. In this study, we used electrophoretic mobility shift assay to analyze 46 arylstibonic acids for their activity to disrupt the DNA binding of three B-ZIP [CCAAT/enhancer-binding protein α, cyclic AMP-response element-binding protein (CREB), and vitellogenin gene-binding protein (VBP)] and two basic helix-loop-helix leucine zipper (B-HLH-ZIP) [USF (upstream stimulating factor) and Mitf] proteins. Twenty-five arylstibonic acids showed activity at micromolar concentrations. The most active compound, P6981 [2-(3-stibonophenyl)malonic acid], had half-maximal inhibition at ∼5 nM for CREB. Circular dichroism thermal denaturation studies indicated that P6981 binds both the B-ZIP domain and the leucine zipper. The crystal structure of an arylstibonic acid, NSC13778, bound to the VBP leucine zipper identified electrostatic interactions between both the stibonic and carboxylic acid groups of NSC13778 [(E)-3-(3-stibonophenyl)acrylic acid] and arginine side chains of VBP, which is also involved in interhelical salt bridges in the leucine zipper. P6981 induced GFP-B-ZIP chimeric proteins to partially localize to the cytoplasm, demonstrating that it is active in cells. P6981 inhibited the growth of a patient-derived clear cell sarcoma cell line whose oncogenic potential is driven by a chimeric protein EWS-ATF1 (Ewings sarcoma protein-activating transcription factor 1), which contains the DNA binding domain of ATF1, a B-ZIP protein. NSC13778 inhibited the growth of xenografted clear cell sarcoma, and no toxicity was observed. These experiments suggest that antimony containing arylstibonic acids are promising leads for suppression of DNA binding activities of B-ZIP and B-HLH-ZIP transcription factors.