Manish K. Tripathi

Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells

The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the non-dividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the –701 to –921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.

Yeast TFIID Serves as a Coactivator for Rap1p by Direct Protein-Protein Interaction

ABSTRACT In vivo studies have previously shown that Saccharomyces cerevisiae ribosomal protein (RP) gene expression is controlled by the transcription factor repressor activator protein 1 (Rap1p) in a TFIID-dependent fashion. Here we have tested the hypothesis that yeast TFIID serves as a coactivator for RP gene transcription by directly interacting with Rap1p. We have found that purified recombinant Rap1p specifically interacts with purified TFIID in pull-down assays, and we have mapped the domains of Rap1p and subunits of TFIID responsible. In vitro transcription of a UASRAP1 enhancer-driven reporter gene requires both Rap1p and TFIID and is independent of the Fhl1p-Ifh1p coregulator. UASRAP1 enhancer-driven transactivation in extracts depleted of both Rap1p and TFIID is efficiently rescued by addition of physiological amounts of these two purified factors but not TATA-binding protein. We conclude that Rap1p and TFIID directly interact and that this interaction contributes importantly to RP gene transcription.

TFIIA and the transactivator Rap1 cooperate to commit TFIID for transcription initiation

Transcription of eukaryotic messenger RNA (mRNA) encoding genes by RNA polymerase II (Pol II) is triggered by the binding of transactivating proteins to enhancer DNA, which stimulates the recruitment of general transcription factors (TFIIA, B, D, E, F, H) and Pol II on the cis-linked promoter, leading to pre-initiation complex formation and transcription. In TFIID-dependent activation pathways, this general transcription factor containing TATA-box-binding protein is first recruited on the promoter through interaction with activators and cooperates with TFIIA to form a committed pre-initiation complex. However, neither the mechanisms by which activation signals are communicated between these factors nor the structural organization of the activated pre-initiation complex are known. Here we used cryo-electron microscopy to determine the architecture of nucleoprotein complexes composed of TFIID, TFIIA, the transcriptional activator Rap1 and yeast enhancer–promoter DNA. These structures revealed the mode of binding of Rap1 and TFIIA to TFIID, as well as a reorganization of TFIIA induced by its interaction with Rap1. We propose that this change in position increases the exposure of TATA-box-binding protein within TFIID, consequently enhancing its ability to interact with the promoter. A large Rap1-dependent DNA loop forms between the activator-binding site and the proximal promoter region. This loop is topologically locked by a TFIIA–Rap1 protein bridge that folds over the DNA. These results highlight the role of TFIIA in transcriptional activation, define a molecular mechanism for enhancer–promoter communication and provide structural insights into the pathways of intramolecular communication that convey transcription activation signals through the TFIID complex.

Mapping the Initiator Binding Taf2 Subunit in the Structure of Hydrated Yeast TFIID

The general transcription factor TFIID is a large multisubunit complex required for the transcription of most protein-encoding genes by RNA polymerase II. Taking advantage of a TFIID preparation partially depleted in the initiator-binding Taf2p subunit, we determined the conformational and biochemical variations of the complex by electron tomography and cryo-electron microscopy of single molecules. Image analysis revealed the extent of conformational flexibility of the complex and the selection of the most homogeneous TFIID subpopulation allowed us to determine an improved structural model at 23 Angstroms resolution. This study also identified two subpopulations of Taf2p-containing and Taf2p-depleted TFIID molecules. By comparing these two TFIID species we could infer the position of Taf2p, which was confirmed by immunolabeling using a subunit-specific antibody. Mapping the position of this crucial subunit in the vicinity of Taf1p and of TBP sheds new light on its role in promoter recognition.

Protein Translocase of Mitochondrial Inner Membrane in Trypanosoma brucei

Background: The trypanosome mitochondrion imports ∼1000 nucleus-encoded proteins. However, its protein translocation machinery remains elusive. Results: We identified several trypanosome-specific members of the translocase of mitochondrial inner membrane (TIM) in T. brucei. Conclusion: The TIM complex in T. brucei is significantly divergent from those of other eukaryotes. Significance: This TIM complex could be a potential drug target in trypanosomatids. Translocases of mitochondrial inner membrane (TIMs) are multiprotein complexes. The only Tim component so far characterized in kinetoplastid parasites such as Trypanosoma brucei is Tim17 (TbTim17), which is essential for cell survival and mitochondrial protein import. Here, we report that TbTim17 is present in a protein complex of about 1,100 kDa, which is much larger than the TIM complexes found in fungi and mammals. Depletion of TbTim17 in T. brucei impairs the mitochondrial import of cytochrome oxidase subunit IV, an N-terminal signal-containing protein. Pretreatment of isolated mitoplasts with the anti-TbTim17 antibody inhibited import of cytochrome oxidase subunit IV, indicating a direct involvement of the TbTim17 in the import process. Purification of the TbTim17-containing protein complex from the mitochondrial membrane of T. brucei by tandem affinity chromatography revealed that TbTim17 associates with seven unique as well as a few known T. brucei mitochondrial proteins. Depletion of three of these novel proteins, i.e. TbTim47, TbTim54, and TbTim62, significantly decreased mitochondrial protein import in vitro. In vivo targeting of a newly synthesized mitochondrial matrix protein, MRP2, was also inhibited due to depletion of TbTim17, TbTim54, and TbTim62. Co-precipitation analysis confirmed the interaction of TbTim54 and TbTim62 with TbTim17 in vivo. Overall, our data reveal that TbTim17, the single homolog of Tim17/22/23 family proteins, is present in a unique TIM complex consisting of novel proteins in T. brucei and is critical for mitochondrial protein import.

Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

BackgroundBRCA2 gene expression is tightly regulated during the cell cycle in human breast cells. The expression of BRCA2 gene is silenced at the G0/G1 phase of cell growth and is de-silenced at the S/G2 phase. While studying the activity of BRCA2 gene promoter in breast cancer cells, we discovered that this promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter at the G0/G1 phase of the cell. Standard techniques like cell synchronization by serum starvation, flow cytometry, N-terminal or C-terminal FLAG epitope-tagged protein expression, immunofluorescence confocal microscopy, dual luciferase assay for promoter evaluation, and chromatin immunoprecipitation assay were employed during this study.ResultsHuman BRCA2 gene promoter is active in both the forward and the reverse orientations. This promoter is 8-20 fold more active in the reverse orientation than in the forward orientation when the cells are in the non-dividing stage (G0/G1). When the cells are in the dividing state (S/G2), the forward activity of the promoter is 5-8 folds higher than the reverse activity. The reverse activity transcribes the ZAR2 mRNA with 966 nt coding sequence which codes for a 321 amino acid protein. ZAR2 has two C4 type zinc fingers at the carboxyl terminus. In the G0/G1 growth phase ZAR2 is predominantly located inside the nucleus of the breast cells, binds to the BRCA2 promoter and inhibits the expression of BRCA2. In the dividing cells, ZAR2 is trapped in the cytoplasm.ConclusionsBRCA2 gene promoter has bi-directional activity, expressing BRCA2 and a novel C4-type zinc finger containing transcription factor ZAR2. Subcellular location of ZAR2 and its expression from the reverse promoter of the BRCA2 gene are stringently regulated in a cell cycle dependent manner. ZAR2 binds to BRCA2/ZAR2 bi-directional promoter in vivo and is responsible, at least in part, for the silencing of BRCA2 gene expression in the G0/G1 phase in human breast cells.

Nuclear Factor of Activated T-cell Activity Is Associated with Metastatic Capacity in Colon Cancer

Metastatic recurrence is the leading cause of cancer-related deaths in patients with colorectal carcinoma. To capture the molecular underpinnings for metastasis and tumor progression, we performed integrative network analysis on 11 independent human colorectal cancer gene expression datasets and applied expression data from an immunocompetent mouse model of metastasis as an additional filter for this biologic process. In silico analysis of one metastasis-related coexpression module predicted nuclear factor of activated T-cell (NFAT) transcription factors as potential regulators for the module. Cells selected for invasiveness and metastatic capability expressed higher levels of NFATc1 as compared with poorly metastatic and less invasive parental cells. We found that inhibition of NFATc1 in human and mouse colon cancer cells resulted in decreased invasiveness in culture and downregulation of metastasis-related network genes. Overexpression of NFATc1 significantly increased the metastatic potential of colon cancer cells, whereas inhibition of NFATc1 reduced metastasis growth in an immunocompetent mouse model. Finally, we found that an 8-gene signature comprising genes upregulated by NFATc1 significantly correlated with worse clinical outcomes in stage II and III colorectal cancer patients. Thus, NFATc1 regulates colon cancer cell behavior and its transcriptional targets constitute a novel, biologically anchored gene expression signature for the identification of colon cancers with high risk of metastatic recurrence.

Down-regulation of 7SL RNA Expression and Impairment of Vesicular Protein Transport Pathways by Leishmania Infection of Macrophages

The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display reverse transcription-PCR and cDNA microarray analyses. The genes that are down-regulated in mouse (J774G8) or human (U937) macrophages upon exposure to Leishmania include small RNA transcripts from the short interspersed element sequences. Among the short interspersed element RNAs that are down-regulated is 7SL RNA, which is the RNA component of the signal recognition particle. Because the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down-regulation of 7SL RNA may be significant in the establishment of infection by Leishmania in macrophage phagolysosomes. To evaluate whether down-regulation of 7SL RNA results in inhibition of signal recognition particle-mediated vesicular protein transport processes, we have tested and found that the targeting of proteins to the endoplasmic reticulum and plasma membrane and the secretion of proteins by macrophages are compromised in Leishmania-infected J774G8 and U937 cells. Knocking down 7SL RNA using small interfering RNA mimicked the effect of exposure of macrophages to Leishmania. The overexpression of 7SL RNA in J774G8 or U937 cells made these cells resistant to Leishmania infection, suggesting the possible biological significance of down-regulation of 7SL RNA synthesis in the establishment of infection by Leishmania. We conclude that Leishmania down-regulates 7SL RNA in macrophages to manipulate the targeting of many proteins that use the vesicular transport pathway and thus favors its successful establishment of infection in macrophages.

p120-Catenin is an obligate haploinsufficient tumor suppressor in intestinal neoplasia

p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an “obligatory” haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3–dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors &agr;-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.

Tannic acid-inspired paclitaxel nanoparticles for enhanced anticancer effects in breast cancer cells

Paclitaxel (PTX) is a gold standard chemotherapeutic agent for breast, ovarian, pancreatic and non-small cell lung carcinoma. However, in clinical use PTX can have adverse side effects or inadequate pharmacodynamic parameters, limiting its use. Nanotechnology is often employed to reduce the therapeutic dosage required for effective therapy, while also minimizing the systemic side effects of chemotherapy drugs. However, there is no nanoformulation of paclitaxel with chemosensitization motifs built in. With this objective, we screened eleven pharmaceutical excipients to develop an alternative paclitaxel nanoformulation using a self-assembly method. Based on the screening results, we observed tannic acid possesses unique properties to produce a paclitaxel nanoparticle formulation, i.e., tannic acid-paclitaxel nanoparticles. This stable TAP nanoformulation, referred to as TAP nanoparticles (TAP NPs), showed a spherical shape of ~ 102 nm and negative zeta potential of ~ -8.85. The presence of PTX in TAP NPs was confirmed by Fourier Transform Infrared (FTIR) spectra, thermogravimetric analyzer (TGA), and X-ray diffraction (XRD). Encapsulation efficiency of PTX in TAP NPs was determined to be ≥96%. Intracellular drug uptake of plain drug PTX on breast cancer cells (MDA-MB-231) shows more or less constant drug levels in 2 to 6 h, suggesting drug efflux by the P-gp transporters, over TAP NPs, in which PTX uptake was more than 95.52 ± 11.01% in 6 h, as analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Various biological assays such as proliferation, clonogenic formation, invasion, and migration confirm superior anticancer effects of TAP NPs over plain PTX at all tested concentrations. P-gp expression, beta-tubulin stabilization, Western blot, and microarray analysis further confirm the improved therapeutic potential of TAP NPs. These results suggest that the TAP nanoformulation provides an important reference for developing a therapeutic nanoformulation affording pronounced, enhanced effects in breast cancer therapy.