Characterization of the novel transcriptional regulator human mesoderm induction early response gene 1 (hMI-ER1): its promoters, interacting proteins and transcriptional regulatory functions

Mesoderm induction early response 1 (mi-er1), previously called er1, was first isolated as a novel fibroblast growth factor-regulated early-response gene from Xenopus embryonic cells induced to differentiate into mesoderm. The human orthologue of xmi-er1, hmi-er1, was found to have ubiquitous, but low level, expression in normal human tissues (Paterno et al., 1998; Paterno et al., 2002). Breast carcinoma cell lines and tumours, on the other hand, showed elevated levels (Paterno et al., 1998), suggesting that hmi-er1 expression is associated with the neoplastic state in human breast carcinoma. Structurally, hMI-ER1 has conserved domains found in a number of transcriptional regulators, including an acid activation domain (Paterno et al., 1997), an ELM2 domain (Solari et al., 1999) and a signature SANT domain (Aasland et al., 1996). -- My hypothesis is that hMI-ER1 may be a potent transcriptional regulator, and deregulation of its expression and/or functions may contribute to tumorigenesis. The purpose of this project was to: (1) isolate and characterize the hmi-er1 promoters; (2) investigate the role of hMI-ER1 in transcriptional regulation; and (3) identify and characterize hMI-ER1-interacting proteins. -- Cloning and sequence analysis of one of the hmi-er1 promoters, P2, showed the absence of a TATA box, but presence of a CpG island, multiple Sp1 binding sites, and other factor binding sites which may be important in the regulation of gene expression. Functional characterization of the promoter revealed that Sp1 binds to the promoter and plays a positive role in the regulation of the P2 promoter activity of hmi-er1. hMI-ER1 was found to function as transcriptional repressor on the G5TKCAT promoter by recruitment of HDAC1 via the ELM2 domain. hMI-ER1α and β also function as transcriptional repressors on their own P2 promoter, through interaction with Sp1 and interference with the Sp1-DNA binding via the SANT domain. In order to better understand the function of hMI-ER1, an attempt was made to identify more hMI-ER1 interacting proteins by yeast two-hybrid cDNA library screening. HSP40 and TRABID were identified as proteins that interact specifically with hMI-ER1α and β. Furthermore, hMI-EMR1α and β were found to interact with the tumour suppressor RB and may participate in cell growth regulation. These results suggest that hMI-ER1 may function as a transcriptional regulator, through distinct mechanisms, to regulate cellular functions

P53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation

Glioblastoma (GBM) is a highly lethal brain tumor presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as high-grade disease that typically harbors EGFR, Pten and Ink4a/Arf mutations, and the secondary GBM subtype evolves from the slow progression of low-grade disease that classically possesses PDGF and p53 events1–3. Here, we show that concomitant CNS-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with striking clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted p53 and Pten mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of p53 as well the expected Pten mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives elevated c-Myc levels and its associated signature. Functional studies validated increased c-Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of p53-Pten null NSCs as well as tumor neurospheres (TNSs) derived from this model. c-Myc also serves to maintain robust tumorigenic potential of p53-Pten null TNSs. These murine modeling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumor suppressor mutation profile in human primary GBM and establish c-Myc as a key target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential

BRAF Activation Initiates but Does Not Maintain Invasive Prostate Adenocarcinoma

Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAFV600E–a mutation found in ∼10% of human prostate tumors–was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAFV600E in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAFV600E is not required for its maintenance

Role of apoptosis in multidrug resistance and tumorigenesis of human cervical cells: implication of BAG-1 and other apoptotic proteins

Recent studies have indicated that inhibition of apoptosis may play an important role m both multistep carcinogenesis and multidrug resistance (.MDR). Apoptosis is controlled through many cellular genes. The pattern of these apoptosis-regulating proteins varied in different cell types. The molecular mechanism of apoptosis in the multistep carcinogenesis and multidrug resistance of cervical cells is still poorly understood. -- To examine the role of apoptosis in tumorigenesis and chemoresistance of human endocervical cells, a cisplatin-resistant endocervical cell line (HEN-16-2/CDDP) was established by treating an HPV16-immortalized human endocervical cell line previously established in this lab, HEN-16-2, with cisplatin. A phenotype of MDR was identified for HEN-16-2/CDDP by clonogenic survival efficiency assay using two structurally and functionally distinct anticancer drugs: cisplatin and paclitaxel. -- The thresholds to undergo apoptosis of HEN-16-2/CDDP cells in response to various apoptotic stimuli was compared with that of its parental HEN-16-2 cells. HEN-16-2/CDDP cells were found to be significantly more resistant to cell death induced by several chemotherapeutic drugs, UV irradiation, anti-Fas antibody and heat shock. Moreover, the dysregulation of apoptosis in HEN-16-2/CDDP cells was found to confer tumorigenicity. Further characterization of HEN-16-2/CDDP cells indicated the following: 1) they displayed distinct morphologies in monolayer; 2) they had an increased rate of proliferation in medium containing physiological calcium levels; 3) they demonstrated anchorage-independent growth in vitro; 4) they expressed similar levels of pro-apoptotic genes, including p53, Bak, Bax and the anti-apoptotic gene Bcl-2, compared to the drug-sensitive cell line, HEN-16-2; and 5) they expressed significantly higher levels of the anti-apoptotic gene Bcl-XL as well as the p50 and p33 isoforms of BAG-1. Overexpression of BAG-1 in cervical carcinoma C33A cell line confers resistance to cisplatin, etoposide and doxorubicin, but not to actinomycin D and paclitaxel BAG-1 also protects C33A cells from apoptosis induced by heat shock and UV irradiation. -- The yeast two-hybrid system was established to screen BAG-1 interacting proteins from a human keratinocyte cDNA library. Eighteen positives were obtained from 2.5 x 106 clones. Further analysis of the interacting clones identified four genes: Hsp 70, Hsp70-2, Hsc70 pseudogene and a putative novel Hsp70Y. Carboxyl-terminal amino acids of BAG-1 were found to be important in the mediation of the interactions. -- Overexpression of Hsp70 or Hsp70-2 in C33A cells conferred the resistance to various apoptotic stimuli, including cisplatin, doxorubicin, etoposide, paclitaxel, actinomycin D, heat shock and UV irradiation. -- In summary, this study provided the first in vitro evidence that inhibition of apoptosis conferred MDR and tumorigenesis in endocervical cells. Increased levels of Bcl-XL and BAG-1 p50 and p33 isoforms were found to be associated with this phenotype. Hsp70s were identified as BAG-1-interacting proteins from a cDNA library using the yeast two-hybrid system, and further studies indicated that they may also contribute to the regulation of apoptosis

Human MI-ER1 Alpha and Beta Function as Transcriptional Repressors by Recruitment of Histone Deacetylase 1 to Their Conserved ELM2 Domain

mi-er1 (previously called er1) was first isolated from Xenopus laevis embryonic cells as a novel fibroblast growth factor-regulated immediate-early gene. Xmi-er1 was shown to encode a nuclear protein with an N-terminal acidic transcription activation domain. The human orthologue of mi-er1 (hmi-er1) displays 91% similarity to the Xenopus sequence at the amino acid level and was shown to be upregulated in breast carcinoma cell lines and tumors. Alternative splicing at the 3′ end of hmi-er1 produces two major isoforms, hMI-ER1α and hMI-ER1β, which contain distinct C-terminal domains. In this study, we investigated the role of hMI-ER1α and hMI-ER1β in the regulation of transcription. Using fusion proteins of hMI-ER1α or hMI-ER1β tethered to the GAL4 DNA binding domain, we show that both isoforms, when recruited to the G5tkCAT minimal promoter, function to repress transcription. We demonstrate that this repressor activity is due to interaction and recruitment of a trichostatin A-sensitive histone deacetylase 1 (HDAC1). Furthermore, deletion analysis revealed that recruitment of HDAC1 to hMI-ER1α and hMI-ER1β occurs through their common ELM2 domain. The ELM2 domain was first described in the Caenorhabditis elegans Egl-27 protein and is present in a number of SANT domain-containing transcription factors. This is the first report of a function for the ELM2 domain, highlighting its role in the regulation of transcription

Utilizing murine inducible telomerase alleles in the studies of tissue degeneration/regeneration and cancer.

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-Lox TERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures of performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells

Role of PDLIM1 in hepatic stellate cell activation and liver fibrosis progression

Abstract Liver fibrosis is caused by chronic hepatic injury and may lead to cirrhosis, and even hepatocellular carcinoma. When hepatic stellate cells (HSCs) are activated by liver injury, they transdifferentiate into myofibroblasts, which secrete extracellular matrix proteins that generate the fibrous scar. Therefore, it is extremely urgent to find safe and effective drugs for HSCs activation treatment to prevent liver against fibrosis. Here, we reported that PDZ and LIM domain protein 1 (PDLIM1), a highly conserved cytoskeleton organization regulator, was significantly up-regulated in fibrotic liver tissues and TGF-β-treated HSC-T6 cells. Through transcriptome analysis, we found that knockdown of PDLIM1 resulted in a significant downregulation of genes related to inflammation and immune-related pathways in HSC-T6 cells. Moreover, PDLIM1 knockdown significantly inhibited the activation of HSC-T6 cells and the trans-differentiation of HSC-T6 cells into myofibroblasts. Mechanistically, PDLIM1 is involved in the regulation of TGF-β-mediated signaling pathways in HSCs activation. Thus, targeting PDLIM1 may provide an alternative method to suppress HSCs activation during liver injury. CCCTC-binding factor (CTCF), a master regulator of genome architecture, is upregulated during HSCs activation. PDLIM1 knockdown also indirectly reduced CTCF protein expression, however, CTCF binding to chromatin was not significantly altered by CUT&Tag analysis. We speculate that CTCF may cooperate with PDLIM1 to activate HSCs in other ways. Our results suggest that PDLIM1 can accelerate the activation of HSCs and liver fibrosis progression and could be a potential biomarker for monitoring response to anti-fibrotic therapy