Transcription factor GATA-1 permits survival and maturation of erythroid precursors by preventing apoptosis.

The transcription factor GATA-1 recognizes a consensus motif present in regulatory regions of numerous erythroid-expressed genes. Mouse embryonic stem cells lacking GATA-1 cannot form mature red blood cells in vivo. In vitro differentiation of GATA-1- embryonic stem cells gives rise to a population of committed erythroid precursors that exhibit developmental arrest and death. We show here that the demise of GATA-1- erythroid cells is accompanied by several features characteristics of apoptosis. This process occurs despite normal expression of all known GATA target genes examined, including the erythropoietin receptor, and independent of detectable accumulation of the tumor suppressor protein p53. Thus, in addition to its established role in regulating genes that define the erythroid phenotype, GATA-1 also supports the viability of red cell precursors by suppressing apoptosis. These results illustrate the multifunctional nature of GATA-1 and suggest a mechanism by which other hematopoietic transcription factors may ensure the development of specific lineages.

Analysis of homeodomain function by structure-based design of a transcription factor.

The homeodomain is a 60-amino acid module which mediates critical protein-DNA and protein-protein interactions for a large family of regulatory proteins. We have used structure-based design to analyze the ability of the Oct-1 homeodomain to nucleate an enhancer complex. The Oct-1 protein regulates herpes simplex virus (HSV) gene expression by participating in the formation of a multiprotein complex (C1 complex) which regulates alpha (immediate early) genes. We recently described the design of ZFHD1, a chimeric transcription factor containing zinc fingers 1 and 2 of Zif268, a four-residue linker, and the Oct-1 homeodomain. In the presence of alpha-transinduction factor and C1 factor, ZFHD1 efficiently nucleates formation of the C1 complex in vitro and specifically activates gene expression in vivo. The sequence specificity of ZFHD1 recruits C1 complex formation to an enhancer element which is not efficiently recognized by Oct-1. ZFHD1 function depends on the recognition of the Oct-1 homeodomain surface. These results prove that the Oct-1 homeodomain mediates all the protein-protein interactions that are required to efficiently recruit alpha-transinduction factor and C1 factor into a C1 complex. The structure-based design of transcription factors should provide valuable tools for dissecting the interactions of DNA-bound domains in other regulatory circuits.

Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction.

The interleukin 2 receptor (IL-2R) consists of three subunits, the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two Janus family protein tyrosine kinases (PTKs), Jak1 and Jak3, were shown to associate with IL-2R beta c and IL-2R gamma c, respectively, and their PTK activities are increased after IL-2 stimulation. A Jak3 mutant with truncation of the C-terminal PTK domain lacks its intrinsic kinase activity but can still associate with IL-2R gamma c. In a hematopoietic cell line, F7, that responds to either IL-2 or IL-3, overexpression of this Jak3 mutant results in selective inhibition of the IL-2-induced activation of Jak1/Jak3 PTKs and of cell proliferation. Of the three target nuclear protooncogenes of the IL-2 signaling, c-fos and c-myc genes, but not the bcl-2 gene, were found to be impaired. On the other hand, overexpression of the dominant negative form of the IL-2R gamma c chain, which lacks most of its cytoplasmic domain, in F7 cells resulted in the inhibition of all three protooncogenes. These results provide a further molecular basis for the critical role of Jak3 in IL-2 signaling and also suggest a Jak PTK-independent signaling pathway(s) for the bcl-2 gene induction by IL-2R.

Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains.

Transcription factor TFIIIB plays a central role in transcription initiation by RNA polymerase III on genes encoding tRNA, 5S rRNA, and other small structural RNAs. We report the purification of a human TFIIIB-derived complex containing only the TATA-binding polypeptide (TBP) and a 90-kDa subunit (TFIIIB90) and the isolation of a cDNA clone encoding the 90-kDa subunit. The N-terminal half of TFIIIB90 exhibits sequence similarity to the yeast TFIIIB70 (BRF) and the class II transcription factor TFIIB and interacts weakly with TBP. The C-terminal half of TFIIIB90 contains a high-mobility-group protein 2 (HMG2)-related domain and interacts strongly with TBP. Recombinant TFIIIB90 plus recombinant human TBP substitute for human TFIIIB in a complementation assay for transcription of 5S, tRNA, and VA1 RNA genes, and both the TFIIB-related domain and the HMG2-related domain are required for this activity. TFIIIB90 is also required for transcription of human 7SK and U6 RNA genes by RNA polymerase III, but apparently within a complex distinct from the TBP/TFIIIB90 complex.

Domains of transcription factor Sp1 required for synergistic activation with sterol regulatory element binding protein 1 of low density lipoprotein receptor promoter.

Feedback regulation of transcription from the low density lipoprotein (LDL) receptor gene is fundamentally important in the maintenance of intracellular sterol balance. The region of the LDL receptor promoter responsible for normal sterol regulation contains adjacent binding sites for the ubiquitous transcription factor Sp1 and the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs). Interestingly, both are essential for normal sterolmediated regulation of the promoter. The cooperation by Sp1 and SREBP-1 occurs at two steps in the activation process. SREBP-1 stimulates the binding of Sp1 to its adjacent recognition site in the promoter followed by enhanced stimulation of transcription after both proteins are bound to DNA. In the present report, we have defined the protein domains of Sp1 that are required for both synergistic DNA binding and transcriptional activation. The major activation domains of Sp1 that have previously been shown to be essential to activation of promoters containing multiple Sp1 sites are required for activation of the LDL receptor promoter. Additionally, the C domain is also crucial. This slightly acidic approximately 120-amino acid region is not required for efficient synergistic activation by multiple Sp1 sites or in combination with other recently characterized transcriptional regulators. We also show that Sp1 domain C is essential for full, enhanced DNA binding by SREBP-1. Taken together with other recent studies on the role of Sp1 in promoter activation, the current experiments suggest a unique combinatorial mechanism for promoter activation by two distinct transcription factors that are both essential to intracellular cholesterol homeostasis.

The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression.

Anchorage-dependent cells that are prevented from attaching to an extracellular matrix substrate stop proliferating and may undergo apoptosis. Cell adhesion to a substrate is mediated by the integrin family of cell surface receptors, which are known to elicit intracellular signals upon cell adhesion. We show here that Chinese hamster ovary cells expressing the alpha 5 beta 1 integrin, which is a fibronectin receptor, do not undergo apoptosis upon serum withdrawal when the cells are plated on fibronectin. However, the alpha v beta 1 integrin, which is also a fibronectin receptor and binds fibronectin on the same RGD motif as alpha 5 beta 1, did not prevent apoptosis on fibronectin of the same cells. The cytoplasmic domain of the integrin alpha 5 subunit was required for the alpha 5 beta 1-mediated cell survival on fibronectin. The fibronectin-mediated survival effect appeared to be independent of the level of tyrosine phosphorylation of the focal adhesion kinase, which is induced by integrin-mediated cell attachment. The expression of the Bcl-2 protein, which counteracts apoptosis, was elevated in cells attaching to fibronectin through alpha 5 beta 1; cells attaching through alpha v beta 1 survived only if exogenous Bcl-2 was provided. Thus, alpha 5 beta 1, but not the closely related alpha v beta 1 integrin, appears to suppress apoptotic cell death through the Bcl-2 pathway.

The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2.

EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general transcription factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted transcription factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo.

Role of heme regulated eIF2α kinase in pancreatic beta cell function and viability

The eukaryotic translation initiation factor 2 alpha (eIF2α) is part of the initiation complex that drives the initiator amino acid methionine to the ribosome, a crucial step in protein translation. In stress conditions such as virus infection, endoplasmic reticulum (ER) stress, amino acid or heme deficiency eIF2α can be phosphorylated and thereby inhibit global protein synthesis. This adaptive mechanism prevents protein accumulation and consequent cytotoxic effects. Heme-regulated eIF2α kinase (HRI) is a member of the eIF2α kinase family that regulates protein translation in heme deficiency conditions. Although present in all tissues, HRI is predominantly expressed in erythroid cells where it remains inactive in the presence of normal heme concentrations. In response to heme deficiency, HRI is activated and phosphorylates eIF2α decreasing globin synthesis. This mechanism is important to prevent accumulation of heme-free globin chains which cause ER stress and apoptosis. RNA sequencing data from our group showed that in human islets and in primary rat beta cells HRI is the most expressed eIF2α kinase compared to the other family members. Despite its high expression levels, little is known about HRI function in beta cells. The aim of this project is to identify the role of HRI in pancreatic beta cells. This was investigated taking a loss-of-function approach. HRI knock down (KD) by RNA interference induced beta cell apoptosis in basal condition. HRI KD potentiated the apoptotic effects of palmitate or proinflammatory cytokines, two in vitro models for type 2 and type 1 diabetes, respectively. Increased cytokine-induced apoptosis was also observed in HRI-deficient primary rat beta cells. Unexpectedly, we observed a mild increase in eIF2α phosphorylation in HRI-deficient cells. The levels of mRNA or protein expression of C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) were not modified. HRI KD cells have decreased spliced X-box binding protein 1 (XBP1s), an important branch of the ER stress response. However, overexpression of XBP1s by adenovirus in HRI KD cells did not protect from HRI siRNA-induced apoptosis. HRI deficiency decreased phosphorylation of Akt and its downstream targets glycogen synthase kinase 3 (GSK3), forkhead box protein O1 (FOXO1) and Bcl-2-associated death promoter (BAD). Overexpression of a constitutively active form of Akt by adenovirus in HRI-deficient beta cells partially decreased HRI KD-mediated apoptosis. Interestingly, BAD silencing protected from apoptosis caused by HRI deficiency. HRI silencing in beta cells also induced JNK activation. These results suggest an important role of HRI in beta cell survival through modulation of the Akt/BAD pathway. Thus, HRI may be an interesting target to modulate beta cell fate in diabetic conditions.

Regulation of MAPK activation, AP-1 transcription factor expression and keratinocyte differentiation in wounded fetal skin

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E-17) in the rat such that embryonic day 19 (E-19) wounds do not re-epithelialize. Moreover, wounds created in E-17 fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E-17 and E-19 skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E-17 and E-19 skin. c-fos and c-jun induction was transient in E-17 skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E-19 skin, AP-11 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E-17 skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.

The TAF5L gene on chromosome 1q42 is associated with type 1 diabetes in Russian affected patients

Type 1 diabetes (T1D) is a multifactorial autoimmune disease, with strong genetic component. Several susceptibility loci contribute to genetic predisposition to T1D. One of these loci have been mapped to chromosome 1q42 in UK and US joined affected family data sets but needs to be replicated in other populations. In this study, we evaluated sixteen microsatellites located on 1q42 for linkage with T1D in 97 Russian affected sibling pairs. A 2.7-cm region of suggestive linkage to T1D between markers D1S1644 and D1S225 was found by multipoint linkage analysis. The peak of linkage was shown for D1S2847 (P = 0.0005). Transmission disequilibrium test showed significant undertransmission of the 156-bp allele of D1S2847 from parents to diabetic children (28 transmissions vs. 68 nontransmissions, P = 0.043) in Russian affected families. A preferential transmission from parents to diabetic offspring was also shown for the T(-25) and T1362 alleles of the C/T(-25) and C/T1362 dimorphisms, both located at the TAF5L gene, which is situated 103 kb from D1S2847. Together with the A/C744 TAF5L SNP, these markers share common T(-25)/A744/T1362 and C(-25)/C744/T1362 haplotypes associated with higher and lower risk of diabetes (Odds Ratio = 2.15 and 0.62, respectively). Our results suggest that the TAF5L gene, encoding TAF5L-like RNA polymerase II p300/CBP associated factor (PCAF)-associated factor, could represent the susceptibility gene for T1D on chromosome 1q42 in Russian affected patients.

The Runx1 transcription factor controls CSF-1-dependent and -independent growth and survival of macrophages

Gene translocations that repress the function of the Runx1 transcription factor play a critical role in the development of myeloid leukemia. In this report, we demonstrate that Runx1 precisely regulates c-fms (CSF-1 receptor) gene expression. Runx1 controlled expression by binding to multiple sites within the mouse c-fms gene, allowing interaction between promoter and downstream enhancer elements. The runx1 and c-fms genes showed an identical pattern of expression in mature macrophages. Runx1 expression was repressed in CSF-1 stimulated, proliferating bone marrow-derived macrophages (BMM) and significantly increased in quiescent, CSF-1 starved cells. The RAW264.7 and Mono-Mac-6, macrophage-like cell lines expressed low levels of Runx1 and both showed growth arrest and cell death with ectopic expression of Runx1. The EM-3 cell line, which represents an early myeloid progenitor cell line, showed growth arrest with Runx1 expression in the absence of any detectable changes in cell differentiation. These findings suggest that Runx1 regulates growth and survival of myeloid cells and provide a novel insight into the role of Runx family gene translocations in leukemogenesis.

Placenta growth factor-1 exerts time-dependent stabilization of adherens junctions following VEGF-induced vascular permeability

Increased vascular permeability is an early event characteristic of tissue ischemia and angiogenesis. Although VEGF family members are potent promoters of endothelial permeability the role of placental growth factor (PlGF) is hotly debated. Here we investigated PlGF isoforms 1 and 2 and present in vitro and in vivo evidence that PlGF-1, but not PlGF-2, can inhibit VEGF-induced permeability but only during a critical window post-VEGF exposure. PlGF-1 promotes VE-cadherin expression via the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter and subsequently stabilizes transendothelial junctions, but only after activation of endothelial cells by VEGF. PlGF-1 regulates vascular permeability associated with the rapid localization of VE-cadherin to the plasma membrane and dephosphorylation of tyrosine residues that precedes changes observed in claudin 5 tyrosine phosphorylation and membrane localization. The critical window during which PlGF-1 exerts its effect on VEGF-induced permeability highlights the importance of the translational significance of this work in that PLGF-1 likely serves as an endogenous anti-permeability factor whose effectiveness is limited to a precise time point following vascular injury. Clinical approaches that would pattern nature's approach would thus limit treatments to precise intervals following injury and bring attention to use of agents only during therapeutic windows.

Regulation of Pygopus (hPygo2) expression in prostate cancer by E74-Like factor 1 (Elf-1) transcription factor

Prostate cancer is a worldwide health concern. Pygopus2 (hPygo2) protein is required for growth in breast, ovarian, cervical and prostate cancer. hPygo2 expression is regulated by the Rb protein via the ETS factor Elf-1 in cervical and breast cancer. Additionally, the ETS family has confirmed roles in carcinogenesis and proliferation. The mechanism of hPygo2 expression has not been elucidated in prostate cancer. My hypothesis proposes that hPygo2 expression is regulated by Elf-1 bound to its promoter region. Prostate cancer cell lines were used to show protein levels of hPygo2, Elf-1 and ETS. ChIP assays confirmed varying binding capability of Elf-1 and ETS factors to the proximal promoter region between cell lines. Elf-1 knockdown experiments were performed, results show no change in hPygo2 protein levels but show reduction in 22Rv1 mRNA levels. These results suggest that Elf-1 might not be exclusively involved in the activation of Pygopus expression in prostate cancer.

Hormonal factors and insulin-like growth factor-1 in North American postmenopausal women

Background: Mechanisms underlying the effect of estrogen exposure on breast cancer risk remain unclear. Insulin-like growth factor-1 (IGF-1) levels have been positively associated with breast cancer and are a potential mechanism. Objectives: The objectives of this thesis are: 1) to explore whether the reproductive risk factors and the lifetime cumulative number of menstrual cycles (LCMC), as measures for long-term estrogen exposure, are associated with IGF-1 levels, and 2) to examine the effect of an aromatase inhibitor (AI) on IGF-1 levels, and the potential interaction with BMI. Methods: A cross sectional study and a randomized controlled trial nested with the MAP.3 chemoprevention trial were used to address objective 1 and 2, respectively. 567 postmenopausal women were selected. Anthropometric measurements, lifestyle factors, reproductive characteristics and serum IGF-1 concentrations were collected at baseline and one year. Objective 1. The LCMC was computed as a composite measure of the reproductive characteristics. Multivariable linear regression models were used to assess the association between IGF-1 levels and LCMC and the hormonal risk factors, while adjusting for potential covariates. Objective 2. Changes in IGF-1 were compared between the exemestane and placebo, and effect modification by BMI was tested with an interaction term. Results: Objective 1. Women aged 55 years or older at menopause had 16.26 ng/mL (95% CI: 1.76, 30.75) higher IGF-1 compared to women aged less than 50 years at menopause. Women in the highest category of menstrual cycles (≥500 cycles) had an average 19.00 ng/mL (95%CI: 5.86, 32.14) higher concentration of IGF-1 compared to women in the lowest category (<350). Exogenous hormones had no effect on postmenopausal IGF-1 levels. Objective 2. Exemestane significantly increased IGF-1 levels by 18% (95% CI: 14%-22%); while, placebo had no effect on IGF-1. The changes in IGF-1 were significantly different between the treatment arms (P<0.0001) and no significant interaction was observed between treatment and BMI on IGF-1 changes (P=0.1327). Conclusion: Objective 1. Larger number of menstrual cycles and a later age at menopause are positively associated with IGF-1. IGF-1 may be one mechanism by which prolonged estrogen exposure increases cancer risk. Objective 2. We conclude that the reduced cancer risk observed with AI therapy likely occurs in an IGF-1 independent mechanism. Further studies exploring the clinical consequences of increased IGF-1 on AI therapy are needed.