In silico comparative genomic analysis of GABAA receptor transcriptional regulation

<p>Abstract</p> <p>Background</p> <p>Subtypes of the GABA_Areceptor subunit exhibit diverse temporal and spatial expression patterns. <it>In silico </it>comparative analysis was used to predict transcriptional regulatory features in individual mammalian GABA_Areceptor subunit genes, and to identify potential transcriptional regulatory components involved in the coordinate regulation of the GABA_Areceptor gene clusters.</p> <p>Results</p> <p>Previously unreported putative promoters were identified for the β2, γ1, γ3, ε, θ and π subunit genes. Putative core elements and proximal transcriptional factors were identified within these predicted promoters, and within the experimentally determined promoters of other subunit genes. Conserved intergenic regions of sequence in the mammalian GABA_Areceptor gene cluster comprising the α1, β2, γ2 and α6 subunits were identified as potential long range transcriptional regulatory components involved in the coordinate regulation of these genes. A region of predicted DNase I hypersensitive sites within the cluster may contain transcriptional regulatory features coordinating gene expression. A novel model is proposed for the coordinate control of the gene cluster and parallel expression of the α1 and β2 subunits, based upon the selective action of putative Scaffold/Matrix Attachment Regions (S/MARs).</p> <p>Conclusion</p> <p>The putative regulatory features identified by genomic analysis of GABA_Areceptor genes were substantiated by cross-species comparative analysis and now require experimental verification. The proposed model for the coordinate regulation of genes in the cluster accounts for the head-to-head orientation and parallel expression of the α1 and β2 subunit genes, and for the disruption of transcription caused by insertion of a neomycin gene in the close vicinity of the α6 gene, which is proximal to a putative critical S/MAR.</p

The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

INTRODUCTION: Overexpression of the ERBB2 oncogene is observed in about 20% of human breast tumors and is the consequence of increased transcription rates frequently associated with gene amplification. Several studies have shown a link between activator protein 2 (AP-2) transcription factors and ERBB2 gene expression in breast cancer cell lines. Moreover, the Yin Yang 1 (YY1) transcription factor has been shown to stimulate AP-2 transcriptional activity on the ERBB2 promoter in vitro. In this report, we examined the relationships between ERBB2, AP-2alpha, and YY1 both in breast cancer tissue specimens and in a mammary cancer cell line. METHODS: ERBB2, AP-2alpha, and YY1 protein levels were analyzed by immunohistochemistry in a panel of 55 primary breast tumors. ERBB2 gene amplification status was determined by fluorescent in situ hybridization. Correlations were evaluated by a chi2 test at a p value of less than 0.05. The functional role of AP-2alpha and YY1 on ERBB2 gene expression was analyzed by small interfering RNA (siRNA) transfection in the BT-474 mammary cancer cell line followed by real-time reverse transcription-polymerase chain reaction and Western blotting. RESULTS: We observed a statistically significant correlation between ERBB2 and AP-2alpha levels in the tumors (p < 0.01). Moreover, associations were found between ERBB2 protein level and the combined high expression of AP-2alpha and YY1 (p < 0.02) as well as between the expression of AP-2alpha and YY1 (p < 0.001). Furthermore, the levels of both AP-2alpha and YY1 proteins were inversely correlated to ERBB2 gene amplification status in the tumors (p < 0.01). Transfection of siRNAs targeting AP-2alpha and AP-2gamma mRNAs in the BT-474 breast cancer cell line repressed the expression of the endogenous ERBB2 gene at both the mRNA and protein levels. Moreover, the additional transfection of an siRNA directed against the YY1 transcript further reduced the ERBB2 protein level, suggesting that AP-2 and YY1 transcription factors cooperate to stimulate the transcription of the ERBB2 gene. CONCLUSION: This study highlights the role of both AP-2alpha and YY1 transcription factors in ERBB2 oncogene overexpression in breast tumors. Our results also suggest that high ERBB2 expression may result either from gene amplification or from increased transcription factor levels

Characterisation of the HSP70-HSP90 organising protein gene and its link to cancer

HOP (Heat shock protein 70/ Heat shock protein 90 organising protein) is a co-chaperone essential for client protein transfer from HSP70 to HSP90 within the HSP90 chaperone machine and has been found to be up-regulated in various cancers. However, minimal in vitro information can be found on the regulation of HOP expression. The aim of this study was to analyse the HOP gene structure across known orthologues, identify and characterise the HOP promoter, and identify the regulatory mechanisms influencing the expression of HOP in cancer. We hypothesized that the expression of HOP in cancer cells is likely regulated by oncogenic signalling pathways linked to cis-elements within the HOP promoter. An initial study of the evolution of the HOP gene speciation was performed across identified orthologues using Mega5.2. The evolutionary pathway of the HOP gene was traced from the unicellular organisms to fish, to amphibian and then to land mammal. The synteny across the orthologues was identified and the co-expression profile of HOP analysed. We identified the putative promoter region for HOP in silico and in vitro. Luciferase reporter assays were utilized to demonstrate promoter activity of the upstream region in vitro. Bioinformatic analysis of the active promoter region identified a large CpG island and a range of putative cis-elements. Many of the cis-elements interact with transcription factors which are activated by oncogenic pathways. We therefore tested the regulation of HOP levels by rat sarcoma viral oncogene homologue (RAS). Cancer cell lines were transfected with mutated RAS to observe the effect of constitutively active RAS expression on the production of HOP using qRT-PCR and Western Blot analyses. Additionally, inhibitors of the RAS signalling pathway were utilised to confirm the regulatory effect of mutated RAS on HOP expression. In cancer cell lines containing mutated RAS (Hs578T), HOP was up-regulated via a mechanism involving the MAPK signalling pathway and the ETS-1 and C/EBPβ cis-elements within the HOP promoter. These findings suggest for the first time that Hop expression in cancer may be regulated by RAS activation of the HOP promoter. Additionally, this study allowed us to determine the murine system to be the most suited genetic model organism with which to study the function of human HOP

Transcriptional Regulation of the Evi-1 Gene

Evi-l shows a temporally and spatially restricted pattern of expression in murine embryonic development and is expressed predominantly in the kidney, lung and developing oocytes of adult mice. The positions of DNAse 1 hypersensitive sites (DHS) within an 18kb region of the 5' Evi-1 locus have been examined to identify putative Evi- 1 gene regulatory regions, in murine kidney and spleen tissues. This analysis identified two DHS sites designated DHS I and DHS II. DHS I is located approximately 2kb upstream of the transcription initiation sites whereas DHS II maps over exon I. The transcriptional activity of the Evi-1 promoter has been investigated by inserting the 5' region of the gene into a luciferase construct and the activity examined by transient transfection of cells which express low levels of Evi-1. A substantial induction of luciferase activity is observed with a 5kb fragment of the 5' Evi-1 locus containing exon I, intron I and exon II which includes DHS I and DHS II. Subsequent deletion mutagenesis has identfied two regions within DHS II, located between -338 to -284 and -284 to -254, which upon removal result in a substantial reduction of promoter activity. One of these, located between -338 to -284, binds several proteins when examined by footprinting and electrophoretic mobility shift assays. Interestingly, the most abundant factor, designated EvBP1, has been shown to bind a 14bp imperfect palindromic sequence, tttccctggggaaa, which is absolutely conserved in the human Evi-1 promoter sequence. This sequence contains homology with putative binding sites for, AP3, AP2 and C/EBP. However, competition studies in EMSA with consensus binding site oligonucleotides failed to identify the components of EvBP1. EvBP1 might be a novel ubiquitous transcription factor which is required for regulation of the Evi-1 promoter. Furthermore, EMSA analysis of the second deleted region between -284 to -254 has identified a CCAAT binding protein, possibly CPI, which may also be important in basal promoter activity. Functional assays have failed to identify either promoter or enhancer activity for DHS I. Since there are no known appropriate high Evi-1 expressing cell lines we have established Evi-1 expressing kidney cultures as a system to examine tissue specific expression. This allowed the identification of a 3kb region necessary for higher activity in the cultures. This activity might correlate with a DHS site which is part of DHS II complex

Arabidopsis leaf mutants reveal conserved and unique proteins involved in light and auxin signaling

Growth and development in plants is steered by the meristems which are established on both poles of the embryo and become active after germination. The activity of the shoot apical meristem (SAM) leads to the formation of lateral organs such as leaves which are responsible for photosynthesis. At flowering time the SAM is transformed to the inflorescence meristem which produces flowers as lateral organs. The root apical meristem is responsible for the growth of the primary root and is important in water- and mineral-uptake. Within the research group “Chromatin and Growth Control” several genes are being cloned of which the mutant phenotype shows a defect in leaf growth. This way the genes coding for components of the Elongator complex were identified. In yeast Elongator was defined as a histone acetyltransferase (HAT) complex associated with RNA polymerase II to facilitate transcription elongation. In this thesis the loci of two other leaf mutants ang3 and ron3 belonging to the angusta and rotunda class respectively were cloned. By transcript profiling of the leaf mutants using micro-array data we try to pinpoint in which biological process the gene is involved. Localization studies and tandem affinity purification of the respective protein helps to identify which molecular pathways are likely to be affected. With detailed phenotypical analysis of the mutants we are often able to link the affected processes to observed phenotypes. Depending on the obtained results we perform specific experiments (ChIP, hormone measurements,…) to verify the true function of the protein. This work aimed at investigating the role of the Elongator complex in plants and several objectives were put forward 1. To prove the role of Elongator in transcription 2. To identify target genes of Elongator using chromatin-immuno-precipitation Besides the functional characterization of the Elongator complex, two other leaf mutants ang3 and ron3 belonging to the angusta and rotunda class respectively were cloned and functionally characterized. The genes were cloned by fine-mapping combined with sequence analysis of candidate genes in the genetic interval around the locus of interest. Molecular analysis of the genes was combined with morphological and cellular analyses of the corresponding mutants to determine the cellular basis of the observed growth defects and to gain deeper insight into the function of the genes

Characterisation of Transcriptional Regulation of the Human Telomerase RNA Gene

The human telomerase core enzyme consists of an essential structural RNA (hTERC) with a template domain for telomeric DNA synthesis and of a catalytic protein (hTERT) with reverse transcriptase activity. Expression of the hTERC and hTERT are essential for telomerase activity. Variation in telomerase activity is correlated with cellular senescence and tumour progression. Recent studies indicate that the regulation of telomerase activity is multifactorial in mammalian cells. The primary mode of control of hTERT appears to be transcriptional regulation but very little is known about the molecular mechanisms involved in the regulation of hTERC transcription. In this study, I have cloned and characterised the genomic sequences and promoter of the hTERC gene to provide evidence that transcriptional mechanisms are involved in hTERC gene regulation. Transient transfection with a series of 5'-deletion mutants demonstrated that between -5.0 kb and -51 by of the hTERC gene is responsible for high promoter activity, the minimal promoter region was defined as 176 by (-107 to +69 bp). With the aid of in vitro DNase I footprinting, electrophoretic mobility shift assays (EMSAs) and mutagenesis analysis, four Sp1 binding sites and one CCAAT-box bound by the transcription factor NF-Y were identified to be involved in regulation of hTERC transcription. Co-transfection experiments showed that Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. Mutation of the CCAAT-box or the coexpression of a dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by pRb and Sp1, suggesting that NF-Y binding is a prerequisite for pRb and Sp1 to activate the hTERC promoter. The different transcriptional regulators appear to act in a species-specific manner. Whilst Sp1 and Sp3 act on the human, bovine and mouse TERC promoters, pRb activates only the human and bovine promoter and NF-Y is important for the human TERC gene. The hTERC gene is expressed during embryogenesis and then down-regulated during normal development but is re-expressed in tumour cells, the hTERC promoter activity was therefore further investigated and a higher promoter activity in immortal cells than in two mortal cell strains (WI38 and IMR90) was shown. In conclusion, hTERC promoter contains sequence elements that allow interactions with several different transcription factors. The interplay between NF-Y, pRb, Sp1 and Sp3 within the architecture of the hTERC promoter may combine to enable a wide variety of cell types from mortal to immortal to regulate hTERC expression through transcriptional control

Characterization of acetylcholinesterase & its promoter region in Tetraodon nigroviridis

Lau Suk Kwan.Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.Includes bibliographical references (leaves 128-150).Abstracts in English and Chinese.Acknowledgment --- p.iTable of content --- p.iiList of Figures --- p.xList of Tables --- p.xivAbbreviation --- p.xvAbstract --- p.xviii論文摘要 --- p.xxChapter 1 --- Chapter 1 Introduction --- p.1Chapter 1.1 --- Tetraodon nigroviridis --- p.1Chapter 1.1.1 --- Background --- p.1Chapter 1.1.2 --- Genomic Sequencing Project --- p.3Chapter 1.1.3 --- Tetraodon nigroviridis as Study Model --- p.4Chapter 1.1.3.1 --- Genomic Comparison --- p.4Chapter 1.1.3.2 --- Gene Order and Structural Studies --- p.5Chapter 1.1.3.3 --- Genomic Evolution --- p.6Chapter 1.2 --- Transcriptional Regulation and Transcription Factors Binding Sites Prediction --- p.7Chapter 1.2.1 --- Transcriptional Regulation --- p.7Chapter 1.2.1.1 --- Chromatin Remodeling --- p.7Chapter 1.2.1.2 --- Locus Control Regions (LCR) and Boundary Elements --- p.8Chapter 1.2.1.3 --- Promoter Structure --- p.9Chapter 1.2.1.4 --- Transcriptional Machinery Assembly --- p.10Chapter 1.2.2 --- Transcription Factors and Their Binding Sites --- p.11Chapter 1.2.3 --- Transcription Factor Binding Site Prediction --- p.12Chapter 1.3 --- Acetylcholinesterase --- p.15Chapter 1.3.1 --- Background --- p.15Chapter 1.3.2 --- Regulation ofAChE --- p.17Chapter 1.3.2.1 --- Transcriptional Level --- p.17Chapter 1.3.2.2 --- Post-transcriptional Level --- p.19Chapter 1.3.2.3 --- Post-translational Level --- p.20Chapter 1.3.2.3.1 --- Oligomerization --- p.20Chapter 1.3.2.3.2 --- Glycosylation --- p.21Chapter 1.3.2.3.3 --- Phosphroylation --- p.22Chapter 1.3.3 --- Functions of AChE --- p.23Chapter 1.3.3.1 --- Hydrolysis Acetylcholine --- p.23Chapter 1.3.3.2 --- Embryonic Development --- p.23Chapter 1.3.3.3 --- Haemotopotesis and Thrombopsiesis --- p.24Chapter 1.3.3.4 --- Neuritogensis --- p.24Chapter 1.3.3.5 --- Amyloid Fibre Assembly --- p.24Chapter 1.3.3.6 --- Apoptosis --- p.25Chapter 1.3.4 --- AChE and Alzheimer's disease --- p.25Chapter 1.3.4.1 --- Treatment for AD Patients --- p.27Chapter 1.4 --- Inducible Cell Expression Systems --- p.28Chapter 1.5 --- Objectives --- p.32Chapter 2 --- Chapter 2 Materials and Methods --- p.33Chapter 2.1 --- Materials --- p.33Chapter 2.2 --- Methods --- p.34Chapter 2.2.1 --- Primer Design --- p.34Chapter 2.2.2 --- Cell Culture --- p.34Chapter 2.2.3 --- Transformation --- p.35Chapter 2.2.4 --- Plasmids Preparation --- p.35Chapter 2.2.5 --- Plasmids Screening --- p.36Chapter 2.2.6 --- RNA Extraction --- p.36Chapter 2.2.7 --- Reverse Transcriptase Polymerase Chain Reaction and Construction tnAChE/pCR4 vector --- p.37Chapter 2.2.8 --- Genomic Analysis --- p.37Chapter 2.2.9 --- Protein Sequence Analysis --- p.38Chapter 2.2.10 --- Genomic DNA Extraction --- p.39Chapter 2.2.11 --- Construction of Reporter Vectors ptnAChE_565/pGL3 and ptnAChK1143/pGL3 --- p.39Chapter 2.2.12 --- Luciferase Assay --- p.40Chapter 2.2.13 --- Transcription Factors and Promoter Prediction --- p.40Chapter 2.2.14 --- Protein Assay --- p.41Chapter 2.2.15 --- AChE Activity Determined by Ellman's Method --- p.41Chapter 2.2.16 --- Histochemistry --- p.42Chapter 2.2.17 --- Protein Extraction from Tissues --- p.42Chapter 2.2.18 --- Construction of Bacterial Expression Vector His-MBP-tnAChEAC/pHISMAL --- p.43Chapter 2.2.19 --- Protein Expression in Bacterial Expression System --- p.43Chapter 2.2.20 --- Purification and Thrombin Cleavage of His-MBP- tnAChEAC --- p.44Chapter 2.2.21 --- SDS Electrophoresis --- p.44Chapter 2.2.22 --- Western Blotting --- p.45Chapter 2.2.23 --- Construction of Tet-Off Expression Vector --- p.45Chapter 2.2.24 --- Transient Expression of tnAChEAC --- p.46Chapter 2.2.25 --- Establishment of Stable Tet-Off CHO Cell Lines Overexpressing tnAChEAC --- p.47Chapter 2.2.26 --- MTT Assay --- p.47Chapter 2.2.27 --- Partial Purification of tnAChEΔC --- p.48Chapter 3 --- Chapter 3 Sequence Analysis of AChE Gene of Tetraodon nigroviridis --- p.49Chapter 3.1 --- Results --- p.49Chapter 3.1.1 --- Cloning of tnAChE from Tetraodon nigroviridis Brain --- p.49Chapter 3.1.2 --- "Comparative genomic analysis of tnAChE with Human, Rat, Mouse, Takifugu rubripes, ZebrafishAChE" --- p.49Chapter 3.1.3 --- Primary Sequence Analysis --- p.52Chapter 3.1.4 --- Promoter and Transcriptional Factors Predictedin tnAChE Promoter Region --- p.60Chapter 3.1.4.1 --- Promoter Region Analysis In Silico --- p.60Chapter 3.1.4.2 --- Promoter Activity Analysis --- p.76Chapter 3.2 --- Discussion --- p.78Chapter 4 --- Characterization of tnAChE in Prokaryotic and Eukaryotic Tet-Off Inducible Expression System --- p.91Chapter 4.1 --- Results --- p.91Chapter 4.1.1 --- AChE Expresses in Tetraodon nigroviridis --- p.91Chapter 4.1.2 --- Expression of recombinant tnAChE in Bacterial Expression System --- p.94Chapter 4.1.2.1 --- Construction of His-MBP-tnAChEΔC/pHISMAL Construct --- p.94Chapter 4.1.2.2 --- His-MBP-tnAChEAC Expression in E. coli Strains BL21 (DE) and C41 --- p.94Chapter 4.1.3 --- Expression of tnAChEAC in Mammalian Expression System --- p.99Chapter 4.1.3.1 --- Construction of tnAChEAC/pTRE2hgyo Mammalian Expression Vector --- p.99Chapter 4.1.3.2 --- Transient Expression of tnAChEAC --- p.99Chapter 4.1.3.3 --- Establishment of Tet-Off CHO Cells Stably Expressing the Inducible tnAChEAC --- p.101Chapter 4.1.3.4 --- Characterization of Tet-Off tnAChEAC Stably Transfected Cell Clones --- p.103Chapter 4.1.3.5 --- Effect of Over Expressed tnAChEAC on cell viability --- p.103Chapter 4.1.3.6 --- Partial Purification of tnAChEAC from Stably Transfected Cells --- p.107Chapter 4.1.3.7 --- tnAChE and tnAChEAC in Different pH Values --- p.112Chapter 4.1.3.8 --- Kinetic Study of tnAChEAC --- p.112Chapter 4.1.3.9 --- Inhibition of AChE Activity of Partial Purified tnAChEAC by Huperzine --- p.112Chapter 4.2 --- Discussion --- p.116Chapter 4.2.1 --- Bacterial Expression System --- p.116Chapter 4.2.2 --- Expression of tnAChEΔC in Mammalian System --- p.119Chapter 5 --- General Discussion --- p.124Chapter 5.1 --- Summaries --- p.124Chapter 5.2 --- Further works --- p.126Chapter 6 --- References --- p.128Appendix 1 internet software and database used in this project --- p.151Appendix 2 tnAChE mRNA sequence --- p.152Appendix 3 ptnAChE-1143 sequence --- p.154Appendix 4 Six open reading frame translation of ptnAChE-1143 --- p.15

Designer Transcription Activator Like Effector - Chromatin Affinity Purification (dTALE-ChAP) a novel in planta method to unravel the protein coverage at a promoter of choice

he novel in vivo method developed in this work, allows to analyze the proteome associated with any promoter of interest and is called dTALE-ChAP. This method makes use of a set of designer Transcription Activator Like Effectors (dTALEs), designed as bait proteins for Chromatin Affinity Purification (ChAP) with subsequent mass spectrometry (MS). To demonstrate the use of the dTALE-ChAP, stable transformed dTALE-expressing Arabidopsis thaliana lines were used. The target of choice to establish the method was the well-known promoter of the Flagellin22 induced Receptor Like Kinase 1 (pFRK1). To establish the method, several pretests had to be performed. First, expression of the dTALEs and their dexamethasone (DEX)-inducible nuclear translocation was confirmed in transgenic Arabidopsis thaliana lines by microscopy. Second, it was demonstrated by promoter-reporter gene assays in Arabidopsis protoplasts, that dTALEs specifically bind to their DNA target sequence, derived from the pFRK1. Third, it was shown by Chromatin Immuno-Precipitation, that a dTALE can precipitate pFRK1 fragments from nuclear extracts of transgenic Arabidopsis lines. Finally, the dTALE-ChAP was performed and several proteins including histones were identified to be associated with pFRK1. Thus, the dTALE-ChAP was successfully established and such a method was used for the first time in plants. This new method allows to analyze the dynamics and post-translational modifications of DNA associated proteins over time in any organism. In future, methods like the dTALE-ChAP will help to better understand transcriptional regulation