Browsing by Subject "Transcription factors"
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Item E2F3a functions as an oncogene and induces DNA damage response pathway mediated apoptosis(2007) Paulson, Qiwei Xia, 1974-; Johnson, David, 1963-; Bratton, Shawn B.Mutation or inactivation of RB occurs in most human tumors and results in the deregulation of several E2F family transcription factors. Among the E2F family, E2F3 has been implicated as a key regulator of cell proliferation and E2f3 gene amplification and overexpression is detected in some human tumors. To study the role of E2F3a in tumor development, we established a transgenic mouse model expressing E2F3a in a number of epithelial tissues via a keratin 5 (K5) promoter. Transgenic expression of E2F3a leads to hyperproliferation, hyperplasia and increased levels of p53-independent apoptosis in transgenic epidermis. Consistent with data from human cancers, the E2f3a transgene is found to have a weak oncogenic activity on its own and to enhance the response to a skin carcinogenesis protocol. While E2F3a induces apoptosis in the absence of p53, the inactivation of both p53 and p73, but not p73 alone, significantly impairs apoptosis induced by E2F3a. This suggests that both p53 and p73 contribute to E2F3a induced apoptosis but that their function is compensatory. Even though data suggest that E2F3a carries out its unique apoptotic activity in part through another E2F family member E2F1, unlike E2F1, the ARF tumor suppressor is required for E2F3a-induced apoptosis. While both E2F3a and E2F1 require ATM for apoptosis, E2F3a activates ATM through a distinct mechanism from E2F1. The overexpression of E2F3a results in the accumulation of DNA damage in K5 transgenic keratinocytes and normal human fibroblasts (NHFs). In response to this, the DNA damage checkpoint kinase ATM is activated, and phosphorylation of the downstream targets p53 and the histone variant H2AX are significantly increased. Additional studies show that increased Cdk activity and aberrant DNA replication contributes to DNA damage, ATM activation and apoptosis in response to deregulated E2F3a, which suggest that aberrant replication imposed by deregulated E2F3a plays an important role in the activation of the ATM DNA damage response pathway. Activation of ATM by E2F3a is not affected by loss of ARF or E2F1. Meanwhile, E2F3a-induced ARF upregulation is not affected by E2F1 loss. The above results indicate that E2F3a engages several parallel pathways involving E2F1, ARF and the ATM kinase, and these pathways cooperate to promote apoptosis.Item Eukaryotic transcriptional regulation : from data mining to transcriptional profiling(2008-12) Morgan, Xochitl Chamorro; Iyer, Vishwanath R.Survival of cells and organisms requires that each of thousands of genes is expressed at the correct time in development, in the correct tissue, and under the correct conditions. Transcription is the primary point of gene regulation. Genes are activated and repressed by transcription factors, which are proteins that become active through signaling, bind, sometimes cooperatively, to regulatory regions of DNA, and interact with other proteins such as chromatin remodelers. Yeast has nearly six thousand genes, several hundred of which are transcription factors; transcription factors comprise around 2000 of the 22,000 genes in the human genome. When and how these transcription factors are activated, as well as which subsets of genes they regulate, is a current, active area of research essential to understanding the transcriptional regulatory programs of organisms. We approached this problem in two divergent ways: first, an in silico study of human transcription factor combinations, and second, an experimental study of the transcriptional response of yeast mutants deficient in DNA repair. First, in order to better understand the combinatorial nature of transcription factor binding, we developed a data mining approach to assess whether transcription factors whose binding motifs were frequently proximal in the human genome were more likely to interact. We found many instances in the literature in which over-represented transcription factor pairs co-regulated the same gene, so we used co-citation to assess the utility of this method on a larger scale. We determined that over-represented pairs were more likely to be co-cited than would be expected by chance. Because proper repair of DNA is an essential and highly-conserved process in all eukaryotes, we next used cDNA microarrays to measure differentially expressed genes in eighteen yeast deletion strains with sensitivity to the DNA cross-linking agent methyl methane sulfonate (MMS); many of these mutants were transcription factors or DNA-binding proteins. Combining this data with tools such as chromatin immunoprecipitation, gene ontology analysis, expression profile similarity, and motif analysis allowed us to propose a model for the roles of Iki3 and of YML081W, a poorly-characterized gene, in DNA repair.Item From developing protein-protein interaction strategies to identifying gene functions: case studies for transcription factor complexes and ribosome biogenesis genes(2007-12) Li, Zhihua, doctor of cell and molecular biology; Marcotte, Edward M.Protein-protein interactions are central to their biological functions in cells. Many approaches have been applied to study protein-protein interactions in a genomic-scale. In an attempt to develop new strategies to study protein-protein interactions, FRET by using ECFP and EYFP as the donor and receptor was evaluated for possible application in protein-protein interaction study in a high-throughput fashion. Due to the intrinsic properties of ECFP and EYFP, FRET-based protein-protein interaction assay is not suitable for large-scale studies. Instead, tandem affinity purification coupled with mass spectrometry approach proved to be a useful strategy to identify protein interacting partners. Several transcription factor complexes in yeast were successfully purified and novel components in the complexes were identified by combining a shotgun mass spectrometry approach and a differential analysis of the mass spectrometry data. In particular, a negative regulator of G1 to S phase transition during cell cycle, Whi5p, was identified to be a component of SBF complex; a regulator of nitrogen metabolism, Gln3p, was identified to be a component of Hap2/3/5 complex that regulates carbon metabolism, suggesting a crosstalk between nitrogen and carbon metabolism. Additionally, one-step purification coupled with shotgun mass spectrometry analysis was applied to simplify and improve the affinity purification approach used for protein-protein interaction studies. In order to map protein complexes in their native state, a sucrose density gradient was used to separate protein complexes in cells. The proteins within each fraction from the sucrose density gradient were analyzed and quantified with mass spectrometry to obtain the protein abundance profiles across the gradient. The known protein complexes were identified by clustering the protein abundance profiles. This method could possibly be improved to become a generic approach to mapping protein complexes. The goal of protein-protein interaction studies is to determine the protein functions. In an effort to identify ribosome biogenesis genes from a yeast gene network reconstructed from diverse large-scale interaction data sets, at least 25 new ribosome biogenesis genes were confirmed by extensive experimental validations, underscoring the value of proteinprotein interaction studies and gene interaction network.Item Functional characterization of the B-cell lymphoma/leukemia 11A (BCL11A) transcription factor(2007-12) Lee, Baeck-seung, 1969-; Tucker, Philip W.Previously a t(2;14)(p13;q32) translocation was characterized in four unusually aggressive cases of B cell chronic lymphocytic leukemia (B-CLL). A gene located near the 2p13 breakpoint, B cell lymphoma/leukemia 11A (BCL11A), was shown to overexpress 3 isoforms (BCL11A-XL, L and S). Bcl11a knockout mice are severely impaired in B cell development at the early (pro-B) stage. I have further characterized BCL11A, focusing on the most abundant and evolutionarily conserved isoform, BCL11A-XL (XL). I demonstrated that XL resides in the nuclear matrix, is modified by ubiquitination, and is destabilized by B cell antigen receptor ligation. I identified domains within XL required for its localization within nuclear paraspeckles and for its transcriptional repression. While BCL11A-XL represses model promoters in non-B cells, its biologically relevant targets in B lymphocytes were unknown. I have identified and confirmed a number of XL targets which are both up- and down-regulated by XL over-expression in B cell lines. A number of these genes have been implicated in B cell function, including the V(D)J recombination activating (RAG) genes. Both RAG1 and RAG2 transcripts were up-regulated by XL. XL binds to the RAG1 promoter and RAG enhancer (Erag) in vivo as well as in vitro. Unexpectedly, XL repressed RAG1 transcription in non-B cells, indicating that additional B cell-specific factors are required for activation. Overexpression of XL in a V(D)J recombination-competent pre-B cell line markedly induced RAG expression and VDJ recombination. IRF4 and IRF8, transcription factors previously shown to be required for early B cell development, were also induced by BCL11A-XL. I propose that the early B cell progenitor block in Bcl11a knockout mice is, at least in part, a direct result of BCL11A-XL regulation of V(D)J recombination. Further experiments are required to establish how other XL targets promote B cell lineage development and how malignant transformation such as in B-CLL may corrupt BCL11A function.Item Functional characterization of two JunD isoforms(Texas Tech University, 2002-08) Yazgan, OyaThe AP-1 transcription factor is involved in many diverse processes such as proliferation, transformation and apoptosis. The ability of AP-1 to rearrange its dimeric composition and DNA-binding activity allows for generation of numerous, highly specific signals. JunD is the predominant component of the AP-1 complex in nondividing cells and has the ability to slow down growth when overexpressed in cells. JunD mRNA generates two isoforms through the use of an alternative translational start site, the full-length isoform (JunD-FL) and a truncated form ΔJunD. We determined the role of two isoforms of JunD in their abilities to alter growth rate, to transactivate promoters, and to induce apoptosis. Our results indicate that JunD-FL has a higher transactivation potential than JunD and that overexpression of ΔJunD in mouse fibroblasts retards growth and sensitizes cells for apoptosis. We also examined their physical interactions with the Jun-N-terminal kinases (JNKs) and the tumor suppressor protein Menin. We found that only JunD-FL interacts with Menin and this interaction leads to inhibitton of the intrinsic transcriptional activity of JunD-FL. This finding suggests that two isoforms of the JunD transcriptions factor are differentially regulated. In addition, Menin inhibits the JNK-mediated acttvatton of both isoforms of JunD and c-Jun in transient transfection assays, suggesting that Menin could be involved in regulation of more diverse JNK substrates in the cell.Item A functional genomics approach to map transcriptional and post-transcriptional gene regulatory networks(2009-08) Bhinge, Akshay Anant; Iyer, Vishwanath R.It has been suggested that organismal complexity correlates with the complexity of gene regulation. Transcriptional control of gene expression is mediated by binding of regulatory proteins to cis-acting sequences on the genome. Hence, it is crucial to identify the chromosomal targets of transcription factors (TFs) to delineate transcriptional regulatory networks underlying gene expression programs. The development of ChIP-chip technology has enabled high throughput mapping of TF binding sites across the genome. However, there are many limitations to the technology including the availability of whole genome arrays for complex organisms such human or mouse. To circumvent these limitations, we developed the Sequence Tag Analysis of Genomic Enrichment (STAGE) methodology that is based on extracting short DNA sequences or “tags” from ChIP-enriched DNA. With improvements in sequencing technologies, we applied the recently developed ChIP-Seq technique i.e. ChIP followed by ultra high throughput sequencing, to identify binding sites for the TF E2F4 across the human genome. We identified previously uncharacterized E2F4 binding sites in intergenic regions and found that several microRNAs are potential E2F4 targets. Binding of TFs to their respective chromosomal targets requires access of the TF to its regulatory element, which is strongly influenced by nucleosomal remodeling. In order to understand nucleosome remodeling in response to transcriptional perturbation, we used ultra high throughput sequencing to map nucleosome positions in yeast that were subjected to heat shock or were grown normally. We generated nucleosome remodeling profiles across yeast promoters and found that specific remodeling patterns correlate with specific TFs active during the transcriptional reprogramming. Another important aspect of gene regulation operates at the post-transcriptional level. MicroRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that suppress translation or mark mRNAs for degradation. MiRNAs regulate TFs and in turn can be regulated by TFs. We characterized a TF-miRNA network involving the oncofactor Myc and the miRNA miR-22 that suppresses the interferon pathway as primary fibroblasts enter a stage of rapid proliferation. We found that miR-22 suppresses the interferon pathway by inhibiting nuclear translocation of the TF NF-kappaB. Our results show how the oncogenic TF Myc cross-talks with other TF regulatory pathways via a miRNA intermediary.Item The induction of apoptosis by the E2F1 transcription factor and the emergence of a role for E2F1 in the DNA double strand break response(2006) Powers, John Thomas; Tucker, Philip W.Item Intersubunit interaction changes resulting from cAMP activation of CRP(Texas Tech University, 1999-05) Riek, Bradley CDimeric cyclic adenosine 3',5' monophosphate receptor protein (CRP) complexes with cAMP to form an asymmetric CRP:(cAMP)1 complex. CRP:(cAMP)1 binds upstream of lacP to activate the promoter via interactions with RNA polymerase. The goal of this work was to determine whether the cAMP-bound subunit of CRP:(c AMP)I must be positioned exclusively in one orientation with respect to RNA polymerase to activate transcription. In vitro transcription experiments were designed to determine whether the cAMP-bound subunit must be oriented in either the promoter-proximal or the promoter-distal position to activate transcription. Site-directed mutagenesis was used to construct a CRP species that had altered affinity for DNA binding and decreased affinity for cAMP. Incubation of this mutant with WT CRP produced a heterodimeric species containing one WT subunit and one mutant subunit. At low concentrations of cAMP, only the WT subunit binds cAMP, producing CRP:(cAMP)i. The differing subunit affinities for specific DNA sequences allowed for orientation of CRP:(cAMP)i on two asymmetric templates. Mutagenesis of the CRP consensus binding sequence produced, via point mutations in either CRPbinding half-site, two templates capable of orienting the cAMP-bound subunit in either the promoter-proximal or promoter-distal position. In vitro transcription assays showed that the heterodimer activated transcription with the cAMP-bound subunit in either orientation. This data indicates the CRP:(cAMP)1 complex is not required to orient its cAMP-bound subunit with respect to RNA polymerase to activate lacP.Item Molecular control mechanisms of myeloid differentiation and proliferation(Texas Tech University, 2000-12) Du, YangStarting from a population of hematopoietic stem cells, myeloid differentiation is a highly regulated process that gives rise to mature myeloid cells including granulocytes and macrophages. The purpose of this study was to gain insights into the molecular control mechanisms imderlying myeloid development through identifying and characterizing regulators of myeloid differentiation and proliferation.Item Structure-functional analyses of Bright, a B cell regulator of immunoglobulin heavy chain transcription(2004) Kim, Dongkyoon; Tucker, Philip W.Item The RING finger binding protein is a nuclear membrane protein that interacts with RUSH transcription factors(Texas Tech University, 2001-05) Mansharamani, Malini; Chilton, Beverly S.; Lee, Vaughan H.; Pfarr, Curt M.; Pressley, Thomas A.; Webster, Daniel R.; Whelley, Sandra M.Molecular regulation of Uteroglobin gene expression by progesterone and prolactin is mediated by RUSH transcription factors. The RUSH family of proteins, which includes rabbit RUSH-la and P and the human, mouse and plant homologs of RUSH lα, are SWI/SNF related chromatin remodeling proteins. These proteins have a novel C3HC4 RING finger, at their -COOH terminus that has been implicated in mediating protein-protein interactions. When this motif was identified in RUSH proteins, it was used to screen an expression library to isolate cDNAs for proteins that complex with it. A single phage clone (~1.6kb insert) was identified. Sequence analysis of this RING Finger Binding Protein (RFBP) clone, revealed a partial cDNA that lacked an initiator codon but contained a stop codon. RACE PCR was then used to extend the 5' and 3' ends of the cDNA. The predicted amino acid sequence from the composite cDNA sequence (4286-bp) is that of a putative Type IV P-type ATPase. P-type ATPases are membrane transporters that use the energy of ATP hydrolysis to transport substrate across the membrane. Genomic cloning and ClustalW alignment indicate that RFBP is an atypical P-type ATPase that has only seven of eight core regions and nine of ten transmembrane domains typical of this family of proteins. Core region D that contains transmembrane domain four is absent from this protein. Western blot analysis, coupled with immunoelectron microscopy data, indicates that RFBP is present in the inner nuclear membrane. Coimmunoprecipitation and GST pulldown experiments showed a direct interaction between RUSH and RFBP. The RUSH binding site lies within aa 612-804 of the RFBP protein. Competitive quantitative RT-PCR indicates that RFBP is ubiquitous in its expression, with the expression pattern correlating with that of RUSH in these same tissues. In addition, expression of RFBP is hormonally regulated in the endometrium, suggesting that RFBP function and expression may be closely linked with the function of the RUSH proteins in regulating gene expression in the reproductive system. Current studies provide important information about RFBP as a RUSH binding interaction between the proteins to shed light on the mechanism of hormone regulation of uteroglobin gene expression.Item Transcription factor involvement in the rat PNS-derived stem cell line family, RT4(Texas Tech University, 1998-05) Reinhart, Adam JThe long-term objectives of this project were twofold: (1) to identify transcription factors (both novel and previously identified) which may influence cell fate and/or maturation in the nervous system, and (2) to investigate the role(s) that these transcription factors may play in either the cell-fate decisions and/or maturation of an in vitro model of peripheral neurogenesis called RT4. In the first phase of this project, five POU-domain genes (Oct-1, Oct-2, Bm-2, Bm- 5 and Tst-1/SCIP), as well as REST/NRSF (a repressor of neuronal-specific gene expression) were shown to be expressed, and their expression levels characterized in the RT4 cell line family. We also reported that 4 candidate transcription factors (Bm-1, Bm-3, Bm-4 and MASH-1) were not expressed in the RT4 cell line family. The second phase of this project involved the elucidation of the possible role(s) of Tst-1/SCIP and REST/NRSF in the RT4 cell line family. We examined whether Tst-1/SCIP influenced the conversion of RT4 stem cells to derivative cell types. The conversion frequency was essentially unchanged when transfected with wild-type Tst-1/SCIP, or a dominant-negative version of Tst-1/SCIP, or pcDNA3 vector alone. Although the conversion frequency among transfection groups (i.e., Tst-1/SCIP; Tst-1/SCIP AN; pcDNA3 vector alone) groups was approximately the same to all of the derivative cell types, the total number of stably transfected colonies within the Tst-1/SCIP transfection group was considerably less than the other transfection groups. The transcription factor REST/NRSF was examined in terms of its possible role(s) in regulating maturation-specific gene expression. Although we were able to demonstrate that the REST/NRSF-mediated repression of an RE 1-CAT reporter was at least partially relieved, we were unable to detect the de novo expression of a set of maturation-specific genes in RT4-B8, which would expect to be expressed if the cell line had been stimulated to mature.