Browsing by Subject "Transcription factors"

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Characterization of underlying transcription factors that regulate betalain pigment formation in beets
(2015-08) Akhavan, Neda Attar; Lloyd, Alan M.; Fischer, Janice A.; Mehdy, Mona; Roux, Stanley J.; Simpson, Beryl B.
The plant kingdom is a colorful place with most vascular plants producing phenylalanine-based red/violet anthocyanin pigment. Only a single order of flowering plants, the Caryophyllales, is known to produce an unusual pigment known as Betalain. Betalains encompasses an entire range of colors between yellow to red, are nitrogen-containing water-soluble compounds derived from tyrosine, and are acidic in nature due to the presence of several carboxyl groups. The betalain and the anthocyanin pigments are mutually exclusive. The pathway and enzymes for betalain biosynthesis, from tyrosine to the end products, red/violet betacyanins and yellow betaxanthins, has largely been determined. Little however is known about the regulation of the biosnynthetic genes. The strong biological correlation between the anthocyanin and the betalains prompted the suggestion that the molecular regulation of betalains and anthocyanins uses the same MYB and bHLH and WD-repeat regulators, the MBW complex. The work described here strives to understand the regulatory mechanisms controlling betalain pigmentation in the Caryophyllales and how they can be controlled and influenced. To understand the pathway, there was a pressing need for analysis at the biochemical, molecular, and genetic levels. Before the work reported here, two pigment biosynthetic genes were identified. The gene/ enzyme responsible for step two was identified as DOPA 4, 5-dioxygenase (DODA) functioning to produce betalamic acid (BA) from LDOPA (Christinet et al., 2004), and later we showed that a novel cytochrome P450, CYP76AD1, is absolutely required for the red pigment in beets by catalyzing the step producing cyclo-DOPA from the LDOPA substrate (Hatlestad et al., 2012). Through this project I have: (1) discovered and characterized Beet MYB1 (BvMYB1), a MYB evolved from the anthocyanin regulating MYBs, and analyzed how BvMYB1 regulates betalain production by its interaction with DNA and other proteins; (2) determined overlapping functional redundancies of BvMYB1 with two other R2R3 BvMYBs, BvMYB2 and BvMYB3; (3) identified a beet bHLH protein, BvbHLH1, and a beet WD-repeat protein, BvTTG1, that function similar to the Arabidopsis thaliana proteins; and finally (4) I worked towards characterizing a novel BvMYB1 Response Element (MRE) that BvMYB1 directly binds to activate betalain biosynthetic genes. By identifying members of the regulatory complex for the betalain pathway I hope to contribute toward understanding the evolutionary replacement of anthocyanins by betalains within a single flowering order, and fill a lack of knowledge about producing, controlling, and influencing this valuable natural pigment.
Dynamic Remodeling of Individual Nucleosomes Across a Eukaryotic Genome in Response to Transcriptional Perturbation
(Public Library of Science, 2008-03-18) Shivaswamy, Sushma; Bhinge, Akshay; Zhao, Yongjun; Jones, Steven; Hirst, Martin; Iyer, Vishwanath R
The eukaryotic genome is packaged as chromatin with nucleosomes comprising its basic structural unit, but the detailed structure of chromatin and its dynamic remodeling in terms of individual nucleosome positions has not been completely defined experimentally for any genome. We used ultra-high–throughput sequencing to map the remodeling of individual nucleosomes throughout the yeast genome before and after a physiological perturbation that causes genome-wide transcriptional changes. Nearly 80% of the genome is covered by positioned nucleosomes occurring in a limited number of stereotypical patterns in relation to transcribed regions and transcription factor binding sites. Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes. Dynamic nucleosome remodeling tends to increase the accessibility of binding sites for transcription factors that mediate transcriptional changes. However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity. Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes.
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.
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.
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.
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.
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.
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.
The E2F1 transcription factor plays an important role in cell cycle progression, largely facilitated by its ability to transcriptionally induce genes involved in cell cycle regulation and DNA replication. E2F1 also has a well documented ability to promote apoptosis in vitro and in vivo, both as a potent inducer and as a mediator. The mechanism by which E2F1 ultimately induces apoptosis has remained elusive, however. Persistent dogma suggests that the p19ARF tumor suppressor, which plays an important role in p53 stabilization and is transcriptionally regulated by E2F1, is the critical mediator of E2F1’s apoptotic capacity. Counter to this hypothesis, data presented here establish that p19ARF is not required for E2F1 to induce apoptosis. Additionally, E2F1 is observed to induce the stabilization and phosphorylation of p53 in a p19ARF independent manner. v This observation led to the discovery that the DNA damage responsive Ataxiatelangiectasia mutated kinase (ATM) is required for E2F1 to induce both p53 phosphorylation and apoptosis. A similar requirement by E2F1 exists for the Nijmegen breakage syndrome protein (NBS1), a component of the Mre11-Rad50-NBS1 (MRN) DNA repair complex and part of the ATM signaling pathway. Despite the ATM signaling pathway being defined as DNA damage responsive, no evidence of E2F1 induction of DNA damage has been observed. Moreover, E2F1 is observed to induce p53 phosphorylation in the absence of both transcription/translation and damaged DNA. Previous work has shown that E2F1 is phosphorylated by ATM in response to DNA damage and that E2F1 associates with NBS1. E2F1 also physically associates with ATM in response to several DNA damaging agents and localizes to discrete foci in response to DNA damage in an ATM and NBS1-dependent manner. Finally, E2F1 deficiency results in reduced phosphorylation of ATM, NBS1, and p53 in response to γ-irradiation. These findings demonstrate that E2F1 associates with key DNA damage-responsive proteins and may play a specific role in the DNA damage response. Such data further suggest that E2F1 induced apoptosis may be both independent of its transcriptional capacity and an extension of a physiologic role in response to DNA damage.
Neuroadaptations in Human Chronic Alcoholics: Dysregulation of the NF-κB System
(Public Library of Science, 2007-09-26) Ökvist, Anna; Johansson, Sofia; Kuzmin, Alexander; Bazov, Igor; Merino-Martinez, Roxana; Ponomarev, Igor; Mayfield, R. Dayne; Harris, R. Adron; Sheedy, Donna; Garrick, Therese; Harper, Clive; Hurd, Yasmin L.; Terenius, Lars; Ekström, Tomas J.; Bakalkin, Georgy; Yakovleva, Tatjana
Background -- Alcohol dependence and associated cognitive impairments apparently result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. Here we investigated whether transcription factors of Nuclear Factor-kappaB (NF-κB) family, controlling neuronal plasticity and neurodegeneration, are involved in these adaptations in human chronic alcoholics. Methods and Findings -- Analysis of DNA-binding of NF-κB (p65/p50 heterodimer) and the p50 homodimer as well as NF-κB proteins and mRNAs was performed in postmortem human brain samples from 15 chronic alcoholics and 15 control subjects. The prefrontal cortex involved in alcohol dependence and cognition was analyzed and the motor cortex was studied for comparison. The p50 homodimer was identified as dominant κB binding factor in analyzed tissues. NF-κB and p50 homodimer DNA-binding was downregulated, levels of p65 (RELA) mRNA were attenuated, and the stoichiometry of p65/p50 proteins and respective mRNAs was altered in the prefrontal cortex of alcoholics. Comparison of a number of p50 homodimer/NF-κB target DNA sites, κB elements in 479 genes, down- or upregulated in alcoholics demonstrated that genes with κB elements were generally upregulated in alcoholics. No significant differences between alcoholics and controls were observed in the motor cortex. Conclusions -- We suggest that cycles of alcohol intoxication/withdrawal, which may initially activate NF-κB, when repeated over years downregulate RELA expression and NF-κB and p50 homodimer DNA-binding. Downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently resulted in derepression of κB regulated genes. Alterations in expression of p50 homodimer/NF-κB regulated genes may contribute to neuroplastic adaptation underlying alcoholism.
Structure-functional analyses of Bright, a B cell regulator of immunoglobulin heavy chain transcription
(2004) Kim, Dongkyoon; Tucker, Philip W.
Bright is a B cell specific trans-activator that regulates IgH gene transcription by binding promoter and enhancer-associated MARs within the IgH gene locus. Domains important for Bright function include the ARID, which mediates sequence-specific binding to DNA and REKLES, a highly conserved but less well understood region within Bright and its two paralogues, Bdp (Bright and dri-like protein) and Bright-like. This thesis further explores features of Bright with respect to the role of these domains in cellular localization, protein-protein interactions, and transcriptional activation. Unexpectedly for a transcription factor, Bright accumulates to significant levels within the cytoplasm as well as the nucleus. Bright appears to be actively exported in a CRM-1 dependent manner. The nuclear localization of Bright is mediated by residues in the N-terminus of the REKLES domain (REKLESa), whereas nuclear export is mediated by the C-terminal region of RECKLES (REKLESb) along with the C-terminal 19 amino acids. Bright accumulates in the nucleus as a result of the loss of its nuclear export activity. This nuclear accumulation also correlates with the cell cycle arrest at G2/M phase. The REKLESb domain also mediates self-association of Bright or heteromeric association of Bright and Bdp. Point mutations in the REKLESb domain abolish the DNA-binding and nuclear export activity of Bright. In addition, Bdp appears to interact with Bright and to facilitate the nuclear localization and retention Bright. DNA binding and trans-activation studies indicate that the promoter-associated MARs repress IgH transcription in non-B cells, and Bright alleviates this effect. Promoter-MAR mediated Bright trans-activation is antagonized by direct competition of MAR DNA binding by the ubiquitously expressed repressor complex NF-mNR. However, it was found that NF-mNR includes Bright in B cells but not in non-B cells. The binding activity of NF-mNR and Bright in B cells is reciprocally altered during the cell division cycle and by the B cell mitogen LPS. LPS treatment had no effect on Bright localization but increased the total amount of Bright in the nucleus and cytoplasm. The increased level of Bright appears to displace NF-mNR from the MARs and to facilitate IgH gene transcription.
Unstable Transcripts in Arabidopsis Allotetraploids Are Associated with Nonadditive Gene Expression in Response to Abiotic and Biotic Stresses
(Public Library of Science, 2011-08-29) Kim, Eun-Deok; Chen, Z. Jeffery
Genome-wide analysis has documented differential gene expression between closely related species in plants and animals and nonadditive gene expression in hybrids and allopolyploids compared to the parents. In Arabidopsis, 15–43% of genes are expressed differently between the related species, Arabidopsis thaliana and Arabidopsis arenosa, the majority of which are nonadditively expressed (differently from mid-parent value) in allotetraploids. Nonadditive gene expression can be caused by transcriptional regulation through chromatin modifications, but the role of posttranscriptional regulation in nonadditive gene expression is largely unknown. Here we reported genome-wide analysis of mRNA decay in resynthesized Arabidopsis allotetraploids. Among ~26,000 annotated genes, over 1% of gene transcripts showed rapid decay with an estimated half-life of less than 60 minutes, and they are called allotetraploid genes with unstable transcripts (AlloGUTs). Remarkably, 30% of alloGUTs matched the nonadditively expressed genes, and their expression levels were negatively correlated with the decay rate. Compared to all genes, these nonadditively expressed alloGUTs were overrepresented 2-6-fold in the Gene Ontology (GOSlim) classifications in response to abiotic and biotic stresses, signal transduction, and transcription. Interestingly, the AlloGUTs include transcription factor genes that are highly inducible under stress conditions and circadian clock regulators that regulate growth in A. thaliana. These data suggest a role of mRNA stability in homoeologous gene expression in Arabidopsis allopolyploids. The enrichment of nonadditively expressed genes in stress-related pathways were commonly observed in Arabidopsis and other allopolyploids such as wheat and cotton, which may suggest a role for stress-mediated growth vigor in hybrids and allopolyploids.