Upstream Promoter Elements Research Articles

Human ACE2 expression, a major tropism determinant for SARS-CoV-2, is regulated by upstream and intragenic elements.

Angiotensin-converting enzyme 2 (ACE2), part of the renin-angiotensin system (RAS), serves as an entry point for SARS-CoV-2, leading to viral proliferation in permissive cell types. Using mouse lines in which the Ace2 locus has been humanized by syntenic replacement, we show that regulation of basal and interferon induced ACE2 expression, relative expression levels of different ACE2 transcripts, and sexual dimorphism in ACE2 expression are unique to each species, differ between tissues, and are determined by both intragenic and upstream promoter elements. Our results indicate that the higher levels of expression of ACE2 observed in the lungs of mice relative to humans may reflect the fact that the mouse promoter drives expression of ACE2 in populous airway club cells while the human promoter drives expression in alveolar type 2 (AT2) cells. In contrast to transgenic mice in which human ACE2 is expressed in ciliated cells under the control of the human FOXJ1 promoter, mice expressing ACE2 in club cells under the control of the endogenous Ace2 promoter show a robust immune response after infection with SARS-CoV-2, leading to rapid clearance of the virus. This supports a model in which differential expression of ACE2 determines which cell types in the lung are infected, and this in turn modulates the host response and outcome of COVID-19.

Structural basis of the complete poxvirus transcription initiation process.

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

Genome-wide characterization and expression profiling of the Phospholipase C (PLC) gene family in three orchids of economic importance

BackgroundPhospholipases hydrolyze glycerophospholipids and generate diverse lipid-derived molecules with secondary messenger activity. Out of these, phospholipase C (PLC) specifically cleaves the phospholipids at ester linkages and yields diacylglycerol (DAG) and phosphorylated head groups. PLCs are classified further as phosphatidylinositol-specific PLCs (PI-PLCs) and non-specific PLCs with biased specificity for phosphatidylcholine (NPC/PC-PLC). ResultsIn the present report, we identified and characterized PLC genes in the genomes of three orchids, Phalaenopsis equestris (seven PePLCs), Dendrobium catenatum (eight DcPLCs), and Apostasia shenzhenica (seven AsPLCs). Multiple sequence alignment analysis confirmed the presence of conserved X and Y catalytic domains, calcium/lipid-binding domain (C2 domain) at the C terminal region, and EF-hand at the N-terminal region in PI-PLC proteins and esterase domain in PC-PLC. Systematic phylogenetic analysis established the relationship of the PLC protein sequences and clustered them into two groups (PI-PLC and PC-PLC) along with those of Arabidopsis thaliana and Oryza sativa. Gene architecture studies showed the presence of nine exons in all PI-PLC genes while the number varied from one to five in PC-PLCs. RNA-seq-based spatio-temporal expression profile for PLC genes was generated, which showed that PePC-PLC1, PePC-PLC2A, DcPC-PLC1A, DcPC-PLC1B, DcPC-PLC2, DcPC-PLC1B, and AsPC-PLC1 had significant expression in all reproductive and vegetative tissues. The expression profile is matched to their upstream cis-regulatory promoter elements, which indicates that PLC genes have a role in various growth and development processes and during stress responses. ConclusionsThe present study unwrapped the opportunity for functional characterization of selected PLC genes in planta for plant improvement.

Activation by TyrR in Escherichia coli K-12 by Interaction between TyrR and the α-Subunit of RNA Polymerase.

A novel selection was developed for mutants of the C-terminal domain of RpoA (α-CTD) altered in activation by the TyrR regulatory protein of Escherichia coli K-12. This allowed the identification of an aspartate to asparagine substitution at residue 250 (DN250) as an activation-defective (Act-) mutation. Amino acid residues known to be close to D250 were altered by in vitro mutagenesis, and the substitutions DR250, RE310, and RD310 were all shown to be defective in activation. None of these mutations caused defects in regulation of the upstream promoter (UP) element. The rpoA mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. The TyrR mutations EK139 and RG119 caused partial suppression of rpoA DN250, and TyrR RC119, RL119, RP119, RA77, and SG100 caused partial suppression of rpoA RE310. Additional activation-defective rpoA mutants (DT250, RS310, and EG288) were also isolated, using the chromosomal rpoA DN250 strain. Several new Act-tyrR mutants were isolated in an rpoA+ strain, adding positions R77, D97, K101, D118, R119, R121, and E141 to known residues S95 and D103 and defining the activation patch on the amino-terminal domain (NTD) of TyrR. These results support a model for activation of TyrR-regulated genes where the activation patch on the TyrR NTD interacts with the TyrR-specific patch on the α-CTD of RNA polymerase. Given known structures, both these sites appear to be surface exposed and suggest a model for activation by TyrR. They also help resolve confusing results in the literature that implicated residues within the 261 and 265 determinants as activator contact sites. IMPORTANCE Regulation of transcription by RNA polymerases is fundamental for adaptation to a changing environment and for cellular differentiation, across all kingdoms of life. The gene tyrR in Escherichia coli is a particularly useful model because it is involved in both activation and repression of a large number of operons by a range of mechanisms, and it interacts with all three aromatic amino acids and probably other effectors. Furthermore, TyrR has homologues in many other genera, regulating many different genes, utilizing different effector molecules, and in some cases affecting virulence and important plant interactions.

Implication of the Type III Effector RipS1 in the Cool-Virulence of Ralstonia solanacearum Strain UW551.

Members of the Ralstonia solanacearum species complex cause a variety of wilting diseases across a wide range of hosts by colonizing and blocking xylem vessels. Of great concern are race 3 biovar 2 strains of R. solanacearum capable of causing brown rot of potato at cool temperatures, which are select agents in the United States. To gain a better understanding of cool-virulence mechanisms, we generated libraries of transposon mutants in the cool-virulent R. solanacearum strain UW551 and screened 10,000 mutants using our seedling assay for significantly reduced virulence at 20°C. We found several mutants that exhibited reduced virulence at 28 and 20°C and also mutants that were only affected at the cooler temperature. One mutant of the latter chosen for further study had the transposon inserted in an intergenic region between a type III secretion system effector gene ripS1 and a major facilitator superfamily (MFS) protein gene. Gene expression analysis showed that expression of ripS1 was altered by the transposon insertion, but not the MFS protein gene. An independent mutant with this insertion upstream of ripS1 was generated and used to confirm virulence and gene expression phenotypes. The effector, RipS1, has unknown function and is part of a family of effectors belonging to the largest known type III effectors. The functional connection between RipS1 and cool-virulence of R. solanacearum UW551 suggests that RipS1 (and/or its upstream promoter element) may serve as a potential target for development of cool-virulence-specific diagnostic tools to differentiate the highly regulated cool-virulent strains from non-cool-virulent strains of R. solanacearum. Our results provide important information for continued work toward a better understanding of cool-virulence of R. solanacearum and development of proper control strategies to combat this important plant pathogen.

The RNA Polymerase α Subunit Recognizes the DNA Shape of the Upstream Promoter Element.

We demonstrate here that the α subunit C-terminal domain of Escherichia coli RNA polymerase (αCTD) recognizes the upstream promoter (UP) DNA element via its characteristic minor groove shape and electrostatic potential. In two compositionally distinct crystallized assemblies, a pair of αCTD subunits bind in tandem to the UP element consensus A-tract that is 6 bp in length (A6-tract), each with their arginine 265 guanidinium group inserted into the minor groove. The A6-tract minor groove is significantly narrowed in these crystal structures, as well as in computationally predicted structures of free and bound DNA duplexes derived by Monte Carlo and molecular dynamics simulations, respectively. The negative electrostatic potential of free A6-tract DNA is substantially enhanced compared to that of generic DNA. Shortening the A-tract by 1 bp is shown to "knock out" binding of the second αCTD through widening of the minor groove. Furthermore, in computationally derived structures with arginine 265 mutated to alanine in either αCTD, either with or without the "knockout" DNA mutation, contact with the DNA is perturbed, highlighting the importance of arginine 265 in achieving αCTD-DNA binding. These results demonstrate that the importance of the DNA shape in sequence-dependent recognition of DNA by RNA polymerase is comparable to that of certain transcription factors.

NER-factor DDB2 regulates HIF1α and hypoxia-response genes in HNSCC.

Cancers in the oral/head & neck region (HNSCC) are aggressive due to high incidence of recurrence and distant metastasis. One prominent feature of aggressive HNSCC is the presence of severely hypoxic regions in tumors and activation of hypoxia-inducible factors (HIFs). In this study, we report that the XPE gene product DDB2 (damaged DNA binding protein 2), a nucleotide excision repair protein, is upregulated by hypoxia. Moreover, DDB2 inhibits HIF1α in HNSCC cells. It inhibits HIF1α in both normoxia and hypoxia by reducing mRNA expression. Knockdown of DDB2 enhances the expression of angiogenic markers and promotes tumor growth in a xenograft model. We show that DDB2 binds to an upstream promoter element in the HIF1Α gene and promotes histone H3K9 trimethylation around the binding site by recruiting Suv39h1. Also, we provide evidence that DDB2 has a significant suppressive effect on expression of the endogenous markers of hypoxia that are also prognostic indicators in HNSCC. Together, these results describe a new mechanism of hypoxia regulation that opposes expression of HIF1Α mRNA and the hypoxia-response genes.

Heat Shock Protein Genes Undergo Dynamic Alteration in Their Three-Dimensional Structure and Genome Organization in Response to Thermal Stress.

Three-dimensional (3D) chromatin organization is important for proper gene regulation, yet how the genome is remodeled in response to stress is largely unknown. Here, we use a highly sensitive version of chromosome conformation capture in combination with fluorescence microscopy to investigate Heat Shock Protein (HSP) gene conformation and 3D nuclear organization in budding yeast. In response to acute thermal stress, HSP genes undergo intense intragenic folding interactions that go well beyond 5'-3' gene looping previously described for RNA polymerase II genes. These interactions include looping between upstream activation sequence (UAS) and promoter elements, promoter and terminator regions, and regulatory and coding regions (gene "crumpling"). They are also dynamic, being prominent within 60 s, peaking within 2.5 min, and attenuating within 30 min, and correlate with HSP gene transcriptional activity. With similarly striking kinetics, activated HSP genes, both chromosomally linked and unlinked, coalesce into discrete intranuclear foci. Constitutively transcribed genes also loop and crumple yet fail to coalesce. Notably, a missense mutation in transcription factor TFIIB suppresses gene looping, yet neither crumpling nor HSP gene coalescence is affected. An inactivating promoter mutation, in contrast, obviates all three. Our results provide evidence for widespread, transcription-associated gene crumpling and demonstrate the de novo assembly and disassembly of HSP gene foci.

Biosynthesis of brain cytoplasmic 200 RNA

Brain cytoplasmic 200 RNA (BC200 RNA), a neuron-specific non-coding RNA, is also highly expressed in a number of tumors of non-neuronal origin. However, the biosynthesis of BC200 RNA remains poorly understood. In this study, we show that the efficient transcription of BC200 RNA requires both internal and upstream promoter elements in cancer cells. The transcription complex seems to interact with a broad range of sequences within the upstream 100-bp region. The cellular levels and half-lives of BC200 RNA were found to differ across various cancer cell types, but there was no significant correlation between these parameters. Exogenously expressed BC200 RNA had a shorter half-life than that observed for the endogenous version in cancer cells, suggesting that BC200 RNA might be protected by some limiting factor(s) in cancer cells. Transient transfection experiments showed that the transcriptional activity of the exogenous BC200 RNA promoter element varied depending on the cancer cell type. However, the promoter activities together with the half-life data could not explain the differences in the levels of BC200 RNA among different cell types, suggesting that there is another level of transcriptional regulation beyond that detected by our transient transfection experiments.

Gal3 Binds Gal80 Tighter than Gal1 Indicating Adaptive Protein Changes Following Duplication.

Derived from the yeast whole-genome duplication, Saccharomyces cerevisiae GAL1 and GAL3 encode the catabolic enzyme galactokinase (Gal1) and its transcriptional coinducer (Gal3), whereas the ancestral, preduplicated GAL1 gene performed both functions. Previous studies indicated that divergence was primarily driven by changes in upstream promoter elements, and changes in GAL3's coding region are assumed to be the result of drift. We show that replacement of GAL3's open-reading-frame with GAL1's results in an extended lag phase upon switching to growth on galactose with up to 2.5-fold differences in the initial cell masses. Accordingly, the binding affinity of Gal3 to Gal80 was found to be greater than 10-folds higher than that of Gal1, with both a higher association rate (ka) and lower dissociation (kd) rate. Thus, while changes in the noncoding, regulatory regions were the initial driving force for GAL3's subfunctionalization as a coinducer, adaptive changes in the protein sequence seem to have followed.

Abstract 3064: MicroRNA-21 enhances the effect of ionizing radiation via alteration of the DNA damage response

Abstract Micro-RNA 21 (miR-21) is a well-known onco-miR found to be up-regulated in over 18 major cancer types and is linked to radiation and chemotherapy resistance. It is hypothesized that dysregulation of miR-21 may contribute to treatment resistance through multiple DNA damage response pathways by enhancing DNA repair and increasing DNA damage tolerance. Given its involvement in multiple resistance pathways, miR-21 appears to be an attractive target for overcoming resistance with radiation therapy. In vitro, we demonstrated that knockdown of miR-21 revealed an increase in tumor cell death via clonogenic cell survival curves following exposure to ionizing radiation in multiple cancer cell lines including triple negative breast cancer cells, GBM, and lung cancer. A miR-21 knockout (KO) mouse model was then generated based on deletion of a miR-21 upstream promoter element. To determine if mir-21 KO would lead to radiation sensitivity, miR-21 deficient mice were exposed to 6.5Gy total body irradiation (TBI) and compared to control wild type (WT) mice. miR-21 KO mice died at an average of 12 days after TBI vs. over 100 days survival in wild-type mice. Tissue analysis revealed an increase in double strand breaks measured by elevated γ-H2AX in miR-21 deficient mice. An associated decrease in DNA repair genes such as MSH2 and STAG2 was also seen. Therefore, we hypothesize deletion or silencing of miR-21 results in a radiation sensitive phenotype due to a decreased DNA repair capacity, particularly in the repair of double-strand breaks. In silico analysis predicted miR-21 targets involved in the DNA repair response including rad21 and stag2, which are both members of the cohesin complex. The cohesion complex is a ring-shaped protein complex involved in homologous repair and the DNA damage-response. When exposed to ionizing radiation, rad21 and stag2 expression appears inversely correlated with miR-21 levels in vivo and in vitro. Similarly, in the setting of miR-21 knockdown, rad21 and stag2 expression are increased, suggesting dysregulation in the function of the cohesin complex may contribute to genomic instability and a DNA damage-permissive phenotype. In the current study, we propose a novel mechanism for increasing radiation sensitivity through miRNA-mediated pathways controlling cellular DNA-repair. Future studies investigating miR-21 expression in patients may identify subgroups of patients who are at risk for resistance to standard cytotoxic therapies and may open avenues for targeted therapies involving mir-21 mediated DNA repair pathways. Citation Format: Tu Dan, Ajay Palagani, Tiziana DeAngelis, Sunny Han, Lance Liotta, Richard Pestell, Nicole Simone. MicroRNA-21 enhances the effect of ionizing radiation via alteration of the DNA damage response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3064. doi:10.1158/1538-7445.AM2015-3064

The rrnA promoter as a tool for the improved expression of heterologous genes in cyanobacteria

The regulatory sequence of ribosomal RNA A (rrnA) operon from Synechococcus PCC7942 was characterized using green fluorescent protein gene (gfp) as a reporter. The PR promoter (nt. −83 to +2) including upstream promoter element and P1 promoter of rrnA exhibited GFP fluorescence intensity about 30-fold higher than full length sequence (nt. −147 to +79). The effects of PR promoter arranged in tandem with consensus-σ70 promoter (PS) of Escherichia coli on the expression of gfp and opd gene encoding organophosphorus hydrolase (OPH) in Synechococcus were investigated. The PS-PR tandem promoter was superior to all of the other promoters; its GFP fluorescence intensity was a 1.8-fold increase when compared to PR-PR tandem promoter, a 2.5-fold, 9.5-fold and a 15-fold increase compared to PR, PS and promoter of tRNApro, respectively. The GFP from PS-PR tandem promoter accounted for about 12% of its total extracted proteins. OPH activity of Synechococcus harboring opd gene under the control of PS-PR tandem promoter was 738±128units/OD730. We demonstrated that the tandem promoters remarkably enhanced the GFP and OPH production which were detected on SDS-PAGE stained with Coomassie blue. The promoter system in this study could be generally applied to production of valuable organic products from cyanobacteria.

Transcriptional directionality of the human insulin-degrading enzyme promoter

Unidirectional promoters dominate among mammalian genomes. However, the mechanism through which the transcriptional directionality of promoters is accomplished remains to be clarified. Insulin-degrading enzyme (IDE) is a ubiquitously expressed zinc metalloprotease, whose promoter contains a CpG island. We previously showed that the basal promoter region of mouse IDE has bidirectional transcriptional activity, but an upstream promoter element blocks its antisense transcription. Therefore, we wonder whether the human IDE promoter contains an analogous element. Similarly, the basal promoter region of human IDE (-102 ~ +173 and -196 ~ +173 relative to the transcription start site) showed bidirectional transcriptional activity. However, the region from -348 to +173 could only be transcribed from the normal orientation, implying that an upstream promoter element between -348 and -196 blocks the antisense transcription of the human IDE promoter. Through promoter deletion and mutagenesis analysis, we mapped this element precisely and found that the upstream promoter element locates between -318 and -304. Furthermore, the transcription-blocking elements in the mouse and human IDE promoters inhibited the transcription of the SV40 promoter when put downstream of it. In conclusion, we identify an upstream promoter element which blocks the antisense transcription of the human IDE promoter. Our studies are helpful to clarify the transcriptional directionality of promoters.

Mitogen- and Stress-Activated Protein Kinases 1 and 2 Are Required for Maximal Trefoil Factor 1 Induction

Mitogen- and stress-activated protein kinases 1 and 2 (MSK1 and MSK2), activated downstream of the ERK- and p38-mitogen-activated protein kinase pathways are involved in cell survival, proliferation and differentiation. Following mitogenic or stress stimuli, they mediate the nucleosomal response, which includes phosphorylation of histone H3 at serine 10 (H3S10ph) coupled with transcriptional activation of immediate-early genes. While MSK1 and MSK2 are closely related, their relative roles may vary with cellular context and/or stimuli. However, our knowledge of MSK2 recruitment to immediate-early genes is limited, as research has primarily focused on MSK1. Here, we demonstrate that both MSK1 and MSK2, regulate the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced expression of the breast cancer marker gene, trefoil factor 1 (TFF1), by phosphorylating H3S10 at its 5′ regulatory regions. The MSK-mediated phosphorylation of H3S10 promotes the recruitment of 14-3-3 isoforms and BRG1, the ATPase subunit of the BAF/PBAF remodeling complex, to the enhancer and upstream promoter elements of TFF1. The recruited chromatin remodeling activity leads to the RNA polymerase II carboxy-terminal domain phosphorylation at the TFF1 promoter, initiating TFF1 expression in MCF-7 breast cancer cells. Moreover, we show that MSK1 or MSK2 is recruited to TFF1 regulatory regions, but as components of different multiprotein complexes.

An upstream promoter element blocks the reverse transcription of the mouse insulin-degrading enzyme gene

Despite the prevalence of bidirectional promoters among the mammalian genomes, the majority of promoters are unidirectional. The mechanism through which unidirectional promoters are prevented from reverse transcription remains to be clarified. Here we investigate the transcriptional directionality of the mouse insulin-degrading enzyme (IDE) promoter, which contains a CpG island and has dispersed transcription initiation sites. Although IDE is unidirectionally transcribed according to its genomic context, the basic promoter region of mouse IDE has bidirectional transcriptional properties. The region between −219 and +133 of mouse IDE relative to its first transcription initiation site has bidirectional transcriptional activities, but the region between −350 and +133 can only be transcribed from the normal direction, implying that an upstream promoter element locating between −350 and −219 blocks the reverse transcription of mouse IDE. We further mapped this upstream promoter element to the region between −243 and −287. Promoter mutation analysis showed that the upstream promoter element contains two functional sub-regions. In conclusion, we identified an upstream promoter element which blocks the reverse transcription of mouse IDE. Our studies are suggestive for the transcriptional mechanism of bidirectional promoters in mammalian genomes.

Mice lacking Sμ tandem repeats maintain RNA polymerase patterns but exhibit histone modification pattern shifts linked to class switch site locations

Antibody switching involves class switch recombination (CSR) events between switch (S) regions located upstream of heavy chain constant (C) genes. Mechanisms targeting CSR to S-regions are not clear. Deletion of Sμ tandem repeat (SμTR) sequences causes CSR to shift into downstream regions that do not undergo CSR in WT B-cells, including the Cμ-region. We now find that, in SμTR−/− B cells, Sμ chromatin histone modification patterns also shift downstream relative to WT and coincide with SμTR−/− CSR locations. Our results suggest that histone H3 acetylation and methylation are involved in accessibility of switch regions and that these modifications are not dependent on the underlying sequence, but may be controlled by the location of upstream promoter or regulatory elements. Our studies also show RNA polymerase II (RNAPII) loading increases in the Eμ/Iμ region in stimulated B cells; these increases are independent of SμTR sequences. Longer Sμ deletions have been reported to eliminate increases in RNAPII density, therefore we suggest that sequences between Iμ and Sμ (possibly the Iμ splicing region as well as G-tracts that are involved in stable RNA:DNA complex formation during transcription) might control the RNAPII density increases.

The coenzyme A disulphide reductase of Borrelia burgdorferi is important for rapid growth throughout the enzootic cycle and essential for infection of the mammalian host

In a microarray analysis of the RpoS regulon in mammalian host-adapted Borrelia burgdorferi, bb0728 (cdr) was found to be dually transcribed by the sigma factors σ(70) and RpoS. The cdr gene encodes a coenzyme A disulphide reductase (CoADR) that reduces CoA-disulphides to CoA in an NADH-dependent manner. Based on the abundance of CoA in B. burgdorferi and the biochemistry of the enzyme, CoADR has been proposed to play a role in the spirochaete's response to reactive oxygen species. To better understand the physiologic function(s) of BbCoADR, we generated a B. burgdorferi mutant in which the cdr gene was disrupted. RT-PCR and 5'-RACE analysis revealed that cdr and bb0729 are co-transcribed from a single transcriptional start site upstream of the bb0729 coding sequence; a shuttle vector containing the bb0729-cdr operon and upstream promoter element was used to complement the cdr mutant. Although the mutant was no more sensitive to hydrogen peroxide than its parent, it did exhibit increased sensitivity to high concentrations of t-butyl-hydroperoxide, an oxidizing compound that damages spirochetal membranes. Characterization of the mutant during standard (15% oxygen, 6% CO(2)) and anaerobic (< 1% O(2) , 9-13% CO(2)) cultivation at 37°C revealed a growth defect under both conditions that was particularly striking during anaerobiosis. The mutant was avirulent by needle inoculation and showed decreased survival in feeding nymphs, but displayed no survival defect in unfed flat nymphs. Based on these results, we propose that BbCoADR is necessary to maintain optimal redox ratios for CoA/CoA-disulphide and NAD(+) /NADH during periods of rapid replication throughout the enzootic cycle, to support thiol-disulphide homeostasis, and to indirectly protect the spirochaete against peroxide-mediated membrane damage; one or more of these functions are essential for infection of the mammalian host by B. burgdorferi.

Specific Expression of a Novel Nodulin GmN479 Gene in the Infected Cells of Soybean (Glycine max) Nodules

Abstract A novel nodulin gene, GmN479 genomic clone composing of 3 630 nucleotides was isolated from mature soybean nodules using GmN479 cDNA as a probe by subtractive hybridization procedure. GmN479 encodes 170 amino acids with 2.09 kb nucleotides promoter region, and contains two important upstream promoter elements, one is a conserved “s-acting sequence motif 5′-AAAGAT-3′ controlling nodulin gene expression, and the other is typical CAAT boxes. GmN479 gene has a single zmc-fmger C 2H 2 domain YS CAF CQRGFSNAQALLGG HMNI H and a conserved motif, QALGGHMN m the zinc-finger with a short leucine repeat in the LDLELRLGL motif closed to C-terminal. These two conserved motifs share respectively higher identity with those in the floral regulator SUPERMAN gene, indicating that GmN479 may function as a transcriptional regulator, and is a likely candidate for playing a role in nodule-morphogenesis. Blotting data showed that GmN479 is a single copy presenting in the genome of soybean nodule, and its expression profile is similar to that of Lb-a, but it is different from that of ENOD2. GUS staining showed that GmN479 promoter just functions in the infected cells of nodules, indicating that the GmN479 is one of the truly symbiotically induced host genes, and belongs to a late nodulin gene. The expression pattern of GmN479 gene seems to imply that it may be closely related to the development of the nodule. In a sense, it may be a useful marker for identifying the development of the infected cell system in the nodules of soybean.

Selective Association of Peroxiredoxin 1 with Genomic DNA and COX-2 Upstream Promoter Elements in Estrogen Receptor Negative Breast Cancer Cells

In a search for proteins differentially cross-linked to DNA by cisplatin or formaldehyde in normal breast epithelial and breast cancer cell lines, we identified peroxiredoxin 1 (PRDX1) as a protein preferentially cross-linked to DNA in estrogen receptor negative (ER-) MDA-MB-231 but not in estrogen receptor positive (ER+) MCF7 breast cancer cells. Indirect immunofluorescence microscopic analyses showed that PRDX1 was located in the cytoplasm and nucleus of normal and breast cancer cells, with nuclear PRDX1 associated with promyelocytic leukemia protein bodies. We demonstrated that PRDX1 association with the transcription factor nuclear factor-kappaB (NF-kappaB) in MDA-MB-231 but not in MCF7 cells contributed to PRDX1-selective recruitment to MDA-MB-231 genomic DNA. Furthermore, PRDX1 was associated with the cyclooxygenase (COX)-2 upstream promoter region at sites occupied by NF-kappaB in ER- but not in ER+ breast cancer cells. PRDX1 knockdown attenuated COX-2 expression by reducing NF-kappaB occupancy at its upstream promoter element in MDA-MB-231 but not in MCF7 cells. A phosphorylated form of PRDX1 was only present in ER- breast cancer cells. Because PRDX1 phosphorylation is known to inhibit its peroxidase activity and to promote PRDX1 oligomerization, we propose that PRDX1 acts as a chaperone to enhance the transactivation potential of NF-kappaB in ER- breast cancer cells.

Estrogen regulated expression of the p21Waf1/Cip1 gene in estrogen receptor positive human breast cancer cells

The cyclin-dependent kinase inhibitor protein p21(Waf1/Cip1) is a potent tumor suppressor. Here, we demonstrate that estradiol regulates the p21(Waf1/Cip1) gene. Estradiol induces p21(Waf1/Cip1) mRNA expression within 30-60 min independent of new protein synthesis in the estrogen receptor alpha (ER alpha) positive human breast cancer cell line MCF-7. Similar to other estradiol responsive promoters, the p21(Waf1/Cip1) upstream promoter region has several estrogen response element (ERE) half-sites nestled in AP-1 binding sites, which are positioned upstream to Sp1 binding sites. Using the chromatin immunoprecipitation (ChIP) assay, we show that estradiol stimulation resulted in the recruitment of transcription factors ER alpha, Sp1, and Sp3 to the p21(Waf1/Cip1) upstream promoter element. The Sp1 inhibitor mithramycin A abrogated Sp1, and to a lesser extent Sp3 binding, and markedly reduced the estradiol stimulated p21(Waf1/Cip1) gene expression. However, ER alpha binding was not affected in the mithramycin A and estradiol treated cells. On closer examination of the half-site ERE/AP-1 sites upstream to the Sp1 sites in a separate ChIP experiment, we found a pronounced association of ER alpha upon estradiol treatment compared to almost negligible binding of Sp1 or Sp3. Together these studies provide evidence that ER alpha is recruited to the half-site ERE/AP-1 sites in the p21(Waf1/Cip1) upstream promoter element. Although Sp1/Sp3 is not involved in the recruitment of ER alpha to the promoter, Sp1 is necessary for estrogen-induced p21(Waf1/Cip1) promoter activity.