Progressive Deletions Research Articles

Glycoengineering of EphA4 Fc leads to a unique, long-acting and broad spectrum, Eph receptor therapeutic antagonist

Eph receptors have emerged as targets for therapy in both neoplastic and non-neoplastic disease, however, particularly in non-neoplastic diseases, redundancy of function limits the effectiveness of targeting individual Eph proteins. We have shown previously that a soluble fusion protein, where the EphA4 ectodomain was fused to IgG Fc (EphA4 Fc), was an effective therapy in acute injuries and demonstrated that EphA4 Fc was a broad spectrum Eph/ephrin antagonist. However, a very short in vivo half-life effectively limited its therapeutic development. We report a unique glycoengineering approach to enhance the half-life of EphA4 Fc. Progressive deletion of three demonstrated N-linked sites in EphA4 progressively increased in vivo half-life such that the triple mutant protein showed dramatically improved pharmacokinetic characteristics. Importantly, protein stability, affinity for ephrin ligands and antagonism of cell expressed EphA4 was fully preserved, enabling it to be developed as a broad spectrum Eph/ephrin antagonist for use in both acute and chronic diseases.

Abstract 5491: Identification of transcriptional controls responsible for differential gene expression of the proton-coupled folate transporter in human solid tumors

Abstract The majority of folate uptake into tissues and tumors involves facilitated carriers, the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). PCFT is highly expressed in a wide range of human tumor cell lines and primary tumor specimens from a variety of lineages, with modest expression in most normal tissues. PCFT transports optimally at pHs characterizing the tumor microenvironment. We discovered novel 6-substituted pyrrolo[2,3-d]pyrimidine folate analogs with substantial selectivity for PCFT over RFC, resulting in potent antitumor efficacy in vitro and in vivo. However, in some cases PCFT levels varied substantially. Studies were performed to characterize the basis for these dramatic differences in PCFT gene expression between HepG2 (elevated) and HT1080 (negligible) tumor cells. Low levels of PCFT gene expression in HT1080 cells were not likely due to methylation of CpG islands in the PCFT promoter since 5-aza-2’-deoxycytidine treatment did not restore PCFT gene expression. A PCFT promoter reporter construct -2005/+96 in pGL3 (transcription starts at +1) was generated and showed high levels of activity in HeLa cells by luciferase reporter assays. For HepG2 and HT1080 cells, wide variations in PCFT transcript levels by real-time RT-PCR were paralleled by reporter gene activities with the -2005/+96 construct, suggesting that these were primarily due to PCFT transcriptional regulation. Progressive deletion analysis of the -2005/+96 construct and reporter assays in HepG2 and HT1080 cells localized the minimal promoter region to between -50 and +96. Additional deletion constructs (-35/+96, -20/+96, -15/+96 and -10/+96) were generated and studied. Whereas the -35/+96, -20/+96, -15/+96 promoter constructs retained ~50% activity for HepG2 cells and ~10-15% activity for HT1080 cells relative to the -50/+96 construct, in both cases the -10/+96 construct was essentially inactive. Two critical cis elements were identified in the PCFT minimal promoter by deletion analysis located between positions -50 and -35, and -15 and -10. Bioinformatics analysis localized putative cis elements for KLF-15 (-50 to -35) and NRF1 (-15 to -10) binding. The functional importance of these elements was confirmed by mutations of core consensus sequences in the -50/+96 construct and reporter gene assays. Binding of NRF1 and KLF15 to the endogenous PCFT promoter in HepG2 and HT1080 cells was confirmed by ChIP assays which paralleled patterns of differential PCFT gene expression. Collectively, our results suggest that NRF1 and KLF15 contribute to dramatic variations in PCFT gene expression between tumor cells. Better understanding the key determinants of PCFT transcriptional control may lead to strategies for modulating PCFT levels in combination with PCFT-targeted 6-pyrrolo[2,3-d]pyrimidine folate analogs for treating solid tumors. Citation Format: Zhanjun Hou, Carrie O'Connor, Steve Orr, Larry H. Matherly. Identification of transcriptional controls responsible for differential gene expression of the proton-coupled folate transporter in human solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2017-5491

Transcription factor Sp1 is necessary and functional in regulating expression of oncogene ZNF703

Zinc finger protein 703 (ZNF703) is a putative oncogene in patients with the luminal B molecular subtype of breast cancer. Although the exact function of ZNF703 protein remains largely unknown, its expression and regulation have been implicated in several physiological and pathological processes. In the current study, for the first time, we identified and characterized the human ZNF703 gene promoter region. As a means of characterizing the transcription elements required for expression of ZNF703 protein at different stages, we cloned the promoter region of ZNF703 then created chimeric reporter plasmids for use in luciferase assays. A progressive deletion analysis of the ZNF703 gene’s 5′ and 3′ -flanking regions revealed that the core promoter is located in a 256-bp region ranging from nt-539 to nt-283. Next, we examined the effects of site-specific mutations and treatment with mithramycin A to identify the functional Sp1 binding site, which was found to be located in a 447 bp region that ranged from nt-509 to nt-76, displayed the characteristics of a CpG island, and overlapped with the promoter region. In conclusion, our data suggest that ZNF703 transcription is regulated by transcription factor Sp1. This finding should facilitate future studies of the mechanism which regulates expression of this important gene.

Effects of Flanking Disorder on the Behaviour of Ordered Domains

Both structured proteins and those containing intrinsic disorder have been the subject of much investigation; especially as disorder relates to protein-protein interactions. However, there has been little exploration into the influence of these elements on each other. Do IDPs regulate the stability and folding mechanisms of connected structured regions and vice versa do ordered domains affect the affinity of IDRs for their ligands e.g. by affecting the association kinetics? To try and answer these questions we use biophysical methods and progressive deletions to vary the length of disordered regions flanking ordered domains in two model systems of one and two globular domains, respectively.

Specific expression and promoter analysis of the albumin gene promoter of the duck (Anas platyrhynchos domesticus)

ABSTRACT1. Albumin (ALB) is a serum protein most highly expressed in liver and regarded as an effective indicator for liver pathologies. The objectives of this study were to determine the expression of duck ALB gene (duALB) in various non-hepatic tissues and identify the potential cis-regulatory elements in the promoter.2. A model was established to assess duALB promoter activity in different cell lines by construction of a duALB promoter-driven GFP (Green Fluorescent Protein)-expressing vector, which exhibited high expression activity in liver-derived cells and lower expression in other cells. Through the firefly luciferase reporter gene driven by a series of constructs carrying progressive deletions, the core transcriptional regulatory region within the duALB promoter was identified. Mutations in candidate-binding sites were made by site-directed mutagenesis.3. The core transcriptional regulatory region was located in the −190/−51 bp region. This region contains three potential transcription factor-binding sites, one each for hepatocyte nuclear factor (HNF-3β) (−158/−149), CCAAT/Enhancer-binding protein element (C/EBPα) (−119/−107) and nuclear factor-1 (HNF-1) (−67/−57). Site-directed mutagenesis of HNF-1 and C/EBPα-binding sites resulted in a significant reduction in duALB promoter activity. Two potential cis-regulatory elements (C/EBPα and HNF-1) were responsible for its transcriptional activity in liver-derived cells.4. These findings contribute to the further understanding of the fundamental mechanisms of ALB gene regulation and the use of tissue-specific gene promoters to regulate tissue-specific expression of exogenous genes in vivo.

Morpho-functional approaches to highlight skeletal muscle response to cell death inducers

Cell death has been long described, with continuously growing interest, in a variety of tissues and models. Its presence in muscle disorders stimulated us to study it in striated muscle tissue, in particular, in “ in vitro” differentiated myotubes. Apoptosis is a regulated mechanism of cell death which occurs in the absence of plasmalemma disruption, but with cell and organellar component swelling. It plays a crucial role in skeletal muscle pathology, in denervation and disuse [1]. Recently, “autophagy”, an intriguing phenomenon characterized by progressive deletion of cell components, could have a role in skeletal muscle death progression, also acting as a survival mechanism. Here, in vitro C2C12 skeletal muscle cells were exposed to etoposide, H2O2 or staurosporine and cell response has been investigated by means of morpho-functional approaches. Myotubes appeared more resistant than myoblasts to apoptotic induction. In particular, etoposide- or H2O2-treated myoblasts showed characteristic apoptotic features, visualized also in etoposide-treated myotubes characterized by a diffuse DNA cleavage presence. After H₂O₂ exposure, necrotic cells could be observed and myotubes exposed to staurosporine, evidenced late apoptotic features and secondary necrosis. The coexistence of normal and apoptotic nuclei within the same fiber has been demonstrated, in particular in the case of etoposide and staurosporine treatments. The deletion of a single nucleus can occur without the death of the entire myotube, evidencing that a multinucleated cell dies ‘more slowly’. After the majority of stimuli, autophagic vacuoles could be diffusely revealed in myotube cytoplasm. They could preserve muscle cell integrity counteracting chemical treatments, or could activate death pathways. It is the case of etoposide drug, which induced skeletal muscle apoptosis in the presence of an autophagic flux impairment. These findings reveal that apoptosis, necrosis and autophagy coexist in muscle biology and, ultrastructural analyses appear a useful approach for highlighting and describing these processes.

Expression of wnt4/5 during reproductive cycle of catfish and wnt5 promoter analysis.

Signaling molecules, Wnt4 and Wnt5, are essential for ovarian growth during developmental stages in mammals. Although these molecules were identified in several teleosts, their precise expression and role in reproductive processes have not yet been explored in any lower vertebrates. In view of this, using catfish, Clarias batrachus as an animal model, cloning and expression analysis of wnt4 and wnt5 were analyzed in different tissues, at various developmental stages, during ovarian reproductive cycle and after gonadotropin induction. These studies indicate a plausible influence of Wnts in ovarian development and recrudescence. Transcript and protein localization revealed their presence in peri-nucleolar, pre-vitellogenic, vitellogenic and follicular layer of post-vitellogenic oocytes. Synchronous expression of pax2 and wnt5 during the ovarian development and recrudescence of catfish led us to analyze the importance of putative binding element of Pax2 in the 5'-promoter motif of wnt5 Promoter activity of wnt5 was analyzed by luciferase assays after transfecting progressive deletion constructs in pGL3 basic vector into the mammalian cell lines (HEK 293 and CHO). The constructs having putative Pax2 motif showed high promoter activity compared with controls. Likewise, the constructs with site-directed mutagenesis showed increased activity after supplementing recombinant Pax2 indicating the prominence of this motif in wnt5 promoter, in vitro Electrophoretic gel mobility shift, supershift and chromatin immunoprecipitation assays confirmed the binding of Pax2 to its corresponding cis-acting element in the upstream of wnt5 This study is the first of its kind to report the critical transcriptional interaction of Pax2 on wnt5 vis-à-vis ovarian development in teleosts.

Sequencing of long stretches of repetitive DNA.

Repetitive DNA is widespread in eukaryotic genomes, in some cases making up more than 80% of the total. SSRs are a type of repetitive DNA formed by short motifs repeated in tandem arrays. In some species, SSRs may be organized into long stretches, usually associated with the constitutive heterochromatin. Variation in repeats can alter the expression of genes, and changes in the number of repeats have been linked to certain human diseases. Unfortunately, the molecular characterization of these repeats has been hampered by technical limitations related to cloning and sequencing. Indeed, most sequenced genomes contain gaps owing to repetitive DNA-related assembly difficulties. This paper reports an alternative method for sequencing of long stretches of repetitive DNA based on the combined use of 1) a linear vector to stabilize the cloning process, and 2) the use of exonuclease III for obtaining progressive deletions of SSR-rich fragments. This strategy allowed the sequencing of a fragment containing a stretch of 6.2 kb of continuous SSRs. To demonstrate that this procedure can sequence other kinds of repetitive DNA, it was used to examine a 4.5 kb fragment containing a cluster of 15 repeats of the 5S rRNA gene of barley.

Characterization of VvPAL-like promoter from grapevine using transgenic tobacco plants.

A 2000-bp 5'-flanking region of VvPAL-like was isolated from 'Summer Black' grapevine by PCR amplification, named pVvPAL-like. To gain a better understanding of the expression and regulatory mechanism of VvPAL-like, a chimeric expression unit consisting of the β-glucuronidase (GUS) reporter gene under the control of a 2000-bp fragment of the VvPAL-like promoter was transformed into tobacco via Agrobacterium tumefaciens. Histochemical staining showed that the full-length promoter directs efficient expression of the reporter gene in cotyledons and hypocotyls, stigma, style, anthers, pollen, ovary, trichomes, and vascular bundles of transgenic plants. A series of 5' progressive deletions of the promoter revealed the presence of a negative regulatory region (-424 to -292) in the VvPAL-like promoter. Exposure of the transgenic tobacco plants to various abiotic stresses demonstrated that the full-length construct could be induced by light, copper (Cu), abscisic acid (ABA), indole-3-acetic (IAA), methyl jasmonate (MeJA) (N-1-naphthylphthalamic acid), ethylene, and drought. Furthermore, the ethylene-responsive region was found to be located in the -1461/-930 fragment, while the element(s) for the MeJA-responsive expression may be present in the -424/-292 region in the VvPAL-like promoter. These findings will help us to better understand the molecular mechanisms by which VvPAL-like participates in biosynthesis of flavonoids and stress responses.

P65 down-regulates DEPTOR expression in response to LPS stimulation in hepatocytes

DEPTOR, a novel endogenous inhibitor of mTOR, plays an important role in regulating the inflammatory response in vascular endothelial cells (ECs) and in mouse skeletal muscle. However, the regulatory mechanism of DEPTOR transcription and its effects on liver inflammation are unknown presently. Here we reported the role of DEPTOR in regulating inflammatory response in mouse liver-derived Hepa1–6 cells and in a mouse model with LPS-induced hepatic inflammation. The results revealed that DEPTOR over-expression in Hepa1–6 liver cells increased the mRNA levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1). Contrasting results were observed in Hepa1–6 cells with DEPTOR interference. Treatment Hepa1–6 cells with rapamycin, a specific inhibitor of mTORC1, increased MCP-1 mRNA, but have no significant effect on IL-6 mRNA. DEPTOR expression was down-regulated in Hepa1–6 cells with the treatment of inflammatory stimuli LPS or the over-expression of p65/NF-κB, a key inflammatory transcription factor. NF-κB antagonist (PDTC) and inhibitor (IκBα) blocked the effect of LPS on DEPTOR expression. The study in vivo showed that DEPTOR mRNA and protein were significantly reduced in a mouse model with LPS-induced hepatic inflammation, which was accompanied by a concurrent activation of the mTOR signaling pathway. Further, the transcriptional regulation of DEPTOR was explored, which revealed that DEPTOR promoter activity was significantly down-regulated by NF-κB. The progressive deletions and mutations demonstrated that the NF-κB binding motif situated at −145/−127 region is an essential component required for the DEPTOR promoter activity. Chromatin immunoprecipitation (ChIP) assays determined that p65 can directly interact with the DEPTOR promoter DNA. Those results indicate DEPTOR regulates liver inflammation at least partially via mTORC1 pathway, and is down-regulated by LPS through p65.

Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly

The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

DiiA is a novel dimorphic cell wall protein of Streptococcus pneumoniae involved in invasive disease

Many outer multidomain proteins play fundamental virulent roles in an allele-dependent manner. We aimed to investigate the influence of the outer SP1992 protein, here renamed DiiA (Dimorphic invasion-involved A), in pneumococcal disease. The presence and type of diiA allele was screened by PCR in 560 clinical isolates. Isogenic mutants carrying progressive diiA deletions were constructed and checked in mouse models of infection. DiiA binding to human molecules was carried out by surface plasmon resonance. The diiA gene is exclusive of Streptococcus pneumoniae and included in the core genome. DiiA variants contain one or two imperfect repeats (R1 and R2), an unstructured region and a cell-wall anchor domain. Clonal complexes carrying both repeats were associated with invasive disease, while those carrying R2 preferentially caused non-invasive syndromes in patients with underlying risk factors. Mutants lacking both repeats were less efficient in nasopharyngeal colonization and dissemination from lungs. Moreover, the ΔdiiA defective strain suffered a severe impairment in bacterial proliferation in blood. Purified DiiA bound to collagen and lactoferrin with high affinity. DiiA is a distinctive pneumococcal virulence factor contributing to colonization and long-term invasion in this pathogen.

Transcriptional modulation of SLC26A3 (DRA) by sphingosine-1-phosphate.

SLC26A3 or Downregulated in adenoma (DRA) is the major Cl(-)/HCO3 (-) exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Alterations in DRA function and expression have been implicated in diarrheal diseases associated with inflammation or infection. Therefore, agents that upregulate DRA activity may serve as potential antidiarrheals. In this regard, sphingosine-1-phosphate (S1P), a member of the bioactive sphingolipid family, has been shown to modulate various cellular processes including improvement of intestinal barrier function. However, the role of S1P in modulating intestinal chloride absorption by regulating DRA is not known. Therefore, the present studies were designed to examine the direct effects of S1P on apical Cl(-)/HCO3 (-) exchange activity and DRA expression. S1P significantly increased Cl(-)/HCO3 (-) exchange activity and also significantly increased DRA mRNA and protein expression. Increased DRA mRNA by S1P was accompanied by enhanced DRA promoter activity, indicating involvement of transcriptional mechanisms. The specific S1P receptor subtype-2 (S1PR2) antagonist JTE-013 blocked the stimulatory effects of S1P on DRA promoter activity, indicating the involvement of S1PR2 S1P-mediated increase in DRA promoter activity involved PI3K/Akt pathway. Progressive deletions of the DRA promoter indicated that the putative S1P-responsive elements are present in the -790/-398 region of the DRA promoter. Furthermore, results obtained from electrophoretic mobility shift assay showed that S1P stimulated DRA promoter activity via increased binding of Ying-Yang1 (YY1) in the S1P-responsive region. In conclusion, transcriptional modulation of DRA expression and function in response to S1P through a PI3/Akt pathway represents a novel role of S1P as a potential proabsorptive agent.

Regulation and mechanism of mouse miR-130a/b in metabolism-related inflammation

Increasing evidence suggests that microRNAs are involved in regulating immune response and metabolism, which are among the most fundamental requirements for survival. Here we investigate the contribution and mechanism of microRNA-130a/b in controlling metabolism-related inflammation. Our findings indicate that miR-130a/b significantly inhibits TNFα and Sp1 expression by directly binding to their 3'-untranslated regions. Overexpressed miR-130a/b decreases the NF-κB mRNA and protein levels by shortening mRNA half-life. In mice primary hepatocytes, over-expressed miR-130a/b ameliorates the up-regulation of TNFα, Sp1, NF-κB and PPARγ translational levels elicited by LPS or FFAs treatment. Further, C/EBPα attenuates the promoter activity of miR-130a, but enhances that of miR-130b. The progressive deletions and mutations show that the C/EBPα binding motif situated at −1033/−1021bp or −130/−116bp region of miR-130a or b promoter respectively is an essential component required for their promoter activity. Chromatin immunoprecipitation (ChIP) assays reveal that C/EBPα can directly interact with miR-130a/b promoter DNA. Conclusively, these data suggest that miR-130a/b, regulated transcriptionally by C/EBPα, can control metabolism-related inflammatory process through inhibiting Sp1-TLR4-NF-κB/P65-TNFα pathway and regulating translational levels of PPARγ and other key genes involved in lipid metabolism.

KLF6 cooperates with NUR77 and SF1 to activate the human INSL3 promoter in mouse MA-10 leydig cells.

Insulin-like 3 (INSL3), a Leydig cell-specific hormone, is essential for testis descent during foetal life and bone metabolism in adults. Despite its essential roles in male reproductive and bone health, very little is known regarding its transcriptional regulation in Leydig cells. To date, few transcription factors have been shown to activate INSL3 promoter activity: the nuclear receptors AR, NUR77, COUP-TFII and SF1. To identify additional regulators, we have isolated and performed a detailed analysis of a 1.1 kb human INSL3 promoter fragment. Through 5' progressive deletions and site-directed mutagenesis, we have mapped a 10 bp element responsible for about 80% of INSL3 promoter activity in Leydig cells. This element is identical to the CPE element of the placental-specific glycoprotein-5 (PSG5) promoter that is recognized by the developmental regulator Krüppel-like factor 6 (KLF6). Using PCR and western blotting, we found that KLF6 is expressed in several Leydig and Sertoli cell lines. Furthermore, immunohistochemistry on adult mouse testis revealed the presence of KLF6 in the nuclei of both Leydig and Sertoli cells. KLF6 binds to the 10 bp KLF element at -108 bp and activates the -1.1 kb human, but not the mouse, INSL3 promoter. KLF6-mediated activation of the human INSL3 promoter required an intact KLF element as well as Leydig/Sertoli-enriched factors because KLF6 did not stimulate the human INSL3 promoter activity in CV-1 fibroblast cells. Consistent with this, we found that KLF6 transcriptionally cooperates with NUR77 and SF1. Collectively, our results identify KLF6 as a regulator of human INSL3 transcription.

The Transmembrane Domain is Sufficient to Direct Junctophilin-1 Localization at the Junctional SR

The sarcoplasmic reticulum (SR) of striated muscle cells is mainly dedicated to Ca2+ homeostasis and regulation of muscle contraction. The SR is organized in longitudinal and junctional SR (j-SR). In skeletal muscle, this latter domain together with the T-tubules form specific junctions called triads, where proteins regulating the excitation-contraction coupling mechanism assemble. Junctophilins (JPs) are directly involved in the formation and maintenance of triads. Basically, they are anchored to the SR via their C-terminal transmembrane domain (TMD), while their N-terminus contains eight MORN motifs, which associate with the phospholipids of the T-tubules. Nevertheless, how JPs are targeted to triads is not known. The roles of the N-terminal and the C-terminal regions of JP1 in this process were investigated. Expression in primary myotubes and/or muscle fibers of JP1 deletion mutants lacking the TMD resulted in protein distribution at both the surface sarcolemma and the T-tubules, confirming that MORN motifs are involved in JP1 interaction with the sarcolemma, but are not sufficient to restrict its localization at the T-tubules.On the other hand, progressive deletion of MORN motifs I-VI, MORN motifs I-VIII or of the entire region 1-635, did no affect JP1 localization at triads, indicating that the presence of the TMD is sufficient for JP1 localization at the j-SR.These results indicate that the localization of JP1 at the triads appears to be mainly directed by mechanisms acting at the level of the SR, rather than at T-tubules. FRAP analysis performed on a GFP-TMD fusion protein expressed in myotubes indicated that this protein has a high mobility, suggesting the absence of strong protein-protein interactions occurring at the j-SR. Further work is needed to better understand the molecular mechanisms driving TMD-mediated JP1 localization at triads.

Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene.

Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) gene plays a crucial role in maintaining genomic stability, tumorigenesis and myogenesis. However, little is known about the regulatory elements governing the transcription of porcine ROCK1 gene. In the current study, the transcription start site (TSS) was identified by 5’-RACE, and was found to differ from the predicted one. The region in ROCK1 promoter which is critical for promoter activity was investigated via progressive deletions. Site-directed mutagenesis indicated that the region from −604 to −554 bp contains responsive elements for Sp1. Subsequent experiments showed that ROCK1 promoter activity is enhanced by Sp1 in a dose-dependent manner, whereas treatment with specific siRNA repressed ROCK1 promoter activity. Electrophoretic mobility shift assay (EMSA), DNA pull down and chromatin immunoprecipitation (ChIP) assays revealed Sp1 can bind to this region. qRT-PCR and Western blotting research followed by overexpression or inhibition of Sp1 indicate that Sp1 can affect endogenous ROCK1 expression at both mRNA and protein levels. Overexpression of Sp1 can promote the expression of myogenic differentiation 1(MyoD), myogenin (MyoG), myosin heavy chain (MyHC). Taken together, we conclude that Sp1 positively regulates ROCK1 transcription by directly binding to the ROCK1 promoter region (from −604 to −532 bp) and may affect the process of myogenesis.

Characterization of the porcine peptidylarginine deiminase type VI gene (PADI6) promoter: Sp1 regulates basal transcription of the porcine PADI6

It is a general consensus that oocyte quality is the key to embryo survival in pig reproduction. Thus, study on regulation of the ovary-associated gene is of great significance in pig breeding. Peptidylarginine deiminases (PADs) are a family of enzymes which catalyze the conversion of arginine to citrulline in proteins. The peptidylarginine deiminases type VI gene (PADI6) is mainly expressed in the ovary, and plays an important role in oocyte growth, fertilization and early embryo development. However, until now, little is known about its transcriptional regulation mechanism. Here, we firstly isolated and characterized the 5′-flanking region of porcine PADI6 gene. We determined the transcription start site using 5′-rapid amplification of cDNA ends (RACE) analysis, and identified the minimal promoter (−85/+68) that drove the basal expression of PADI6 by constructing various progressive deletions. Mutational analysis and electrophoretic mobility shift assays demonstrated Sp1 bound to the −56/−47 region of the PADI6 promoter. Furthermore, overexpression of Sp1 significantly increased the promoter activity and promoted PADI6 gene expression, and accordingly, inhibition of Sp1 expression with specific siRNA significantly reduced the promoter activity and suppressed the PADI6 expression. In addition, inhibition of Sp1 binding by Mithramycin A treatment reduced the transcriptional activity of PADI6 in a dose-dependent manner. Taken together, these data indicate that Sp1 is essential for the transcriptional regulation of PADI6.

Influence of Isoforms and Carboxyl-Terminal Truncations on the Capacity of Apolipoprotein E To Associate with and Activate Phospholipid Transfer Protein.

Phospholipid transfer protein (PLTP), a main protein in lipid and lipoprotein metabolism, exists in high-activity (HA-PLTP) and low-activity (LA-PLTP) forms in human plasma. Proper phospholipid transfer activity of PLTP is modulated by interactions with various apolipoproteins (apo) including apoE. The domains of apoE involved in interactions with PLTP are not known. Here we analyzed the capacity of recombinant apoE isoforms and apoE4 mutants with progressive carboxyl-terminal deletions to bind to and activate HA-PLTP and LA-PLTP. Our analyses demonstrated that lipid-free apoE isoforms bind to both HA-PLTP and LA-PLTP, resulting in phospholipid transfer activation, with apoE3 inducing the highest PLTP activation. The isoform-specific differences in apoE/PLTP binding and PLTP activation were abolished following apoE lipidation. Lipid-free apoE4[Δ(260-299)], apoE4[Δ(230-299)], apoE4[Δ(203-299)], and apoE4[Δ(186-299)] activated HA-PLTP by 120-160% compared to full-length apoE4. Lipid-free apoE4[Δ(186-299)] also activated LA-PLTP by 85% compared to full-length apoE4. All lipidated truncated apoE4 forms displayed a similar effect on HA-PLTP and LA-PLTP activity as full-length apoE4. Strikingly, lipid-free or lipidated full-length apoE4 and apoE4[Δ(186-299)] demonstrated similar binding capacity to LA-PLTP and HA-PLTP. Biophysical studies showed that the carboxyl-terminal truncations of apoE4 resulted in small changes of the structural or thermodynamic properties of lipidated apoE4, that were much less pronounced compared to changes observed previously for lipid-free apoE4. Overall, our findings show an isoform-dependent binding to and activation of PLTP by lipid-free apoE. Furthermore, the domain of apoE4 required for PLTP activation resides within its amino-terminal 1-185 region. The apoE/PLTP interactions can be modulated by the conformation and lipidation state of apoE.

Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans.

In animals, microRNAs frequently form families with related sequences. The functional relevance of miRNA families and the relative contribution of family members to target repression have remained, however, largely unexplored. Here, we used the Caenorhabditis elegans miR-58 miRNA family, composed primarily of the four highly abundant members miR-58.1, miR-80, miR-81, and miR-82, as a model to investigate the redundancy of miRNA family members and their impact on target expression in an in vivo setting. We found that miR-58 family members repress largely overlapping sets of targets in a predominantly additive fashion. Progressive deletions of miR-58 family members lead to cumulative up-regulation of target protein and RNA levels. Phenotypic defects could only be observed in the family quadruple mutant, which also showed the strongest change in target protein levels. Interestingly, although the seed sequences of miR-80 and miR-58.1 differ in a single nucleotide, predicted canonical miR-80 targets were efficiently up-regulated in the mir-58.1 single mutant, indicating functional redundancy of distinct members of this miRNA family. At the aggregate level, target binding leads mainly to mRNA degradation, although we also observed some degree of translational inhibition, particularly in the single miR-58 family mutants. These results provide a framework for understanding how miRNA family members interact to regulate target mRNAs.