Combining chromatin immunoprecipitation and DNA footprinting: a novel method to analyze protein–DNA interactions in vivo

A variety of methods are available to analyze protein–DNA interactions in vivo. Two of the most prominent of these methods are chromatin immunoprecipitation (ChIP) and in vivo footprinting. Both of these procedures have specific limitations. For example, the ChIP assay fails to document where exactly a protein binds in vivo. The precipitation of a specific segment of DNA with antibodies directed against DNA-binding proteins does not necessarily indicate that the protein directly interacts with a sequence in the precipitate but could rather reflect protein–protein interactions. Furthermore, the results of in vivo footprinting studies are inconclusive if a DNA sequence is analyzed that is bound by a specific protein in only a certain fraction of cells. Finally, in vivo footprinting does not indicate which protein is bound at a specific site. We have developed a new procedure that combines the ChIP assay and DMS footprinting techniques. Using this method we show here that antibodies specific for USF1 and NF-E2 precipitate the murine β-globin promoter in MEL cells. DMS footprinting analysis of the DNA precipitated with NF-E2 antibodies revealed a protection over a partial NF-E2-binding site in the β-globin downstream promoter region. We believe that this novel method will generally benefit investigators interested in analyzing protein–DNA interactions in vivo.     

Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti

A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus. Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999     

Homologous constitutive expression of Xyn III in Trichoderma reesei QM9414 and its characterization

Xylanase III (Xyn III), a specific endoxylanase that belongs to family 10 of the glycoside hydrolases, was overexpressed in Trichoderma reesei QM9414 using a constitutive strong promoter of the gene encoding pyruvate decarboxylase (pdc). The maximum recombinant xylanase activity achieved was 817.2?±?65.2 U/mL in the transformant fermentation liquid. The productivities of Xyn III accounted for approximately 53 % of the total protein secreted by the recombinant. The enzyme was optimally active at 60 °C and pH 6. The recombinant Xyn III was stable at pH 5–8. This is the first report on the homologous expression of xyn3 in T. reesei QM9414. The properties of Xyn III make it promising in a variety of industrial use.     

Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation

Constitutive and induced protein SUMOylation is involved in the regulation of a variety of cellular processes, such as regulation of gene expression and protein transport, and proceeds mainly in the nucleus of the cell. So far, several hundred SUMOylation targets have been identified, but presumably they represent only a part of the total of proteins which are regulated by SUMOylation. Here, we used the Ubc9 fusion-dependent SUMOylation system (UFDS) to screen for constitutive and induced SUMOylation of 46 randomly chosen proteins with proven or potential nuclear localization. Fourteen new UFDS-substrate proteins were identified of which eight could be demonstrated to be SUMOylated in a UFDS-independent manner in vivo. Of these, three were constitutively SUMOylated (FOS, CRSP9 and CDC37) while the remaining five substrates (CSNK2B, TAF10, HSF2BP, PSMC3 and DRG1) showed a stimulation-dependent SUMOylation induced by the MAP3 kinase MEKK1. Hence, UFDS is appropriate for the identification and characterization of constitutive and, more importantly, induced protein SUMOylation in vivo.     

Characterization of Paenibacillus curdlanolyticus Intracellular Xylanase Xyn10B Encoded by the xyn10B Gene

The Paenibacillus curdlanolyticus xyn10B gene encoding a family-10 xylanase was cloned and expressed in Escherichia coli, and the recombinant enzyme rXyn10B was characterized. Immunological analysis suggested that Xyn10B is an intracellular enzyme. rXyn10B hydrolyzed birch-wood xylan and xylooligosaccharides to produce mainly xylobiose, suggesting that it is an endoxylanase. Its properties were significantly different from those of some homologous enzymes.     

Increased Production of Xylanase by Expression of a Truncated Version of the xyn11A Gene from Nonomuraea flexuosa in Trichoderma reesei

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3′ to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter⁻¹ of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.     

Two New Xylanases with Different Substrate Specificities from the Human Gut Bacterium Bacteroides intestinalis DSM 17393

Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X₅) and xylohexaose (X₆) to a mixture of xylobiose (X₂) and xylotriose (X₃), while Rex8A hydrolyzed X₃ through X₆ to a mixture of xylose (X₁) and X₂. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit β-xylosidase activity and would be able to further hydrolyze X₂ to X₁. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.     

CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival

Homeostatic chemokines, such as CXCL12, can affect neuronal activity by the regulation of inhibitory and excitatory neurotransmission, but the mechanisms involved are still undefined. Our previous studies have shown that CXCL12 protects cortical neurons from excitotoxicity by promoting the function of the gene-repressor protein Rb, which is involved in the recruitment of chromatin modifiers (such as histone deacetylases (HDACs)) to gene promoters. In neurons, Rb controls activity-dependent genes essential to neuronal plasticity and survival, such as the N-methyl--aspartic acid (NMDA) receptor''s subunit NR2B, the expression of which in the tetrameric ion channel largely affects calcium signaling by glutamate. In this study, we report that CXCL12 differentially modulates intracellular responses after stimulation of synaptic and extrasynaptic NMDA receptors, by a specific regulation of the NR2B gene that involves HDACs. Our results show that CXCL12 selectively inhibits NR2B expression in vitro and in vivo altering NMDA-induced calcium responses associated with neuronal death, while promoting prosurvival pathways that depend on stimulation of synaptic receptors. Along with previous studies, these findings underline the role of CXCL12/CXCR4 in the regulation of crucial components of glutamatergic transmission. These novel effects of CXCL12 may be involved in the physiological function of the chemokine in both developing and mature brains.     

Sequence analysis,cloning and over-expression of an endoxylanase from the alkaliphilic <Emphasis Type="BoldItalic">Bacillus halodurans</Emphasis>

The BhMIR32 xyn11A gene, encoding an extracellular endoxylanase of potential interest in bio-bleaching applications, was amplified from Bacillus halodurans MIR32 genomic DNA. The protein encoded is an endo-1,4-β-xylanase belonging to family 11 of glycosyl hydrolases. Its nucleotide sequence was analysed and the mature peptide was subcloned into pET22b(+) expression vector. The enzyme was over-expressed in a high density Escherichia coli culture as a soluble and active protein, and purified in a single step by immobilised metal ion affinity chromatography with a specific activity of 3073 IU mg⁻¹.     

Drosophila architectural protein CTCF is not essential for fly survival and is able to function independently of CP190

CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates and Drosophila, CTCF is involved in the regulation of homeotic genes. Previously, it was found that null mutations in the dCTCF gene died as pharate adults, which failed to eclose from their pupal case, or shortly after hatching of adults. Here, we obtained several new null dCTCF mutations and found that the complete inactivation of dCTCF appears is limited mainly to phenotypic manifestations of the Abd-B gene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the null dCTCF mutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation in Drosophila. We also mapped the 715–735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not important for the functional activity of dCTCF in vivo.     

Doxycyline regulation in a single retroviral vector by an autoregulatory loop facilitates controlled gene expression in liver cells

The tetracycline system has limitations in liver cells, such as toxic effects and low controllability. We generated different retroviral vectors for controlled gene expression in liver cells, in which the regulatory elements were arranged in different patterns. Only the organization of the tetracycline system in an autoregulatory loop in the sense orientation results in high retroviral titres and in tight regulation of gene expression in highly differentiated hepatoma cells. Because of the toxicity of the transactivator tTA, it was impossible to establish doxycycline-dependent stable HepG2 cell lines. To avoid sequelching-related toxicity in liver cells, we replaced tTA with new non-toxic transactivators. By using tTA2, tTA3 and tTA4, we observed tight doxycycline-dependent gene expression in 23, 49 and 45% of the isolated clones. The tTA4 vector was used to transduce hepatocytes of mice in vivo. Tight doxycycline-controllable gene regulation was also observed in the liver of mice, confirming our hypothesis that retroviral vectors with autoregulatory loops of the tetracycline system facilitate inducible gene expression in the liver in vivo. Our new retroviral vector system allows rapid isolation of controllable clones in a very high yield and should make the tetracycline system more applicable to liver-derived cells and in liver gene therapy in vivo.