Lysyl oxidase regulates MMTV promoter: indirect evidence of histone H1 involvement

2011 ◽  
Vol 89 (6) ◽  
pp. 522-532 ◽  
Author(s):  
Roberta Oleggini ◽  
Armando Di Donato
Keyword(s):  
Lysyl Oxidase ◽  
Specific Binding ◽  
Indirect Evidence ◽  
Histone H1 ◽  
Gene Promoters ◽  
Collagen Iii ◽  
Lysine Residues ◽  
Tumor Virus ◽  
Mmtv Promoter

Lysyl oxidase (LOX) is the enzyme that facilitates the cross-linking of collagen and elastin, although other functions for this enzyme have been indicated. Of these other functions, we describe herein the ability of LOX to regulate several gene promoters, like collagen III, elastin, and cyclin D1. We have previously demonstrated a specific binding between LOX and histone H1, in vitro. Therefore, we investigated whether LOX would affect the mouse mammary tumor virus (MMTV) promoter and its glucocorticoid regulation, which depends on the phophorylation status of histone H1. Our results show that the over-expression of recombinant human LOX was able to trigger MMTV activity, both in the presence and absence of glucocorticoids. Moreover, we demonstrated that histone H1 from cells expressing recombinant LOX contained isodesmosine and desmosine, indicating specific lysyl-oxidase-dependent lysine modifications. Finally, we were able to co-immunoprecipitate the exogenous LOX and histone H1 from the LOX transfected cells. The data are compatible with a decreased positive charge of histone H1, owing to deamination by LOX of its lysine residues. This event would favor H1 detachment from the target DNA, and consequent opening of the MMTV promoter structure to the activating transcription factors. The presented data, therefore, suggest a possible histone-H1-dependent mechanism for the modulation of MMTV promoter by LOX.

scholarly journals Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas.

1987 ◽  
Vol 105 (3) ◽  
pp. 1463-1469 ◽  
Author(s):  
C Fornieri ◽  
M Baccarani-Contri ◽  
D Quaglino ◽  
I Pasquali-Ronchetti
Keyword(s):  
Hydrophobic Interactions ◽  
Isonicotinic Acid ◽  
Lysyl Oxidase ◽  
Acid Hydrazide ◽  
Elastic Fibers ◽  
Amino Groups ◽  
Lysine Residues ◽  
Oxidase Inhibition

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

Cloning of rat and mouse aquaporin-2 gene promoters and identification of a negative cis-regulatory element

1997 ◽  
Vol 273 (2) ◽  
pp. F264-F273 ◽  
Author(s):  
T. Rai ◽  
S. Uchida ◽  
F. Marumo ◽  
S. Sasaki
Keyword(s):  
Specific Binding ◽  
Regulatory Element ◽  
Gene Promoter ◽  
Nuclear Extract ◽  
Gene Promoters ◽  
Specific Expression ◽  
Cis Element ◽  
Aquaporin 2 ◽  
Gene Assay

The promoters of rat and mouse aquaporin-2 (AQP-2) genes were cloned and compared with that of human genes. Nucleotide identity up to -593 bp was 62%, and consensus sequences such as TATA box and adenosine 3',5'-cyclic monophosphate responsive element were conserved. Deoxyribonuclease I footprint assay revealed a footprinted region at -210 to -184 bp in rat AQP-2 gene promoter produced by nuclear extract from nonexpressing (liver) tissue. The sequence of this region included a GATA motif but otherwise showed no homology with any other previously known cis-elements. Electromobility shift assay and ultraviolet cross-linking analysis confirmed that specific binding proteins to this element were present in kidney, spleen, and liver and that these proteins were distinct from GATA factors. Both deletion and mutation of this cis-element abolished the protein DNA binding and increased promoter activity in in vitro reporter gene assay using rat cultured hepatocyte Ac2F cells, suggesting the negative regulatory role of this cis-element. These results indicate that tissue-specific expression of AQP-2 gene may in part be regulated by this novel negative acting cis-element.

scholarly journals Androgen responsiveness of the pituitary gonadotrope cell line LbetaT2

2001 ◽  
Vol 170 (3) ◽  
pp. 601-607 ◽  
Author(s):  
MA Lawson ◽  
D Li ◽  
CA Glidewell-Kenney ◽  
FJ Lopez
Keyword(s):  
Cell Line ◽  
Rank Order ◽  
Pcr Analysis ◽  
Androgen Action ◽  
Steroid Dependent ◽  
Tumor Virus ◽  
Mmtv Promoter ◽  
Mouse Pituitary

Androgens have a profound effect on the hypothalamic-pituitary axis by reducing the synthesis and release of the pituitary gonadotropin LH. The effect on LH is partly a consequence of a direct, steroid-dependent action on pituitary function. Although androgen action has been well studied in vivo, in vitro cell models of androgen action on pituitary gonadotropes have been scarce. Recently, an LH-expressing cell line, LbetaT2, was generated by tumorigenesis targeted to the LH-producing cells of the mouse pituitary. The purpose of these studies was to determine the presence of androgen receptor (AR) and establish its function in this cell line. RT-PCR analysis indicated that the LbetaT2 cell line expresses AR mRNA. Transient transfection assays, using the mouse mammary tumor virus (MMTV) promoter, showed that a functional AR is also present. Testosterone (TEST), dihydrotestosterone (DHT), 7alpha-methyl-19-nortestosterone (MENT), and fluoxymesterone (FLUOXY) increased reporter gene activity in the rank order of potencies MENT>DHT> TEST>FLUOXY. Additionally, activation of MMTV promoter activity by DHT in LbetaT2 cells was diminished by the AR antagonists casodex and 2-hydroxy-flutamide, indicating that the effects of DHT are mediated through AR. In summary, these studies showed that the LbetaT2 cell line is a useful model for the evaluation and molecular characterization of androgen action in pituitary gonadotropes.

scholarly journals Histone H1 Phosphorylation by Cdk2 Selectively Modulates Mouse Mammary Tumor Virus Transcription through Chromatin Remodeling

2001 ◽  
Vol 21 (16) ◽  
pp. 5417-5425 ◽  
Author(s):  
Rabindra N. Bhattacharjee ◽  
Geoffrey C. Banks ◽  
Kevin W. Trotter ◽  
Huay-Leng Lee ◽  
Trevor K. Archer
Keyword(s):  
Chromatin Remodeling ◽  
Mammary Tumor ◽  
Mouse Mammary Tumor Virus ◽  
Histone H1 ◽  
Necessary Condition ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Mmtv Promoter

ABSTRACT Transcriptional activation of the mouse mammary tumor virus (MMTV) promoter by ligand-bound glucocorticoid receptor (GR) is transient. Previously, we demonstrated that prolonged hormone exposure results in displacement of the transcription factor nuclear factor 1 (NF1) and the basal transcription complex from the promoter, the dephosphorylation of histone H1, and the establishment of a repressive chromatin structure. We have explored the mechanistic link between histone H1 dephosphorylation and silencing of the MMTV promoter by describing the putative kinase responsible for H1 phosphorylation. Both in vitro kinase assays and in vivo protein expression studies suggest that in hormone-treated cells the ability of cdk2 to phosphorylate histone H1 is decreased and the cdk2 inhibitory p21 protein level is increased. To address the role of cdk2 and histone H1 dephosphorylation in the silencing of the MMTV promoter, we used potent cdk2 inhibitors, Roscovitine and CVT-313, to generate an MMTV promoter which is associated predominantly with the dephosphorylated form of histone H1. Both Roscovitine and CVT-313 block phosphorylation of histone H1 and, under these conditions, the GR is unable to remodel chromatin, recruit transcription factors to the promoter, or stimulate MMTV mRNA accumulation. These results suggest a model where cdk2-directed histone H1 phosphorylation is a necessary condition to permit GR-mediated chromatin remodeling and activation of the MMTV promoter in vivo.

scholarly journals Unusual DNA-binding properties of the Arabidopsis thaliana WRKY50 transcription factor at target gene promoters

2020 ◽  
Author(s):  
Konstantin Kanofsky ◽  
Jendrik Rusche ◽  
Lea Eilert ◽  
Fabian Machens ◽  
Reinhard Hehl
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Target Gene ◽  
Target Genes ◽  
Specific Binding ◽  
Gene Promoters ◽  
Molecular Pattern ◽  
High Flexibility ◽  
Site Recognition

Abstract Key message WRKY50 from A. thaliana requires WT-boxes at target gene promoters for activation and binding. Abstract Based on the genome-wide prediction of WRKY50 target genes and the similarity of a WRKY50 binding site to WT-boxes in microbe-associated molecular pattern (MAMP)-responsive cis-regulatory modules (CRM), four WT-box containing CRMs from the promoter region of three WRKY50 target genes were investigated for their interaction with WRKY50. These target genes are DJ1E, WRKY30 and ATBBE4. Two of the four CRMs, one from DJ1E and one from WRKY30, were able to activate reporter gene expression in the presence of WRKY50. Activation requires the WT-boxes GGACTTTT, GGACTTTG from DJ1E and GGACTTTC from WRKY30. WRKY50 does not activate a second CRM from WRKY30 and the CRM from ATBBE4, both containing the WT-box TGACTTTT. In vitro gel-shift assays demonstrate WT-box-specific binding of the WRKY50 DNA-binding domain to all four CRMs. This work shows a high flexibility of WRKY50 binding site recognition beyond the classic W-box TTGACC/T.

scholarly journals In Vitro Oxidative Crosslinking of Recombinant Barnacle Cyprid Cement Gland Proteins

2021 ◽  
Author(s):  
Cleverley RM ◽  
Webb DS ◽  
Middlemiss S ◽  
Duke PW ◽  
Clare AS ◽  
...  
Keyword(s):  
Lysyl Oxidase ◽  
Inhibitory Effect ◽  
Cement Gland ◽  
E Coli ◽  
Fouling Control ◽  
Lysine Residues ◽  
Specific Proteins ◽  
Secretory Tissue ◽  
Control Technologies

AbstractBarnacle adhesion is a focus for fouling-control technologies as well as the development of bioinspired adhesives, although the mechanisms remain very poorly understood. The barnacle cypris larva is responsible for surface colonisation. Cyprids release cement from paired glands that contain proteins, carbohydrates and lipids, although further compositional details are scant. Several genes coding for cement gland-specific proteins were identified, but only one of these showed database homology. This was a lysyl oxidase-like protein (lcp_LOX). LOX-like enzymes have been previously identified in the proteome of adult barnacle cement secretory tissue. We attempted to produce recombinant LOX in E. coli, in order to identify its role in cyprid cement polymerisation. We also produced two other cement gland proteins (lcp3_36k_3B8 and lcp2_57k_2F5). lcp2_57k_2F5 contained 56 lysine residues and constituted a plausible substrate for LOX. While significant quantities of soluble lcp3_36k_3B8 and lcp2_57k_2F5 were produced in E. coli, production of stably soluble lcp_LOX failed. A commercially sourced human LOX catalysed the crosslinking of lcp2_57k_2F5 into putative dimers and trimers, and this reaction was inhibited by lcp3_36k_3B8. Inhibition of the lcp_LOX:lcp2_57k_2F5 reaction by lcp3_36k_3B8 appeared to be substrate specific, with no inhibitory effect on the oxidation of cadaverine by LOX. The results demonstrate a possible curing mechanism for barnacle cyprid cement and, thus, provide a basis for a more complete understanding of larval adhesion for targeted control of marine biofouling and adhesives for niche applications.

scholarly journals Cell-free system for assembly of transcriptionally repressed chromatin from Drosophila embryos

1992 ◽  
Vol 12 (5) ◽  
pp. 2241-2249
Author(s):  
P B Becker ◽  
C Wu
Keyword(s):  
Dna Synthesis ◽  
Histone H1 ◽  
Chromatin Assembly ◽  
Assembly System ◽  
Gene Promoters ◽  
Nucleosome Assembly ◽  
Free System ◽  
Cell Free System ◽  
Drosophila Embryos

We describe a cell-free system, derived from preblastoderm Drosophila embryos, for the efficient assembly of cloned DNA into chromatin. The chromatin assembly system utilizes endogenous core histones and assembly factors and yields long arrays of regularly spaced nucleosomes with a repeat length of 180 bp. The assembly system is also capable of complementary-strand DNA synthesis accompanied by rapid nucleosome formation when the starting template is single-stranded circular DNA. Chromatin assembled with the preblastoderm embryo extract is naturally deficient in histone H1, but exogenous H1 can be incorporated during nucleosome assembly in vitro. Regular spacing of nucleosomes with or without histone H1 is sufficient to maximally repress transcription from hsp70 and fushi tarazu gene promoters. The Drosophila assembly system should be particularly useful for in vitro studies of chromatin assembly during DNA synthesis and for elucidating the action of transcription factors in the context of native chromatin.

scholarly journals Ligand-Specific Dynamics of the Progesterone Receptor in Living Cells and during Chromatin Remodeling In Vitro

2005 ◽  
Vol 25 (6) ◽  
pp. 2406-2418 ◽  
Author(s):  
Geetha V. Rayasam ◽  
Cem Elbi ◽  
Dawn A. Walker ◽  
Ronald Wolford ◽  
Terace M. Fletcher ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Chromatin Remodeling ◽  
Reproductive Function ◽  
Living Cells ◽  
Nuclear Dynamics ◽  
Tumor Virus ◽  
Mmtv Promoter ◽  
Natural Target

ABSTRACT Progesterone receptor (PR), a member of the nuclear receptor superfamily, is a key regulator of several processes in reproductive function. We have studied the dynamics of the interaction of PR with a natural target promoter in living cells through the use of fluorescence recovery after photobleaching (FRAP) analysis and also have characterized the dynamics of the interaction of PR with the mouse mammary tumor virus (MMTV) promoter reconstituted into chromatin in vitro. In photobleaching experiments, PR in the presence of the agonist R5020 exhibits rapid exchange with the MMTV promoter in living cells. Two PR antagonists, RU486 and ZK98299, have opposite effects on receptor dynamics in vivo. In the presence of RU486, PR binds to the promoter and is exchanged more slowly than the agonist-activated receptor. In contrast, PR bound to ZK98299 is not localized to the promoter and exhibits higher mobility in the nucleoplasm than the agonist-bound receptor. Significantly, PR bound to R5020 or RU486 can recruit the SWI/SNF chromatin remodeling complex to the promoter, but PR activated with ZK98299 cannot. Furthermore, we found ligand-specific active displacement of PR from the MMTV promoter during chromatin remodeling in vitro and conclude that the interaction of PR with chromatin is highly dynamic both in vivo and in vitro. We propose that factor displacement during chromatin remodeling is an important component of receptor mobility and that ligand-specific interactions with remodeling complexes can strongly influence receptor nuclear dynamics and rates of exchange with chromatin in living cells.

scholarly journals Cell-free system for assembly of transcriptionally repressed chromatin from Drosophila embryos.

1992 ◽  
Vol 12 (5) ◽  
pp. 2241-2249 ◽  
Author(s):  
P B Becker ◽  
C Wu
Keyword(s):  
Dna Synthesis ◽  
Histone H1 ◽  
Chromatin Assembly ◽  
Assembly System ◽  
Gene Promoters ◽  
Nucleosome Assembly ◽  
Free System ◽  
Cell Free System ◽  
Drosophila Embryos

We describe a cell-free system, derived from preblastoderm Drosophila embryos, for the efficient assembly of cloned DNA into chromatin. The chromatin assembly system utilizes endogenous core histones and assembly factors and yields long arrays of regularly spaced nucleosomes with a repeat length of 180 bp. The assembly system is also capable of complementary-strand DNA synthesis accompanied by rapid nucleosome formation when the starting template is single-stranded circular DNA. Chromatin assembled with the preblastoderm embryo extract is naturally deficient in histone H1, but exogenous H1 can be incorporated during nucleosome assembly in vitro. Regular spacing of nucleosomes with or without histone H1 is sufficient to maximally repress transcription from hsp70 and fushi tarazu gene promoters. The Drosophila assembly system should be particularly useful for in vitro studies of chromatin assembly during DNA synthesis and for elucidating the action of transcription factors in the context of native chromatin.

scholarly journals Rap1 in Candida albicans: an Unusual Structural Organization and a Critical Function in Suppressing Telomere Recombination

2009 ◽  
Vol 30 (5) ◽  
pp. 1254-1268 ◽  
Author(s):  
Eun Young Yu ◽  
Wei-Feng Yen ◽  
Olga Steinberg-Neifach ◽  
Neal F. Lue
Keyword(s):  
Candida Albicans ◽  
Telomere Length ◽  
Specific Binding ◽  
Repeat Unit ◽  
Gene Promoters ◽  
Sequence Specificity ◽  
Null Mutant ◽  
Ribosomal Protein Genes

ABSTRACT Rap1 (repressor activator protein 1) is a conserved multifunctional protein initially identified as a transcriptional regulator of ribosomal protein genes in Saccharomyces cerevisiae but subsequently shown to play diverse functions at multiple chromosomal loci, including telomeres. The function of Rap1 appears to be evolutionarily plastic, especially in the budding yeast lineages. We report here our biochemical and molecular genetic characterizations of Candida albicans Rap1, which exhibits an unusual, miniaturized domain organization in comparison to the S. cerevisiae homologue. We show that in contrast to S. cerevisiae, C. albicans RAP1 is not essential for cell viability but is critical for maintaining normal telomere length and structure. The rap1 null mutant exhibits drastic telomere-length dysregulation and accumulates high levels of telomere circles, which can be largely attributed to aberrant recombination activities at telomeres. Analysis of combination mutants indicates that Rap1 and other telomere proteins mediate overlapping but nonredundant roles in telomere protection. Consistent with the telomere phenotypes of the mutant, C. albicans Rap1 is localized to telomeres in vivo and recognizes the unusual telomere repeat unit with high affinity and sequence specificity in vitro. The DNA-binding Myb domain of C. albicans Rap1 is sufficient to suppress most of the telomere aberrations observed in the null mutant. Notably, we were unable to detect specific binding of C. albicans Rap1 to gene promoters in vivo or in vitro, suggesting that its functions are more circumscribed in this organism. Our findings provide insights on the evolution and mechanistic plasticity of a widely conserved and functionally critical telomere component.

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