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Hervé Vaucheret - One of the best experts on this subject based on the ideXlab platform.

  • second site mutagenesis of a hypomorphic argonaute1 allele identifies superkiller3 as an endogenous suppressor of Transgene posttranscriptional gene silencing
    Plant Physiology, 2015
    Co-Authors: Agnes Yu, Baptiste Saudemont, Nathalie Bouteiller, Jean Sébastien Parent, Jean-benoit Morel, Emilie Elviramatelot, Gersende Lepère, Taline Elmayan, Hervé Vaucheret
    Abstract:

    Second-site mutagenesis was performed on the argonaute1-33 (ago1-33) hypomorphic mutant, which exhibits reduced sense Transgene posttranscriptional gene silencing (S-PTGS). Mutations in FIERY1, a positive regulator of the cytoplasmic 5′-to-3′ EXORIBONUCLEASE4 (XRN4), and in SUPERKILLER3 (SKI3), a member of the SKI complex that threads RNAs directly to the 3′-to-5′ exoribonuclease of the cytoplasmic exosome, compensated AGO1 partial deficiency and restored S-PTGS with 100% efficiency. Moreover, xrn4 and ski3 single mutations provoked the entry of nonsilenced Transgenes into S-PTGS and enhanced S-PTGS on partially silenced Transgenes, indicating that cytoplasmic 5′-to-3′ and 3′-to-5′ RNA degradation generally counteract S-PTGS, likely by reducing the amount of Transgene aberrant RNAs that are used by the S-PTGS pathway to build up small interfering RNAs that guide Transgene RNA cleavage by AGO1. Constructs generating improperly terminated Transgene messenger RNAs (mRNAs) were not more sensitive to ski3 or xrn4 than regular constructs, suggesting that improperly terminated Transgene mRNAs not only are degraded from both the 3′ end but also from the 5′ end, likely after decapping. The facts that impairment of either 5′-to-3′ or 3′-to-5′ RNA degradation is sufficient to provoke the entry of Transgene RNA into the S-PTGS pathway, whereas simultaneous impairment of both pathways is necessary to provoke the entry of endogenous mRNA into the S-PTGS pathway, suggest poor RNA quality upon the transcription of Transgenes integrated at random genomic locations.

  • mutations in the arabidopsis h3k4me2 3 demethylase jmj14 suppress posttranscriptional gene silencing by decreasing Transgene transcription
    The Plant Cell, 2012
    Co-Authors: Ivan Le Masson, Nathalie Bouteiller, Vincent Jauvion, Maud Rivard, Taline Elmayan, Hervé Vaucheret
    Abstract:

    Posttranscriptional gene silencing (PTGS) mediated by sense Transgenes (S-PTGS) results in RNA degradation and DNA methylation of the transcribed region. Through a forward genetic screen, a mutant defective in the Histone3 Lysine4 di/trimethyl (H3K4me2/3) demethylase Jumonji-C (JmjC) domain-containing protein14 (JMJ14) was identified. This mutant reactivates various Transgenes silenced by S-PTGS and shows reduced Histone3 Lysine9 Lysine14 acetylation (H3K9K14Ac) levels, reduced polymerase II occupancy, reduced Transgene transcription, and increased DNA methylation in the promoter region, consistent with the hypothesis that high levels of transcription are required to trigger S-PTGS. The jmj14 mutation also reduces the expression of Transgenes that do not trigger S-PTGS. Moreover, expression of Transgenes that undergo S-PTGS in a wild-type background is reduced in jmj14 sgs3 double mutants compared with PTGS-deficient sgs3 mutants, indicating that JMJ14 is required for high levels of transcription in a PTGS-independent manner. Whereas endogenous loci regulated by JMJ14 exhibit increased H3K4me2 and H3K4me3 levels in the jmj14 mutant, Transgene loci exhibit unchanged H3K4me2 and decreased H3K4me3 levels. Because jmj14 mutations impair PTGS of Transgenes expressed under various plant or viral promoters, we hypothesize that JMJ14 demethylation activity is prevented by antagonistic epigenetic marks specifically imposed at Transgene loci. Removing JMJ14 likely allows other H3K4 demethylases encoded by the Arabidopsis thaliana genome to act on Transgenes and reduce transcription levels, thus preventing the triggering of S-PTGS.

  • a branched pathway for Transgene induced rna silencing in plants
    Current Biology, 2002
    Co-Authors: Christophe Beclin, Stephanie Boutet, Peter M Waterhouse, Hervé Vaucheret
    Abstract:

    In plants, RNA silencing can be induced by highly transcribed sense Transgenes (S-PTGS) or by Transgene loci producing double-stranded RNA (dsRNA) due to the presence of inverted repeats (IR-PTGS). Both phenomena correlate with accumulation of 21-25 nt sense and anti-sense RNA homologous to the silent gene and with methylation of the coding sequence. We have challenged IR-PTGS with four viruses known to inhibit S-PTGS: CMV, TuMV, TVCV, and TCV ( this work) and in sgs2, sgs3, and ago1 mutants impaired in S-PTGS. Surprisingly, whereas the four viruses inhibit IR-PTGS, IR-PTGS and methylation of a GUS trangene and IR-PTGS of three endogeneous genes occur in the sgs2, sgs3, and ago1 mutations. Based on these results, we propose a branched pathway for RNA silencing in plants. RNA silencing would occur via the action of dsRNA produced either via the action of SGS2 (also known as SDE1), SGS3, and AGO1 on the S-PTGS branch or by Transgenes arranged as inverted repeats on the IR-PTGS branch. Moreover, Transgene methylation would result from production or action of dsRNA, since it does not require SGS2/SDE1, SGS3, and AGO1.

  • systemic acquired silencing Transgene specific post transcriptional silencing is transmitted by grafting from silenced stocks to non silenced scions
    The EMBO Journal, 1997
    Co-Authors: Jeanchristophe Palauqui, Taline Elmayan, Jeanmarie Pollien, Hervé Vaucheret
    Abstract:

    Using grafting procedures, we investigated the transmission of co-suppression of nitrate reductase and nitrite reductase host genes and Transgenes and of post-transcriptional silencing of a uidA Transgene encoding glucuronidase in tobacco. We demonstrate that silencing is transmitted with 100% efficiency from silenced stocks to non-silenced scions expressing the corresponding Transgene. Transmission is unidirectional from stock to scion, Transgene specific, locus independent and requires the presence of a transcriptionally active Transgene in the target scion. The transmission of co-suppression occurs when silenced stocks and non-silenced target scions are physically separated by up to 30 cm of stem of a non-target wild-type plant. Taken together, these results suggest that a non-metabolic, Transgene-specific, diffusable messenger mediates the propagation of de novo post-transcriptional silencing through the plant.

  • molecular and genetic analysis of nitrite reductase co suppression in transgenic tobacco plants
    Molecular Genetics and Genomics, 1995
    Co-Authors: Hervé Vaucheret, Taline Elmayan, Jeanchristophe Palauqui, Barbara Moffatt
    Abstract:

    Silencing ofNia host genes and Transgenes (encoding nitrate reductase) was previously achieved by introducing into tobacco plants the tobaccoNia2 cDNA cloned downstream of the cauliflower mosaic virus (CaMV) 35S promoter. To check whetherNii host genes and Transgenes (encoding nitrite reductase, the second enzyme of the nitrate assimilation pathway) were also susceptible to silencing, a Transgene consisting of the tobaccoNii1 gene with two copies of the enhancer of the 35S promoter cloned 1 kb upstream of theNii promoter region was introduced into tobacco plants. Among nine independent transformants analysed, two showed silencing ofNii host genes and Transgenes in some descendants after selfing, but never after back-crossing with wild-type plants, suggesting that silencing depends on the number of Transgene loci and/or on certain allelic or ectopic combinations of Transgene loci. In one transformant carrying a single Transgene locus in a homozygous state, silencing was triggered in all progeny plants of each generation, 20 to 50 days after germination. Field trial analysis confirmed that silencing was not triggered when the Transgene locus of this latter line was present in a hemizygous state. In addition, it was revealed that silencing can be triggered, albeit at low frequency and later during the development, when this Transgene locus is brought into the presence of a non-allelic Transgene locus by crossing, suggesting that a homozygous state is not absolutely required.

Megan K Pugach - One of the best experts on this subject based on the ideXlab platform.

  • dose dependent rescue of ko amelogenin enamel by Transgenes in vivo
    Frontiers in Physiology, 2017
    Co-Authors: Felicitas B Bidlack, Yan Xia, Megan K Pugach
    Abstract:

    Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP Transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin Transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin Transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double Transgene and M180Tg, CTRNCTg, LRAPTg triple Transgene mice with Transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- versus hemizygosity was determined by qPCR and relative Transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different Transgenes, but mice homozygous for a given Transgene had significantly thinner enamel than mice hemizygous for the Transgene (p<0.05).The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin Transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.

  • dose dependent rescue of ko amelogenin enamel by Transgenes in vivo
    Frontiers in Physiology, 2017
    Co-Authors: Felicitas B Bidlack, Yan Xia, Megan K Pugach
    Abstract:

    Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP Transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin Transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin Transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double Transgene and M180Tg, CTRNCTg, LRAPTg triple Transgene mice with Transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative Transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different Transgenes, but mice homozygous for a given Transgene had significantly thinner enamel than mice hemizygous for the Transgene (p < 0.05). The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin Transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.

Felicitas B Bidlack - One of the best experts on this subject based on the ideXlab platform.

  • dose dependent rescue of ko amelogenin enamel by Transgenes in vivo
    Frontiers in Physiology, 2017
    Co-Authors: Felicitas B Bidlack, Yan Xia, Megan K Pugach
    Abstract:

    Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP Transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin Transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin Transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double Transgene and M180Tg, CTRNCTg, LRAPTg triple Transgene mice with Transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- versus hemizygosity was determined by qPCR and relative Transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different Transgenes, but mice homozygous for a given Transgene had significantly thinner enamel than mice hemizygous for the Transgene (p<0.05).The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin Transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.

  • dose dependent rescue of ko amelogenin enamel by Transgenes in vivo
    Frontiers in Physiology, 2017
    Co-Authors: Felicitas B Bidlack, Yan Xia, Megan K Pugach
    Abstract:

    Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP Transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin Transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin Transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double Transgene and M180Tg, CTRNCTg, LRAPTg triple Transgene mice with Transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative Transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different Transgenes, but mice homozygous for a given Transgene had significantly thinner enamel than mice hemizygous for the Transgene (p < 0.05). The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin Transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.

Taline Elmayan - One of the best experts on this subject based on the ideXlab platform.

  • second site mutagenesis of a hypomorphic argonaute1 allele identifies superkiller3 as an endogenous suppressor of Transgene posttranscriptional gene silencing
    Plant Physiology, 2015
    Co-Authors: Agnes Yu, Baptiste Saudemont, Nathalie Bouteiller, Jean Sébastien Parent, Jean-benoit Morel, Emilie Elviramatelot, Gersende Lepère, Taline Elmayan, Hervé Vaucheret
    Abstract:

    Second-site mutagenesis was performed on the argonaute1-33 (ago1-33) hypomorphic mutant, which exhibits reduced sense Transgene posttranscriptional gene silencing (S-PTGS). Mutations in FIERY1, a positive regulator of the cytoplasmic 5′-to-3′ EXORIBONUCLEASE4 (XRN4), and in SUPERKILLER3 (SKI3), a member of the SKI complex that threads RNAs directly to the 3′-to-5′ exoribonuclease of the cytoplasmic exosome, compensated AGO1 partial deficiency and restored S-PTGS with 100% efficiency. Moreover, xrn4 and ski3 single mutations provoked the entry of nonsilenced Transgenes into S-PTGS and enhanced S-PTGS on partially silenced Transgenes, indicating that cytoplasmic 5′-to-3′ and 3′-to-5′ RNA degradation generally counteract S-PTGS, likely by reducing the amount of Transgene aberrant RNAs that are used by the S-PTGS pathway to build up small interfering RNAs that guide Transgene RNA cleavage by AGO1. Constructs generating improperly terminated Transgene messenger RNAs (mRNAs) were not more sensitive to ski3 or xrn4 than regular constructs, suggesting that improperly terminated Transgene mRNAs not only are degraded from both the 3′ end but also from the 5′ end, likely after decapping. The facts that impairment of either 5′-to-3′ or 3′-to-5′ RNA degradation is sufficient to provoke the entry of Transgene RNA into the S-PTGS pathway, whereas simultaneous impairment of both pathways is necessary to provoke the entry of endogenous mRNA into the S-PTGS pathway, suggest poor RNA quality upon the transcription of Transgenes integrated at random genomic locations.

  • mutations in the arabidopsis h3k4me2 3 demethylase jmj14 suppress posttranscriptional gene silencing by decreasing Transgene transcription
    The Plant Cell, 2012
    Co-Authors: Ivan Le Masson, Nathalie Bouteiller, Vincent Jauvion, Maud Rivard, Taline Elmayan, Hervé Vaucheret
    Abstract:

    Posttranscriptional gene silencing (PTGS) mediated by sense Transgenes (S-PTGS) results in RNA degradation and DNA methylation of the transcribed region. Through a forward genetic screen, a mutant defective in the Histone3 Lysine4 di/trimethyl (H3K4me2/3) demethylase Jumonji-C (JmjC) domain-containing protein14 (JMJ14) was identified. This mutant reactivates various Transgenes silenced by S-PTGS and shows reduced Histone3 Lysine9 Lysine14 acetylation (H3K9K14Ac) levels, reduced polymerase II occupancy, reduced Transgene transcription, and increased DNA methylation in the promoter region, consistent with the hypothesis that high levels of transcription are required to trigger S-PTGS. The jmj14 mutation also reduces the expression of Transgenes that do not trigger S-PTGS. Moreover, expression of Transgenes that undergo S-PTGS in a wild-type background is reduced in jmj14 sgs3 double mutants compared with PTGS-deficient sgs3 mutants, indicating that JMJ14 is required for high levels of transcription in a PTGS-independent manner. Whereas endogenous loci regulated by JMJ14 exhibit increased H3K4me2 and H3K4me3 levels in the jmj14 mutant, Transgene loci exhibit unchanged H3K4me2 and decreased H3K4me3 levels. Because jmj14 mutations impair PTGS of Transgenes expressed under various plant or viral promoters, we hypothesize that JMJ14 demethylation activity is prevented by antagonistic epigenetic marks specifically imposed at Transgene loci. Removing JMJ14 likely allows other H3K4 demethylases encoded by the Arabidopsis thaliana genome to act on Transgenes and reduce transcription levels, thus preventing the triggering of S-PTGS.

  • systemic acquired silencing Transgene specific post transcriptional silencing is transmitted by grafting from silenced stocks to non silenced scions
    The EMBO Journal, 1997
    Co-Authors: Jeanchristophe Palauqui, Taline Elmayan, Jeanmarie Pollien, Hervé Vaucheret
    Abstract:

    Using grafting procedures, we investigated the transmission of co-suppression of nitrate reductase and nitrite reductase host genes and Transgenes and of post-transcriptional silencing of a uidA Transgene encoding glucuronidase in tobacco. We demonstrate that silencing is transmitted with 100% efficiency from silenced stocks to non-silenced scions expressing the corresponding Transgene. Transmission is unidirectional from stock to scion, Transgene specific, locus independent and requires the presence of a transcriptionally active Transgene in the target scion. The transmission of co-suppression occurs when silenced stocks and non-silenced target scions are physically separated by up to 30 cm of stem of a non-target wild-type plant. Taken together, these results suggest that a non-metabolic, Transgene-specific, diffusable messenger mediates the propagation of de novo post-transcriptional silencing through the plant.

  • molecular and genetic analysis of nitrite reductase co suppression in transgenic tobacco plants
    Molecular Genetics and Genomics, 1995
    Co-Authors: Hervé Vaucheret, Taline Elmayan, Jeanchristophe Palauqui, Barbara Moffatt
    Abstract:

    Silencing ofNia host genes and Transgenes (encoding nitrate reductase) was previously achieved by introducing into tobacco plants the tobaccoNia2 cDNA cloned downstream of the cauliflower mosaic virus (CaMV) 35S promoter. To check whetherNii host genes and Transgenes (encoding nitrite reductase, the second enzyme of the nitrate assimilation pathway) were also susceptible to silencing, a Transgene consisting of the tobaccoNii1 gene with two copies of the enhancer of the 35S promoter cloned 1 kb upstream of theNii promoter region was introduced into tobacco plants. Among nine independent transformants analysed, two showed silencing ofNii host genes and Transgenes in some descendants after selfing, but never after back-crossing with wild-type plants, suggesting that silencing depends on the number of Transgene loci and/or on certain allelic or ectopic combinations of Transgene loci. In one transformant carrying a single Transgene locus in a homozygous state, silencing was triggered in all progeny plants of each generation, 20 to 50 days after germination. Field trial analysis confirmed that silencing was not triggered when the Transgene locus of this latter line was present in a hemizygous state. In addition, it was revealed that silencing can be triggered, albeit at low frequency and later during the development, when this Transgene locus is brought into the presence of a non-allelic Transgene locus by crossing, suggesting that a homozygous state is not absolutely required.

John D. Bartlett - One of the best experts on this subject based on the ideXlab platform.

  • Matrix Metalloproteinase-20 Over-Expression Is Detrimental to Enamel Development: A Mus musculus Model
    2016
    Co-Authors: Masashi Shin, Jerry V. Antone, Xiaomu Guan, Craig C Deagle, James P. Simmer, Coralee E. Tye, Charles E. Smith, John D. Bartlett
    Abstract:

    Background: Matrix metalloproteinase-20 (Mmp20) ablated mice have enamel that is thin and soft with an abnormal rod pattern that abrades from the underlying dentin. We asked if introduction of Transgenes expressing Mmp20 would revert this Mmp20 null phenotype back to normal. Unexpectedly, for Transgenes expressing medium or high levels of Mmp20, we found opposite enamel phenotypes depending on the genetic background (Mmp202/2 or Mmp20+/+) in which the Transgenes were expressed. Methodology/Principal Findings: Amelx-promoter-Mmp20 transgenic founder mouse lines were assessed for Transgene expression and those expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Regardless of expression level, each Transgene brought the null enamel back to full thickness. However, the high and medium expressing Mmp20 Transgenes in the Mmp20 null background had significantly harder more mineralized enamel than did the low Transgene expresser. Strikingly, when the high and medium expressing Mmp20 Transgenes were present in the wild-type background, the enamel was significantly less well mineralized than normal. Protein gel analysis of enamel matrix proteins from the high and medium expressing Transgenes present in the wild-type background demonstrated that greater than normal amounts of cleavage products and smaller quantities of higher molecular weight proteins were present within their enamel matrices

  • matrix metalloproteinase 20 over expression is detrimental to enamel development a mus musculus model
    PLOS ONE, 2014
    Co-Authors: Masashi Shin, Jerry V. Antone, Yuanyuan Hu, Xiaomu Guan, Craig C Deagle, James P. Simmer, John D. Bartlett
    Abstract:

    Background Matrix metalloproteinase-20 (Mmp20) ablated mice have enamel that is thin and soft with an abnormal rod pattern that abrades from the underlying dentin. We asked if introduction of Transgenes expressing Mmp20 would revert this Mmp20 null phenotype back to normal. Unexpectedly, for Transgenes expressing medium or high levels of Mmp20, we found opposite enamel phenotypes depending on the genetic background (Mmp20−/− or Mmp20+/+) in which the Transgenes were expressed. Methodology/Principal Findings Amelx-promoter-Mmp20 transgenic founder mouse lines were assessed for Transgene expression and those expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Regardless of expression level, each Transgene brought the null enamel back to full thickness. However, the high and medium expressing Mmp20 Transgenes in the Mmp20 null background had significantly harder more mineralized enamel than did the low Transgene expresser. Strikingly, when the high and medium expressing Mmp20 Transgenes were present in the wild-type background, the enamel was significantly less well mineralized than normal. Protein gel analysis of enamel matrix proteins from the high and medium expressing Transgenes present in the wild-type background demonstrated that greater than normal amounts of cleavage products and smaller quantities of higher molecular weight proteins were present within their enamel matrices. Conclusions/Significance Mmp20 expression levels must be within a specific range for normal enamel development to occur. Creation of a normally thick enamel layer may occur over a wider range of Mmp20 expression levels, but acquisition of normal enamel hardness has a narrower range. Since over-expression of Mmp20 results in decreased enamel hardness, this suggests that a balance exists between cleaved and full-length enamel matrix proteins that are essential for formation of a properly hardened enamel layer. It also suggests that few feedback controls are present in the enamel matrix to prevent excessive MMP20 activity.