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Heterozygosity for a hypomorphic Polβ mutation reduces the expansion frequency in a mouse model of the Fragile X-related disorders.

Lokanga RA, Senejani AG, Sweasy JB, Usdin K - PLoS Genet. (2015)

Bottom Line: The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene.Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals.This may have interesting implications for the mechanism by which BER generates expansions.

View Article: PubMed Central - PubMed

Affiliation: Section on Gene Structure and Disease, Laboratory of Cell and molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America; Department of Biochemistry, University of Cape Town Medical School, Cape Town, South Africa.

ABSTRACT
The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polβ, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.

No MeSH data available.


Related in: MedlinePlus

Expression of various BER proteins in different mouse organs.Total protein was extracted from different organs of 3 different FXD mice as described in the Materials and Methods. Since in our experience proteins used as “normalizing controls” including β-actin and α-tubulin and GAPDH differ significantly in different organs, we took care to analyze equal amounts of protein as assessed by the Bradford Assay. Ten micrograms of protein from the organs of each animal were pooled and loaded onto 3–8% Tris-Acetate gels, resolved by gel electrophoresis and subjected to Western blotting as described in the Materials and Methods.
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pgen.1005181.g006: Expression of various BER proteins in different mouse organs.Total protein was extracted from different organs of 3 different FXD mice as described in the Materials and Methods. Since in our experience proteins used as “normalizing controls” including β-actin and α-tubulin and GAPDH differ significantly in different organs, we took care to analyze equal amounts of protein as assessed by the Bradford Assay. Ten micrograms of protein from the organs of each animal were pooled and loaded onto 3–8% Tris-Acetate gels, resolved by gel electrophoresis and subjected to Western blotting as described in the Materials and Methods.

Mentions: The propensity of the CAG/CTG repeat to expand more in striatum than in the cerebellum of a mouse model for HD has been attributed to differences in the stoichiometry of the proteins involved in BER that are expressed in these two brain regions [39,40]. To evaluate the role of these proteins in determining the tissue specificity of expansion in the Fragile PM mouse we compared the expression levels of the key BER enzymes APE1, DNA ligase 1, DNA ligase 3, FEN1, OGG1, NEIL1 and Polβ in a selection of different tissues. Expansion levels in these tissues follow the trend: testis (SII = 19) > liver (SII = 15) > brain (SII = 12) > kidney (SII = 4) > heart (SII = 0). As can be seen from Fig 6, some of the proteins tested do not show a good correlation with the propensity to expand. For example, DNA ligase 3 and OGG1 show the highest levels of expression in heart, an organ in which the repeat is stable whilst NEIL1 is expressed at its lowest levels in brain, liver and testis, organs that show the highest levels of expansion. Furthermore, organs that have very different propensities to expand, like brain and heart, have similar levels of Polβ. A low level of FEN1 relative to Polβ has been suggested to be an important determinant of the predisposition of cells of the striatum to expand relative to cells of the cerebellum in a HD mouse model [41]. However, in our mouse background this relationship does not appear to hold, with the FEN1/Polβ ratio being lower in heart, an organ that shows no expansion, than it is in brain. Of all the proteins tested, only APE1 and FEN1 showed a general correlation between the level of expression and the SII of all 5 organs tested.


Heterozygosity for a hypomorphic Polβ mutation reduces the expansion frequency in a mouse model of the Fragile X-related disorders.

Lokanga RA, Senejani AG, Sweasy JB, Usdin K - PLoS Genet. (2015)

Expression of various BER proteins in different mouse organs.Total protein was extracted from different organs of 3 different FXD mice as described in the Materials and Methods. Since in our experience proteins used as “normalizing controls” including β-actin and α-tubulin and GAPDH differ significantly in different organs, we took care to analyze equal amounts of protein as assessed by the Bradford Assay. Ten micrograms of protein from the organs of each animal were pooled and loaded onto 3–8% Tris-Acetate gels, resolved by gel electrophoresis and subjected to Western blotting as described in the Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401650&req=5

pgen.1005181.g006: Expression of various BER proteins in different mouse organs.Total protein was extracted from different organs of 3 different FXD mice as described in the Materials and Methods. Since in our experience proteins used as “normalizing controls” including β-actin and α-tubulin and GAPDH differ significantly in different organs, we took care to analyze equal amounts of protein as assessed by the Bradford Assay. Ten micrograms of protein from the organs of each animal were pooled and loaded onto 3–8% Tris-Acetate gels, resolved by gel electrophoresis and subjected to Western blotting as described in the Materials and Methods.
Mentions: The propensity of the CAG/CTG repeat to expand more in striatum than in the cerebellum of a mouse model for HD has been attributed to differences in the stoichiometry of the proteins involved in BER that are expressed in these two brain regions [39,40]. To evaluate the role of these proteins in determining the tissue specificity of expansion in the Fragile PM mouse we compared the expression levels of the key BER enzymes APE1, DNA ligase 1, DNA ligase 3, FEN1, OGG1, NEIL1 and Polβ in a selection of different tissues. Expansion levels in these tissues follow the trend: testis (SII = 19) > liver (SII = 15) > brain (SII = 12) > kidney (SII = 4) > heart (SII = 0). As can be seen from Fig 6, some of the proteins tested do not show a good correlation with the propensity to expand. For example, DNA ligase 3 and OGG1 show the highest levels of expression in heart, an organ in which the repeat is stable whilst NEIL1 is expressed at its lowest levels in brain, liver and testis, organs that show the highest levels of expansion. Furthermore, organs that have very different propensities to expand, like brain and heart, have similar levels of Polβ. A low level of FEN1 relative to Polβ has been suggested to be an important determinant of the predisposition of cells of the striatum to expand relative to cells of the cerebellum in a HD mouse model [41]. However, in our mouse background this relationship does not appear to hold, with the FEN1/Polβ ratio being lower in heart, an organ that shows no expansion, than it is in brain. Of all the proteins tested, only APE1 and FEN1 showed a general correlation between the level of expression and the SII of all 5 organs tested.

Bottom Line: The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene.Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals.This may have interesting implications for the mechanism by which BER generates expansions.

View Article: PubMed Central - PubMed

Affiliation: Section on Gene Structure and Disease, Laboratory of Cell and molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America; Department of Biochemistry, University of Cape Town Medical School, Cape Town, South Africa.

ABSTRACT
The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polβ, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.

No MeSH data available.


Related in: MedlinePlus