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HP1gamma function is required for male germ cell survival and spermatogenesis.

Brown JP, Bullwinkel J, Baron-Lühr B, Billur M, Schneider P, Winking H, Singh PB - Epigenetics Chromatin (2010)

Bottom Line: While targeting the Cbx3 gene (encoding the HP1gamma protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1gamma protein.Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype).The Cbx3hypo/hypo spermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunoepigenetics, Department of Immunology and Cell Biology, Research Center Borstel, D-23845 Borstel, Germany. psingh@fz-borstel.de.

ABSTRACT

Background: HP1 proteins are conserved components of eukaryotic constitutive heterochromatin. In mammals, there are three genes that encode HP1-like proteins, termed HP1alpha, HP1beta and HP1gamma, which have a high degree of homology This paper describes for the first time, to our knowledge, the physiological function of HP1gamma using a gene-targeted mouse.

Results: While targeting the Cbx3 gene (encoding the HP1gamma protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1gamma protein. Homozygotes for the hypomorphic allele (Cbx3hypo/hypo) are rare, with only 1% of Cbx3hypo/hypo animals reaching adulthood. Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype). The percentage of seminiferous tubules that are positive for L1 ORF1 protein (ORF1p) in Cbx3hypo/hypo testes is greater than that for wild-type testes, indicating that L1 retrotransposon silencing is reversed, leading to ectopic expression of ORF1p in Cbx3hypo/hypo germ cells.

Conclusions: The Cbx3 gene product (the HP1gamma protein) has a non-redundant function during spermatogenesis that cannot be compensated for by the other two HP1 isotypes. The Cbx3hypo/hypo spermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants. The Cbx3 gene-targeted mice generated in this study provide an appropriate model for the study of HP1gamma in transposon silencing and parental imprinting.

No MeSH data available.


Related in: MedlinePlus

Presence of the neo-tk cassette results in a hypomorphic (Cbx3hypo) allele. (a) Row 1, expression of HP1γ in MEFs. HP1γ expression was dramatically reduced to almost undetectable levels in MEFs derived from embryos that are homozygous for the targeted allele shown in row 3 of Figure 1 (Cbx3hypo/hypo MEFs). After expression of FlpE in the Cbx3hypo/hypo MEFs (FlpE-treated MEFs), the expression of HP1γ returned to wild-type levels. HP1γ protein expression in the FlpE-treated MEFs could be extinguished by expression of Cre recombinase, giving rise to Cbx3-/- MEFs. Hnrnpa2b1 protein expression was not affected by the presence or absence of the neo-tk cassette, as shown in row 2. Row 3, actin loading control. (b) Row 1, immunofluorescent HP1γ staining of MEFs. The typical punctate HP1γ pattern of expression in wild-type MEFs was reduced to very faint, almost background, levels in Cbx3hypo/hypo MEFs. Strikingly, HP1γ staining levels returned to wild-type levels after the neo-tk cassette was excised after Flpe treatment (FlpE-treated MEFs). HP1γ staining was lost after expression of Cre recombinase giving rise to Cbx3-/- MEFs. Row 2, DAPI staining; row 3, merged images. Bar = 20 μm.
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Figure 2: Presence of the neo-tk cassette results in a hypomorphic (Cbx3hypo) allele. (a) Row 1, expression of HP1γ in MEFs. HP1γ expression was dramatically reduced to almost undetectable levels in MEFs derived from embryos that are homozygous for the targeted allele shown in row 3 of Figure 1 (Cbx3hypo/hypo MEFs). After expression of FlpE in the Cbx3hypo/hypo MEFs (FlpE-treated MEFs), the expression of HP1γ returned to wild-type levels. HP1γ protein expression in the FlpE-treated MEFs could be extinguished by expression of Cre recombinase, giving rise to Cbx3-/- MEFs. Hnrnpa2b1 protein expression was not affected by the presence or absence of the neo-tk cassette, as shown in row 2. Row 3, actin loading control. (b) Row 1, immunofluorescent HP1γ staining of MEFs. The typical punctate HP1γ pattern of expression in wild-type MEFs was reduced to very faint, almost background, levels in Cbx3hypo/hypo MEFs. Strikingly, HP1γ staining levels returned to wild-type levels after the neo-tk cassette was excised after Flpe treatment (FlpE-treated MEFs). HP1γ staining was lost after expression of Cre recombinase giving rise to Cbx3-/- MEFs. Row 2, DAPI staining; row 3, merged images. Bar = 20 μm.

Mentions: The greatly reduced numbers of Cbx3hypo/hypo adults prompted us to investigate whether the targeting event itself had affected Cbx3 expression, and if this was the likely cause of the reduced numbers of Cbx3hypo/hypo adults. To explore this hypothesis further, we generated primary mouse fibroblasts from E13.5 wild-type and Cbx3hypo/hypo littermates and compared the expression levels of HP1γ by Western blotting. As shown in Figure 2, there was a dramatic reduction in HP1γ expression levels in Cbx3hypo/hypo compared with wild-type mouse embryonic fibroblasts (MEFs) (Figure 2a, top row: wild-type to hypo/hypo) indicating that the Cbx3hypo allele was a hypomorph. The effect of the targeting event was specific to the Cbx3 gene, as protein expression of the closely linked Hnrnpa2b1 gene was not changed (Figure 2a, middle row). Given this unexpected result, we were prompted to investigate whether the presence of the neo-tk selection cassette itself was interfering with Cbx3 expression. Previous work has shown that knockdown of target gene expression can result from the presence of a neo gene in the targeting vector [14]. The mechanism for such a knockdown is not fully understood but may involve transcriptional interference, by which the presence of one transcriptional unit interferes with another that is in cis [15].


HP1gamma function is required for male germ cell survival and spermatogenesis.

Brown JP, Bullwinkel J, Baron-Lühr B, Billur M, Schneider P, Winking H, Singh PB - Epigenetics Chromatin (2010)

Presence of the neo-tk cassette results in a hypomorphic (Cbx3hypo) allele. (a) Row 1, expression of HP1γ in MEFs. HP1γ expression was dramatically reduced to almost undetectable levels in MEFs derived from embryos that are homozygous for the targeted allele shown in row 3 of Figure 1 (Cbx3hypo/hypo MEFs). After expression of FlpE in the Cbx3hypo/hypo MEFs (FlpE-treated MEFs), the expression of HP1γ returned to wild-type levels. HP1γ protein expression in the FlpE-treated MEFs could be extinguished by expression of Cre recombinase, giving rise to Cbx3-/- MEFs. Hnrnpa2b1 protein expression was not affected by the presence or absence of the neo-tk cassette, as shown in row 2. Row 3, actin loading control. (b) Row 1, immunofluorescent HP1γ staining of MEFs. The typical punctate HP1γ pattern of expression in wild-type MEFs was reduced to very faint, almost background, levels in Cbx3hypo/hypo MEFs. Strikingly, HP1γ staining levels returned to wild-type levels after the neo-tk cassette was excised after Flpe treatment (FlpE-treated MEFs). HP1γ staining was lost after expression of Cre recombinase giving rise to Cbx3-/- MEFs. Row 2, DAPI staining; row 3, merged images. Bar = 20 μm.
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Related In: Results  -  Collection

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Figure 2: Presence of the neo-tk cassette results in a hypomorphic (Cbx3hypo) allele. (a) Row 1, expression of HP1γ in MEFs. HP1γ expression was dramatically reduced to almost undetectable levels in MEFs derived from embryos that are homozygous for the targeted allele shown in row 3 of Figure 1 (Cbx3hypo/hypo MEFs). After expression of FlpE in the Cbx3hypo/hypo MEFs (FlpE-treated MEFs), the expression of HP1γ returned to wild-type levels. HP1γ protein expression in the FlpE-treated MEFs could be extinguished by expression of Cre recombinase, giving rise to Cbx3-/- MEFs. Hnrnpa2b1 protein expression was not affected by the presence or absence of the neo-tk cassette, as shown in row 2. Row 3, actin loading control. (b) Row 1, immunofluorescent HP1γ staining of MEFs. The typical punctate HP1γ pattern of expression in wild-type MEFs was reduced to very faint, almost background, levels in Cbx3hypo/hypo MEFs. Strikingly, HP1γ staining levels returned to wild-type levels after the neo-tk cassette was excised after Flpe treatment (FlpE-treated MEFs). HP1γ staining was lost after expression of Cre recombinase giving rise to Cbx3-/- MEFs. Row 2, DAPI staining; row 3, merged images. Bar = 20 μm.
Mentions: The greatly reduced numbers of Cbx3hypo/hypo adults prompted us to investigate whether the targeting event itself had affected Cbx3 expression, and if this was the likely cause of the reduced numbers of Cbx3hypo/hypo adults. To explore this hypothesis further, we generated primary mouse fibroblasts from E13.5 wild-type and Cbx3hypo/hypo littermates and compared the expression levels of HP1γ by Western blotting. As shown in Figure 2, there was a dramatic reduction in HP1γ expression levels in Cbx3hypo/hypo compared with wild-type mouse embryonic fibroblasts (MEFs) (Figure 2a, top row: wild-type to hypo/hypo) indicating that the Cbx3hypo allele was a hypomorph. The effect of the targeting event was specific to the Cbx3 gene, as protein expression of the closely linked Hnrnpa2b1 gene was not changed (Figure 2a, middle row). Given this unexpected result, we were prompted to investigate whether the presence of the neo-tk selection cassette itself was interfering with Cbx3 expression. Previous work has shown that knockdown of target gene expression can result from the presence of a neo gene in the targeting vector [14]. The mechanism for such a knockdown is not fully understood but may involve transcriptional interference, by which the presence of one transcriptional unit interferes with another that is in cis [15].

Bottom Line: While targeting the Cbx3 gene (encoding the HP1gamma protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1gamma protein.Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype).The Cbx3hypo/hypo spermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunoepigenetics, Department of Immunology and Cell Biology, Research Center Borstel, D-23845 Borstel, Germany. psingh@fz-borstel.de.

ABSTRACT

Background: HP1 proteins are conserved components of eukaryotic constitutive heterochromatin. In mammals, there are three genes that encode HP1-like proteins, termed HP1alpha, HP1beta and HP1gamma, which have a high degree of homology This paper describes for the first time, to our knowledge, the physiological function of HP1gamma using a gene-targeted mouse.

Results: While targeting the Cbx3 gene (encoding the HP1gamma protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1gamma protein. Homozygotes for the hypomorphic allele (Cbx3hypo/hypo) are rare, with only 1% of Cbx3hypo/hypo animals reaching adulthood. Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype). The percentage of seminiferous tubules that are positive for L1 ORF1 protein (ORF1p) in Cbx3hypo/hypo testes is greater than that for wild-type testes, indicating that L1 retrotransposon silencing is reversed, leading to ectopic expression of ORF1p in Cbx3hypo/hypo germ cells.

Conclusions: The Cbx3 gene product (the HP1gamma protein) has a non-redundant function during spermatogenesis that cannot be compensated for by the other two HP1 isotypes. The Cbx3hypo/hypo spermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants. The Cbx3 gene-targeted mice generated in this study provide an appropriate model for the study of HP1gamma in transposon silencing and parental imprinting.

No MeSH data available.


Related in: MedlinePlus