Greciano Patricia G, Ruiz Maria Fernanda, Kremer Leonor, Goday Clara
Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Chromosoma. 2009 Jun;118(3):361-76. doi: 10.1007/s00412-009-0203-y. Epub 2009 Feb 10.
We report here the molecular and cytological characterization of two proteins, ScoHET1 and ScoHET2 (for Sciara coprophila heterochromatin), which associate to constitutive heterochromatin in the dipteran S. coprophila. Both proteins, ScoHET1 of 37 kDa and ScoHET2 of 44 kDa, display two chromodomain motifs that contain the conserved residues essential for the recognition of methylated histone H3 at lysine 9. We raised antibodies to analyze the chromosomal location of ScoHET1 and ScoHET2 in somatic and germline cells. In S. coprophila polytene chromosomes, both proteins associate to the pericentromeric regions and to the heterochromatic subterminal bands of the chromosomes. In germinal nuclei, ScoHET1 and ScoHET2 proteins distribute to the heterochromatic regions of the regular chromosome complement and are abundantly present along the heterochromatic germline-limited "L" chromosomes. We investigated histone methylation modifications and found that all heterochromatic regions enriched in ScoHET1/ScoHET2 proteins exhibit high levels of di- and tri-methylated histone H3 at lysine 9. Taken together, our results support that the association of ScoHET1/ScoHET2 to heterochromatin is mediated by histone H3K9 methylation. Using 5-methylcytosine antibodies, we proved the cytological detection of DNA methylation in S. coprophila. From our observations in L germline chromosomes, heterochromatin in S. coprophila is highly enriched in DNA 5-methylcytosine residues.
我们在此报告了两种蛋白质ScoHET1和ScoHET2（嗜粪Sciara的异染色质）的分子和细胞学特征，它们与双翅目昆虫嗜粪Sciara中的组成型异染色质相关联。这两种蛋白质，37 kDa的ScoHET1和44 kDa的ScoHET2，都显示出两个色域基序，其中包含识别赖氨酸9处甲基化组蛋白H3所必需的保守残基。我们制备了抗体，以分析ScoHET1和ScoHET2在体细胞和生殖细胞中的染色体定位。在嗜粪Sciara的多线染色体中，这两种蛋白质都与着丝粒周围区域以及染色体的异染色质亚末端带相关联。在生殖细胞核中，ScoHET1和ScoHET2蛋白质分布在正常染色体组的异染色质区域，并大量存在于异染色质的生殖系受限“L”染色体上。我们研究了组蛋白甲基化修饰，发现所有富含ScoHET1/ScoHET2蛋白质的异染色质区域都显示出赖氨酸9处高水平的二甲基化和三甲基化组蛋白H3。综上所述，我们的结果支持ScoHET1/ScoHET2与异染色质的关联是由组蛋白H3K9甲基化介导的。使用5-甲基胞嘧啶抗体，我们证明了在嗜粪Sciara中对DNA甲基化的细胞学检测。根据我们在L生殖系染色体中的观察，嗜粪Sciara中的异染色质高度富含DNA 5-甲基胞嘧啶残基。
