Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance.

Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.

Ino2, activator of yeast phospholipid biosynthetic genes, interacts with basal transcription factors TFIIA and Bdf1.

Binding of general transcription factors TFIID and TFIIA to basal promoters is rate-limiting for transcriptional initiation of eukaryotic protein-coding genes. Consequently, activator proteins interacting with subunits of TFIID and/or TFIIA can drastically increase the rate of initiation events. Yeast transcriptional activator Ino2 interacts with several Taf subunits of TFIID, among them the multifunctional Taf1 protein. In contrast to mammalian Taf1, yeast Taf1 lacks bromodomains which are instead encoded by separate proteins Bdf1 and Bdf2. In this work, we show that Bdf1 not only binds to acetylated histone H4 but can also be recruited by Ino2 and unrelated activators such as Gal4, Rap1, Leu3 and Flo8. An activator-binding domain was mapped in the N-terminus of Bdf1. Subunits Toa1 and Toa2 of yeast TFIIA directly contact sequences of basal promoters and TFIID subunit TBP but may also mediate the influence of activators. Indeed, Ino2 efficiently binds to two separate structural domains of Toa1, specifically with its N-terminal four-helix bundle structure required for dimerization with Toa2 and its C-terminal ß-barrel domain contacting TBP and sequences of the TATA element. These findings complete the functional analysis of yeast general transcription factors Bdf1 and Toa1 and identify them as targets of activator proteins.

Clear evidence of the carcinogenic potential of anthracene: A 2-year feeding study in rats and mice.

Anthracene is an environmental pollutant and its adverse effects on human health have long been a concern due to its persistence and bioaccumulation properties. However, there is insufficient evidence for its chronic toxicity, especially carcinogenicity, in either humans or experimental animals. In this study, its carcinogenicity and chronic toxicity were investigated in compliance with the OECD test guideline 451 (OECD TG 451) and Good Laboratory Practice Standards. Fifty male and 50 female F344 rats and 50 female BDF1 mice were administrated 0, 8000, 20000, or 50000 ppm anthracene in the diet for 104 weeks, and 50 male BDF1 mice were fed diets containing anthracene at 0, 3200, 8000, or 20000 ppm. Anthracene treatment had no adverse effect on either the survival rate or general condition of the rats or mice during the study period. Body weights were lower or tended to be lower in the anthracene-treated groups than in the control groups. Increased incidence of hepatocellular carcinoma and hepatocellular adenoma was observed in male rats and female mice. Renal cell carcinoma and renal cell adenoma, fibroadenoma in the mammary gland, and uterine endometrial stromal sarcoma were increased in female rats. Transitional cell carcinoma and transitional cell papilloma in the urinary bladder were also increased in male and female rats. In addition, several different pre-neoplastic lesions were increased in the anthracene-treated male and female rats and female mice. These results provide clear evidence that oral administration of anthracene for 104 weeks has a carcinogenic effect in male and female rats and female mice.

Differences in resistance against osmotic challenge among C57BL/6, DBA/2 and their hybrid mice metaphase II (MII) stage oocytes.

Oocytes of B6D2F1 (BDF1) mice are often used as recipients for intracytoplasmic sperm injection because of their cell membrane resistance against capillary penetration. It is assumed that oocytes of BDF1 mice have superior traits because of their hybrid vigour. However, the mechanisms of hybrid vigour are unclear. In this study, we focused on the membrane resistance of MII stage oocytes against changes in extracellular osmotic pressure. As a result, MII stage oocytes of inbred C57BL/6 and DBA/2 mice showed high tolerance in either a hypertonic or a hypotonic environment. Conversely, MII stage oocytes of hybrid BDF1 and D2B6F1 mice showed high tolerance in both hypertonic and hypotonic environments. Therefore, it is considered that MII stage oocytes of hybrid mice have superior traits than those of inbred mice. Our findings demonstrated that the hybrid vigour exists in the form of resistance to extracellular osmotic environment in hybrid MII stage oocytes.

Yeast Bromodomain Factor 1 and Its Human Homolog TAF1 Play Conserved Roles in Promoting Homologous Recombination.

Histone acetylation is a key histone post-translational modification that shapes chromatin structure, dynamics, and function. Bromodomain (BRD) proteins, the readers of acetyl-lysines, are located in the center of the histone acetylation-signaling network. How they regulate DNA repair and genome stability remains poorly understood. Here, a conserved function of the yeast Bromodomain Factor 1 (Bdf1) and its human counterpart TAF1 is reported in promoting DNA double-stranded break repair by homologous recombination (HR). Depletion of either yeast BDF1 or human TAF1, or disruption of their BRDs impairs DNA end resection, Replication Protein A (RPA) and Rad51 loading, and HR repair, causing genome instability and hypersensitivity to DNA damage. Mechanistically, it is shown that Bdf1 preferentially binds the DNA damage-induced histone H4 acetylation (H4Ac) via the BRD motifs, leading to its chromatin recruitment. Meanwhile, Bdf1 physically interacts with RPA, and this interaction facilitates RPA loading in the chromatin context and the subsequent HR repair. Similarly, TAF1 also interacts with H4Ac or RPA. Thus, Bdf1 and TAF1 appear to share a conserved mechanism in linking the HR repair to chromatin acetylation in preserving genome integrity.