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Tunicate cytostatic factor TC14-3 induces a polycomb group gene and histone modification through Ca(2+) binding and protein dimerization.

Kawamura K, Takakura K, Mori D, Ikeda K, Nakamura A, Suzuki T - BMC Cell Biol. (2012)

Bottom Line: When Phe(65) of TC14-3 was mutated to an acidic amino acid, the resultant mutant protein failed to dimerize.PmEed knockdown by RNA interference rescued cultured cells from the growth-inhibitory effects of TC14-3.These results show that in P. misakiensis, the cytostatic activity of TC14-3 is mediated by PmEed and resultant histone modification, and that the gene expression requires both the protein dimerization and Ca(2+)-binding of TC14-3.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular and Molecular Biotechnology, Faculty of Science, Kochi University, Kochi 780-8520, Japan. kazuk@kochi-u.ac.jp

ABSTRACT

Background: As many invertebrate species have multipotent cells that undergo cell growth and differentiation during regeneration and budding, many unique and interesting homeostatic factors are expected to exist in those animals. However, our understanding of such factors and global mechanisms remains very poor. Single zooids of the tunicate, Polyandrocarpa misakiensis, can give off as many as 40 buds during the life span. Bud development proceeds by means of transdifferentiation of very limited number of cells and tissues. TC14-3 is one of several different but closely related polypeptides isolated from P. misakiensis. It acts as a cytostatic factor that regulates proliferation, adhesion, and differentiation of multipotent cells, although the molecular mechanism remains uncertain. The Polycomb group (PcG) genes are involved in epigenetic control of genomic activity in mammals. In invertebrates except Drosophila, PcG and histone methylation have not been studied so extensively, and genome-wide gene regulation is poorly understood.

Results: When Phe(65) of TC14-3 was mutated to an acidic amino acid, the resultant mutant protein failed to dimerize. The replacement of Thr(69) with Arg(69) made dimers unstable. When Glu(106) was changed to Gly(106), the resultant mutant protein completely lost Ca(2+) binding. All these mutant proteins lacked cytostatic activity, indicating the requirement of protein dimerization and calcium for the activity. Polyandrocarpa Eed, a component of PcG, is highly expressed during budding, like TC14-3. When wild-type and mutant TC14-3s were applied in vivo and in vitro to Polyandrocarpa cells, only wild-type TC14-3 could induce Eed without affecting histone methyltransferase gene expression. Eed-expressing cells underwent trimethylation of histone H3 lysine27. PmEed knockdown by RNA interference rescued cultured cells from the growth-inhibitory effects of TC14-3.

Conclusion: These results show that in P. misakiensis, the cytostatic activity of TC14-3 is mediated by PmEed and resultant histone modification, and that the gene expression requires both the protein dimerization and Ca(2+)-binding of TC14-3. This system consisting of a humoral factor, PcG, and histone methylation would contribute to the homeostatic regulation of cell growth and terminal differentiation of invertebrate multipotent cells.

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Electrophoretic mobility of wild-type and mutant TC14s on SDS-PAGE. Odd lanes and even lanes show heat-denatured samples and non-heated samples, respectively. Lanes 1,2, wild TC14-3. Lanes 3,4, wild TC14-2. Lanes 5,6, TC14-2(21-60)/TC14-3(61-145). Lanes 7,8, TC14-3F65D. Lanes 9,10, TC14-3T69R. Lanes 11,12, TC14-2R69T.
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Figure 4: Electrophoretic mobility of wild-type and mutant TC14s on SDS-PAGE. Odd lanes and even lanes show heat-denatured samples and non-heated samples, respectively. Lanes 1,2, wild TC14-3. Lanes 3,4, wild TC14-2. Lanes 5,6, TC14-2(21-60)/TC14-3(61-145). Lanes 7,8, TC14-3F65D. Lanes 9,10, TC14-3T69R. Lanes 11,12, TC14-2R69T.

Mentions: TC14-3 exhibited a relative electrophoretic mobility of 15 kDa (Figure 4, lane 1) on SDS-PAGE following heat denaturation, while under non-heated conditions, more than 99% of the total protein exhibited a relative mobility of 30 kDa (Figure 4, lane 2; Table 2). In contrast, TC14-2 exhibited a single band of 18 kDa following heat denaturation (Figure 4, lane 3) and separated into 2 bands of 18 and 28 kDa under non-heated conditions (Figure 4, lane 4). The 28-kDa form of TC14-2 accounted for approximately 61% of the total amount of protein (Table 2). The chimeric protein, TC14-221-60/TC14-361-145 exhibited an electrophoretic pattern similar to that of wild-type TC14-3 (Figure 4, lanes 5, 6). These results strongly suggest that wild-type TC14-3 may form more stable dimers than wild-type TC14-2.


Tunicate cytostatic factor TC14-3 induces a polycomb group gene and histone modification through Ca(2+) binding and protein dimerization.

Kawamura K, Takakura K, Mori D, Ikeda K, Nakamura A, Suzuki T - BMC Cell Biol. (2012)

Electrophoretic mobility of wild-type and mutant TC14s on SDS-PAGE. Odd lanes and even lanes show heat-denatured samples and non-heated samples, respectively. Lanes 1,2, wild TC14-3. Lanes 3,4, wild TC14-2. Lanes 5,6, TC14-2(21-60)/TC14-3(61-145). Lanes 7,8, TC14-3F65D. Lanes 9,10, TC14-3T69R. Lanes 11,12, TC14-2R69T.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293724&req=5

Figure 4: Electrophoretic mobility of wild-type and mutant TC14s on SDS-PAGE. Odd lanes and even lanes show heat-denatured samples and non-heated samples, respectively. Lanes 1,2, wild TC14-3. Lanes 3,4, wild TC14-2. Lanes 5,6, TC14-2(21-60)/TC14-3(61-145). Lanes 7,8, TC14-3F65D. Lanes 9,10, TC14-3T69R. Lanes 11,12, TC14-2R69T.
Mentions: TC14-3 exhibited a relative electrophoretic mobility of 15 kDa (Figure 4, lane 1) on SDS-PAGE following heat denaturation, while under non-heated conditions, more than 99% of the total protein exhibited a relative mobility of 30 kDa (Figure 4, lane 2; Table 2). In contrast, TC14-2 exhibited a single band of 18 kDa following heat denaturation (Figure 4, lane 3) and separated into 2 bands of 18 and 28 kDa under non-heated conditions (Figure 4, lane 4). The 28-kDa form of TC14-2 accounted for approximately 61% of the total amount of protein (Table 2). The chimeric protein, TC14-221-60/TC14-361-145 exhibited an electrophoretic pattern similar to that of wild-type TC14-3 (Figure 4, lanes 5, 6). These results strongly suggest that wild-type TC14-3 may form more stable dimers than wild-type TC14-2.

Bottom Line: When Phe(65) of TC14-3 was mutated to an acidic amino acid, the resultant mutant protein failed to dimerize.PmEed knockdown by RNA interference rescued cultured cells from the growth-inhibitory effects of TC14-3.These results show that in P. misakiensis, the cytostatic activity of TC14-3 is mediated by PmEed and resultant histone modification, and that the gene expression requires both the protein dimerization and Ca(2+)-binding of TC14-3.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular and Molecular Biotechnology, Faculty of Science, Kochi University, Kochi 780-8520, Japan. kazuk@kochi-u.ac.jp

ABSTRACT

Background: As many invertebrate species have multipotent cells that undergo cell growth and differentiation during regeneration and budding, many unique and interesting homeostatic factors are expected to exist in those animals. However, our understanding of such factors and global mechanisms remains very poor. Single zooids of the tunicate, Polyandrocarpa misakiensis, can give off as many as 40 buds during the life span. Bud development proceeds by means of transdifferentiation of very limited number of cells and tissues. TC14-3 is one of several different but closely related polypeptides isolated from P. misakiensis. It acts as a cytostatic factor that regulates proliferation, adhesion, and differentiation of multipotent cells, although the molecular mechanism remains uncertain. The Polycomb group (PcG) genes are involved in epigenetic control of genomic activity in mammals. In invertebrates except Drosophila, PcG and histone methylation have not been studied so extensively, and genome-wide gene regulation is poorly understood.

Results: When Phe(65) of TC14-3 was mutated to an acidic amino acid, the resultant mutant protein failed to dimerize. The replacement of Thr(69) with Arg(69) made dimers unstable. When Glu(106) was changed to Gly(106), the resultant mutant protein completely lost Ca(2+) binding. All these mutant proteins lacked cytostatic activity, indicating the requirement of protein dimerization and calcium for the activity. Polyandrocarpa Eed, a component of PcG, is highly expressed during budding, like TC14-3. When wild-type and mutant TC14-3s were applied in vivo and in vitro to Polyandrocarpa cells, only wild-type TC14-3 could induce Eed without affecting histone methyltransferase gene expression. Eed-expressing cells underwent trimethylation of histone H3 lysine27. PmEed knockdown by RNA interference rescued cultured cells from the growth-inhibitory effects of TC14-3.

Conclusion: These results show that in P. misakiensis, the cytostatic activity of TC14-3 is mediated by PmEed and resultant histone modification, and that the gene expression requires both the protein dimerization and Ca(2+)-binding of TC14-3. This system consisting of a humoral factor, PcG, and histone methylation would contribute to the homeostatic regulation of cell growth and terminal differentiation of invertebrate multipotent cells.

Show MeSH
Related in: MedlinePlus