Apoptosis Is Induced in BHK Cells by the tsBN462/13 Mutation in the CCG1/TAFII250 Subunit of the TFIID Basal Transcription Factor

doi:10.1006/excr.1995.1183

Experimental Cell Research

Volume 218, Issue 2, June 1995, Pages 490-498

https://doi.org/10.1006/excr.1995.1183 Get rights and content

Abstract

A temperature-sensitive (ts) mutant of the BHK21 cell line derived from golden hamsters, tsBN462 has a mutation in the gene encoding the largest subunit of the TFIID complex, TAF_II250/p230/CCG1, and arrests in the G1 phase at the nonpermissive temperature, 39.5°C. We found that tsBN462 cells underwent apoptosis following growth arrest at 39.5°C, suggesting a role for CCG1 as a repressor of apoptosis. By electron microscopic observation, tsBN462 cells at 39.5°C showed characteristic features of apoptosis. Apoptosis was not suppressed by expression of Bc1-2 or the adenovirus E1B 19 kDa protein. Cell death was suppressed completely by expression of wild-type CCG1 and partially by wild-type p53, a growth suppressor protein. Cell cycle arrest induced by p53 may help survival of tsBN462 cells at 39.5°C. Apoptosis was accelerated in SV40 large T antigen-transformed tsBN462 cells at 39.5°C where SV40 large T antigen formed a complex with p53, implying that the apoptosis of tsBN462 cells at 39.5°C occurred in a p53-independent manner. Our results suggest that CCG1/TAF_II250 is required for the expression of factors regulating apoptosis.

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Cited by (36)

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Due to the pleiotropic activities of Taf1, it is difficult to selectively knock out the UBAC activity to establish whether Taf1 is the only enzyme responsible for the monoubiquitination of Pax3. To attest to this difficulty, the ts13 or tsBN462 cell lines, which lack the acetyltransferase activity of Taf1, display cell-cycle arrest and apoptosis (Sekiguchi et al., 1995). Based on a mutation in Drosophila with reduced UBAC activity (Pham and Sauer, 2000), we generated homologous mutations in the mammalian Taf1 protein.
Pax3 plays critical roles during developmental and postnatal myogenesis. We have previously shown that levels of Pax3 protein are regulated by monoubiquitination and proteasomal degradation during postnatal myogenesis, but none of the key regulators of the monoubiquitination process were known. Here we show that Pax3 monoubiquitination is mediated by the ubiquitin-activating/conjugating activity of Taf1, a component of the core transcriptional machinery that was recently reported to be downregulated during myogenic differentiation. We show that Taf1 binds directly to Pax3 and overexpression of Taf1 increases the level of monoubiquitinated Pax3 and its degradation by the proteasome. A decrease of Taf1 results in a decrease in Pax3 monoubiquitination, an increase in the levels of Pax3 protein, and a concomitant increase in Pax3-mediated inhibition of myogenic differentiation and myoblast migration. These results suggest that Taf1 regulates Pax3 protein levels through its ability to mediate monoubiquitination, revealing a critical interaction between two proteins that are involved in distinct aspects of myogenic differentiation. Finally, these results suggest that the components of the core transcriptional are integrally involved in the process of myogenic differentiation, acting as nodal regulators of the differentiation program.
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2004, Molecular Cell
The largest subunit of TFIID, TAF1, possesses an intrinsic protein kinase activity and is important for cell G1 progression and apoptosis. Since p53 functions by inducing cell G1 arrest and apoptosis, we investigated the link between TAF1 and p53. We found that TAF1 induces G1 progression in a p53-dependent manner. TAF1 interacts with and phosphorylates p53 at Thr-55 in vivo. Substitution of Thr-55 with an alanine residue (T55A) stabilizes p53 and impairs the ability of TAF1 to induce G1 progression. Furthermore, both RNAi-mediated TAF1 ablation and apigenin-mediated inhibition of the kinase activity of TAF1 markedly reduced Thr-55 phosphorylation. Thus, phosphorylation and the resultant degradation of p53 provide a mechanism for regulation of the cell cycle by TAF1. Significantly, the Thr-55 phosphorylation was reduced following DNA damage, suggesting that this phosphorylation contributes to the stabilization of p53 in response to DNA damage.
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In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast—reactive oxygen species (ROS)—can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.
Transcription initiation factor IID-interactive histone chaperone CIA-II implicated in mammalian spermatogenesis
2003, Journal of Biological Chemistry
Citation Excerpt :
The activity of CIA-II might also be controlled by protein modification of its interactive proteins such as core histones H3/H4 (37). The role of CIA-II in the control of apoptotic cell death is intriguing because the hamster tsBN462 cell, which has a mutation in the gene encoding CCG1, undergoes apoptosis following growth arrest at the nonpermissive temperature 39.5 °C (62). In addition, the S. cerevisiae mutant for the CIA-II counterpart (asf1) spontaneously dies because of preferential arrest at the G2/M phase during the cell cycle (31).
Histones are thought to have specific roles in mammalian spermatogenesis, because several subtypes of histones emerge that are post-translationally modified during spermatogenesis. Though regular assembly of nucleosome is guaranteed by histone chaperones, their involvement in spermatogenesis is yet to be characterized. Here we identified a histone chaperone-related factor, which we designated as CCG1-interacting factor A-II (CIA-II), through interaction with bromodomains of TAF_II250/CCG1, which is the largest subunit of human transcription initiation factor IID (TFIID). We found that human CIA-II (hCIA-II) localizes in HeLa nuclei and is highly expressed in testis and other proliferating cell-containing tissues. Expression of mouse CIA-II (mCIA-II) does not occur in the germ cell-lacking testes of adult WBB6F1-W/W^v mutant mice, indicating its expression in testis to be specific to germ cells. Fractionation of testicular germ cells revealed that mCIA-II transcripts accumulate in pachytene spermatocytes but not in spermatids. In addition, the mCIA-II transcripts in testis were present as early as 4 days after birth and decreased at 56 days after birth. These findings indicate that mCIA-II expression in testis is restricted to premeiotic to meiotic stages during spermatogenesis. Also, we found that hCIA-II interacts with histone H3 in vivo and with histones H3/H4 in vitro and that it facilitates supercoiling of circular DNA when it is incubated with core histones and topoisomerase I in vitro. These data suggest that CIA-II is a histone chaperone and is implicated in the regulation of mammalian spermatogenesis.
The cell cycle regulatory factor TAF1 stimulates ribosomal DNA transcription by binding to the activator UBF
2002, Current Biology
Citation Excerpt :
The clone was sequenced, and a nucleotide database search revealed that it is identical to human TAF1 (previously known as TAFII250) [12, 13]. TAF1 is the largest subunit of the TFIID complex and is identical to a factor that has been implicated in the regulation of G1-to-S progression (CCG1) [14–16]. The clone isolated from the yeast two-hybrid screen encodes for the carboxy-terminal portion of TAF1, amino acids 1096–1872.
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Transcription factor TFIID is a multiprotein complex composed of the TATA binding protein and its associated factors, and is required for accurate and regulated initiation of transcription by RNA polymerase II. The subunit composition of this factor is highly conserved from yeast to mammals. X-ray crystallography and biochemical experiments have shown that the histone fold motif mediates many of the subunit interactions within this complex. These results, together with electron microscopy and yeast genetics, provide insights into the overall organization of this complex.

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Regular ArticleApoptosis Is Induced in BHK Cells by the tsBN462/13 Mutation in the CCG1/TAFII250 Subunit of the TFIID Basal Transcription Factor

Abstract

Regular Article
Apoptosis Is Induced in BHK Cells by the tsBN462/13 Mutation in the CCG1/TAF_II250 Subunit of the TFIID Basal Transcription Factor