In Vivo Functional Analysis of the Histone 3-like TAF9 and a TAF9-related Factor, TAF9L

Abstract

The majority of the TATA-binding protein (TBP)-associated factors (TAFs) that constitute transcription factor II D (TFIID) contain histone fold motifs (HFMs). Our previous results utilizing DT40 cells containing a conditional TAF9 allele indicated that the histone 3-like TAF9 is essential for cell viability but largely dispensable for general transcription. In this study, we investigated further the role of TAF9 structural domains in TFIID integrity and cell growth and the functions of a TAF9-related factor, TAF9L. We first show that TAF9 depletion severely disrupts TFIID, indicating that the observed ongoing transcription is initiated with at least partially TAF-free TATA-binding protein. We also provide evidence for specific roles of TAF HFMs, highlighting the functional significance of HFM specificity observed in vitro and, importantly, of the TAF9-histone 3 similarity. Although we provide evidence that TAF9 and TAF9L are partly redundant, RNA interference experiments suggest that TAF9L is essential for HeLa cell growth. Strikingly, we provide evidence that TAF9L plays a role in transcriptional repression and/or silencing.Initiation of transcription mediated by RNA polymerase II (RNAP II) 1 requires a number of general transcription factors (GTFs), among which TFIID is the major core promoter recognition factor. TFIID is composed of highly conserved subunits, including the TATA-binding protein (TBP) and about 12-14 TBP-associated factors (TAFs) (1-4). Despite their diverse sequence and structural features, most TAFs (9 of 14 in yeast) contain domains known as histone fold motifs (HFMs) that display varying degrees of similarity with canonical histone core domains (5). TAF HFMs have been shown to mediate highly specific pairwise TAF-TAF interactions in cocrystal structures, in vitro interaction assays, and coexpression analyses (5-9). Furthermore, histone-like TAFs were shown in immunolabeling/electron microscopy analysis to colocalize to specific TFIID subdomains (10, 11) in a pattern consistent with the above pairwise TAF interactions (12). Additionally, four yeast histone-like TAFs, TAF6/9 (H4/H3-like) and TAF4/12 (H2A/ H2B-like), were reconstituted into a histone octamer-like complex in vitro (13). However, the roles of the HFMs in TFIID structure and function in vivo are still unclear for most TAFs, especially in vertebrates. For example, the highly conserved arginine residues in histones that contact DNA are missing in TAFs (14), suggesting that histones and histone-like TAFs contact DNA differently.A subset of the histone-like TAFs were also found to be integral components of histone acetyltransferase (HAT) protein complexes, including SAGA in yeast and GCN5, PCAF, TFTC, and STAGA in vertebrates (2, 3, 15, 16). These HAT complexes are larger than TFIID, containing GCN5 or PCAF HAT activity and other factors likely involved in diverse nuclear processes (17, 18). Importantly, however, histone-like TAFs that are missing in the vertebrate HAT complexes are replaced with a different set of HFM-containing sub...

TAF9b (Formerly TAF9L) Is a Bona Fide TAF That Has Unique and Overlapping Roles with TAF9

“…8B), further suggesting that only TAF9-or TAF9b-regulated genes exist and that the two proteins regulate only partially overlapping transcription programs. In agreement with several previous studies (11,20,27,28,32,53), our results also demonstrate that TAF depletion (either TAF9 or TAF9b) causes both up-and down-regulation of gene expression.…”

“…Our results also demonstrate that TAF depletion (either TAF9 or TAF9b) causes both up-and down-regulation of gene expression, further suggesting that TAFs can act as both positive and negative regulatory factors. This finding together with other TAF knockdown studies carried out mainly in yeast (11,20,27,28,32,53) raise an interesting hypothesis that TAFs, when binding to promoters in the different TAF-containing complexes (TFIID and TFTC type), would be involved at certain promoters as repressors and at others as positive cofactors of transcription.…”

“…The finding that there are genes regulated only by TAF9 or TAF9b suggests that in HeLa cells, TAF9 cannot structurally and/or functionally replace TAF9b and vice versa. Surprisingly, human TAF9b (formerly hTAF9L) could restore the viability of the chicken DT40 TAF9 Ϫ/Ϫ cells (11), suggesting that in chicken cells, only one TAF9 gene exists and that at this stage of evolution, TAF9 and TAF9b functions were not yet separated. Our results also demonstrate that TAF depletion (either TAF9 or TAF9b) causes both up-and down-regulation of gene expression, further suggesting that TAFs can act as both positive and negative regulatory factors.…”

“…Furthermore, human TAF9L was suggested to play a role in transcriptional repression and/or silencing (11). However, the association of TAF9L with TBP was not demonstrated.…”

“…Previously, it was shown that TAF9b (formerly TAF9L) is essential for cell growth in human cells (11) and that TAF9 depletion leads to apoptotic cell death in chicken cells (12). To further characterize the functional differences between human TAF9 and TAF9b during normal cell growth and in the same system, we specifically knocked down either TAF9 or TAF9b expression by using siRNAs.…”