Core promoter recognition complex changes accompany liver development Joseph A. D’Alessioa,b,c,1, Raymond Ngd, Holger Willenbringd,e, and Robert Tjianb,c,f,2 aJanelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147; bDepartment of Molecular and Cell Biology, and cHoward Hughes Medical Institute, University of California, Berkeley, CA 94720; dEli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA 94143; eDepartment of Surgery, Division of Transplantation, University of California, San Francisco, CA 94143; and fLi Ka Shing Center for Biomedical and Health Sciences, University of California, Berkeley, CA 94720 Contributed by Robert Tjian, January 19, 2011 (sent for review October 28, 2010) Recent studies of several key developmental transitions have muscle differentiation can proceed normally in the near absence brought into question the long held view of the basal transcrip- of intact TFIID provided an additional clue that core promoter tional apparatus as ubiquitous and invariant. In an effort to better complex switching may be more widespread than initially appre- understand the role of core promoter recognition and coactivator ciated (9, 10). In myotubes, an alternative core component, TAF3, complex switching in cellular differentiation, we have examined is retained and required for myogenic differentiation. An addi- changes in transcription factor IID (TFIID) and cofactor required tional recent example of the relative dispensability of TFIID for Sp1 activation/Mediator during mouse liver development. Here was the finding that inactivation of TAF10 has little or no affect we show that the differentiation of fetal liver progenitors to adult on adult hepatic gene expression (11). Thus, the notion of switch- hepatocytes involves a wholesale depletion of canonical cofactor ing or “remodeling” of the core promoter machinery may be more required for Sp1 activation/Mediator and TFIID complexes at both widespread than initially appreciated. the RNA and protein level, and that this alteration likely involves Almost simultaneously with the discovery of “atypical” TAFs silencing of transcription factor promoters as well as protein such as TAF4b, TBP-related factors (TRFs) or TBP-like proteins degradation. It will be intriguing for future studies to determine (TBPLs) were identified, further expanding potential core pro- if a novel and as yet unknown core promoter recognition complex moter recognition complex diversity. The first of these subunits, takes the place of TFIID in adult hepatocytes and to uncover TRF1, is restricted to Drosophila where it was shown to direct the mechanisms that down-regulate TFIID during this critical transcription by RNA polymerase III (12). The evolutionarily developmental transition. conserved TRF2 has been primarily examined during spermato- genesis, although its expression appears to be widespread TATA box-binding protein-associated factor ∣ hepatoblast ∣ hepatogenesis (13–15). Most recently, the vertebrate-specific TRF3 has been examined in oogenesis, hematopoiesis, and myogenesis. The ear- he precise and orderly differentiation of embryonic progeni- liest reports suggested that TRF3 may be widely expressed, albeit Ttors to committed adult cell types requires exquisite spatial at a low level, but significantly enriched in the developing oocyte and temporal control of gene expression. Until recently, the (16, 17). More detailed and simultaneous studies found that a dynamic changes in transcriptional output that accompany devel- unique TAF3-TRF3 complex may in some instances largely opmental transitions were assumed to depend exclusively on replace canonical TFIID in the regulation of hematopoietic and regulatory DNA and its associated sequence-specific activators myogenic genes, and that this change in core promoter recogni- and repressors, whereas core promoter recognition, coactivator, tion complexes was essential for both the myogenic transition in and chromatin modifier complexes were generally taken to be mice and hematopoietic differentiation in zebrafish and mouse ubiquitous and invariant from one cell type to the next. Indeed, cells (10, 18, 19). Importantly, the extensive down-regulation these so-called core factors are highly conserved from yeast to of canonical TFIID that accompanies myogenic development man (1). However, recent evidence suggests that several key de- is accompanied by the finding of a wholesale depletion of the velopmental transitions are accompanied by, and actually require, CRSP/Med coactivator complex (9). Most recently, ablation of dramatic changes in components of this general machinery includ- the TRF3 locus has revealed its essential role in germ cell devel- ing transcription factor IID (TFIID) composed of the TATA opment and the transcription of oogenic genes (20). As develop- box-binding protein (TBP) and its associated factors (TAFs), mental changes in the core promoter recognition machinery the cofactor required for Sp1 activation/Mediator (CRSP/Med) continue to be uncovered, yet other studies have revealed the complex, and the Brg1/Brm-associated factor (BAF) complex developmental dynamics of the chromatin remodeling machinery (for review, see 2). (for review, see ref. 21). The first evidence that some cell types may contain alterations Collectively these recent examples suggest that the long held “ ” of the general machinery came with the identification of germ- view of TFIID, BAF, and CRSP/Med complexes as general cell-specific TFIID subcomplexes in which key subunits are transcription factors that function at a majority of promoters replaced by paralogous components. Initially oogenesis and sper- in every cell type needs to be reconsidered. Historically these fac- matogenesis were found to require an altered TFIID in which tors have been studied in transformed or highly proliferative cell paralogous TAF4b replaces the canonical TAF4 (3, 4). Later types such as HeLa, and recent advancements in the purification spermatocytes were found to also express paralogous TAF7L that of more relevant primary cells have improved our ability to study may cooperate with TBP and TAF1 in the regulation of sperma- the dynamics of such factors in a physiologically and developmen- togenesis genes (5, 6). Although unique paralogs of other cano- nical TAFs have been identified, their tissue-specific expression Author contributions: J.A.D., H.W., and R.T. designed research; J.A.D. and R.N. performed and functional significance remain to be elucidated (7). The find- research; J.A.D. contributed new reagents/analytic tools; J.A.D., H.W., and R.T. analyzed ing that TAF8 is significantly up-regulated and may be required data; J.A.D. and R.T. wrote the paper. during adipogenesis is one of the few examples to date of a The authors declare no conflict of interest. canonical TAF that is normally associated with TFIID but Freely available online through the PNAS open access option. which may also have an independent developmental function (8). 1Present address: Igenica, Inc., Burlingame, CA 94010. The striking finding that some developmental programs such as 2To whom correspondence should be addressed. E-mail: [email protected]. 3906–3911 ∣ PNAS ∣ March 8, 2011 ∣ vol. 108 ∣ no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1100640108 tally relevant context. To further broaden our understanding of of the cells in the fetal liver, whereas hepatocytes make up how core machinery switching contributes to developmental tran- approximately 60% of the adult liver mass. Toinvestigate whether sitions and to establish the role of TFIID and CRSP/Med changes differences in the core machinery existed between the distinct during the development of multiple cell types, we have examined stages of hepatogenesis represented by these two cell popula- the expression of TFIID and CRSP/Med subunits in additional tions, we established a protocol for the purification of hepato- primary cells and their associated developmental programs. Pre- blasts from E13.5 mouse embryos based on enzymatic digestion vious reports suggested that the adult mammalian liver might of individually dissected fetal livers followed by magnetic- contain unusually low levels of both TBP and TAF4, critical com- bead-based depletion of TER119+ erythrocytes and CD45+ leu- ponents of canonical TFIID (3, 17). Here we report dramatic kocytes. Both fluorescence-activated cell sorting and immuno- changes in the cellular levels of both TFIID and CRSP/Med fluorescence staining of CD45, TER119, E-Cadherin, and DLK1 during mouse liver development. showed these preparations to contain greater than 90% pure hepatoblasts, consistent with previous results (23). Adult hepato- Results and Discussion cytes from 8 to 10-wk-old mice were purified using established in Differential Expression of TFIID in Hepatoblasts and Hepatocytes. As a situ collagenase perfusion protocols (24). first step toward identifying additional developmental programs We compared the levels of TBP, multiple TAFs, and other gen- that may involve down-regulation of TFIID, we generated whole eral transcription factors in these highly purified hepatoblasts to cell extracts from adult mouse tissues and compared the TBP, those in hepatocytes by immunoblot (Fig. 1 A and B). Strikingly, TAF1, and TAF4 levels to those in myoblasts by immunoblotting. protein levels of TBP and every TAF subunit examined are Consistent with previous results, we find that the adult liver con- dramatically lower in adult hepatocytes