J. Mol. Biol. (1990) 214, 865-874
A Ubiquitous CCAAT Factor is Required for Efficient irz Vitro Transcription from the Mouse Albumin Promoter ExBme Wuarin, Department Quai
Christopher
Mueller
and Ueli Schibler
of Molecular Biology, Sciences II, University of Geneva Ernest Ansermet 30, CH-1211 Geneva-4, Switzerland
(Received 13 March
1990; accepted 2 Nay
1990)
Among the various factors binding to DNA elements within the mouse albumin promoter, NF-Y is the only one present at identical concentrations in the nuclei of all examined tissues. NF-Y binds to albumin promoter element C, which contains the sequence CCAAT. To determine whether this factor augments in vitro transcription from the albumin promoter, an extensive point-mutation analysis was performed wit’hin the promoter element C. In liver extracts, six out of the ten mutations result in a strong inhibition of NF-Y decrease in promoter activity. Two mut,ations that increase binding and in a concomitant the affinity of the C-element for NF-Y also augment, the transcription efficiency from the albumin promoter. A similarly strong correlation of NF-Y-binding with transcription efficiency has also been observed in spleen nuclear extracts. The liver-enriched CCAAT and enhancer binding factor C/EBP also recognizes the C element. In contrast to NF-Y; no correlation between the affinity of mutant C-elements for C/EBP and transcript’ional activity could be observed in liver nuclear extracts.
reached by Heard et al. (1987) on the basis of DNA transfection studies. This promoter region contains six distinct elements (A to F) that are protected from DNase I digestion by proteins present in a rat et al., 1987). Most liver nuclear extract (Lichtsteiner of the factors interacting with the various albumin promoter elements have been identified (Lichtsteiner et al., 1987; Schorpp et al., 1988: Courtois et al., 1987; Baumhueter et al., 1988; Babiss et al., 1987; Costa et al., 1988a,b; Cereghini et al.: 198’7; Raymondjean et al., 1988; Lichtsteiner & Schibler, 1989; Mueller et al., 1990). Figure 1 shows the inventory of transcription factors with affinity to the various k-acting elements found within t’he albumin promoter. C/EBP (Graves et al., 1986; Johnson et al., 1987; Landschulz et al., 1988a,b; Vinson et al., 1988) has a high affinity for site D, lower affinities for sites A and F, and an even lower but readily measurable affinity for site C (see below; and see Lichtsteiner et al., 1987; Mueller et al., 1990). Site E can be filled with several members of the CTF/NFl factor family, NF-Y (Dorn et al., 1987; Raymondjean et al., 1988), one of several CAAT factors, recognizes site C which bears the common promoter motif CCAAT. Finally, site B is a highaffinity site for HNFl, a factor binding to promoter elements of a number of liver-specific genes (Lichtsteiner & Schibler, 1989; Cereghini et al., 1988; Courtois et al., 1988). Most of these factors, such as HNFl (Courtois et al., 1987; Baumhueter et al.; 1988; Lichtsteiner &. Schibler, 1989), C-EBP
1. Introduction The tissue-specific activation of many genes is controlled primarily at the level of transcription initiation (e.g. see Derman et al., 1981; Schibler et al., 1983; Tilghman & Belayew, 1982; Shaw et al., 1985). For several cases; it has been suggested that the combinatorial interplay of trans-acting transcription factors with &s-acting promoter elements plays an important role in controlling differential transcription (for a review, see Maniatis et al., 1983). Therefore, a detailed knowledge of these interactions is essential for understanding the molecular basis of cell-type specific gene expression. The developmentally regulat’ed expression of the serum albumin gene provides an excellent model system for studies of this nature (Tilghman & Belayew, 1982; Krumlauf et al., 1985; Godbout et al., 1986; Ott et al., 1984; Heard et aZ., 1987; Gorski et al., 1986). Furthermore, the elaboration in our laboratory of a, tissue-specific in vitro transcription system differential accurately reproduces the that, expression of the albumin gene renders this system amenable to biochemical dissection (Gorski et al., 1986: Lichtsteiner et al., 1987; Lichtsteiner & Schibler, 1989; Maire et al., 1989). Previously reported studies from our laboratory indicated that the minimal albumin promoter sequences required for optimal in, vitro transcription span about’ 170 nucleotides of 5’ flanking region et al., 1986). A similar conclusion was (Gorski
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A Ubiquitous CCAAT Factor is Required for Efficient irz Vitro Transcription from the Mouse Albumin Promoter ExBme Wuarin, Department Quai
Christopher
Mueller
and Ueli Schibler
of Molecular Biology, Sciences II, University of Geneva Ernest Ansermet 30, CH-1211 Geneva-4, Switzerland
(Received 13 March
1990; accepted 2 Nay
1990)
Among the various factors binding to DNA elements within the mouse albumin promoter, NF-Y is the only one present at identical concentrations in the nuclei of all examined tissues. NF-Y binds to albumin promoter element C, which contains the sequence CCAAT. To determine whether this factor augments in vitro transcription from the albumin promoter, an extensive point-mutation analysis was performed wit’hin the promoter element C. In liver extracts, six out of the ten mutations result in a strong inhibition of NF-Y decrease in promoter activity. Two mut,ations that increase binding and in a concomitant the affinity of the C-element for NF-Y also augment, the transcription efficiency from the albumin promoter. A similarly strong correlation of NF-Y-binding with transcription efficiency has also been observed in spleen nuclear extracts. The liver-enriched CCAAT and enhancer binding factor C/EBP also recognizes the C element. In contrast to NF-Y; no correlation between the affinity of mutant C-elements for C/EBP and transcript’ional activity could be observed in liver nuclear extracts.
reached by Heard et al. (1987) on the basis of DNA transfection studies. This promoter region contains six distinct elements (A to F) that are protected from DNase I digestion by proteins present in a rat et al., 1987). Most liver nuclear extract (Lichtsteiner of the factors interacting with the various albumin promoter elements have been identified (Lichtsteiner et al., 1987; Schorpp et al., 1988: Courtois et al., 1987; Baumhueter et al., 1988; Babiss et al., 1987; Costa et al., 1988a,b; Cereghini et al.: 198’7; Raymondjean et al., 1988; Lichtsteiner & Schibler, 1989; Mueller et al., 1990). Figure 1 shows the inventory of transcription factors with affinity to the various k-acting elements found within t’he albumin promoter. C/EBP (Graves et al., 1986; Johnson et al., 1987; Landschulz et al., 1988a,b; Vinson et al., 1988) has a high affinity for site D, lower affinities for sites A and F, and an even lower but readily measurable affinity for site C (see below; and see Lichtsteiner et al., 1987; Mueller et al., 1990). Site E can be filled with several members of the CTF/NFl factor family, NF-Y (Dorn et al., 1987; Raymondjean et al., 1988), one of several CAAT factors, recognizes site C which bears the common promoter motif CCAAT. Finally, site B is a highaffinity site for HNFl, a factor binding to promoter elements of a number of liver-specific genes (Lichtsteiner & Schibler, 1989; Cereghini et al., 1988; Courtois et al., 1988). Most of these factors, such as HNFl (Courtois et al., 1987; Baumhueter et al.; 1988; Lichtsteiner &. Schibler, 1989), C-EBP
1. Introduction The tissue-specific activation of many genes is controlled primarily at the level of transcription initiation (e.g. see Derman et al., 1981; Schibler et al., 1983; Tilghman & Belayew, 1982; Shaw et al., 1985). For several cases; it has been suggested that the combinatorial interplay of trans-acting transcription factors with &s-acting promoter elements plays an important role in controlling differential transcription (for a review, see Maniatis et al., 1983). Therefore, a detailed knowledge of these interactions is essential for understanding the molecular basis of cell-type specific gene expression. The developmentally regulat’ed expression of the serum albumin gene provides an excellent model system for studies of this nature (Tilghman & Belayew, 1982; Krumlauf et al., 1985; Godbout et al., 1986; Ott et al., 1984; Heard et aZ., 1987; Gorski et al., 1986). Furthermore, the elaboration in our laboratory of a, tissue-specific in vitro transcription system differential accurately reproduces the that, expression of the albumin gene renders this system amenable to biochemical dissection (Gorski et al., 1986: Lichtsteiner et al., 1987; Lichtsteiner & Schibler, 1989; Maire et al., 1989). Previously reported studies from our laboratory indicated that the minimal albumin promoter sequences required for optimal in, vitro transcription span about’ 170 nucleotides of 5’ flanking region et al., 1986). A similar conclusion was (Gorski
865 002%2836/90/160865-10
$03.00/O
0
1990 Academic
Press Limited
J. Wuarin et al
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