Human replication-dependent histone H3 genes are activated by a tandemly arranged pair of two CCAAT boxes

Abstract

We have analysed the transcriptional regulation of the human histone H3 genes using promoter deletion series, scanning mutagenesis, specific mutagenesis and electrophoretic mobility-shift assay experiments. The promoters of five of the six examined histone H3 genes showed near-maximal activity at lengths of 133-227 bp: H3/d 198 bp, H3/h 147 bp, H3/k 133 bp, H3/m 227 bp, H3/n 140 bp (exception H3/i). To search for functional cis-elements within these regions, we performed scanning mutagenesis of the two histone H3 promoters H3/k and H3/m. Mutagenesis revealed that the functional framework of the histone H3 promoters consists of a TATA box and two tandemly arranged CCAAT boxes in relatively fixed positions. Alterations of the distance between the CCAAT boxes and of the distance between the CCAAT boxes and the TATA box resulted in significant loss of activity. In electrophoretic mobility-shift assay experiments, the factor CBF (CCAAT-binding factor)/NF-Y (nuclear factor-Y) bound to isolated CCAAT boxes of the H3/k promoter. This suggests that an initiation complex is formed on the histone H3 promoter that has a defined structure and limited flexibility, consisting of two molecules of CBF/NF-Y and further (general or specific) transcription factors.

Figure 1. Reporter gene analysis of histone H3 promoter deletion series in the HEK-293 cell line

The reporter gene activity of the indicated constructs is shown as a statistical result (relative to the longest construct, which is set to the value of 1.0; error bars, S.E.M.; c, control construct pGL3-basic) of five independent experiments. A diagram of the promoters with the lengths of the constructs indicated is shown below each graph. Arrow, transcription start site; numbers, distance to transcription start site in bp; luc⁺, firefly luciferase coding sequence; black boxes, TATA boxes; white boxes, CCAAT boxes; grey boxes, ATF boxes; fusion site, ATG start codon.

Figure 2. Scanning mutagenesis of the promoters of histone H3 genes H3/k (A) and H3/m (B)

Top panels: to scale diagram of the promoters. luc⁺, firefly luciferase coding sequence; arrow, transcription start site; black boxes, TATA box; white boxes, CCAAT boxes; grey box, ATF box. The mutated areas and the names of the mutated constructs are indicated at the bottom of each diagram. The positions of the deletion constructs are shown on top of each diagram for orientation. Middle panels: nucleotide sequences of the promoters. The regions that were mutated in the respective mutation constructs are underlined and the mutated sequence is shown above in bold italic style. The start codon of the firefly luciferase coding sequence, the CCAAT boxes, the TATA boxes and the ATF box are shown in bold. Bottom panels: the reporter gene activity of the constructs in the HEK-293 cell line is shown as a statistical mean for four independent experiments [relative to the wild-type promoter K5 (wt) or M4 (wt) respectively, which is set to the value of 1.0; error bars, S.E.M.].

Figure 3. Specific mutagenesis of the CCAAT boxes of the histone H3/k promoter

To scale diagrams of the histone H3/k promoter: luc⁺, firefly luciferase coding sequence; arrow, transcription start site; black boxes, TATA box; white boxes, CCAAT boxes. At the bottom of each diagram, the wild-type sequence of the region surrounding the proximal CCAAT box (upper panel) and the distal CCAAT box (lower panel) and the mutated sequence of the mutation constructs are shown (mutated bases are inversely shaded). The reporter gene activity of the constructs in the HEK-293 cell line is shown below each diagram as a statistical mean for four independent experiments [relative to the wild-type promoter K5 (wt), which is set to the value of 1.0; error bars, S.E.M.].

Figure 4. Autoradiograms of the EMSA experiments

(A) Competition experiments with the proximal CCAAT box of the histone H3/k promoter (KProx). Composition of the individual incubation mixtures: labelled oligonucleotides are marked with an *, proteins added (HNE, HeLa nuclear extract; NF-Y, isolated NF-Y) are indicated by ±, competitors (80-fold molar excess) and antibody are indicated in the lower part of the heading. The NF-Y double bands are marked by black arrowheads. It may be noted that the specific activity of the NF-Y * probe used was much lower than the specific activity of the KProx* probe. (B) Supershift and complex-binding experiments with HNE and recombinant NF-Y. The experimental set-up and the heading is the same as for (A). To the incubation mixture indicated by ab CBF-A, 1 μg of antibody raised against NF-YB was added. Approx. 100 ng of isolated NF-Y complex was added to the labelled oligonucletides where indicated. The supershift band is indicated by the open arrow. (C) Titration competition experiments. The binding of labelled double-stranded oligonucleotides containing the consensus sequence of the binding site of NF-Y was competed by increasing concentrations of unlabelled double-stranded oligonucleotides (NF-Y, KProx and KDist). The molar excess of unlabelled competitor oligonucleotide is indicated. The intensities of the signals were quantified with respect to the PhosphoImager signals by the Aida 2.2 software from Raytest and expressed as the percentage ratio to the uncompeted signal intensities.

Figure 5. Changes in the distances between the relevant cis-elements in the histone H3/k promoter

To scale diagrams of the histone H3/k promoter: luc⁺, firefly luciferase coding sequence; arrow, transcription start site; black boxes, TATA boxes; white boxes, CCAAT boxes. At the bottom of each diagram, the positions of inserted or deleted sequences are shown: grey boxes, inserted sequences; white boxes with pair of scissors, deleted sequences. The reporter gene activity of the constructs in the HEK-293 cell line is shown as a statistical mean for four independent experiments [relative to the wild-type promoter K5 (wt), which is set to the value of 1.0; error bars, S.E.M.].