Nucleosome positioning by the winged helix transcription factor HNF3

Abstract

Nucleosome positioning at genetic regulatory sequences is not well understood. The transcriptional enhancer of the mouse serum albumin gene is active in liver, where regulatory factors occupy their target sites on three nucleosome-like particles designated N1, N2, and N3. The winged helix transcription factor HNF3 binds to two sites near the center of the N1 particle. We created dinucleosome templates using the albumin enhancer sequence and found that site-specific binding of HNF3 protein resulted in nucleosome positioning in vitro similar to that seen in liver nuclei. Thus, binding of a transcription factor can position an underlying nucleosome core.

Figure 1

Assembly and characterization of albumin enhancer dinucleosomes. (A) A 428-bp albumin enhancer fragment used for nucleosome assembly; end positions on the enhancer sequence (Liu et al. 1991) are shown (top). Transcription factor binding sites eD to eX are indicated. Arrows below ovals (core particles) indicate random positioning. (B) Agarose gel analysis of gradient fractions. (C) DNA from MNase digestion of free DNA, mononucleosomes (1N), and dinuclueosomes (2N). (M) φX174 marker; (1N) mononucleosome-sized intermediates; (2N) additional material in the dinucleosome fraction. (D) Electron microscopic analysis of dinucleosome samples.

Figure 2

DNase I footprinting of HNF3 bound to dinucleosomes. (A) Wild-type enhancer templates. (Lane 1) G sequencing reaction. Brackets indicate positions of the N1 and N2 particles in liver nuclei (McPherson et al. 1993). Numbers at left indicate enhancer positions of liver particle boundaries; numbers at top and bottom indicate extent of enhancer sequence. (B) Templates containing mutations of the HNF3-binding sites. At 100 nm HNF3, some nonspecific hypersensitive cleavages on the templates is observed, but no footprints.

Figure 3

Nucleosome positioning by HNF3. (A–C) 4 nm concentrations of free DNA or dinucleosomes were incubated with HNF3 and digested with MNase for five min. Double-stranded MNase cleavage sites were mapped by LM–PCR. The labeled primer position is depicted in D. (A) (Lane 1) G cleavage; (lanes 2,3) 0.015 U/ml of MNase; (lane 4) 0.05 U/ml. (B,C) (Lanes 1,3) 0.025 U/ml; (lanes 2,4,5) 0.05 U/ml. The oval at the right of B marks the region of MNase protection upon HNF3 binding to the wild-type dinucleosomes. Data in all panels are from the same gel.

Figure 4

Positioning of a nucleosome array. (A) (Lane 1) G cleavage; (lane 2) no MNase; (lanes 3,4) 0.075 and 0.15 U/ml of MNase, respectively, for 1 min digestion. (B,C) (Lanes 1,3) 0.075 U/ml; (lanes 2,4) 0.15 U/ml; (lane 5) 0.25 U/ml; all for 1 min digestion. The ovals at the right of B mark MNase-protected regions. • and ○ at right in B indicate enhanced and protected sites, respectively, of MNase cleavage. Data in all panels are from the same gel. (D) Priming strategy.