Nucleosome-mediated synergism between transcription factors on the mouse mammary tumor virus promoter

Abstract

In unstimulated mammalian cells and in Saccharomyces cerevisiae, the mouse mammary tumor virus (MMTV) promoter is silent and organized into positioned nucleosomes, one of which encompasses the binding sites for glucocorticoid receptor (GR) and nuclear factor I (NFI). Glucocorticoid induction in vivo involves a functional synergism between GR and NFI and simultaneous occupancy of the promoter sites for both proteins that cannot be reproduced on naked DNA. The role of chromatin in the process of induction was investigated by manipulating the nucleosome density in yeast strains carrying a regulated histone H4 gene. Following depletion of nucleosomes, independent transactivation by NFI or by GR, as well as binding of the individual proteins to the MMTV promoter, were enhanced, in agreement with a repressive function of nucleosomes. In contrast, NFI-dependent hormone induction of the promoter and the simultaneous binding of receptor and NFI were compromised by nucleosome depletion. This effect could be partly mediated by a cryptic binding site for the receptor that is functional only in the nucleosomal context. Thus, positioned nucleosomes do not only account for constitutive repression of the MMTV promoter, but also participate in induction by mediating cooperative binding and functional synergism between GR and NFI.

Figure 1

Repression of histone H4 gene results in nucleosome depletion over the MMTV promoter. (A) Schematic representation of histone H4-mediated nucleosome depletion, according to the method developed by Grunstein and collaborators (26). (B) Superhelicity of episomes carrying the MMTV–lacZ reporter in SChY50 cells grown in galactose- or glucose-containing media (17). The modal value of the distribution of topoisomers is indicated by arrows. The difference between cells grown in galactose (Gal) and in glucose (Glu) corresponds to 40% nucleosome depletion. Superhelicity analysis was performed as described (32) but hybridizing with an Escherichia coli lacZ probe. (C) Micrococcal nuclease (MNase) sensitivity of MMTV promoter in normal (galactose) or histone H4-depleted chromatin (glucose) of SChY50 cells (17). Note that in glucose medium the nuclease digestion is more effective and leads to appearance of shorter polynucleosomes and mononucleosomes. MMTV sensitivity to micrococcal nuclease was assayed as described (17) with a probe extending from −173 to −78 in the MMTV promoter.

Figure 2

Nucleosome depletion derepresses NFI-dependent transcription of MMTV promoter. (A) Yeast strains with the histone H4 gene under its own promoter or under galactose (Gal) control, and containing a complete MMTV–lacZ reporter with a positioned nucleosome, were transformed with an expression vector for NFI, NFI–VP16, or empty vector (23). β-Galactosidase activities of cultures switched from galactose to glucose (Glu) or kept in galactose are shown. (B) A similar yeast strain with the histone H4 gene under galactose control and carrying a truncated MMTV–lacZ reporter, depicted at the bottom, was assayed as in A. Note the difference in scale between A and B.

Figure 4

Genomic footprinting over the MMTV promoter. (A Left) At low nucleosome density (glucose; Glu) there is a better access of GR to the HRE5 (marked by a triangle on the right) in response to ligand. The asterisks on the left mark two bands detected under all conditions, which do not correspond to guanines in the DNA sequence. Their identity is unknown. (Right) In the presence of NFI, at low nucleosome density, a cluster of guanines within the NFI site (triangle) is more protected before hormone treatment (lane 8) than after hormone induction (lane 7). The relevant guanine on HRE4 is marked by a triangle. (B) PhosphorImager quantitation of the protection over the NFI site and the HRE2, -3, and -4, derived from three experiments similar to that shown in A Right. The values were normalized for variations in loading using the strong band within the octamer distal site (OctD) at the bottom of the gel. The mean and standard deviation are shown.

Figure 3

Nucleosome depletion does not inhibit GR function but hinders the synergistic activation of MMTV promoter by GR and NFI. (A) A yeast strain with the histone H4 gene under Gal control was transformed with the episomal GRE–lacZ reporter described in the scheme, together with an expression vector for rat GR (27). Cultures were incubated with ligand or vehicle after switching cells from galactose (Gal) to glucose (Glu) or keeping them in galactose. β-Galactosidase activities are shown. (B Left) β-Galactosidase activities obtained in experiments similar to those described in A but using the MMTV–lacZ reporter depicted in Fig. 2A. (Right) Results obtained from a yeast strain with the histone H4 gene driven by its own promoter. (C) Functional synergism between GR and NFI. Synergism is defined as the ratio of the activity found in the presence of GR/DAC and NFI over the sum of the activity found with GR/DAC in the absence of NFI and the activity measured with NFI in the absence of GR/DAC (Fig. 2A Right). (D) Similar experiments to those described in B but in strains additionally transformed with an NFI-expressing vector.

Figure 5

HRE5 participates in synergistic induction by ligand activated GR. (A) Influence of mutations in the HRE5 site on the activity of the MMTV promoter. The mutation is described in Materials and Methods. (B) Schematic array of sites: distance between HRE5 and the NFI site. (C) Hypothetical model of GR and NFI bound to their cognate sites on the surface of a nucleosome. A GR (bold) bound to HRE5 would be close to the DNA-bound NFI. The model and the positions of the cis elements are not drawn to scale.