Mathias Truss

Hormone induces binding of receptors and transcription factors to a rearranged nucleosome on the MMTV promoter in vivo.

Hormonal induction of the mouse mammary tumour virus (MMTV) promoter is mediated by interactions between hormone receptors and other transcription factors bound to a complex array of sites. Previous results suggested that access to these sites is modulated by their precise organization into a positioned regulatory nucleosome. Using genomic footprinting, we show that MMTV promoter DNA is rotationally phased in intact cells containing either episomal or chromosomally integrated proviral fragments. Prior to induction there is no evidence for factors bound to the promoter. Following progesterone induction of cells with high levels of receptor, genomic footprinting detects simultaneous protection over the binding sites for hormone receptors, NF‐I and the octamer binding proteins. Glucocorticoid or progestin induction leads to a characteristic chromatin remodelling that is independent of ongoing transcription. The centre of the regulatory nucleosome becomes more accessible to DNase I and restriction enzymes, but the limits of the nucleosome are unchanged and the 145 bp core region remains protected against micrococcal nuclease digestion. Thus, the nucleosome covering the MMTV promoter is neither removed nor shifted upon hormone induction, and all relevant transcription factors bind to the surface of the rearranged nucleosome. Since these factors cannot bind simultaneously to free DNA, maintainance of the nucleosome may be required for binding of factors to contiguous sites.

Transcriptional regulation by steroid hormones.

Steroid hormones influence the transcription of a large number of genes by virtue of their interaction with intracellular receptors, which are modular proteins composed of a ligand binding domain, a DNA binding domain, and several transactivation functions distributed along the molecule. The DNA binding domain is organized around two zinc ions and allows the receptors to bind as homodimers to palindromic DNA sequences, the hormones responsive elements (HRE), is such a way that each homodimer contacts one half of the palindrome. Since the two halves are separated by three base pairs, the two homodimers contact the same face of the double helix. Before hormone binding, the receptors are part of a complex with multiple chaperones which maintain the receptor in its steroid binding conformation. Following hormone binding, the complex dissociates and the receptors bind to HREs in chromatin. Regulation of gene expression by hormones involves an interaction of the DNA-bound receptors with other sequence-specific transcription factors and with the general transcription factors, which is partly mediated by co-activators and co-repressors. The specific array of cis regulatory elements in a particular promoter/enhancer region, as well as the organization of the DNA sequences in nucleosomes, specifies the network of receptor interactions. Depending on the nature of these interactions, the final outcome can be induction or repression of transcription. The various levels at which these interactions are modulated are discussed using as an example the promoter of the Mouse Mammary Tumor Virus and its organization in chromatin.

Periodic cdc25C transcription is mediated by a novel cell cycle-regulated repressor element (CDE).

We show that the cell cycle‐regulated transcription of the TATA‐less cdc25C gene in late S/G2 is largely mediated by a novel promoter element (CDE) located directly 5′ to one of the two major transcription initiation sites. Genomic dimethylsulfate footprinting experiments, using either synchronized or sorted normally cycling cells, show the formation in vivo of a CDE‐protein complex in both G0 and G1 cells and its dissociation in G2. Mutation of the CDE severely impairs cell cycle regulation of the cdc25C promoter and results in high expression in G0/G1, indicating that the CDE functions as a cell cycle‐regulated cis‐acting repressor element. Cell cycle regulation is also lost upon removal of the enhancer region located immediately upstream of the CDE, but is largely restored when this enhancerless minimal cdc25C promoter fragment is linked to the constitutive SV40 early enhancer. This indicates that the CDE is dependent on the presence of a transcriptional enhancer to effect cell cycle regulation. Our observations suggest that the periodic activation of the cdc25C gene in late S/G2 is brought about, at least in part, by a unique regulatory mechanism involving the cell cycle‐regulated dissociation of a repressor from the CDE.

Structural features of a regulatory nucleosome

DNA sequences from the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR) position nucleosomes both in vivo and in vitro. Here, were present chromatin reconstitution experiments showing that MMTV-LTR sequences from -236 to +204 accommodate two histone octamers in positions compatible with the in vivo data. This positioning is not influenced by the length of the DNA fragment and occurs in linear as well as in closed circular DNA molecules. MMTV-LTR DNA sequences show an intrinsic bendability that closely resembles its wrapping around the histone octamer. We propose that bendability is responsible for the observed rotational nucleosome positioning. Translational nucleosome positioning seems also to be determined by the DNA sequence. These data, along with the results from reconstitution experiments with insertion mutants, support a modular model of nucleosome phasing on MMTV-LTR, where the actual positioning of the histone octamer results from the additive effect of multiple features of the DNA sequence.

Constitutive repression and nuclear factor I-dependent hormone activation of the mouse mammary tumor virus promoter in Saccharomyces cerevisiae.

To study the influence of various transactivators and the role of nucleosomal structure in gene regulation by steroid hormones, we have introduced mouse mammary tumor virus (MMTV) promoter sequences along with expression vectors for the glucocorticoid receptor (GR) and nuclear factor I (NFI) in Saccharomyces cerevisiae, an organism amenable to genetic manipulation. Both in the context of an episomal multicopy vector and in a centromeric single-copy plasmid, the MMTV promoter was virtually silent in the absence of inducer, even in yeast strains expressing GR and NFI. Induction was optimal with deacylcortivazol and required both GR and NFI. The transactivation function AF1 in the N-terminal half of GR is required for ligand-dependent induction and acts constitutively in truncated GR lacking the ligand binding domain. A piece of the MMTV long terminal repeat extending from -236 to +111 is sufficient to position a nucleosome, B, over the regulatory region of the promoter from -45 to -190 and another nucleosome over the transcription start region. The rotational orientation of the DNA on the surface of nucleosome B is the same as that previously found in animal cells and in reconstitution experiments. This orientation is compatible with binding of GR to two sites, while it should preclude binding of NFI and hence be responsible for constitutive repression. Upon ligand induction, there is no major chromatin rearrangement, but the proximal linker DNA, including the TATA box, becomes hypersensitive to nucleases. The transcriptional behavior of the MMTV promoter was unaffected by deletions of the genes for zuotin or SIN1/SPT2, two proteins which have been claimed to assume some of the functions of linker histones. Thus, despite the lack of histone H1, yeast cells could be a suitable system to study the contribution of nucleosomal organization to the regulated expression of the MMTV promoter.

Interaction of steroid hormone receptors with transcription factors involves chromatin remodelling.

The mechanism by which steroid hormones modulate promoter utilization is not clear. Evidence from transfection studies and cell-free assays points to an interaction of the hormone receptors with general transcription factors, as well as with sequence-specific transcription factors. Moreover co-activators or transcription intermediary factors, have been identified which could mediate some of the transcriptional effects of the hormone-receptor complex. However, in addition to this interaction of receptors with proteins directly involved in transcription, a participation of chromatin structure in gene regulation by steroid hormones is becoming increasingly evident. In the case of the MMTV promoter, the nucleosomal organization seems to be responsible for transcriptional repression prior to hormonal stimulation. This effect is due to occlusion by a nucleosome positioned on the MMTV promoter sequences in such a way that essential transcription factors cannot access their recognition sites. Following hormone induction, a remodelling of the nucleosome structure takes place which enables a whole complement of sequence specific transcription factors to assemble on the promoter. Since a complete occupancy of binding sites does not take place when the promoter is present as naked DNA, the nucleosomal organization appears to be required for the proper synergism between transcription factors following hormonal induction. According to this model, the positioning of a nucleosome sets the stage for constitutive repression and hormone induction of the MMTV promoter.

Transcriptional control by steroid hormones.

Gene regulation by steroid hormones leads to induction or repression of particular sets of genes. These effects are mediated by intracellular hormone receptors that, in the unliganded state, are maintained in an inactive form by unknown mechanisms possibly involving association with other cellular proteins. Induction of the mouse mammary tumor virus (MMTV) requires binding of the hormone receptor to a complex hormone-responsive element (HRE) located between 75 and 190 bp upstream from the start of transcription. The interaction of several receptor molecules with the four receptor binding sites in the HRE is highly cooperative on circular DNA molecules and each individual site is needed for optimal induction. In chromatin the HRE is precisely organized in phased nucleosomes. Following hormone treatment and receptor binding, changes in chromatin structure are detected that correlate with binding of transcription factors, including nuclear factor I, to the MMTV promoter. However, though nuclear factor I acts as a basal transcription factor on the MMTV promoter it does not cooperate with the hormone receptors in terms of binding to free DNA, and mutation of the nuclear factor I binding site does not eliminate hormonal stimulation. This residual induction is mediated by octamer motifs, upstream of the TATA box, that bind the ubiquitous transcription factor OTF-1. Mutation of these octamer motifs does not influence basal transcription in vitro, but completely abolishes the stimulatory effect of progesterone receptor.

Chromatin structure modulates transcription factor binding to the mouse mammary tumor virus (MMTV) promoter

The MMTV promoter contains a complex hormone responsive region (HRR) upstream of a binding site for the transcription factor nuclear factor I (NFI). Hormonal induction of MMTV expression requires the integrity of both the HRR and the NFI binding site. However, in vitro NFI acts as a basal transcription factor on the MMTV promoter that does not cooperate but rather competes with the hormone receptors in terms of binding to MMTV-DNA. Fragments that contain the HRR and the NFI binding site have been reconstituted into mononucleosomes. Steroid hormone receptors bind efficiently to these nucleosomes, NFI does not. Therefore it has been postulated that the chromatin structure may be responsible for the inability of NFI to bind to the chromosomally organized inactive MMTV promoter. In vivo DNaseI and methidium-propyl-EDTA-Fe(II) (MPE) digestion pattern indicate the presence of a nucleosome covering the HRR and the NFI binding site. Genomic footprinting shows that in vivo the rotational setting of the MMTV promoter DNA in this nucleosome is identical to that previously reported for reconstituted nucleosomes in which the major grooves of the NFI half palindromes are facing towards the histone octamer and appear not to be accessible to NFI. These results indicate that MMTV promoter sequences are determining nucleosome positioning in vivo and supports the concept that rotational positioning of DNA in this nucleosome constitutively represses the MMTV promoter.

Chromatin structure of the MMTV promoter and its changes during hormonal induction

Summary1. The packaging of nuclear DNA in chromatin determines the conversion of the genetic information into a defined phenotype by influencing the availability of DNA sequences for interactions with regulatory proteins and transcription factors.2. We have studied the influence of the first level of chromatin organization, the nucleosome, on the activity of the mouse mammary tumor virus (MMTV) promoter. The MMTV promoter is strongly transcribed in response to steroid hormones but is virtually silent in the absence of hormonal stimuli. Full hormonal induction requires binding of the hormone receptors to four hormone-responsive elements (HREs), as well as binding of nuclear factor I (NFI) and the octamer transcription factor 1 (OTF-1 or Oct-1) to sites located between the HREs and the TATA box. A full loading with transcription factors cannot be achieved on free DNA due to steric hindrance between hormone receptor and NFI and between NFI and OTF-1.3. The low basal activity of the MMTV promoter is most likely due to its organization in a positioned nucleosome. In the intact cell, as well in reconstituted chromatin, the regulatory region of the MMTV promoter is wrapped around a histone octamer in a precise rotational orientation, which permits access of the hormone receptors to only two of the four HREs, while precluding binding of NFI and OTF-1 to their respective sites. Upon hormone induction, the nucleosome is remodeled and the path of its DNA altered in a way which makes the nucleosomal dyad axis more accessible to DNase I and enables occupancy of all relevant sites: the four HREs, as well as the binding sites for NFI and OTF-1.4. These results suggest that the nucleosomal organization of the MMTV promoter not only is responsible for the low activity prior to hormone treatment, but also may be a prerequisite for full loading with transcription factors after hormone induction. We conclude that the DNA contains topological information which modulates the expression of the genetic program.

Interaction of Steroid Hormone Receptors with DNA

Modulation of gene expression frequently takes place at the level of transcription, and is mediated by the interaction of regulatory proteins with specific DNA sequences near the regulated promoters. Among the best-characterized examples of such regulatory proteins are the steroid hormone receptors. These proteins are members of a large family of nuclear proteins called the steroid/thyroid hormone receptor family or the nuclear receptor family. Most members of this gene family share a double specificity; on the one hand, they interact with small ligands, such as steroid hormones, thyroid hormones, vitamin D, and retinoic acid; on the other hand, they bind to regulatory elements in the DNA that mediate modulation of gene activity in cis. During the last decade the DNA complementary to the mRNAs encoding most of the members of the hormone receptor gene family have been cloned. A comparison of the primary structure of the various members of the family permits one to derive a general scheme for the modular architecture of these regulatory proteins (Fig. 1). The proteins are composed of three separate domains, one of which interacts with the hormone-ligand, the second binds to DNA, and the third domain modulates transcriptional activity. The most conserved region of the protein is located in the central domain, which consists of a short stretch of about 70 amino acids containing the DNA binding activity. This region of the protein exhibits an array of cysteine residues compatible with an organization of the domain into two so-called zinc fingers, in which two zinc ions are each tetrahedrally coordinated with four cysteines (for a review, see Evans 1988).