Miguel Beato

The nuclear receptor superfamily: The second decade

David J. Mangelsdorf,’ Carl Thummel,2 Miguel Beato,3 Peter Herrlich,4 Giinther Schiitq5 Kazuhiko Umesono,6 Bruce Blumberg,’ Philippe Kastner,’ Manuel Mark,* Pierre Chambon,8 and Ronald M. Evan&‘* ‘Howard Hughes Medical Institute University of Texas Southwestern Medical Center Dallas, Texas 75235-9050 *Howard Hughes Medical Institute University of Utah Salt Lake City, Utah 84112 31nstutut fiir Molekularbiologie und Tumorforschung 35037 Marburg Federal Republic of Germany 4Forschungszentrum Karlsruhe lnstitut Genetik 76021 Karlsruhe Federal Republic of Germany 5Deutsches Krebsforschungszentrum 69120 Heidelberg Federal Republic of Germany GAdvanced Institute of Science and Technology Graduate School of Biological Sciences Nara 630-01 Japan ‘The Salk Institute for Biological Studies La Jolla, California 92037-5800 Blnstitut de Genetique et de Biologie Moleculaire et Cellulaire Centre National de la Recherche Scientifique lnstitut National de la Sante et de la Recherche M6dicale 67404 lllkirch Cedex Strasbourg France gHoward Hughes Medical Institute The Salk Institute for Biological Studies La Jolla, California 92037-5800

Gene Regulation by Steroid Hormones

The location, orientation, and structure of the hormone regulatory elements (HRE) in nine hormonally modulated genes is described. Based on analysis of the contact points between the glucocorticoid receptor (GR) and the DNA double helix within the HREs, a model for the interaction is proposed in which a dimer of the receptor in head-to-head orientation binds to the inverted symmetry element of the HRE. The relationship between the regulatory elements for glucocorticoids and progesterone in the long terminal repeat region (LTR) of mouse mammary tumor virus (MMTV), and in the promoter region of the chicken lysozyme gene, indicates that the recognition mechanism for both receptors is similar but not identical. Curiously, the hormone ligand is not an absolute requirement for the GR to bind its HRE, though it influences the kinetics of the interaction. Other possible functions of the hormone in vivo are discussed, as well as the molecular mechanism responsible for transcriptional regulation after receptor binding to the HRE.

Steroid hormone receptors: Many Actors in search of a plot

Miguel Beato,’ Peter Herrlich,t and Giinther Schlitz* *Institut ftir Molekularbiologie und Tumorforschung Philipps-Universitat Marburg Emil-Mannkopff-Strasse 2 D-35037 Marburg Federal Republic of Germany tForschungszentrum Karlsruhe lnstitut fiir Genetik D-76021 Karlsruhe Federal Republic of Germany *Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280 D-691 20 Heidelberg Federal Republic of Germany

Sp1-mediated transcriptional activation is repressed by Sp3.

Sp1, Sp3 (SPR‐2) and Sp4 (SPR‐1) are human sequence‐specific DNA binding proteins with very similar structural features. In this report, we have analyzed Sp3 in direct comparison with Sp1. We have raised antibodies against both Sp1 and Sp3, and show that Sp3 protein, like Sp1, is expressed in various cell lines. Co‐transfection experiments in different mammalian cell lines reveal that in contrast to Sp1 and Sp4, Sp3 is not able to activate several Sp1 responsive promoters. In addition, Sp3 also fails to activate reporter constructs in Drosophila SL2 cells lacking endogenous Sp factors. Instead, we find that Sp3 represses Sp1‐mediated activation in a linear dose‐dependent manner. A mutant of Sp3 lacking the DNA binding domain does not affect activation by Sp1, suggesting that the inhibition is most likely due to the competition with Sp1 for their common binding sites. To determine if any structurally similar domain of Sp3 is able to replace partially homologous domains of Sp1, we have generated chimeric proteins and tested their activation characteristics in gene transfer experiments. It appears that neither the glutamine‐rich domains A and B nor the D domain of Sp1 can be replaced by the homologous regions of Sp3. Our results suggest that Sp3 is an inhibitory member of the Sp family.

Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross‐talk with estrogen receptor

The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal‐transducing Src/p21ras/Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c‐Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti‐progestins but also by anti‐estrogens. In Cos‐7 cells transfected with the B isoform of progesterone receptor (PRB), progestin activation of the MAP kinase pathway depends on co‐transfection of ER. A transcriptionally inactive PRB mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PRB does not interact with c‐Src but associates via the N‐terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21ras/Erk pathway signals received from the agonist‐activated PRB. These findings reveal a hitherto unrecognized cross‐talk between ovarian hormones which could be crucial for their growth‐promoting effects on cancer cells.

Nucleosome positioning as a determinant of exon recognition

Chromatin structure influences transcription, but its role in subsequent RNA processing is unclear. Here we present analyses of high-throughput data that imply a relationship between nucleosome positioning and exon definition. First, we have found stable nucleosome occupancy within human and Caenorhabditis elegans exons that is stronger in exons with weak splice sites. Conversely, we have found that pseudoexons—intronic sequences that are not included in mRNAs but are flanked by strong splice sites—show nucleosome depletion. Second, the ratio between nucleosome occupancy within and upstream from the exons correlates with exon-inclusion levels. Third, nucleosomes are positioned central to exons rather than proximal to splice sites. These exonic nucleosomal patterns are also observed in non-expressed genes, suggesting that nucleosome marking of exons exists in the absence of transcription. Our analysis provides a framework that contributes to the understanding of splicing on the basis of chromatin architecture.

Nucleosome positioning modulates accessibility of regulatory proteins to the mouse mammary tumor virus promoter

Minichromosomes containing the MMTV hormone responsive element (HRE) exhibit precisely positioned nucleosomes. Chromatin reconstitution of short HRE DNA fragments also results in precise positioning of nucleosomes as revealed by footprinting, which suggests that information for nucleosome phasing is contained within this short sequence. While hormone receptors bind naked DNA and reconstituted nucleosomes with similar affinities (3- to 5-fold difference), NFI, a transcription factor essential for efficient utilization of the MMTV promoter, binds naked DNA very tightly but does not bind the nucleosomally organized promoter. Hormone receptor binding to the MMTV nucleosome does not dissociate the nucleosome but leads to greater accessibility of the promoter-proximal end to exonuclease III. Precise positioning of one nucleosome over the MMTV promoter could repress transcription by preventing NFI binding in the absence of hormone, while still allowing interaction of activated hormone receptor with HRE.

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.

The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction

Sequences within the long terminal repeat region (LTR) of mouse mammary tumour virus (MMTV) confer progestin inducibility to either the tk‐promoter or the MMTV‐promoter in T47D cells, a human mammary tumour cell line which possesses high constitutive levels of progesterone receptor. In a clone of MCF7 cells, another human mammary tumour cell line with a low level of progesterone receptor, as well as in rat fibroblasts, glucocorticoid but not progestin induction is observed. The effect of the progesterone analogue R5020 is much more pronounced than the effect of dexamethasone, and at the concentrations required for maximal induction, R5020 does not significantly compete with binding of dexamethasone to the glucocorticoid receptor. In conjunction with previous results on the DNA binding of the glucocorticoid and progesterone receptors, these data show that two different steroid hormones, acting through their respective receptors, can mediate the induction of gene expression by interacting with the same DNA sequences. Our results suggest that the hormone regulatory element of MMTV may primarily be a progesterone‐responsive element in mammary cells.

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.