Philippe Kastner

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

Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B.

The human progesterone receptor (hPR) cDNA, synthesized from T47D breast cancer cells, and the hPR gene 5′‐flanking region were cloned and sequenced. Comparison of the cDNA‐deduced amino acid sequence with other PR homologues demonstrated the modular structure characteristic of nuclear receptors. As in the case of the chicken homologue, there are two hPR forms, A and B, which originate from translational initiation at AUG2 (codon 165) and AUG1, respectively. Northern blot analysis of T47D mRNA using various cDNA derived probes identified two classes of hPR mRNAs, one of which could code for hPR form B, while the other one lacked the 5′ region upstream of AUG1. S1 nuclease mapping and primer extension analyses confirmed that the second class of hPR transcripts are initiated between +737 and +842 and thus encode hPR form A, but not form B. By using the hPR gene 5′‐flanking sequences as promoter region in chimeric genes, we show that a functional promoter (located between ‐711 and +31) directs initiation of hPR mRNAs from the authentic start sites located at +1 and +15. Most importantly, initiation of transcription from chimeric genes demonstrated the existence of a second promoter located between +464 and +1105. Transient co‐transfection experiments with vectors expressing the human estrogen receptor showed that both promoters were estrogen inducible, although no classical estrogen responsive element was detected in the corresponding sequences. When transiently expressed, the two hPR forms similarly activated transcription from reporter genes containing a single palindromic progestin responsive element (PRE), while form B was more efficient at activating the PRE of the mouse mammary tumor virus long terminal repeat. Transcription from the ovalbumin promoter, however, was induced by hPR form A, but not by form B.

NONSTEROID NUCLEAR RECEPTORS : WHAT ARE GENETIC STUDIES TELLING US ABOUT THEIR ROLE IN REAL LIFE ?

Introduction Many of the cloned vertebrate nuclear receptors have been extensively studied in vitro in transfected cultured cells. Unfortunately, this approach isfarfrom being physiological (e.g., the amount of receptor may greatly exceed the normal cellular amount) and does not necessarily reflect what is occurring in vivo. Ligand “manipulation” may provide some clues about functions of the cognate receptor. However, the actual involvement of a particular receptor in the known functions of its ligand needs to be demonstrated in animals in which the functions of the receptor have been abrogated by genetic means. This is particularly true in cases when several receptors bind the same ligand or when the ligand also interacts with other proteins (e.g., cellular retinoic acid-binding proteins [CRABPs] in the case of retinoic acid [RA]). Genetic studies are even more crucial for assigning functions to orphan receptors. Targeted disruption (knockout) via homologous recombination has recently been used to generate mice lacking receptors (Table 1). Expression of dominant negative receptors or antisense mRNA has also been employed. Together with some nuclear receptor mutations associated with pathological conditions, knockouts have led to significant advances in our knowledge of the physiological functions of several nuclear receptors, but have also raised unexpected problems. These studies and their implications are summarized and discussed here except those concerning steroid hormone receptors (see Beato et al., 1995 [this issue of Cell]).

Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently

We have purified and cloned a HeLa cell nuclear protein that strongly stimulates binding of retinoic acid and thyroid hormone receptors (RARs and TRs) to response elements. The purified protein is a human retinoid X receptor beta (hRXR beta). Three murine members of the RXR family (mRXR alpha, beta, and gamma) have also been cloned, and their interactions with RARs and TRs have been investigated. Under conditions where RAR, RXR, and TR bound poorly as homodimers to various response elements, strongly cooperative RAR-RXR and TR-RXR binding was observed. The binding efficiency was dependent on the sequence, relative orientation, and spacing of the repeated motifs of response elements. We show also that unstable RAR-RXR heterodimers were formed in solution, and that C-terminal sequences and the DNA-binding domains of both receptors were required for efficient formation of stable heterodimers on response elements. These findings suggest a convergence of the signaling pathways of some members of the nuclear receptor superfamily.

Multiplicity generates diversity in the retinoic acid signalling pathways.

Complexity in the retinoid signalling system arises from a combination of several forms of retinoic acid, multiple cytoplasmic binding proteins and nuclear receptors, and the existence of polymorphic retinoic acid response elements. Additional diversity appears to be generated by heterodimeric interactions between two families of nuclear retinoic acid receptors, and between nuclear retinoic acid receptors and other members of the nuclear receptor superfamily. Thus, a complex array of combinatorial effects is beginning to emerge which may account for the pleiotropic effects of retinoids.

Genetic analysis of RXRα developmental function: Convergence of RXR and RAR signaling pathways in heart and eye morphogenesis

A null mutation was generated in the mouse RXR alpha gene by targeted disruption. Growth deficiency occurred in heterozygote mice. Null mutants died in utero and displayed myocardial and ocular malformations. These malformations belong to the fetal vitamin A deficiency syndrome, supporting the idea that RXR alpha is involved in retinoid signaling in vivo. A phenotypic synergy was observed when the RXR alpha mutation was introduced into RAR alpha or RAR gamma mutant backgrounds: RXR alpha null mutants and RXR alpha +/-/RAR gamma-/- double mutants displayed similar ocular defects, which became more severe in RXR alpha-/-/RAR gamma+/- and RXR alpha-/-/RAR gamma-/- mutants. Furthermore, RXR alpha/RAR double mutants exhibited several malformations not seen in single mutants. This functional convergence strongly suggests that RXR alpha/RAR heterodimers mediate retinoid signaling in vivo.

Function of retinoic acid receptor γ in the mouse

Abstract Null mutant mice for retinoic acid receptor γ2 (RARγ2) or all RARγ isoforms were generated. RARγ2 mutants appeared normal, whereas RARγ mutants exhibited growth deficiency, early lethality, and male sterility due to squamous metaplasia of the seminal vesicles and prostate. These defects were previously observed in vitamin A-deficient animals and could be prevented by RA administration, demonstrating that RARγ mediates some of the retinoid signal in vivo. Congenital defects included Harderian gland agenesis, tracheal cartilage malformations, and homeotic transformations along the rostral axial skeleton, establishing a direct link between RA and patterning of the axial skeleton. We also show that in utero RA-induced lumbosacral truncations are mediated by RARγ. The observed RARγ null phenotype suggests a high degree of functional redundancy among the RARs. The variable penetrance of some of the observed defects is discussed in light of this redundancy and stochastic variation of gene activity.

Fuzzy C-means method for clustering microarray data

MOTIVATION Clustering analysis of data from DNA microarray hybridization studies is essential for identifying biologically relevant groups of genes. Partitional clustering methods such as K-means or self-organizing maps assign each gene to a single cluster. However, these methods do not provide information about the influence of a given gene for the overall shape of clusters. Here we apply a fuzzy partitioning method, Fuzzy C-means (FCM), to attribute cluster membership values to genes. RESULTS A major problem in applying the FCM method for clustering microarray data is the choice of the fuzziness parameter m. We show that the commonly used value m = 2 is not appropriate for some data sets, and that optimal values for m vary widely from one data set to another. We propose an empirical method, based on the distribution of distances between genes in a given data set, to determine an adequate value for m. By setting threshold levels for the membership values, genes which are tigthly associated to a given cluster can be selected. Using a yeast cell cycle data set as an example, we show that this selection increases the overall biological significance of the genes within the cluster. AVAILABILITY Supplementary text and Matlab functions are available at http://www-igbmc.u-strasbg.fr/fcm/

Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling

BackgroundDendritic cells (DCs) are a complex group of cells that play a critical role in vertebrate immunity. Lymph-node resident DCs (LN-DCs) are subdivided into conventional DC (cDC) subsets (CD11b and CD8α in mouse; BDCA1 and BDCA3 in human) and plasmacytoid DCs (pDCs). It is currently unclear if these various DC populations belong to a unique hematopoietic lineage and if the subsets identified in the mouse and human systems are evolutionary homologs. To gain novel insights into these questions, we sought conserved genetic signatures for LN-DCs and in vitro derived granulocyte-macrophage colony stimulating factor (GM-CSF) DCs through the analysis of a compendium of genome-wide expression profiles of mouse or human leukocytes.ResultsWe show through clustering analysis that all LN-DC subsets form a distinct branch within the leukocyte family tree, and reveal a transcriptomal signature evolutionarily conserved in all LN-DC subsets. Moreover, we identify a large gene expression program shared between mouse and human pDCs, and smaller conserved profiles shared between mouse and human LN-cDC subsets. Importantly, most of these genes have not been previously associated with DC function and many have unknown functions. Finally, we use compendium analysis to re-evaluate the classification of interferon-producing killer DCs, lin-CD16+HLA-DR+ cells and in vitro derived GM-CSF DCs, and show that these cells are more closely linked to natural killer and myeloid cells, respectively.ConclusionOur study provides a unique database resource for future investigation of the evolutionarily conserved molecular pathways governing the ontogeny and functions of leukocyte subsets, especially DCs.

Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing.

Using anchored PCR, three different cDNA isoforms of the mouse retinoic acid receptor beta [mRAR‐beta 1, mRAR‐beta 2 (formerly mRAR‐beta 0) and mRAR‐beta 3], generated from the same gene by differential promoter usage and alternative splicing, were isolated. These three isoforms encode RAR proteins with different N‐terminal A regions and identical B ‐ F regions. The sequence encoding the first 59 amino acids of the mRAR‐beta 3 A region is identical with the entire A region of mRAR‐beta 1. However, the sequence of mRAR‐beta 3 region A differs from that of mRAR‐beta 1 by an additional 27 C‐terminal amino acids encoded in an 81 nucleotide‐long putative exon which is spliced in between the exons encoding the A and B regions of mRAR‐beta 1. Both mRAR‐beta 1 and beta 3 cDNAs differ entirely from mRAR‐beta 2 in their 5′‐untranslated (5′‐UTR) and A region coding sequences. This N‐terminal variability, in a region which was shown to be important for cell‐type specific differential target gene trans‐activation by other nuclear receptors, suggests that the three mRAR‐beta isoforms may be functionally distinct. The conservation of RAR‐beta isoform sequences from mouse to human, as seen by cross‐hybridization on Southern blots or DNA sequence analysis, as well as their differential patterns of expression in various mouse tissues, corroborates this view. Additionally, the mRNA analysis data suggest that mRAR‐beta 2, whose expression predominates in RA‐treated embryonal carcinoma (EC) and embryonic stem (ES) cells, may be important during early stages of development. mRAR‐beta 1 and beta 3, on the other hand, which are predominantly expressed in fetal and adult brain, may play some specific role in the development of the central nervous system.