Udo Moehren

VDR-Alien: a novel, DNA-selective vitamin D3 receptor-corepressor partnership

The vitamin D receptor (VDR) is a transcription factor that transmits incoming 1,25‐dihy‐droxyvitamin D3 (1α,25(OH)2D3) signaling via combined contact with coactivator proteins and specific DNA binding sites (VDREs), which ultimately results in activation of transcription. In contrast, the mechanisms of transcriptional repression via the VDR are less well understood. This study documents VDR‐dependent transcriptional repression largely via histone deacety‐lase (HDAC) activity. Direct, ligand‐sensitive protein‐protein interaction of the VDR with the nuclear receptor corepressor (NCoR) and a novel corepressor, called Alien, was demonstrated to be comparable but independent of the VDR AF‐2 trans‐activation domain. Functional assays indicated that Alien, but not NCoR, displays selectivity for different VDRE structures for transferring these repressive effects into gene regulatory activities. Moreover, superrepression via Alien was found to be affected only in part by HDAC inhibitors such as trichostatin A. Finally, for a dissociation of VDR‐Alien complexes in vitro and in vivo, higher ligand concentrations were needed than for a dissociation of VDR‐NCoR complexes. This suggests that Alien and NCoR are using different interfaces for interaction with the VDR and different pathways for mediating superrepression, which in turn characterizes Alien as a representative of a new class of corepressors. Taken together, association of the VDR with corepressor proteins provides a further level of transcriptional regulation, which is emerging as a complex network of protein‐protein interaction‐mediated control.—Polly, P., Herdick, M., Moehren, U., Baniahmad, A., Heinzel, T., Carlberg, C. VDR‐Alien: a novel, VDR‐Alien: a novel, DNA‐selective vitamin D3 receptor‐corepressor partnership. FASEBJ. 14, 1455–1463 (2000)

The minimal repression domain of MBD2b overlaps with the methyl-CpG-binding domain and binds directly to Sin3A.

Different mechanisms mediating methylation-dependent repression have been demonstrated. Two of these mechanisms play a role in the context of the granulocyte/macrophage-specific lysozyme gene: direct interference with DNA binding of the transcription factor GA-binding protein and deacetylation of histones. Besides enhancement in the unmethylated state, and transcriptional repression upon DNA methylation, the lysozyme downstream enhancer confers tissue-specific demethylation. Because both demethylation activity and repression ability have been attributed to the methyl-CpG-binding domain-containing protein MBD2, we analyzed this protein. The short form MBD2b binds to the methylated lysozyme enhancer and mediates transcriptional repression. MBD2b is capable of binding to the transcriptional repressor Sin3A. The interaction domain of Sin3A required for binding to MBD2b contains the paired amphipathic helix 3. We identified a minimal functional domain that confers both transcriptional repression as well as the interaction to Sin3A. In contrast to the functionally related proteins MeCP2 and MBD1, the repression domain of MBD2b overlaps with the methyl-CpG-binding domain.

Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model

Androgens influence transcription of their target genes through the activation of the androgen receptor (AR) that subsequently interacts with specific DNA motifs in these genes. These DNA motifs, called androgen response elements (AREs), can be classified in two classes: the classical AREs, which are also recognized by the other steroid hormone receptors; and the AR-selective AREs, which display selectivity for the AR. For in vitro interaction with the selective AREs, the androgen receptor DNA-binding domain is dependent on specific residues in its second zinc-finger. To evaluate the physiological relevance of these selective elements, we generated a germ-line knockin mouse model, termed SPARKI (SPecificity-affecting AR KnockIn), in which the second zinc-finger of the AR was replaced with that of the glucocorticoid receptor, resulting in a chimeric protein that retains its ability to bind classical AREs but is unable to bind selective AREs. The reproductive organs of SPARKI males are smaller compared with wild-type animals, and they are also subfertile. Intriguingly, however, they do not display any anabolic phenotype. The expression of two testis-specific, androgen-responsive genes is differentially affected by the SPARKI mutation, which is correlated with the involvement of different types of response elements in their androgen responsiveness. In this report, we present the first in vivo evidence of the existence of two functionally different types of AREs and demonstrate that AR-regulated gene expression can be targeted based on this distinction.

Wild-type but not mutant androgen receptor inhibits expression of the hTERT telomerase subunit: a novel role of AR mutation for prostate cancer development

Androgens play a central role in prostate development and prostate cancer proliferation. Induction of telomerase is an early event in prostate carcino‐genesis and is considered as a marker for both primary tumors and metastases. Interestingly, several reports suggest that telomerase activity is regulated by andro‐gens in vivo. Here, we show that the wild‐type (WT) human androgen receptor (AR) inhibits the expression of the human telomerase reverse transcriptase (hTERT) and telomerase activity via inhibition of hTERT promoter activity in the presence of androgen receptor agonists. However, pure androgen antagonists failed to repress hTERT transcription. The androgen‐mediated repression of hTERT is abrogated in a human prostate cancer cell line exhibiting hormone‐dependent growth, which expresses a mutant AR (T877A) frequently occurring in prostate cancer. We reveal that this single amino acid exchange is sufficient for the lack of transrepression. Interestingly, chromatin immunopre‐cipitation data suggest that, in contrast to the WT AR, the mutant AR is recruited less efficiently to the hTERT promoter in vivo, indicating that loss of transrepression results from reduced chromatin recruitment. Thus, our findings suggest that the WT AR inhibits expression of hTERT, which is indicative of a protective mechanism, whereas the T877A mutation of AR not only broadens the ligand spectrum of the receptor but abrogates this inhibitory mechanism in prostate cancer cells. This novel role of AR mutations in prostate cancer development suggests the benefit to a search for new AR antagonists that inhibit transactivation but allow transrepression. Moehren, U., Papaioannou, M., Reeb, C. A., Grasselli, A., Nanni, S., Asim, M., Roell, D., Prade, I., Farsetti, A., Baniahmad, A. Wild‐type but not mutant androgen receptor inhibits expression of the hTERT telomerase subunit: a novel role of AR mutation for prostate cancer development. FASEB J. 22, 1258–1267 (2008)

Agonist–antagonist induced coactivator and corepressor interplay on the human androgen receptor

The human androgen receptor (AR) is a member of the nuclear hormone receptor superfamily. However, in contrast to other members of this family the amino-(N)-terminus of AR harbors the major transactivation function. Previously we have shown that hormone antagonists that bind to the carboxy-terminal ligand-binding domain repress AR through recruitment of corepressors that are recruited to the receptor N-terminus. Here we show by a modified mammalian two-hybrid system that both the AR interacting domains of the coactivator SRC1 and of the corepressor SMRT compete for interaction with the AR N-terminus. In contrast to other members of the nuclear receptor superfamily the LXXLL motifs of SRC1e are not required for this interaction, instead a stretch of 135 amino acids of the glutamine rich region (Qr) of SRC1e is essential to bind to the AR N-terminus. We show that the Qr-region of SRC1 is able to inhibit the interaction of SMRT with AR. Also, we demonstrate that the corepressor mediated repression decreases the antagonist-induced transactivation while, surprisingly, it increases the agonist-induced transactivation. This may indicate that coactivators and corepressors act in concert to dictate the overall receptor-mediated action dependent on the type of ligand.

Gene silencing by the thyroid hormone receptor

The thyroid hormone receptors (TR) are able to bind DNA and to repress transcription in the absence of thyroid hormone. This repression function is an important feature of TRs as aberrant silencing can lead to severe diseases and developmental abnormalities. TR utilizes different mechanisms to achieve repression of target genes including the recruitment of cofactors called corepressors and interference with the basal transcriptional machinery. Recent studies have revealed an important role of chromatin in TR silencing involving different histone modifications and the responsible enzymes. Furthermore, the transcriptional properties of TR depend on the type of the TR DNA-binding elements. This review will focus on the molecular basis of gene silencing by TR and diseases caused by aberrant functioning.

The androgen receptor DNA-binding domain determines androgen selectivity of transcriptional response

The AR (androgen receptor) is a hormone-dependent transcription factor that translates circulating androgen hormone levels into a physiological cellular response by directly regulating the expression of its target genes. It is the key molecule in e.g. the development and maintenance of the male sexual characteristics, spermatocyte production and prostate gland development and growth. It is also a major factor in the onset and maintenance of prostate cancer and a first target for pharmaceutical action against the further proliferation of prostate cancer cells. The AR is a member of the steroid hormone receptors, a group of steroid-inducible transcription factors sharing an identical consensus DNA-binding motif. The problem of how specificity in gene activation is achieved among the different members of this nuclear receptor subfamily is still unclear. In this report, we describe our investigations on how the AR can specifically activate its target genes, while the other steroid hormone receptors do not, despite having the same consensus monomeric DNA-binding motif. In this respect, we describe how the AR interacts with a newly identified class of steroid-response elements to which only the AR and not, for example, the glucocorticoid receptor can bind.

Molecular biology of the androgen responses

The androgen receptor is a ligand-inducible transcription factor with very specific target genes. This definition implies the activation by the cognate ligand through the ligand-binding domain, the recognition of the target genes by means of the DNA-binding domain and the transcriptional activation through different activation functions. When the first androgen-responsive genes were cloned, we identified receptor-binding sites by means of a DNAcellulose competition assay with partially purified androgen receptor from rat prostate (Claessens et al., 1990). Once the receptor cDNA was cloned, the separate DNAbinding domain was expressed and shown to have similar, if not identical DNA recognition properties as the full size receptor. The binding sites were proven functional in transient transfection experiments with reporter genes cloned downstream of these sites (Claessens et al., 1993). The motifs which are recognized by the receptor are called androgen response elements (ARE), and a consensus of the first identified AREs pointed out that it is very similar to the glucocorticoid/progesterone response element (GRE/PRE) consensus 5¢-GGTACAnnnTGTTCT-3¢. Not surprisingly, these AREs also act as GRE/PRE in transient transfections. The probasin promoter region also contains two AR-binding sites, but in contrast to what was observed for the earlier AREs, these are not recognized by the glucocorticoid receptor. Later on, several other selective AREs were characterized in the slp and sc enhancers (Verrijdt et al., 2000). A comparison of the DNA-binding domains of the androgen and glucocorticoid receptors revealed specific residues which are involved in the recognition of these selective AREs, but not in the recognition of the classical AREs. These residues are not situated within the first zinc-coordinated module or zinc finger, but rather in the second one, as well as in a carboxy-terminal extension of the DNA-binding motif (Schoenmakers et al., 2000). This hinted to us that the recognition of the selective AREs occurs through an alternative dimerization of the DNA-binding domain that would be specific for the androgen receptor. Indeed, when the direct repeat nature of the selective AREs was changed into inverted repeat nature, the selectivity of the AREs and of the enhancers, of which they form part, was lost (Verrijdt et al., 2000). The silico screening of human genome has led to the definition of several additional selective AREs. In collaboration with the group of Daniel Gewirth, we were able to solve a crystal structure of the DNA-binding domain of the androgen receptor complexed to a perfect direct repeat of the 5¢TGTTCT-3¢ hexamer (Shaffer et al., 2004). This revealed that the domain is folded into two zinc-coordinated modules very similar to what has been described for other nuclear receptors. The two monomers are organized in a head-to-head configuration. Specific for the androgen receptor is the increased strength of the dimerisation interface due to an enlarged contact surface as well as to three additional hydrogen bonds. A functional analysis of the carboxyterminal extension of the DBD, which is part of the hinge region, revealed that it has more functions besides contributing to selective DNA binding. It overlaps with part of a nuclear localization signal and it is involved in the control of transactivation. Indeed, opposite to what is expected, deletions within this region result in a superactive androgen receptor, even when DNA binding in band shifts becomes difficult to demonstrate. The transcription activation by the androgen receptor is complex in the sense that different domains are contributing to it. For all steroid receptors, two activation functions have been described: the activation function 1 (AF1) in the amino-terminal domain and activation function 2 (AF2) in the ligand-binding domain. The androgen receptor is an exception since the AF2 is weak and in most experiments difficult to demonstrate. A possible explanation for this was found in a strong interaction between the ligand-binding domain and the amino-terminal domain of the androgen receptor. This occurs through a motif at the amino-terminal end of the receptor that interacts with AF2, described as a hydrophobic cleft on the surface of the ligand-binding domain. This interaction seems to prevent recruitment of the known p160 co-activators to