Liesbeth Clinckemalie

Structural basis for nuclear hormone receptor DNA binding

The gene family of nuclear receptors is characterized by the presence of a typical, well conserved DNA-binding domain. In general, two zinc coordinating modules are folded such that an α-helix is inserted in the major groove of the DNA-helix displaying a sequence similar to one of two hexameric consensus motifs. Both zinc molecules coordinate four cysteines. Although the DNA-binding domains as well as the hormone response elements are very similar, each nuclear receptor will affect transcription of a specific set of target genes. This is in part due to some important receptor-specific variations on the general theme of DNA interaction. For most nuclear receptors, the DNA-binding domain dimerizes on DNA, which explains why most hormone response elements consist of a repeat of two hexamers. The hexamer dimers can be organized either as direct, inverted or everted repeats with spacers of varying lengths. The DNA can be bound by homodimers, heterodimers and for some orphan receptors, as monomer. Another key element for DNA binding by nuclear receptors is the carboxy-terminal extension of the DNA-binding domain extending into the hinge region. This part not only co-determines sequence specificity, but also affects other functions of the receptors like nuclear translocation, intranuclear mobility and transactivation potential. Moreover, allosteric signals passing through towards other receptor domains, explain why to some extent, the DNA elements can also be considered as controlling ligands.

The hinge region in androgen receptor control.

The region between the DNA-binding domain and the ligand-binding domain of nuclear receptors is termed the hinge region. Although this flexible linker is poorly conserved, diverse functions have been ascribed to it. For the androgen receptor (AR), the hinge region and in particular the (629)RKLKKL(634) motif, plays a central role in controlling AR activity, not only because it acts as the main part of the nuclear translocation signal, but also because it regulates the transactivation potential and intranuclear mobility of the receptor. It is also a target site for acetylation, ubiquitylation and methylation. The interplay between these different modifications as well as the phosphorylation at serine 650 will be discussed here. The hinge also has an important function in AR binding to classical versus selective androgen response elements. In addition, the number of coactivators/corepressors that might act via interaction with the hinge region is still growing. The importance of the hinge region is further illustrated by the different somatic mutations described in patients with androgen insensitivity syndrome and prostate cancer. In conclusion, the hinge region serves as an integrator for signals coming from different pathways that provide feedback to the control of AR activity.

A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle

Androgens have well‐established anabolic actions on skeletal muscle, although the direct effects of the androgen receptor (AR) in muscle remain unclear. We generated satellite cell‐specific AR‐knockout (satARKO) mice in which the AR is selectively ablated in satellite cells, the muscle precursor cells. Total‐limb maximal grip strength is decreased by 7% in satARKO mice, with soleus muscles containing ~10% more type I fibers and 10% less type IIa fibers than the corresponding control littermates. The weight of the perineal levator ani muscle is markedly reduced (–52%). Thus, muscle AR is involved in fiber‐type distribution and force production of the limb muscles, while it is a major determinant of the perineal muscle mass. Surprisingly, myostatin (Mstn), a strong inhibitor of skeletal muscle growth, is one of the most androgen‐responsive genes (6‐fold reduction in satARKO) through direct transcription activation by the AR. Consequently, muscle hypertrophy in response to androgens is augmented in Mstn‐knockout mice. Our finding that androgens induce Mstn signaling to restrain their own anabolic actions has implications for the treatment of muscle wasting disorders.—Dubois, V., Laurent, M. R., Sinnesael, M., Cielen, N., Helsen, C., Clinckemalie, L., Spans, L., Gayan‐Ramirez, G., Deldicque, L., Hespel, P., Carmeliet, G., Vanderschueren, D., and Claessens, F. A satellite cell‐specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle. FASEB J. 28, 2979–2994 (2014). www.fasebj.org

Expression of Tubb3, a Beta-Tubulin Isotype, Is Regulated by Androgens in Mouse and Rat Sertoli Cells

Our previous analysis of Sertoli cell androgen receptor (AR) knockout (SCARKO) mice revealed that several cytoskeletal components are a potential target of androgen action. Here, we found that one of these components, the beta-tubulin isotype Tubb3, is differentially regulated in testes from SCARKO mice (relative to littermate controls) from Postnatal Day 10 to adulthood. The Tubb3 gene is unique in this respect, as at Day 10, no other beta-tubulin genes are significantly regulated by AR. We further characterized androgen regulation of Tubb3 in vivo and in vitro and demonstrated that it is a conserved feature in both mice and rats. To investigate whether androgens directly regulate Tubb3 expression, we screened for androgen response elements (AREs) in the Tubb3 gene. In silico analysis revealed the presence of four ARE motifs in Tubb3 intron 1, two of which bind to AR in vitro. Mutation of one of these (ARE1) strongly reduced androgen-dependent reporter gene expression. These results, coupled with the finding that the AR binds to the Tubb3 ARE region in vivo, suggest that Tubb3 is a direct target of AR. Our data strengthen the contention that androgens exert their effects on spermatogenesis, in part, through modulation of the Sertoli cell cytoskeleton. Androgen regulation of beta-tubulin has also been described in neurons, fortifying the already known similarity in microtubule organization in Sertoli cell processes and neurons, the only other cell type in which Tubb3 is known to be expressed.

The Role of Single Nucleotide Polymorphisms in Predicting Prostate Cancer Risk and Therapeutic Decision Making

Prostate cancer (PCa) is a major health care problem because of its high prevalence, health-related costs, and mortality. Epidemiological studies have suggested an important role of genetics in PCa development. Because of this, an increasing number of single nucleotide polymorphisms (SNPs) had been suggested to be implicated in the development and progression of PCa. While individual SNPs are only moderately associated with PCa risk, in combination, they have a stronger, dose-dependent association, currently explaining 30% of PCa familial risk. This review aims to give a brief overview of studies in which the possible role of genetic variants was investigated in clinical settings. We will highlight the major research questions in the translation of SNP identification into clinical practice.

The Genomic Landscape of Prostate Cancer

By the age of 80, approximately 80% of men will manifest some cancerous cells within their prostate, indicating that prostate cancer constitutes a major health burden. While this disease is clinically insignificant in most men, it can become lethal in others. The most challenging task for clinicians is developing a patient-tailored treatment in the knowledge that this disease is highly heterogeneous and that relatively little adequate prognostic tools are available to distinguish aggressive from indolent disease. Next-generation sequencing allows a description of the cancer at an unprecedented level of detail and at different levels, going from whole genome or exome sequencing to transcriptome analysis and methylation-specific immunoprecipitation, followed by sequencing. Integration of all these data is leading to a better understanding of the initiation, progression and metastatic processes of prostate cancer. Ultimately, these insights will result in a better and more personalized treatment of patients suffering from prostate cancer. The present review summarizes current knowledge on copy number changes, gene fusions, single nucleotide mutations and polymorphisms, methylation, microRNAs and long non-coding RNAs obtained from high-throughput studies.

A role for selective androgen response elements in the development of the epididymis and the androgen control of the 5α reductase II gene

The androgen receptor (AR) recognizes two types of DNA elements that are dimers of 5′‐AGAACA‐3′‐like hexamers, either organized as inverted or direct repeats. We developed a mouse model [(specificity affecting AR knock‐in (SPARKI)] in which the AR DNA‐binding domain was mutated such that it lost binding to direct repeats but not to inverted elements. The impaired fertility of the male SPARKI mice correlates with the reduced motility of the spermatozoa, a characteristic that is developed during transit through the epididymis. Comparative transcriptome analyses revealed that the expression of 39 genes is changed in SPARKI epididymis. Remarkably, the expression of the steroid 5α‐reductase type II (Srd5α2) gene, which metabolizes testosterone into the more potent dihydrotestosterone, is reduced 4‐fold in SPARKI vs. wild type. The comparison of the SPARKI phenotype with that of Srd5α2‐knockout mice shows, however, that the reduced Srd5α2 expression cannot explain all defects of the SPARKI epididymis. Moreover, we describe three new selective androgen response elements (AREs), which control the androgen responsiveness of the Srd5α2 gene. We conclude that the SPARKI model can be considered a knockout model for AR functioning via selective AREs and that this has a dramatic effect on sperm maturation in the epididymis.—Kerkhofs, S., Dubois, V., De Gendt, K., Helsen, C., Clinckemalie, L., Spans, L., Schuit, F., Boonen, S., Vanderschueren, D., Saunders, P. T. K., Verhoeven, G., Claessens, F. A role for selective androgen response elements in the development of the epididymis and the androgen control of the 5α reductase II gene. FASEB J. 26, 4360–4372 (2012). www.fasebj.org

Androgen Regulation of the TMPRSS2 Gene and the Effect of a SNP in an Androgen Response Element

More than 50% of prostate cancers have undergone a genomic reorganization that juxtaposes the androgen-regulated promoter of TMPRSS2 and the protein coding parts of several ETS oncogenes. These gene fusions lead to prostate-specific and androgen-induced ETS expression and are associated with aggressive lesions, poor prognosis, and early-onset prostate cancer. In this study, we showed that an enhancer at 13 kb upstream of the TMPRSS2 transcription start site is crucial for the androgen regulation of the TMPRSS2 gene when tested in bacterial artificial chromosomal vectors. Within this enhancer, we identified the exact androgen receptor binding sequence. This newly identified androgen response element is situated next to two binding sites for the pioneer factor GATA2, which were identified by DNase I footprinting. Both the androgen response element and the GATA-2 binding sites are involved in the enhancer activity. Importantly, a single nucleotide polymorphism (rs8134378) within this androgen response element reduces binding and transactivation by the androgen receptor. The presence of this SNP might have implications on the expression and/or formation levels of TMPRSS2 fusions, because both have been shown to be influenced by androgens.

Comparative Genomic and Transcriptomic Analyses of LNCaP and C4-2B Prostate Cancer Cell Lines

The LNCaP and C4-2B cell lines form an excellent preclinical model to study the development of metastatic castration-resistant prostate cancer, since C4-2B cells were derived from a bone metastasis that grew in nude mice after inoculation with the LNCaP-derived, castration-resistant C4-2 cells. Exome sequencing detected 2188 and 3840 mutations in LNCaP and C4-2B cells, respectively, of which 1784 were found in both cell lines. Surprisingly, the parental LNCaP cells have over 400 mutations that were not found in the C4-2B genome. More than half of the mutations found in the exomes were confirmed by analyzing the RNA-seq data, and we observed that the expressed genes are more prone to mutations than non-expressed genes. The transcriptomes also revealed that 457 genes show increased expression and 246 genes show decreased expression in C4-2B compared to LNCaP cells. By combining the list of C4-2B-specific mutations with the list of differentially expressed genes, we detected important changes in the focal adhesion and ECM-receptor interaction pathways. Integration of these pathways converges on the myosin light chain kinase gene (MLCK) which might contribute to the metastatic potential of C4-2B cells. In conclusion, we provide extensive databases for mutated genes and differentially expressed genes in the LNCaP and C4-2B prostate cancer cell lines. These can be useful for other researchers using these cell models.

The transcription intermediary factor 1β coactivates the androgen receptor.

The androgen receptor (AR) is a ligand-inducible transcription factor. Its transcription activation domain consists of the two transcription activation units called Tau-1 and Tau-5. Tau-5 interacts with p160 coactivators like the transcription intermediary factor 2 (TIF2), which in their turn recruit histone modifiers and chromatin-remodelling complexes. The mechanism of action of Tau-1, however, remains elusive. Here, we demonstrate that transcription intermediary factor 1β (TIF1β) can induce the activity of the AR up to five fold when tested in vitro. Although there is no evidence for direct interactions between TIF1β and AR, mutation studies show that the activity of TIF1β depends on the integrity of Tau-1 in AR on the one hand, and the so-called tripartite motif domain in TIF1β on the other. Surprisingly, the coactivation by TIF1β via Tau-1 seems additive rather than cooperative with the AR coactivation by TIF2. Some mutations naturally occurring in androgen-insensitivity syndrome patients that reside in Tau-1 seem to impair the TIF1β coactivation of the AR, indicating that TIF1β could also be relevant for the in vivo androgen response in humans. Moreover, since TIF1β is well expressed in prostate cancer cells, its functional interaction with androgen signalling could in the long run be a therapeutic target for this disease.