Lien Spans

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.

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

Emerging mechanisms of enzalutamide resistance in prostate cancer

The majority of prostate cancers are hormone-dependent at diagnosis highlighting the central role of androgen signalling in this disease. Surprisingly, most forms of castration-resistant prostate cancer (CRPC) are still dependent on the androgen receptor (AR) for survival. Therefore, the advent of new AR-targeting drugs, such as enzalutamide, is certainly beneficial for the many patients with metastatic CRPC. Indeed, this compound provides a substantial survival benefit—but it is not curative. This Perspectives article describes the different ways through which cancer cells can become resistant to enzalutamide, such as AR truncation and other mutations, as well as by-pass of the AR dependence of prostate cancer cells through expression of the glucocorticoid receptor. The clinical relevance of these mechanisms and emerging questions concerning new therapeutic regimens in the treatment of metastatic CRPC are being discussed.

A 629RKLKK633 motif in the hinge region controls the androgen receptor at multiple levels

The androgen receptor protein has specific domains involved in DNA binding, ligand binding, and transactivation, whose activities need to be integrated during transcription activation. The hinge region, more particular a 629RKLKK633 motif, seems to play a crucial role in this process. Indeed, although the motif is not part of the DNA-binding domain, its positive residues are involved in optimal DNA binding and nuclear translocation as shown by mutation analysis. When the mutated ARs are forced into the nucleus, however, the residues seem to play different roles in transactivation. Moreover, we show by FRAP analysis that during activation, the AR is distributed in the nucleus in a mobile and two immobile fractions, and that mutations in the 629RKLKK633 motif affect the distribution of the AR over these three intranuclear fractions. Taken together, the 629RKLKK633 motif is a multifunctional motif that integrates nuclear localization, receptor stability, DNA binding, transactivation potential and intranuclear mobility.

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.

Dynamic Switching of Active Promoter and Enhancer Domains Regulates Tet1 and Tet2 Expression during Cell State Transitions between Pluripotency and Differentiation

ABSTRACT The Tet 5-methylcytosine dioxygenases catalyze DNA demethylation by producing 5-hydroxymethylcytosine and further oxidized products. Tet1 and Tet2 are highly expressed in mouse pluripotent cells and downregulated to different extents in somatic cells, but the transcriptional mechanisms are unclear. Here we defined the promoter and enhancer domains in Tet1 and Tet2. Within a 15-kb “superenhancer” of Tet1, there are two transcription start sites (TSSs) with different activation patterns during development. A 6-kb promoter region upstream of the distal TSS is highly active in naive pluripotent cells, autonomously reports Tet1 expression in a transgenic system, and rapidly undergoes DNA methylation and silencing upon differentiation in cultured cells and native epiblast. A second TSS downstream, associated with a constitutively weak CpG-rich promoter, is activated by a neighboring enhancer in naive embryonic stem cells (ESCs) and primed epiblast-like cells (EpiLCs). Tet2 has a CpG island promoter with pluripotency-independent activity and an ESC-specific distal intragenic enhancer; the latter is rapidly downregulated in EpiLCs. Our study reveals distinct modes of transcriptional regulation at Tet1 and Tet2 during cell state transitions of early development. New transgenic reporters using Tet1 and Tet2 cis-regulatory domains may serve to distinguish nuanced changes in pluripotent states and the underlying epigenetic variations.

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.

Variations in the exome of the LNCaP prostate cancer cell line

The LNCaP cell line is widely used as a model for prostate cancer. However, information on protein‐changing mutations, genetic heterogeneity and genetic (in)stability is largely lacking for these cells.

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.