Aria Baniahmad

Modular structure of a chicken lysozyme silencer: Involvement of an unusual thyroid hormone receptor binding site

Silencer elements, by analogy to enhancer elements, function independently of their position and orientation. We show that the chicken lysozyme silencer S-2.4 kb has many other characteristics in common with enhancer elements. The silencer is comprised of modules that independently repress gene activity--repression being increased synergistically when different or identical modules are combined. Repression is effective both on a complete and on a minimal promoter consisting of a TATA box only. One silencer module is bound in vitro by a 75-93 kd protein, termed NeP1; the other can be bound either by the product of the oncogene v-erbA or by the thyroid hormone receptor. This erbA binding site is unusual in that the palindromic sequence is inverted.

A transferable silencing domain is present in the thyroid hormone receptor, in the v-erbA oncogene product and in the retinoic acid receptor

Inhibition of gene transcription is brought about by several mechanisms. The least understood mechanism is probably silencing, the analogue to transcriptional enhancing. We provide evidence that the silencing function of the oncogene product v‐ERBA or the cellular counterpart, the thyroid hormone receptor (TR, c‐erbA) is located in the C‐terminal part and is transferable to a heterologous DNA binding domain. Deletion analyses suggest an important role for a basic and hydrophilic amino acid stretch on both ends of the domain. In addition we show that the related retinoic acid receptor (RAR) also contains a functional silencing domain similar in size and amino acid sequence. However, the activity of this domain can be neutralized by an additional domain in the C‐terminus which functions cell specifically.

The tau 4 activation domain of the thyroid hormone receptor is required for release of a putative corepressor(s) necessary for transcriptional silencing.

The C terminus of nuclear hormone receptors is a complex structure that contains multiple functions. We are interested in the mechanism by which thyroid hormone converts its receptor from a transcriptional silencer to an activator of transcription. Both regulatory functions are localized in the ligand binding domain of this receptor superfamily member. In this study, we have identified and characterized several functional domains within the ligand binding domain of the human thyroid hormone receptor (TR beta) conferring transactivation. Interestingly, these domains are localized adjacent to hormone binding sites. One activation domain, designated tau 4, is only 17 amino acids in length and is localized at the extreme C terminus of TR. Deletion of six amino acids of tau 4 resulted in a receptor that could still bind hormone but acted as a constitutive silencer, indicating that tau 4 is required for both transactivation and relief of the silencing functions. In addition, we performed in vivo competition experiments, the results of which suggest that in the absence of tau 4 or hormone, TR is bound by a corepressor protein(s) and that one role of hormone is to release corepressor from the receptor. We propose a general model in which the role of hormone is to induce a conformational change in the receptor that subsequently affects the action of tau 4, leading to both relief of silencing and transcriptional activation.

Mouse Germline Restriction of Oct4 Expression by Germ Cell Nuclear Factor

The POU-domain transcription factor Oct4 is essential for the maintenance of the mammalian germline. In this study, we show that the germ cell nuclear factor (GCNF), an orphan nuclear receptor, represses Oct4 gene activity by specifically binding within the proximal promoter. GCNF expression inversely correlates with Oct4 expression in differentiating embryonal cells. GCNF overexpression in embryonal cells represses Oct4 gene and transgene activities, and we establish a link to transcriptional corepressors mediating repression by GCNF. In GCNF-deficient mouse embryos, Oct4 expression is no longer restricted to the germ cell lineage after gastrulation. Our studies suggest that GCNF is critical in repressing Oct4 gene activity as pluripotent stem cells differentiate and in confining Oct4 expression to the germline.

Alien, a Highly Conserved Protein with Characteristics of a Corepressor for Members of the Nuclear Hormone Receptor Superfamily

ABSTRACT Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the hormone. Corepressors, which bind to the receptor’s silencing domain, are involved in this repression. Hormone binding leads to dissociation of corepressors and binding of coactivators, which in turn mediate gene activation. Here, we describe the characteristics of Alien, a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of Alien from the receptor, as shown by the yeast two-hybrid system, glutathioneS-transferase pull-down, and coimmunoprecipitation experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows that Alien is localized in the cell nucleus. Alien is a highly conserved protein showing 90% identity between human and Drosophila. Drosophila Alien shows similar activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homologue of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily.

Co-repressors 2000

In the last 5 years, many co‐repressors have been identified in eukaryotes that function in a wide range of species, from yeast to Drosophila and humans. Co‐repressors are coregulators that are recruited by DNA‐bound transcriptional silencers and play essential roles in many pathways including differentiation, proliferation, programmed cell death, and cell cycle. Accordingly, it has been shown that aberrant interactions of co‐repressors with tran‐scriptional silencers provide the molecular basis of a variety of human diseases. Co‐repressors mediate transcriptional silencing by mechanisms that include direct inhibition of the basal transcription machinery and recruitment of chromatin‐modifying enzymes. Chromatin modification includes histone deacetylation, which is thought to lead to a compact chromatin structure to which the accessibility of transcriptional activators is impaired. In a general mechanistic view, the overall picture suggests that transcriptional si‐lencers and co‐repressors act in analogy to transcriptional activators and coactivators, but with the oppo‐site effect leading to gene silencing. We provide a comprehensive overview of the currently known higher eukaryotic co‐repressors, their mechanism of action, and their involvement in biological and pathophysiological pathways. We also show the dif‐ferent pathways that lead to the regulation of co‐repressor–silencer complex formation.—Burke, L. J., Baniahmad, A. Co‐repressors 2000. FASEB J. 14, 1876–1888 (2000)

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)

Activity of two different silencer elements of the chicken lysozyme gene can be compensated by enhancer elements.

The chicken lysozyme gene is constitutively expressed in macrophages. Transfection of recombinant genes containing different portions of the lysozyme 5′ upstream region revealed the existence of two negative transcriptional elements within 1 kb upstream of the start sites. Both elements placed upstream or downstream of a heterologous promoter‐gene unit repress transcription independent of their orientation and are therefore called silencer elements, although their repressing activities 3′ of the gene are reduced. One silencer (N‐1.0 kb) at position −1 kb consists of the central region of the chicken middle repetitive sequence element CR1 and can be divided into two functional domains. N‐1.0 kb is active in all cell types tested. The other silencer (N‐0.25 kb) at position −0.25 kb shows reduced activity in primary macrophages. Despite their different specificities, the activity of both silencer elements can be influenced similarly. An inverse linear relationship between the transcriptional activity of the tested constructs and the potential inhibition by the silencer elements was found: weak transcription units can be strongly repressed, whereas strong transcription units can be only weakly repressed. Such a mechanism may help to turn off completely a particular gene in situations or tissues where strong positive regulators are inactive.

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target

Background: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. Methods: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. Results: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. Conclusions: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.

The glucocorticoid receptor is associated with the RNA-binding nuclear matrix protein hnRNP U.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that is able to modulate gene activity by binding to its response element, interacting with other transcription factors, and contacting several accessory proteins such as coactivators. Here we show that GRIP120, one of the factors we have identified to interact with the glucocorticoid receptor, is identical to the heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix protein binding to RNA as well as to scaffold attachment regions. GR·hnRNP U complexes were identified by blotting and coimmunoprecipitation. The subnuclear distribution of GR and hnRNP U was characterized by indirect immunofluorescent labeling and confocal laser microscopy demonstrating a colocalization of both proteins. Using a nuclear transport-deficient deletion of hnRNP U, nuclear translocation was seen to be dependent on GR and dexamethasone. Transient transfections were used to identify possible interaction domains. Overexpressed hnRNP U interfered with glucocorticoid induction, and the COOH-terminal domains of both proteins were sufficient in mediating the transcriptional interference. A possible functional role for this GR binding-protein in addition to its binding to the nuclear matrix, to RNA, and to scaffold attachment regions is discussed.