BASP1 down-regulates RANKL-induced osteoclastogenesis.

The cytokine RANKL (Receptor Activator of NFκB Ligand) is the key driver of differentiation of monocytes/macrophages to form multi-nucleated, bone-resorbing osteoclasts, a process that is accompanied by significant changes in gene expression. We show that exposure to RANKL rapidly down-regulates expression of Brain Acid Soluble Protein 1 (BASP1) in cultured primary mouse bone marrow macrophages (BMMs), and that this reduced expression is causally linked to the osteoclastogenic process in vitro. Knocking down BASP1 expression in BMMs or eliminating its expression in these cells or in RAW 264.7 cells enhanced RANKL-induced osteoclastogenesis, promoted cell-cell fusion, and generated cultures containing larger osteoclasts with increased mineral degrading abilities relative to controls. Expression of exogenous BASP1 in BMMs undergoing osteoclastogenic differentiation produced the opposite effects. Upon exposure to RANKL, primary mouse BMMs in which BASP1 had been knocked down exhibited increased expression of the key osteoclastogenic transcription factor Nfatc1and of its downstream target genes Dc-stamp, Ctsk, Itgb3, and Mmp9 relative to controls. The knock-down cells also exhibited increased sensitivity to the pro-osteoclastogenic effects of RANKL. We conclude that BASP1 is a negative regulator of RANKL-induced osteoclastogenesis, which down-regulates the pro-osteoclastogenic gene expression pattern induced by this cytokine. Decreased expression of BASP1 upon exposure of BMMs to RANKL removes a negative regulator of osteoclastogenesis and promotes this process.

Cholesterol is required for transcriptional repression by BASP1.

Lipids are present within the cell nucleus, where they engage with factors involved in gene regulation. Cholesterol associates with chromatin in vivo and stimulates nucleosome packing in vitro, but its effects on specific transcriptional responses are not clear. Here, we show that the lipidated Wilms tumor 1 (WT1) transcriptional corepressor, brain acid soluble protein 1 (BASP1), interacts with cholesterol in the cell nucleus through a conserved cholesterol interaction motif. We demonstrate that BASP1 directly recruits cholesterol to the promoter region of WT1 target genes. Mutation of BASP1 to ablate its interaction with cholesterol or the treatment of cells with drugs that block cholesterol biosynthesis inhibits the transcriptional repressor function of BASP1. We find that the BASP1-cholesterol interaction is required for BASP1-dependent chromatin remodeling and the direction of transcription programs that control cell differentiation. Our study uncovers a mechanism for gene-specific targeting of cholesterol where it is required to mediate transcriptional repression.

A transcription factor IIA-binding site differentially regulates RNA polymerase II-mediated transcription in a promoter context-dependent manner.

RNA polymerase II (pol II) is required for the transcription of all protein-coding genes and as such represents a major enzyme whose activity is tightly regulated. Transcriptional initiation therefore requires numerous general transcriptional factors and cofactors that associate with pol II at the core promoter to form a pre-initiation complex. Transcription factor IIA (TFIIA) is a general cofactor that binds TFIID and stabilizes the TFIID-DNA complex during transcription initiation. Previous studies showed that TFIIA can make contact with the DNA sequence upstream or downstream of the TATA box, and that the region bound by TFIIA could overlap with the elements recognized by another factor, TFIIB, at adenovirus major late core promoter. Whether core promoters contain a DNA motif recognized by TFIIA remains unknown. Here we have identified a core promoter element upstream of the TATA box that is recognized by TFIIA. A search of the human promoter database revealed that many natural promoters contain a TFIIA recognition element (IIARE). We show that the IIARE enhances TFIIA-promoter binding and enhances the activity of TATA-containing promoters, but represses or activates promoters that lack a TATA box. Chromatin immunoprecipitation assays revealed that the IIARE activates transcription by increasing the recruitment of pol II, TFIIA, TAF4, and P300 at TATA-dependent promoters. These findings extend our understanding of the role of TFIIA in transcription, and provide new insights into the regulatory mechanism of core promoter elements in gene transcription by pol II.

The Wilms' tumor suppressor protein WT1 is processed by the serine protease HtrA2/Omi.

The Wilms' tumor suppressor protein WT1 functions as a transcriptional regulator of genes controlling growth, apoptosis, and differentiation. It has become clear that WT1 can act as an oncogene in many tumors, primarily through the inhibition of apoptosis. Here, we identify the serine protease HtrA2 as a WT1 binding partner and find that it cleaves WT1 at multiple sites following the treatment of cells with cytotoxic drugs. Ablation of HtrA2 activity either by chemical inhibitor or by siRNA prevents the proteolysis of WT1 under apoptotic conditions. Moreover, the apoptosis-dependent cleavage of WT1 is defective in HtrA2 knockout cells. Proteolysis of WT1 by HtrA2 causes the removal of WT1 from its binding sites at gene promoters, leading to alterations in gene regulation that enhance apoptosis. Our findings provide insights into the function of HtrA2 in the regulation of apoptosis and the oncogenic activities of WT1.

WT1 regulates the development of the posterior taste field.

Despite the importance of taste in determining nutrient intake, our understanding of the processes that control the development of the peripheral taste system is lacking. Several early regulators of taste development have been identified, including sonic hedgehog, bone morphogenetic protein 4 and multiple members of the Wnt/ß-catenin signaling pathway. However, the regulation of these factors, including their induction, remains poorly understood. Here, we identify a crucial role for the Wilms' tumor 1 protein (WT1) in circumvallate (CV) papillae development. WT1 is a transcription factor that is important in the normal development of multiple tissues, including both the olfactory and visual systems. In mice, WT1 expression is detectable by E12.5, when the CV taste placode begins to form. In mice lacking WT1, the CV fails to develop normally and markers of early taste development are dysregulated compared with wild type. We demonstrate that expression of the WT1 target genes Lef1, Ptch1 and Bmp4 is significantly reduced in developing tongue tissue derived from Wt1 knockout mice and that, in normal tongue, WT1 is bound to the promoter regions of these genes. Moreover, siRNA knockdown of WT1 in cultured taste cells leads to a reduction in the expression of Lef1 and Ptch1. Our data identify WT1 as a crucial transcription factor in the development of the CV through the regulation of multiple signaling pathways that have established roles in the formation and patterning of taste placodes.

Control of epigenetic states by WT1 via regulation of de novo DNA methyltransferase 3A.

Although tumour suppressor gene hypermethylation is a universal feature of cancer cells, little is known about the necessary molecular triggers. Here, we show that Wilms' tumour 1 (WT1), a developmental master regulator that can also act as a tumour suppressor or oncoprotein, transcriptionally regulates the de novo DNA methyltransferase 3A (DNMT3A) and that cellular WT1 levels can influence DNA methylation of gene promoters genome-wide. Specifically, we demonstrate that depletion of WT1 by short-interfering RNAs leads to reduced DNMT3A in Wilms' tumour cells and human embryonal kidney-derived cell lines. Chromatin immunoprecipitation assays demonstrate WT1 recruitment to the DNMT3A promoter region and reporter assays confirm that WT1 directly transactivates DNMT3A expression. Consistent with this regulatory role, immunohistochemical analysis shows co-expression of WT1 and DNMT3A proteins in nuclei of blastemal cells in human fetal kidney and Wilms' tumours. Using genome-wide promoter methylation arrays, we show that human embryonal kidney cells over-expressing WT1 acquire DNA methylation changes at specific gene promoters where DNMT3A recruitment is increased, with hypermethylation being associated with silencing of gene expression. Elevated DNMT3A is also demonstrated at hypermethylated genes in Wilms' tumour cells, including a region of long-range epigenetic silencing. Finally, we show that depletion of WT1 in Wilms' tumour cells can lead to reactivation of gene expression from methylated promoters, such as TGFB2, a key modulator of epithelial-mesenchymal transitions. Collectively, our work defines a new regulatory modality for WT1 involving elicitation of epigenetic alterations which is most likely crucial to its functions in development and disease.

Mechanisms of transcriptional regulation by WT1 (Wilms' tumour 1).

The WT1 (Wilms' tumour 1) gene encodes a zinc finger transcription factor and RNA-binding protein that direct the development of several organs and tissues. WT1 manifests both tumour suppressor and oncogenic activities, but the reasons behind these opposing functions are still not clear. As a transcriptional regulator, WT1 can either activate or repress numerous target genes resulting in disparate biological effects such as growth, differentiation and apoptosis. The complex nature of WT1 is exemplified by a plethora of isoforms, post-translational modifications and multiple binding partners. How WT1 achieves specificity to regulate a large number of target genes involved in diverse physiological processes is the focus of the present review. We discuss the wealth of the growing molecular information that defines our current understanding of the versatility and utility of WT1 as a master regulator of organ development, a tumour suppressor and an oncogene.

TFIIB dephosphorylation links transcription inhibition with the p53-dependent DNA damage response.

The general transcription factor II B (TFIIB) plays a central role in both the assembly of the transcription complex at gene promoters and also in the events that lead to transcription initiation. TFIIB is phosphorylated at serine-65 at the promoters of several endogenous genes, and this modification is required to drive the formation of gene promoter-3' processing site contacts through the cleavage stimulation factor 3' (CstF 3')-processing complex. Here we demonstrate that TFIIB phosphorylation is dispensable for the transcription of genes activated by the p53 tumor suppressor. We find that the kinase activity of TFIIH is critical for the phosphorylation of TFIIB serine-65, but it is also dispensable for the transcriptional activation of p53-target genes. Moreover, we demonstrate that p53 directly interacts with CstF independent of TFIIB phosphorylation, providing an alternative route to the recruitment of 3'-processing complexes to the gene promoter. Finally, we show that DNA damage leads to a reduction in the level of phospho-ser65 TFIIB that leaves the p53 transcriptional response intact, but attenuates transcription at other genes. Our data reveal a mode of phospho-TFIIB-independent transcriptional regulation that prioritizes the transcription of p53-target genes during cellular stress.

The transcription cycle in eukaryotes: from productive initiation to RNA polymerase II recycling.

The cycle of eukaryotic transcription, from initiation to elongation and termination is regulated at multiple steps. Coordinated action of regulatory factors keeps in check the transcriptional competence of RNA polymerase II (RNAPII) at different stages. Productive transcription requires the escape of the paused RNAPII from the promoter and transition to rapid elongation of the transcript. Numerous studies have identified diverse mechanisms of initiating transcription by overriding inhibitory signals at the gene promoter. The general theme that has emerged is that the balance between positive and negative regulatory factors determines the overall rate of transcription. Recently transcription termination has emerged as an important area of transcriptional regulation that is coupled with the efficient recycling of RNAPII. The factors associated with transcription termination can also mediate gene looping and thereby determine the efficiency of re-initiation. This review highlights these regulatory steps, the key modulators involved in transcription dynamics, and the emerging tools to analyze them.

WT1 and its transcriptional cofactor BASP1 redirect the differentiation pathway of an established blood cell line.

The Wilms' tumour suppressor WT1 (Wilms' tumour 1) is a transcriptional regulator that plays a central role in organogenesis, and is mutated or aberrantly expressed in several childhood and adult malignancies. We previously identified BASP1 (brain acid-soluble protein 1) as a WT1 cofactor that suppresses the transcriptional activation function of WT1. In the present study we have analysed the dynamic between WT1 and BASP1 in the regulation of gene expression in myelogenous leukaemia K562 cells. Our findings reveal that BASP1 is a significant regulator of WT1 that is recruited to WT1-binding sites and suppresses WT1-mediated transcriptional activation at several WT1 target genes. We find that WT1 and BASP1 can divert the differentiation programme of K562 cells to a non-blood cell type following induction by the phorbol ester PMA. WT1 and BASP1 co-operate to induce the differentiation of K562 cells to a neuronal-like morphology that exhibits extensive arborization, and the expression of several genes involved in neurite outgrowth and synapse formation. Functional analysis revealed the relevance of the transcriptional reprogramming and morphological changes, in that the cells elicited a response to the neurotransmitter ATP. Taken together, the results of the present study reveal that WT1 and BASP1 can divert the lineage potential of an established blood cell line towards a cell with neuronal characteristics.

The BASP1 transcriptional corepressor modifies chromatin through lipid-dependent and lipid-independent mechanisms.

The transcriptional corepressor BASP1 requires N-terminal myristoylation for its activity and functions through interactions with nuclear lipids. Here we determine the role of BASP1 lipidation in histone modification and the modulation of chromatin accessibility. We find that the removal of the active histone modifications H3K9ac and H3K4me3 by BASP1 requires the N-terminal myristoylation of BASP1. In contrast, the placement of the repressive histone modification, H3K27me3, by BASP1 does not require BASP1 lipidation. RNA-seq and ATAC-seq analysis finds that BASP1 regulates the activity of multiple transcription factors and induces extensive changes in chromatin accessibility. We find that ∼50% of BASP1 target genes show lipidation-dependent chromatin compaction and transcriptional repression. Our results suggest that BASP1 elicits both lipid-dependent and lipid-independent functions in histone modification and transcriptional repression. In accordance with this, we find that the tumor suppressor activity of BASP1 is also partially dependent on its myristoylation.

Dynamic interaction between WT1 and BASP1 in transcriptional regulation during differentiation.

The Wilms' tumour suppressor protein WT1 plays a central role in the development of the kidney and also other organs. WT1 can act as a transcription factor with highly context-specific activator and repressor functions. We previously identified Brain Acid Soluble Protein 1 (BASP1) as a transcriptional cosuppressor that can block the transcriptional activation function of WT1. WT1 and BASP1 are co-expressed during nephrogenesis and both proteins ultimately become restricted to the podocyte cells of the adult kidney. Here, we have analysed the WT1/BASP1 complex in a podocyte precursor cell line that can be induced to differentiate. Chromatin immunoprecipitation revealed that WT1 and BASP1 occupy the promoters of the Bak, c-myc and podocalyxin genes in podocyte precursor cells. During differentiation-dependent upregulation of podocalyxin expression BASP1 occupancy of the podocalyxin promoter is reduced compared to that of WT1. In contrast, the repressive WT1/BASP1 occupancy of the c-myc and Bak promoters is maintained and these genes are downregulated during the differentiation process. We provide evidence that the regulation of BASP1 promoter occupancy involves the sumoylation of BASP1. Our results reveal a dynamic cooperation between WT1 and BASP1 in the regulation of gene expression during differentiation.

Differential Effects of Diet and Weight on Taste Responses in Diet-Induced Obese Mice.

OBJECTIVE: Previous studies have reported that individuals with obesity have reduced taste perception, but the relationship between obesity and taste is poorly understood. Earlier work has demonstrated that diet-induced obesity directly impairs taste. Currently, it is not clear whether these changes to taste are due to obesity or to the high-fat diet exposure. The goal of the current study was to determine whether diet or excess weight is responsible for the taste deficits induced by diet-induced obesity. METHODS: C57BL/6 mice were placed on either high-fat or standard chow in the presence or absence of captopril. Mice on captopril did not gain weight when exposed to a high-fat diet. Changes in the responses to different taste stimuli were evaluated using live cell imaging, brief-access licking, immunohistochemistry, and real-time polymerase chain reaction. RESULTS: Diet and weight gain each affected taste responses, but their effects varied by stimulus. Two key signaling proteins, α-gustducin and phospholipase Cß2, were significantly reduced in the mice on the high-fat diet with and without weight gain, identifying a potential mechanism for the reduced taste responsiveness to some stimuli. CONCLUSIONS: Our data indicate that, for some stimuli, diet alone can cause taste deficits, even without the onset of obesity.

Transcriptional regulation by WT1 in development.

The Wilms' tumour suppressor protein, WT1, plays a central role in the development of the genitourinary system and also other organs and tissues. WT1 can act as a transcriptional regulator or as an RNA processing factor in an isoform-dependent manner. The mechanisms that are used by WT1 to regulate transcription, and its associated target genes have been difficult to study, in part because the transcription function of WT1 is highly context-dependent. Recent studies have provided new insights into how WT1 achieves this specificity and have uncovered new target genes that are regulated by WT1 during development. In addition, ongoing studies of transgenic animals and analyses in kidney explant systems have revealed further roles for WT1 in development.

IDPpi: Protein-Protein Interaction Analyses of Human Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are characterized by the lack of a fixed tertiary structure and are involved in the regulation of key biological processes via binding to multiple protein partners. IDPs are malleable, adapting to structurally different partners, and this flexibility stems from features encoded in the primary structure. The assumption that universal sequence information will facilitate coverage of the sparse zones of the human interactome motivated us to explore the possibility of predicting protein-protein interactions (PPIs) that involve IDPs based on sequence characteristics. We developed a method that relies on features of the interacting and non-interacting protein pairs and utilizes machine learning to classify and predict IDP PPIs. Consideration of both sequence determinants specific for conformational organizations and the multiplicity of IDP interactions in the training phase ensured a reliable approach that is superior to current state-of-the-art methods. By applying a strict evaluation procedure, we confirm that our method predicts interactions of the IDP of interest even on the proteome-scale. This service is provided as a web tool to expedite the discovery of new interactions and IDP functions with enhanced efficiency.

Core promoter-dependent TFIIB conformation and a role for TFIIB conformation in transcription start site selection.

The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5' and 3' parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitope-specific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.

BASP1 is a transcriptional cosuppressor for the Wilms' tumor suppressor protein WT1.

The Wilms' tumor suppressor protein WT1 is a transcriptional regulator that plays a key role in the development of the kidneys. The transcriptional activation domain of WT1 is subject to regulation by a suppression region within the N terminus of WT1. Using a functional assay, we provide direct evidence that this requires a transcriptional cosuppressor, which we identify as brain acid soluble protein 1 (BASP1). WT1 and BASP1 associate within the nuclei of cells that naturally express both proteins. BASP1 can confer WT1 cosuppressor activity in transfection assays, and elimination of endogenous BASP1 expression augments transcriptional activation by WT1. BASP1 is present in the developing nephron structures of the embryonic kidney and, coincident with that of WT1, its expression is restricted to the highly specialized podocyte cells of the adult kidney. Taken together, our results show that BASP1 is a WT1-associated factor that can regulate WT1 transcriptional activity.

The modulation of WTI transcription function by cofactors.

Wilms' tumour is a paediatric malignancy of the kidneys that affects one in every 10,000 live births, making it the most common solid tumour in the young. This cancer arises due to a failure of the metanephric mesenchyme to differentiate and form the kidney filtration units and tubules, which instead undergo uncontrolled proliferation. WT1 (Wilms' tumour 1) was identified as a factor that is frequently mutated in Wilms' tumours. WT1 plays a central role in the development of the genito-urinary organs and also other regions of the embryo. A major function of WT1 is to act as a regulator of transcription, controlling the expression of genes that are involved in proliferation and differentiation. WT1 can either activate or repress transcription of its target genes. Thus the transcription function of WT1 is highly context-specific, and can be modulated by a number of cofactors. Here, the known interaction partners of WT1 and the mechanisms by which they modulate WT1 transcription function will be discussed.

A conformational change in TFIIB is required for activator-mediated assembly of the preinitiation complex.

TFIIB plays a pivotal role during assembly of the RNA polymerase II transcription preinitiation complex. TFIIB is composed of two domains that engage in an intramolecular interaction that can be disrupted by the VP16 activation domain. In this study, we describe a novel human TFIIB derivative harbouring two point mutations in the highly conserved N-terminal charged cluster domain. This mutant, TFIIB R53E:R66E, exhibits an enhanced affinity in its intramolecular interaction when compared with wild-type TFIIB. Consistent with this, the mutant displays a significantly reduced affinity for VP16. However, its ability to complex with TATA-binding protein at a model promoter is equivalent to that of wild-type TFIIB. Furthermore, this TFIIB derivative is able to support high order preinitiation complex assembly in the absence of an activator. Strikingly though, an activator fails to recruit the TFIIB mutant to the promoter. Taken together, our results show that a TFIIB conformational change is critical for the formation of activator-dependent transcription complexes.

In Vitro Transcription to Study WT1 Function.

In vitro transcription methods using mammalian nuclear extracts have been available for over 30 years and have allowed sophisticated biochemical analyses of the transcription process. This method has been extensively used to study the basic mechanisms of transcription, allowing the identification of the general transcription factors and elucidation of their mechanisms of action. Gene-specific transcriptional regulators have also been studied using in vitro transcription. This has facilitated the identification of their cofactors and provided information on their function that is invaluable to facilitate their study in a more physiological setting. Here we describe the application of in vitro transcription methods to study the mechanism of action of WT1. Coupling transcription assays with methods to purify transcription complexes, and protein affinity chromatography, has provided insights into how WT1 can both positively and negatively regulate transcription.