DNA-binding Partner Research Articles

Dose-dependent responses to canonical Wnt transcriptional complexes in the regulation of mammalian nephron progenitors.

In vivo and in vitro studies argue that concentration-dependent Wnt signaling regulates mammalian nephron progenitor cell (NPC) programs. Canonical Wnt signaling is regulated through the stabilization of β-catenin, a transcriptional co-activator when complexed with Lef/Tcf DNA-binding partners. Using the GSK3β inhibitor CHIR99021 (CHIR) to block GSK3β-dependent destruction of β-catenin, we examined dose-dependent responses to β-catenin in mouse NPCs, using mRNA transduction to modify gene expression. Low CHIR-dependent proliferation of NPCs was blocked on β-catenin removal, with evidence of NPCs arresting at the G2-M transition. While NPC identity was maintained following β-catenin removal, mRNA-seq identified low CHIR and β-catenin dependent genes. High CHIR activated nephrogenesis. Nephrogenic programming was dependent on Lef/Tcf factors and β-catenin transcriptional activity. Molecular and cellular features of early nephrogenesis were driven in the absence of CHIR by a mutated stabilized form of β-catenin. Chromatin association studies indicate low and high CHIR response genes are likely direct targets of canonical Wnt transcriptional complexes. Together, these studies provide evidence for concentration-dependent Wnt signaling in the regulation of NPCs and provide new insight into Wnt targets initiating mammalian nephrogenesis.

Netrin-1 and UNC5B Cooperate with Integrins to Mediate YAP-Driven Cytostasis.

Netrins are widely perceived as procancer proteins; however, we uncover an anticancer function for Netrin-1 and its receptor UNC5B.

Hippo pathway effectors YAP, TAZ and TEAD are associated with EMT master regulators ZEB, Snail and with aggressive phenotype in phyllodes breast tumors

BackgroundPhyllodes tumors (PTs) of the breast are uncommon fibroepithelial neoplasms that tend to recur locally and may have metastatic potential. Their pathogenesis is poorly understood. Hippo signaling pathway plays an essential role in organ size control, tumor suppression, tissue regeneration and stem cell self-renewal. Hippo signaling dysfunction has been implicated in cancer. Recent evidence suggests that there is cross-talk between the Hippo signaling key proteins YAP/TAZ and the epithelial-mesenchymal transition (EMT) master regulators Snail and ZEB. In this study we aimed to investigate the expression of Hippo signaling pathway components and EMT regulators in PTs, in relation to tumor grade. MethodsExpression of Hippo signaling effector proteins YAP, TAZ and their DNA binding partner TEAD was evaluated by immunohistochemistry in paraffin-embedded tissue specimens from 86 human phyllodes breast tumors (45 benign, 21 borderline, 20 malignant), in comparison with tumor grade and with the expression of EMT-related transcription factors ZEB and Snail. ResultsNuclear immunopositivity for YAP, TAZ and TEAD was detected in both stromal and epithelial cells in PTs and was significantly higher in high grade tumors. Interestingly, there was a significant correlation between the expression of YAP, TAZ, TEAD and the expression of ZEB and SNAIL. ConclusionsOur results originally implicate Hippo signaling pathway in PTs pathogenesis and suggest that an interaction between Hippo signaling key components and EMT regulators may promote the malignant features of PTs.

Stabilization of β-Catenin Directs HEB to Limit Thymic Selection.

Activation of β-catenin in CD4+CD8+ double-positive (DP) thymocytes halts development before the thymic selection stage and predisposes to transformation. Leukemogenesis, but not the developmental block, depends on TCF-1, β-catenin's DNA-binding partner. In this study, we show that β-catenin activation directs the DNA-binding protein HEB to block DP thymocyte development. Conditional loss of HEB in DP thymocytes with stabilized β-catenin restores the frequencies of postselection TCRβhi/CCR7+ and TCRβhi/CD69+ DPs and their cell-cycle profile. This recovery is associated with significant reversal of β-catenin-induced expression changes, particularly those related to the CD69+ DP cell signature and to cell-cycle pathways. Stabilizing β-catenin in DP thymocytes directs HEB binding to ≈11,000 novel DNA sites throughout the genome. Novel HEB sites mark most CD69+ DP cell signature genes that change expression upon activation of β-catenin and then revert after loss of HEB. Moreover, many of the novel HEB sites occupy promoter regions of genes enriched in mitotic cell cycle pathways. HEB binding to those regions correlates with downregulation of the associated genes, and HEB inactivation restores expression to physiologic levels. These findings highlight a molecular interplay between HEB and β-catenin that can impair thymic development.

Multi-signal regulation of the GSK-3β homolog Rim11 controls meiosis entry in budding yeast

Starvation in diploid budding yeast cells triggers a cell-fate program culminating in meiosis and spore formation. Transcriptional activation of early meiotic genes (EMGs) hinges on the master regulator Ime1, its DNA-binding partner Ume6, and GSK-3β kinase Rim11. Phosphorylation of Ume6 by Rim11 is required for EMG activation. We report here that Rim11 functions as the central signal integrator for controlling Ume6 phosphorylation and EMG transcription. In nutrient-rich conditions, PKA suppresses Rim11 levels, while TORC1 retains Rim11 in the cytoplasm. Inhibition of PKA and TORC1 induces Rim11 expression and nuclear localization. Remarkably, nuclear Rim11 is required, but not sufficient, for Rim11-dependent Ume6 phosphorylation. In addition, Ime1 is an anchor protein enabling Ume6 phosphorylation by Rim11. Subsequently, Ume6-Ime1 coactivator complexes form and induce EMG transcription. Our results demonstrate how various signaling inputs (PKA/TORC1/Ime1) converge through Rim11 to regulate EMG expression and meiosis initiation. We posit that the signaling-regulatory network elucidated here generates robustness in cell-fate control.

Stabilization of β-Catenin directs HEB to limit Thymic Selection

Abstract Activation of β-catenin in CD4+CD8+ (DP) thymocytes stalls progression through thymic selection and predisposes to transformation. Leukemogenesis but not the developmental block depends on Tcf-1, β-catenin’s DNA binding partner. Here we show that β-catenin deregulation directs the DNA-binding protein HEB to stall thymocyte development at the thymic selection stage. Conditional loss of HEB along with β-catenin activation restores the frequency of TCRβ+/CCR7+ DP thymocytes, with reduced CXCR4 expression indicating their potential to migrate to the thymic medulla. It also restores the emergence of TCRβ+/CD69+ DPs and enhances the overall CD69+DP expression profile that were diminished by activating β-catenin, indicating a functional response to TCR stimulation. β-Catenin stabilization redirects the binding of HEB to ~11,000 novel DNA sites throughout the genome. A major fraction of these sites occupy promoter regions of genes associated with cell cycle regulation and mitotic processes. HEB binding to those regions corelates with downregulation of the associated genes, and HEB inactivation restores their expression to physiologic levels. These findings highlight the complex molecular interplay between HEB and β-catenin in impairing thymic development.

Cardiomyocyte proliferation is suppressed by ARID1A-mediated YAP inhibition during cardiac maturation

The inability of adult human cardiomyocytes to proliferate is an obstacle to efficient cardiac regeneration after injury. Understanding the mechanisms that drive postnatal cardiomyocytes to switch to a non-regenerative state is therefore of great significance. Here we show that Arid1a, a subunit of the switching defective/sucrose non-fermenting (SWI/SNF) chromatin remodeling complex, suppresses postnatal cardiomyocyte proliferation while enhancing maturation. Genome-wide transcriptome and epigenome analyses revealed that Arid1a is required for the activation of a cardiomyocyte maturation gene program by promoting DNA access to transcription factors that drive cardiomyocyte maturation. Furthermore, we show that ARID1A directly binds and inhibits the proliferation-promoting transcriptional coactivators YAP and TAZ, indicating ARID1A sequesters YAP/TAZ from their DNA-binding partner TEAD. In ischemic heart disease, Arid1a expression is enhanced in cardiomyocytes of the border zone region. Inactivation of Arid1a after ischemic injury enhanced proliferation of border zone cardiomyocytes. Our study illuminates the pivotal role of Arid1a in cardiomyocyte maturation, and uncovers Arid1a as a crucial suppressor of cardiomyocyte proliferation.

Chemogenetic Minitool for Dissecting the Roles of Protein Phase Separation.

Biomolecular condensate is an emerging structural entity that regulates various cellular processes. Recent studies have discovered the phase-separation (PS) capability of several transcription factors (TFs) including YAP/TAZ upon biological stimuli, which provide new mechanisms of gene regulation. However, it remains mostly unanswered as to whether PS from a diffuse state to a phase-separated state promotes gene transcription. To address this question, we have designed a chemogenetic tool, dubbed SPARK-ON, which manipulates the PS of YAP and TAZ without a biological stimulus, forming condensates that are transcriptionally active, containing the DNA-binding partner TEAD, genomic DNA, transcriptional machinery, and nascent RNA. Most importantly, PS of TAZ increases the transcription of its target genes. Therefore, our data indicate that PS promotes gene transcription of TAZ. SPARK-ON is advantageous to current mutagenesis-based approaches that are often problematic when mutagenesis affects the transcriptional activity of a TF. Furthermore, protein abundance levels also affect gene transcription, but PS depends on protein abundance because PS occurs only when the protein level is above a saturation concentration. SPARK-ON decouples PS from protein abundance levels without introducing mutations and thus will find important applications in understanding the biological roles of PS for many TFs and other biomolecular condensates.

Depicting pseudotime-lagged causality across single-cell trajectories for accurate gene-regulatory inference.

Identifying the causal interactions in gene-regulatory networks requires an accurate understanding of the time-lagged relationships between transcription factors and their target genes. Here we describe DELAY (short for Depicting Lagged Causality), a convolutional neural network for the inference of gene-regulatory relationships across pseudotime-ordered single-cell trajectories. We show that combining supervised deep learning with joint probability matrices of pseudotime-lagged trajectories allows the network to overcome important limitations of ordinary Granger causality-based methods, for example, the inability to infer cyclic relationships such as feedback loops. Our network outperforms several common methods for inferring gene regulation and, when given partial ground-truth labels, predicts novel regulatory networks from single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) data sets. To validate this approach, we used DELAY to identify important genes and modules in the regulatory network of auditory hair cells, as well as likely DNA-binding partners for two hair cell cofactors (Hist1h1c and Ccnd1) and a novel binding sequence for the hair cell-specific transcription factor Fiz1. We provide an easy-to-use implementation of DELAY under an open-source license at https://github.com/calebclayreagor/DELAY.

AMPLIFICATION AND HIGHER EXPRESSION OF THE ZNF281 GENE IN PANCREATIC CANCER CORRELATES WITH POOR PROGNOSIS

Establishing precise gene expression patterns in different cell types is essential for the proper differentiation and development of multicellular organisms. The resulting level of transcription is determined both by the genetic nucleotide sequence of DNA and by epigenetic factors that modify chromatin. Epigenetic repressors of the Polycomb group (PcG) are regulatory proteins that repress gene transcription and maintain correct pattern of gene expression in multicellular organisms. PcG proteins form two main complexes: Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2) that possess ubiquitin ligase and histone methyltransferase enzymatic activities, respectively. Several mechanisms have been suggested to account for the recruitment of Polycomb proteins in mammals, one of which involves interactions with specific DNA-binding factors. While deregulation main PcG genes in cancer have been well documented, the role of PcG DNA-binding partners in oncology remains elusive. In the present study, we analyzed genomic and transcriptomic databases of clinical tumor samples (cBioPortal, TNMplot, KMplot) to evaluate clinical correlations of Polycomb-associated DNA-binding proteins. We found that amplifications and higher expression of the ZNF281 gene are often found in pancreatic cancer and correlate with poor prognosis of overall survival.

The LEF-1 high-mobility group box adopts residual structure in its DNA-free form

Intrinsically disordered regions (IDRs) lack stable tertiary structure and instead rapidly interconvert between different conformations. This structural plasticity enables IDRs to act as key players in cellular signaling pathways. Transcription factors are enriched in IDRs, many of which are stabilized by or acquire tertiary structure in the presence of DNA or other binding partners. Using the T-cell factor/lymphoid enhancer binding factor 1 (TCF/LEF-1) transcription factor as a model system, we characterized the structure and dynamics of the high-mobility group (HMG) domain in the absence of DNA. Inclusion of the IDRs that flank the HMG domain led to enhanced solubility of the protein. Secondary 13Cα chemical shifts, 1H nuclear Overhauser effects, 15N spin relaxation, and 1HN solvent paramagnetic relaxation enhancements indicate that the three helices in the HMG domain are oriented similarly to the DNA-bound form of the protein. By contrast, the flanking IDRs do not show evidence of structure. Helix 1 and helix 3 appear to be less stable in the DNA-free conformation, indicating some form of conformational exchange or local motion in the absence of DNA. Given the high degree of sequence conservation in the TCF/LEF family of transcription factors, our results should apply to other members of the family.

RPD3 and UME6 are involved in the activation of PDR5 transcription and pleiotropic drug resistance in \u03c10 cells of Saccharomyces cerevisiae

BackgroundIn Saccharomyces cerevisiae, the retrograde signalling pathway is activated in ρ0/− cells, which lack mitochondrial DNA. Within this pathway, the activation of the transcription factor Pdr3 induces transcription of the ATP-binding cassette (ABC) transporter gene, PDR5, and causes pleiotropic drug resistance (PDR). Although a histone deacetylase, Rpd3, is also required for cycloheximide resistance in ρ0/− cells, it is currently unknown whether Rpd3 and its DNA binding partners, Ume6 and Ash1, are involved in the activation of PDR5 transcription and PDR in ρ0/− cells. This study investigated the roles of RPD3, UME6, and ASH1 in the activation of PDR5 transcription and PDR by retrograde signalling in ρ0 cells.Resultsρ0 cells in the rpd3∆ and ume6∆ strains, with the exception of the ash1∆ strain, were sensitive to fluconazole and cycloheximide. The PDR5 mRNA levels in ρ0 cells of the rpd3∆ and ume6∆ strains were significantly reduced compared to the wild-type and ash1∆ strain. Transcriptional expression of PDR5 was reduced in cycloheximide-exposed and unexposed ρ0 cells of the ume6∆ strain; the transcriptional positive response of PDR5 to cycloheximide exposure was also impaired in this strain.ConclusionsRPD3 and UME6 are responsible for enhanced PDR5 mRNA levels and PDR by retrograde signalling in ρ0 cells of S. cerevisiae.

Tramtrack acts during late pupal development to direct ant caste identity.

A key question in the rising field of neuroepigenetics is how behavioral plasticity is established and maintained in the developing CNS of multicellular organisms. Behavior is controlled through systemic changes in hormonal signaling, cell-specific regulation of gene expression, and changes in neuronal connections in the nervous system, however the link between these pathways is unclear. In the ant Camponotus floridanus, the epigenetic corepressor CoREST is a central player in experimentally-induced reprogramming of caste-specific behavior, from soldier (Major worker) to forager (Minor worker). Here, we show this pathway is engaged naturally on a large genomic scale during late pupal development targeting multiple genes differentially expressed between castes, and central to this mechanism is the protein tramtrack (ttk), a DNA binding partner of CoREST. Caste-specific differences in DNA binding of ttk co-binding with CoREST correlate with caste-biased gene expression both in the late pupal stage and immediately after eclosion. However, we find a unique set of exclusive Minor-bound genes that show ttk pre-binding in the late pupal stage preceding CoREST binding, followed by caste-specific gene repression on the first day of eclosion. In addition, we show that ttk binding correlates with neurogenic Notch signaling, and that specific ttk binding between castes is enriched for regulatory sites associated with hormonal function. Overall our findings elucidate a pathway of transcription factor binding leading to a repressive epigenetic axis that lies at the crux of development and hormonal signaling to define worker caste identity in C. floridanus.

High-Resolution SNP-Array Profiling of “Ultra-High Risk” CLL Cases Reveals New Recurrent Genomic Alterations

High-Resolution SNP-Array Profiling of “Ultra-High Risk” CLL Cases Reveals New Recurrent Genomic Alterations

Abstract IA04: Regulation of the Hippo pathway in cancer

Abstract The Hippo pathway is crucial in organ size control and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include the protein kinases of MST1/2, MAP4Ks, LATS1/2, the transcription co-activators YAP/TAZ, and their DNA binding partners TEADs. LATS phosphorylates YAP/TAZ to promote cytoplasmic localization and degradation, thereby inhibiting YAP/TAZ and cell growth. The Hippo pathway is regulated by a wide range of signals, including cell density, GPCR, cellular energy levels, and mechanical cues. We will present our recent progresses on Hippo pathway regulation and its role in cancer. The emerging role of the Hippo pathway in tumorigenesis suggests the potential therapeutic value of targeting this pathway for cancer treatment. Citation Format: Kun-Liang Guan. Regulation of the Hippo pathway in cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA04.

Immunoprecipitation of DNA:RNA Hybrids Using the S9.6 Antibody.

Formation of DNA:RNA hybrids or R-loops contributes to numerous biologic processes. The development of the S9.6 antibody makes the analysis of R-Loops (DNA:RNA hybrids) possible through immunoprecipitation. Here, we describe the isolation of DNA:RNA hybrid structures using the S9.6 antibody. Using this protocol, both the DNA and RNA binding partners of the R-loop can be analyzed via qPCR, whole genome sequencing, or other methods.

OsLUGL is involved in the regulating auxin level and OsARFs expression in rice (Oryza sativa L.)

Specification of floral organ identity is critical for floral morphology and inflorescence architecture. Floral organ identity in plants is controlled by floral homeotic A/B/C/D/E-class genes. Although multiple genes regulate floral organogenesis, our understanding of the regulatory network remains fragmentary. Here, we characterized a rice floral organ gene KAIKOUXIAO (KKX), mutation of which produces an uncharacteristic open hull, abnormal seed and semi-sterility. KKX encodes a putative LEUNIG-like (LUGL) transcriptional regulator OsLUGL. OsLUGL is preferentially expressed in young panicles and its protein can interact with OsSEU, which functions were reported as an adaptor for LEUNIG. OsLUGL-OsSEU functions together as a transcriptional co-regulatory complex to control organ identity. SEP3 (such as OsMADS8) and AP1 (such as OsMADS18) serve as the DNA-binding partner of OsLUGL-OsSEU complex. Further studies indicated that OsMADS8 and OsMADS18 could bind to the promoter of OsGH3-8. The altered expression of OsGH3-8 might cause the increased auxin level and the decreased expression of OsARFs. Overall, our results demonstrate a possible pathway whereby OsLUGL-OsSEU-OsAP1-OsSEP3 complex as a transcriptional co-regulator by targeting the promoter of OsGH3-8, then affecting auxin level, OsARFs expression and thereby influencing floral development. These findings provide a valuable insight into the molecular functions of OsLUGL in rice floral development.

Abstract SY40-01: The hippo pathway in growth, regeneration, and cancer

Abstract The Hippo pathway is crucial in organ size control and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include the protein kinases of MST1/2, MAP4Ks, LATS1/2, the transcription co-activators YAP/TAZ and their DNA binding partners TEADs. LATS phosphorylates YAP/TAZ to promote cytoplasmic localization and degradation, thereby inhibiting YAP/TAZ and cell growth. The Hippo pathway is regulated by a wide range of signals, including cell density, GPCR, cellular energy levels, and mechanical cues. We will present our recent progresses on Hippo pathway regulation and its role in cancer. The emerging role of the Hippo pathway in tumorigenesis suggests potential therapeutic value of targeting this pathway for cancer treatment. Citation Format: Kun-Liang Guan. The hippo pathway in growth, regeneration, and cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY40-01.

Assessment of total, ligand-induced peroxisome proliferator activated receptor \u03b3 ligand activity in serum

BackgroundHumans are exposed to a complex mixture of environmental chemicals that impact bone and metabolic health, and traditional exposure assessments struggle to capture these exposure scenarios. Peroxisome proliferator activated receptor-gamma (PPARγ) is an essential regulator of metabolic and bone homeostasis, and its inappropriate activation by environmental chemicals can set the stage for adverse health effects. Here, we present the development of the Serum PPARγ Activity Assay (SPAA), a simple and cost-effective method to measure total ligand activity in small volumes of serum.MethodsFirst, we determined essential components of the bioassay. Cos-7 cells were transfected with combinations of expression vectors for human PPARγ and RXRα, the obligate DNA-binding partner of PPARγ, along with PPRE (DR1)-driven luciferase and control eGFP reporter constructs. Transfected cells were treated with rosiglitazone, a synthetic PPARγ ligand and/or LG100268, a synthetic RXR ligand, to characterize the dose response and determine the simplest and most efficacious format. Following optimization of the bioassay, we assessed the cumulative activation of PPARγ by ligands in serum from mice treated with a PPARγ ligand and commercial human serum samples.ResultsCos-7 cells endogenously express sufficient RXR to support efficacious activation of transfected PPARγ. Co-transfection of an RXR expression vector with the PPARγ expression vector did not increase PPRE transcriptional activity induced by rosiglitazone. Treatment with an RXR ligand marginally increased PPRE transcriptional activity in the presence of transfected PPARγ, and co-treatment with an RXR ligand reduced rosiglitazone-induced PPRE transcriptional activity. Therefore, the final bioassay protocol consists of transfecting Cos-7 cells with a PPARγ expression vector along with the reporter vectors, applying rosiglitazone standards and/or 10 μL of serum, and measuring luminescence and fluorescence after a 24 h incubation. Sera from mice dosed with rosiglitazone induced PPRE transcriptional activity in the SPAA in a dose-dependent and PPARγ-dependent manner. Additionally, human serum from commercial sources induced a range of PPRE transcriptional activities in a PPARγ-dependent manner, demonstrating the ability of the bioassay to detect potentially low levels of ligands.ConclusionsThe SPAA can reliably measure total PPRE transcriptional activity in small volumes of serum. This system provides a sensitive, straightforward assay that can be reproduced in any cell culture laboratory.

Investigating the Mechanisms by which the Methyl‐CpG Binding Protein ZBTB38 Identifies Interacting Partners and Mediates Transcription

Cytosine methylation in CpG dinucleotides is an essential epigenetic modification utilized for regulating transcriptional processes within the eukaryotic genome. Consequently, misappropriation of DNA methylation patterns is associated with many diseases, including cancer. At many gene promoters, DNA methylation mediates expression through recruitment of methyl‐CpG binding proteins (MBPs). These specialized transcription factors read the methylated DNA signal and subsequently recruit chromatin remodeling complexes; leading to alterations in chromatin architecture and transcriptional responses. Thus, MBPs represent important functional intermediaries between these two key epigenetic events. ZBTB38 is the largest member of the ZBTB family of MBPs and contains two sets of five Cys2His2 zinc finger (ZF) domains distributed into N‐ and C‐terminal clusters, as well as an N‐terminal BTB/POZ protein interaction domain. There is increasing evidence that ZBTB38 regulates transcription for several core cellular processes in both normal cells as well as disease states. However, full mechanistic insight for how ZBTB38 identifies binding partners to elicit cellular function remains to be determined. Using combined structural and biochemical approaches, specific DNA binding partners have been identified and characterized for both the N‐ and C‐terminal ZF domains of ZBTB38. Importantly, we recently discovered that in addition to the N‐terminal ZFs, the C‐terminal ZFs also exhibit methyl‐selective DNA binding through the use of an alternative mode for methylated DNA recognition. Cell based studies have been utilized to begin delineating how these two ZF domains may function together or independently to modulate transcription. In addition to DNA interactions, we have begun to evaluate protein interacting partners for ZBTB38 using a combination of biophysical and cell based methodologies. Together, these findings expand our understanding for ZF recognition of methylated DNA, as well as illuminate the molecular basis by which ZBTB38 preferentially recognizes its biomolecular binding partners to translate transcriptional outcomes into specific cellular responses.Support or Funding InformationThis work was supported in part by MCB‐1715370 from the National Science Foundation and by RSG‐14‐185‐01‐DMC from the American Cancer Society.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.