Target Of Repression Research Articles

The Zinc Finger Transcription Factor Fts2 Represses the Yeast-to-Filament Transition in the Dimorphic Yeast Yarrowia lipolytica.

The yeast-to-filament transition is an important cellular response to environmental stimulations in dimorphic fungi. In addition to activators, there are repressors in the cells to prevent filament formation, which is important to keep the cells in the yeast form when filamentation is not necessary. However, very few repressors of filamentation are known so far. Here, we identify a novel repressor of filamentation in the dimorphic yeast Yarrowia lipolytica, Fts2, which is a C2H2-type zinc finger transcription factor. We show that fts2Δ cells exhibited increased filamentation under mild filament-inducing conditions and formed filaments under non-filament-inducing conditions. We also show that Fts2 interacts with YlSsn6, component of the Tup1-Ssn6 transcriptional corepressor, and Fts2-LexA represses a lexAop-PYlACT1-lacZ reporter in a Tup1-Ssn6-dependent manner, suggesting that Fts2 has transcriptional repressor activity and represses gene expression via Tup1-Ssn6. In addition, we show that Fts2 represses a large number of cell wall protein genes and transcription factor genes, some of which are implicated in the filamentation response. Interestingly, about two-thirds of Fts2-repressed genes are also repressed by Tup1-Ssn6, suggesting that Fts2 may repress the bulk of its target genes via Tup1-Ssn6. Lastly, we show that Fts2 expression is downregulated in response to alkaline pH and the relief of negative control by Fts2 facilitates the induction of filamentation by alkaline pH. IMPORTANCE The repressors of filamentation are important negative regulators of the yeast-to-filament transition. However, except in Candida albicans, very few repressors of filamentation are known in dimorphic fungi. More importantly, how they repress filamentation is often not clear. In this paper, we report a novel repressor of filamentation in Y. lipolytica. Fts2 is not closely related in amino acid sequence to CaNrg1 and Rfg1, two major repressors of filamentation in C. albicans, yet it represses gene expression via the transcriptional corepressor Tup1-Ssn6, similar to CaNrg1 and Rfg1. Using transcriptome sequencing, we determined the whole set of genes regulated by Fts2 and identified the major targets of Fts2 repression, which provide clues to the mechanism by which Fts2 represses filamentation. Our results have important implications for understanding the negative control of the yeast-to-filament transition in dimorphic fungi.

Przemilczane ludobójstwo – „operacja polska NKWD” 1937–1938

The Order No. 00485 that was given by the head of the NKVD, Nikolai Yezhov, resulted in carring out the Polish Operation during the Great Purge. Political repression against Poles that took place in 1937-1938 in the Soviet Union, resulted in executions of 111 thousand Poles and sentencing nearly 30 thousand people to labor camps. Institutions associated with Polishness, such as the Roman Catholic Church, became the target of repression. According to international definitions, it was an act qualified as genocide and resulted in depolonization of further Easter Borderlands.

GATA-type transcriptional factor SpGAT1 interacts with SpMIG1 and promotes lipid accumulation in the oleaginous yeast Saitozyma podzolica zwy-2-3

BackgroundIn oleaginous yeast, nitrogen limitation is a critical parameter for lipid synthesis. GATA-family transcriptional factor GAT1, a member of the target of rapamycin (TOR) pathway and nitrogen catabolite repression (NCR), regulates nitrogen uptake and utilization. Therefore, it is significant to study the SpGAT1 regulatory mechanism of lipid metabolism for conversion of biomass to microbial oil in Saitozyma podzolica zwy-2-3.ResultsCompared with WT, Delta gat1, and OE::gat1, the lipid yield of OE::gat1 increased markedly in the low carbon and nitrogen ratio (C/N ratio) mediums, while the lipid yield and residual sugar of Delta gat1 decreased in the high C/N ratio medium. According to yeast two-hybrid assays, SpGAT1 interacted with SpMIG1, and its deletion drastically lowered SpMIG1 expression on the high C/N ratio medium. MIG1 deletion has been found in earlier research to affect glucose metabolic capacity, resulting in a prolonged lag period. Therefore, we speculated that SpGAT1 influenced glucose consumption rate across SpMIG1. Based on yeast one-hybrid assays and qRT-PCR analyses, SpGAT1 regulated the glyoxylate cycle genes ICL1, ICL2, and pyruvate bypass pathway gene ACS, irrespective of the C/N ratio. SpGAT1 also could bind to the ACAT2 promoter in the low C/N medium and induce sterol ester (SE) accumulation.ConclusionOur findings indicated that SpGAT1 positively regulated lipid metabolism in S.podzolica zwy-2-3, but that its regulatory patterns varied depending on the C/N ratio. When the C/N ratio was high, SpGAT1 interacted with SpMIG1 to affect carbon absorption and utilization. SpGAT1 also stimulated lipid accumulation by regulating essential lipid anabolism genes. Our insights might spur more research into how nitrogen and carbon metabolism interact to regulate lipid metabolism.

A MYC-ZNF148-ID1/3 regulatory axis modulating cancer stem cell traits in aggressive breast cancer

The MYC proto-oncogene (MYC) is one of the most frequently overexpressed genes in breast cancer that drives cancer stem cell-like traits, resulting in aggressive disease progression and poor prognosis. In this study, we identified zinc finger transcription factor 148 (ZNF148, also called Zfp148 and ZBP-89) as a direct target of MYC. ZNF148 suppressed cell proliferation and migration and was transcriptionally repressed by MYC in breast cancer. Depletion of ZNF148 by short hairpin RNA (shRNA) and CRISPR/Cas9 increased triple-negative breast cancer (TNBC) cell proliferation and migration. Global transcriptome and chromatin occupancy analyses of ZNF148 revealed a central role in inhibiting cancer cell de-differentiation and migration. Mechanistically, we identified the Inhibitor of DNA binding 1 and 3 (ID1, ID3), drivers of cancer stemness and plasticity, as previously uncharacterized targets of transcriptional repression by ZNF148. Silencing of ZNF148 increased the stemness and tumorigenicity in TNBC cells. These findings uncover a previously unknown tumor suppressor role for ZNF148, and a transcriptional regulatory circuitry encompassing MYC, ZNF148, and ID1/3 in driving cancer stem cell traits in aggressive breast cancer.

W.E.B. Du Bois Against U.S. Capitalist Racism: Durable Peace and the Fulfillment of People(s)-Centered Human Rights

This article argues that W.E.B. Du Bois’s struggle for a durable peace was inextricably linked to his rejection of the United States’ racialized economic order that emplaced Blacks at the bottom of society within its borders and, given its imperial ambitions, threatened perpetual war abroad, with particularly dire consequences for colonized peoples. The first section re-interprets Du Bois’s writings and speeches about “caste” as, more accurately, explicating a system of “U.S. capitalist racism” in the United States that was inexorably tied to imperialism and war. Such clarity opens up an understanding of Du Bois’s conception of peace not as the absence of conflict, but rather as a necessary condition for the eradication of political, economic, and social inequality and injustice on a world scale. The next section argues that Du Bois’s analysis and critique of U.S. capitalist racism undergirded his advocacy of “People(s)-Centered Human Rights” (PCHR). Coined by Black Alliance for Peace National Organizer Ajamu Baraka, PCHR emanate from and address the everyday realities, needs, and challenges of racialized and colonized people. For Du Bois, PCHR could only be achieved through a durable peace, and durable peace could only be maintained through the extension of PCHR to all persons irrespective of race, class, or nation. The final section explicates how this anticapitalist, antiracist, anti-imperial, and anticolonial conception of durable peace drew the ire of the U.S. government and made Du Bois, the Peace Information Center, and his fellow peace activists targets of Black Scare and Red Scare repression.

Maternal H3K36 and H3K27 HMTs protect germline development via regulation of the transcription factor LIN-15B

Maternally synthesized products play critical roles in the development of offspring. A premier example is the Caenorhabditis elegans H3K36 methyltransferase MES-4, which is essential for germline survival and development in offspring. How maternal MES-4 protects the germline is not well understood, but its role in H3K36 methylation hinted that it may regulate gene expression in primordial germ cells (PGCs). We tested this hypothesis by profiling transcripts from nascent germlines (PGCs and their descendants) dissected from wild-type and mes-4 mutant (lacking maternal and zygotic MES-4) larvae. mes-4 nascent germlines displayed downregulation of some germline genes, upregulation of some somatic genes, and dramatic upregulation of hundreds of genes on the X chromosome. We demonstrated that upregulation of one or more genes on the X is the cause of germline death by generating and analyzing mes-4 mutants that inherited different endowments of X chromosome(s). Intriguingly, removal of the THAP transcription factor LIN-15B from mes-4 mutants reduced X misexpression and prevented germline death. lin-15B is X-linked and misexpressed in mes-4 PGCs, identifying it as a critical target for MES-4 repression. The above findings extend to the H3K27 methyltransferase MES-2/3/6, the C. elegans version of polycomb repressive complex 2. We propose that maternal MES-4 and PRC2 cooperate to protect germline survival by preventing synthesis of germline-toxic products encoded by genes on the X chromosome, including the key transcription factor LIN-15B.

The dynamics of t1 adenosine binding on human Argonaute 2: Understanding recognition with conformational selection.

The control of expression in genetic regulation is a fundamental process for cell life. In RNA‐mediated silencing, human Argonaute‐2 protein (hAgo2) uses sequence information encoded in small RNAs (guide) to identify complementary sites in messenger RNAs (target) for repression. The specificity of this molecular recognition lies at the basis of the mechanisms that control the expression of thousands of genes, which necessarily requires a fine tuning of complex events. Among these, the binding of the first nucleotide of the target RNA (t1) is emerging as an important modulator of hAgo2‐mediated machinery. Using atomistic molecular dynamics‐derived analyses, we address the mechanism behind t1‐dependent regulation and study the impact of different t1 nucleotides (t1A, t1C, t1G, t1U) on the conformational dynamics of both hAgo2 and guide–target RNAs. Only when an adenine is found at this position, t1 directly interacts with a specific hAgo2 binding pocket, favoring the stabilization of target binding. Our findings show that hAgo2 exploits a dynamic recognition mechanism of the t1‐target thanks to a modulation of RNA conformations. Here, t1‐adenine is the only nucleobase endowed with a dual binding mode: a T‐shape and a co‐planar conformation, respectively, orthogonal and parallel to the following base‐pairs of guide–target duplex. This triggers a composite set of molecular interactions that stabilizes distinctive conformational ensembles. Our comparative analyses show characteristic traits of local and global dynamic interplay between hAgo2 and the RNA molecules and highlight how t1A binding acts as a molecular switch for target recognition and complex stabilization. Implications for future mechanistic studies are discussed.

The Impact of Domestic Surveillance on Political Imprisonment: Evidence from the German Democratic Republic

How does domestic surveillance affect the frequency of political imprisonments in autocratic states? In contrast to conventional wisdom, I argue that surveillance reduces the frequency of political imprisonments in power-maximizing autocracies. Surveillance decreases uncertainty about the correct targets of repression, allowing for more selective detentions and shifts to silent instruments of repression. To investigate these claims, I draw on a unique county-level dataset of political imprisonment in the German Democratic Republic between 1984 and 1988. I proxy the number of monitored individuals with newly collected county-level data on surveillance operations. I use ordinary least squares (OLS) regression, random effects, and instrumental variable models to investigate the impact of surveillance on political imprisonment. I find that higher shares of spies per monitored individual were associated with a reduction of political imprisonment. Further, increasing levels of spy infiltration were linked to a systematic shift to silent instruments of repression.

Dysregulation of Lipid and Glucose Homeostasis in Hepatocyte-Specific SLC25A34 Knockout Mice

Nonalcoholic fatty liver disease (NAFLD) is an epidemic affecting 30% of the US population. It is characterized by insulin resistance, and by defective lipid metabolism and mitochondrial dysfunction in the liver. SLC25A34 is a major repressive target of miR-122, a miR that has a central role in NAFLD and liver cancer. However, little is known about the function of SLC25A34. To investigate SLC25A34 invitro, mitochondrial respiration and bioenergetics were examined using hepatocytes depleted of Slc25a34 or overexpressing Slc25a34. To test the function of SLC25A34 invivo, a hepatocyte-specific knockout mouse was generated, and loss of SLC25A34 was assessed in mice maintained on a chow diet and a fast-food diet (FFD), a model for NAFLD. Hepatocytes depleted of Slc25a34 displayed increased mitochondrial biogenesis, lipid synthesis, and ADP/ATP ratio; Slc25a34 overexpression had the opposite effect. In the knockout model on chow diet, SLC25A34 loss modestly affected liver function (altered glucose metabolism was the most pronounced defect). RNA-sequencing revealed changes in metabolic processes, especially fatty acid metabolism. After 2 months on FFD, knockouts had a more severe phenotype, with increased lipid content and impaired glucose tolerance, which was attenuated after longer FFD feeding (6 months). This work thus presents a novel model for studying SLC25A34 invivo in which SLC25A34 plays a role in mitochondrial respiration and bioenergetics during NAFLD.

The CDK1/TFCP2L1/ID2 cascade offers a novel combination therapy strategy in a preclinical model of bladder cancer

Aberrant activation of embryogenesis-related molecular programs in urothelial bladder cancer (BC) is associated with stemness features related to oncogenic dedifferentiation and tumor metastasis. Recently, we reported that overexpression of transcription factor CP2-like protein-1 (TFCP2L1) and its phosphorylation at Thr177 by cyclin-dependent kinase-1 (CDK1) play key roles in regulating bladder carcinogenesis. However, the clinical relevance and therapeutic potential of this novel CDK1-TFCP2L1 molecular network remain elusive. Here, we demonstrated that inhibitor of DNA binding-2 (ID2) functions as a crucial mediator by acting as a direct repressive target of TFCP2L1 to modulate the stemness features and survival of BC cells. Low ID2 and high CDK1 expression were significantly associated with unfavorable clinical characteristics. TFCP2L1 downregulated ID2 by directly binding to its promoter region. Consistent with these findings, ectopic expression of ID2 or treatment with apigenin, a chemical activator of ID2, triggered apoptosis and impaired the proliferation, suppressed the stemness features, and reduced the invasive capacity of BC cells. Combination treatment with the specific CDK1 inhibitor RO-3306 and apigenin significantly suppressed tumor growth in an orthotopic BC xenograft animal model. This study demonstrates the biological role and clinical utility of ID2 as a direct target of the CDK1-TFCP2L1 pathway for modulating the stemness features of BC cells.

Family Members Additively Repress the Ectopic Expression of BASIC PENTACYSTEINE3 to Prevent Disorders in Arabidopsis Circadian Vegetative Development.

BARLEY B-RECOMBINANT/BASIC PENTACYSTEINE (BBR/BPC) family members are plant-specific GAGA-motif binding factors (GAFs) controlling multiple developmental processes of growth and propagation. BPCs recruit histone remodeling factors for transcriptional repression of downstream targets. It has been revealed that BPCs have an overlapping and antagonistic relationship in regulating development. In this study, we showed disturbances interfering with the homeostasis of BPC expressions impede growth and development. The ectopic expression of BPC3 results in the daily growth defect shown by higher-order bpc mutants. Oscillations of multiple circadian clock genes are phase-delayed in the quadruple mutant of bpc1 bpc2 bpc4 bpc6 (bpc1,2,4,6). By introducing the overexpression of BPC3 into wild-type Arabidopsis, we found that BPC3 is a repressor participating in its repression and repressing multiple regulators essential to the circadian clock. However, the induction of BPC3 overexpression did not fully replicate clock defects shown by the quadruple mutant, indicating that in addition to the BPC3 antagonization, BPC members also cofunction in the circadian clock regulation. A leaf edge defect similar to that shown by bpc1,2,4,6 is also observed under BPC3 induction, accompanied by repression of a subset of TCPs required for the edge formation. This proves that BPC3 is a repressor that must be confined during the vegetative phase. Our findings demonstrate that BPCs form a meticulous repressor network for restricting their repressive functions to molecular mechanisms controlling plant growth and development.

H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats.

Six methyltransferases divide labor in establishing genomic profiles of histone H3 lysine 9 methylation (H3K9me), an epigenomic modification controlling constitutive heterochromatin, gene repression, and silencing of retroelements. Among them, SETDB1 is recruited to active chromatin domains to silence the expression of endogenous retroviruses. In the context of experiments aimed at determining the impact of SETDB1 on stimulus-inducible gene expression in macrophages, we found that loss of H3K9me3 caused by SETDB1 depletion was associated with increased recruitment of CTCF to >1600 DNA binding motifs contained within SINE B2 repeats, a previously unidentified target of SETDB1-mediated repression. CTCF is an essential regulator of chromatin folding that restrains DNA looping by cohesin, thus creating boundaries among adjacent topological domains. Increased CTCF binding to SINE B2 repeats enhanced insulation at hundreds of sites and increased loop formation within topological domains containing lipopolysaccharide-inducible genes, which correlated with their impaired regulation in response to stimulation. These data indicate a role of H3K9me3 in restraining genomic distribution and activity of CTCF, with an impact on chromatin organization and gene regulation.

Motvilliga vittnen

Reluctant Witnesses: Swedish Memoirs from the Soviet Karelian Purges Several hundred Swedes emigrated to Soviet Karelia in the beginning of the 1930s. Many of them remained there when the Great Terror began, and they became targets of repression. As of now, there is no coherent public narrative in Sweden that acknowledge the Swedish survivors; most survivors never talked publicly about their experiences, let alone wrote about them, and the few who did, did so reluctantly. This article discusses four memoirs written by Swedish survivors of the Soviet repression and the silence that still surrounds these memoirs. The narratives themselves, as well as the reception of the narratives, bear witness to the difficulty of narrating experiences that have not yet been publicly acknowledged. Based on the Swedish witness accounts, this article aims to examine 1) the difficulty of testifying in the absence of a public narrative, and 2) the ability of witness literature to challenge and change established historical narratives.

Silencing of Ago-2 Interacting Protein SERBP1 Relieves KCC2 Repression by miR-92 in Neurons.

RNA-binding proteins (RBPs) play important roles in modulating miRNA-mediated mRNA target repression. Argonaute2 (Ago2) is an essential component of the RNA-induced silencing complex (RISC) that plays a central role in silencing mechanisms via small non-coding RNA molecules known as siRNAs and miRNAs. Small RNAs loaded into Argonaute proteins catalyze endoribonucleolytic cleavage of target RNAs or recruit factors responsible for translational silencing and mRNA target destabilization. In previous studies we have shown that KCC2, a neuronal Cl (−) extruding K (+) Cl (−) co-transporter 2, is regulated by miR-92 in neuronal cells. Searching for Ago2 partners by immunoprecipitation and LC-MS/MS analysis, we isolated among other proteins the Serpine mRNA binding protein 1 (SERBP1) from SH-SY5Y neuroblastoma cells. Exploring the role of SERBP1 in miRNA-mediated gene silencing in SH-SY5Y cells and primary hippocampal neurons, we demonstrated that SERBP1 silencing regulates KCC2 expression through the 3′ untranslated region (UTR). In addition, we found that SERBP1 as well as Ago2/miR-92 complex bind to KCC2 3′UTR. Finally, we demonstrated the attenuation of miR-92-mediated repression of KCC2 3′UTR by SERBP1 silencing. These findings advance our knowledge regarding the miR-92-mediated modulation of KCC2 translation in neuronal cells and highlight SERBP1 as a key component of this gene regulation.

MicroRNA-139, an Emerging Gate-Keeper in Various Types of Cancer.

Mounting data show that MIR139 is commonly silenced in solid cancer and hematological malignancies. MIR139 acts as a critical tumor suppressor by tuning the cellular response to different types of stress, including DNA damage, and by repressing oncogenic signaling pathways. Recently, novel insights into the mechanism of MIR139 silencing in tumor cells have been described. These include epigenetic silencing, inhibition of POL-II transcriptional activity on gene regulatory elements, enhanced expression of competing RNAs and post-transcriptional regulation by the microprocessor complex. Some of these MIR139-silencing mechanisms have been demonstrated in different types of cancer, suggesting that these are more general oncogenic events. Reactivation of MIR139 expression in tumor cells causes inhibition of tumor cell expansion and induction of cell death by the repression of oncogenic mRNA targets. In this review, we discuss the different aspects of MIR139 as a tumor suppressor gene and give an overview on different transcriptional mechanisms regulating MIR139 in oncogenic stress and across different types of cancer. The novel insights into the expression regulation and the tumor-suppressing activities of MIR139 may pave the way to new treatment options for cancer.

ScanMiR: a biochemically based toolkit for versatile and efficient microRNA target prediction.

microRNAs are important post-transcriptional regulators of gene expression, but the identification of functionally relevant targets is still challenging. Recent research has shown improved prediction of microRNA-mediated repression using a biochemical model combined with empirically-derived k-mer affinity predictions; however, these findings are not easily applicable. We translate this approach into a flexible and user-friendly bioconductor package, scanMiR, also available through a web interface. Using lightweight linear models, scanMiR efficiently scans for binding sites, estimates their affinity and predicts aggregated transcript repression. Moreover, flexible 3'-supplementary alignment enables the prediction of unconventional interactions, such as bindings potentially leading to target-directed microRNA degradation or slicing. We showcase scanMiR through a systematic scan for such unconventional sites on neuronal transcripts, including lncRNAs and circRNAs. Finally, in addition to the main bioconductor package implementing these functions, we provide a user-friendly web application enabling the scanning of sequences, the visualization of predicted bindings and the browsing of predicted target repression. scanMiR and companion packages are implemented in R, released under the GPL-3 and accessible on Bioconductor (https://bioconductor.org/packages/release/bioc/html/scanMiR.html) as well as through a shiny web server (https://ethz-ins.org/scanMiR/). Supplementary data are available at Bioinformatics online.

Engineering miRNA features into siRNAs: Guide-strand bulges are compatible with gene repression

Synthetic siRNA guide strands are typically designed with perfect complementarity to the passenger strand and the target mRNA. We examined whether siRNAs with intentional guide-strand bulges are functional in vitro and in vivo. Importantly, this was done by systematic shortening of the passenger strand, evaluating identical 19-mer guide-strand sequences but forcing them into conformations with 1- to 4-nt bulges after annealing. We demonstrate that guide-strand bulges can be well tolerated at several positions of unmodified and modified siRNAs. Beyond that, we show that GalNAc-conjugated siRNAs with bulges at certain positions of the guide strand repress transthyretin in murine primary hepatocytes and in vivo in mice. In vivo, a GalNAc-conjugated siRNA with a 1-nt bulge at position 14 of the guide strand was as active as the perfectly complementary siRNA. Finally, in a luciferase reporter system, mRNA target sequences were systematically shortened so that RNA-induced silencing complex activity could only occur with a guide-strand bulge. Here, luciferase reporters were repressed when 1- and 2-nt deletions of the reporter were applied to the edges of the sequence. We conclude that some guide-strand bulges versus target transcript can result in target repression and therefore should be evaluated as off-target risks.

Integrative analysis reveals multiple modes of LXR transcriptional regulation in liver

The nuclear receptors liver X receptor (LXR) α and β play crucial roles in hepatic metabolism. Many genes induced in response to pharmacologic LXR agonism have been defined; however, the transcriptional consequences of loss of LXR binding to its genomic targets are less well characterized. Here, we addressed how deletion of both LXRα and LXRβ from mouse liver (LXR double knockout [DKO]) affects the transcriptional regulatory landscape by integrating changes in LXR binding, chromatin accessibility, and gene expression. Many genes involved in fatty acid metabolism showed reduced expression and chromatin accessibility at their intergenic and intronic regions in LXRDKO livers. Genes that were up-regulated with LXR deletion had increased chromatin accessibility at their promoter regions and were enriched for functions not linked to lipid metabolism. Loss of LXR binding in liver reduced the activity of a broad set of hepatic transcription factors, inferred through changes in motif accessibility. By contrast, accessibility at promoter nuclear factor Y (NF-Y)motifs was increased in the absence of LXR. Unexpectedly, we also defined a small set of LXR targets for direct ligand-dependent repression. These genes have LXR-binding sites but showed increased expression in LXRDKO liver and reduced expression in response to the LXR agonist. In summary, the binding of LXRs to the hepatic genome has broad effects on the transcriptional landscape that extend beyond its canonical function as an activator of lipid metabolic genes.

Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins.

The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

Genome-Wide ChIPseq Analysis of AhR, COUP-TF, and HNF4 Enrichment in TCDD-Treated Mouse Liver.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known for mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the canonical mechanism of AhR activation involves heterodimerization with the aryl hydrocarbon receptor nuclear translocator, other transcriptional regulators that interact with AhR have been identified. Enrichment analysis of motifs in AhR-bound genomic regions implicated co-operation with COUP transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF4). The present study investigated AhR, HNF4α and COUP-TFII genomic binding and effects on gene expression associated with liver-specific function and cell differentiation in response to TCDD. Hepatic ChIPseq data from male C57BL/6 mice at 2 h after oral gavage with 30 µg/kg TCDD were integrated with bulk RNA-sequencing (RNAseq) time-course (2–72 h) and dose–response (0.01–30 µg/kg) datasets to assess putative AhR, HNF4α and COUP-TFII interactions associated with differential gene expression. Functional enrichment analysis of differentially expressed genes (DEGs) identified differential binding enrichment for AhR, COUP-TFII, and HNF4α to regions within liver-specific genes, suggesting intersections associated with the loss of liver-specific functions and hepatocyte differentiation. Analysis found that the repression of liver-specific, HNF4α target and hepatocyte differentiation genes, involved increased AhR and HNF4α binding with decreased COUP-TFII binding. Collectively, these results suggested TCDD-elicited loss of liver-specific functions and markers of hepatocyte differentiation involved interactions between AhR, COUP-TFII and HNF4α.