Repression Loop Research Articles

High-Resolution Characterization of DNA/Protein Complexes in Living Bacteria.

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of the Escherichia coli lactose (lac) operon. Here we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example, we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.

ZmMS1/ZmLBD30-orchestrated transcriptional regulatory networks precisely control pollen exine development

Because of its significance for plant male fertility and, hence, direct impact on crop yield, pollen exine development has inspired decades of scientific inquiry. However, the molecular mechanism underlying exine formation and thickness remains elusive. In this study, we identified that a previously unrecognized repressor, ZmMS1/ZmLBD30, controls proper pollen exine development in maize. Using an ms1 mutant with aberrantly thickened exine, we cloned a male-sterility gene, ZmMs1, which encodes a tapetum-specific lateral organ boundary domain transcription factor, ZmLBD30. We showed that ZmMs1/ZmLBD30 is initially turned on by a transcriptional activation cascade of ZmbHLH51-ZmMYB84-ZmMS7, and then it serves as a repressor to shut down this cascade via feedback repression to ensure timely tapetal degeneration and proper level of exine. This activation-feedback repression loop regulating male fertility is conserved in maize and sorghum, and similar regulatory mechanism may also exist in other flowering plants such as rice and Arabidopsis. Collectively, these findings reveal a novel regulatory mechanism of pollen exine development by which a long-sought master repressor of upstream activators prevents excessive exine formation.

Subtype-specific 3D genome alteration in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

FOXF2 deficiency accelerates the visceral metastasis of basal-like breast cancer by unrestrictedly increasing TGF-β and miR-182-5p.

The mesenchymal transcription factor forkhead box F2 (FOXF2) is a critical regulator of embryogenesis and tissue homeostasis. Our previous studies demonstrated that FOXF2 is ectopically expressed in basal-like breast cancer (BLBC) cells and that FOXF2 deficiency promotes the epithelial-mesenchymal transition and aggressiveness of BLBC cells. In this study, we found that FOXF2 controls transforming growth factor-beta (TGF-β)/SMAD signaling pathway activation through transrepression of TGF-β-coding genes in BLBC cells. FOXF2-deficient BLBC cells adopt a myofibroblast-/cancer-associated fibroblast (CAF)-like phenotype, and tend to metastasize to visceral organs by increasing autocrine TGF-β signaling and conferring aggressiveness to neighboring cells by increasing paracrine TGF-β signaling. In turn, TGF-β silences FOXF2 expression through upregulating miR-182-5p, a posttranscriptional regulator of FOXF2 and inducer of metastasis. In addition to mediating a reciprocal repression loop between FOXF2 and TGF-β through direct transrepression by SMAD3, miR-182-5p forms a reciprocal repression loop with FOXF2 that directly transrepresses MIR182 expression. Therefore, FOXF2 deficiency accelerates the visceral metastasis of BLBC through unrestricted increases in autocrine and paracrine TGF-β signaling, and miR-182-5p expression. Our findings provide novel mechanisms underlying the roles of TGF-β, miR-182-5p, and FOXF2 in accelerating BLBC dissemination and metastasis, and may facilitate the development of therapeutic strategies for aggressive BLBC.

Characterizing the Stability of an Engineered Regulatory DNA Loop in Living E. coli Cells

DNA looping has evolved as a regulatory mechanism in both eukaryotic and prokaryotic organisms. Cellular functions including replication, DNA packaging, recombination, and gene regulation utilize DNA looping. In prokaryotes, genes are typically available for expression unless switched off by repressor protein binding at or near the transcriptional start site. Repression may be further enhanced by formation of a DNA loop encompassing the transcriptional start site. The lac operon offers a classic example of a prokaryotic repression loop. The tetrameric LacI protein simultaneously binds two operators within relatively close proximity resulting in a DNA loop, repressing transcription of the downstream genes. Using the lac repression loop as a model, we have created a dimeric fusion protein capable of binding DNA in a sequence-specific manner to create tunable DNA loops that regulate gene expression in vivo. The fusion protein utilizes a transcription activator-like effector (TALE) protein as a programmable DNA binding domain fused to FK506 binding protein (FKBP) as a regulatable dimerization domain. The resulting TALE-FKBP fusion binds DNA in a bidentate manner, reminiscent to LacI, resulting in a closed loop. We utilize our established lac promoter looping system to assess a TALE-FKBP protein fusion targeted to the O2 operator. This system creates a single loop orientation. We optimize this engineered system to account for the directionality of operator binding by the designed protein, determining optimal operator spacings for transcriptional repression. Relative to binding of the fusion protein at the promoter without DNA looping, we report ∼5-fold enhancement of gene repression upon DNA looping. We find that repression enhancement is dependent on both the orientation and the spacing between the DNA operators. We will adapt this system to create an approach for artificial regulation of any promoter.

Copy number gain of ZEB1 mediates a double-negative feedback loop with miR-33a-5p that regulates EMT and bone metastasis of prostate cancer dependent on TGF-β signaling.

Background: The reciprocal repressive loop between ZEB1 and miRNAs has been extensively reported to play an important role in tumor progression and metastasis of various human tumor types. The aim of this study was to elucidate the role and the underlying mechanism of the double-negative feedback loop between ZEB1and miR-33a-5p in bone metastasis of prostate cancer (PCa).Methods: miR-33a-5p expression was examined in 40 bone metastatic and 165 non-bone metastatic PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-33a-5p expression and clinicopathological characteristics, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-33a-5p in bone metastasis of PCa were investigated both by EMT and the Transwell assay in vitro, and by a mouse model of left cardiac ventricle inoculation in vivo. siRNA library, real-time PCR and chromatin immunoprecipitation (ChIP) were used to identify the underlying mechanism responsible for the decreased expression of miR-33a-5p in PCa. Bioinformatics analysis, Western blotting and luciferase reporter analysis were employed to examine the relationship between miR-33a-5p and its potential targets. Clinical correlation of miR-33a-5p with its targets was examined in human PCa tissues and primary PCa cells.Results: miR-33a-5p expression was downregulated in PCa tissues with bone metastasis and bone-derived cells, and low expression of miR-33a-5p strongly and positively correlated with advanced clinicopathological characteristics, and shorter overall and bone metastasis-free survival in PCa patients. Upregulating miR-33a-5p inhibited, while silencing miR-33a-5p promoted EMT, invasion and migration of PCa cells. Importantly, upregulating miR-33a-5p significantly repressed bone metastasis of PC-3 cells in vivo. Our results further revealed that recurrent ZEB1 upregulation induced by copy number gains transcriptionally inhibited miR-33a-5p expression, contributing to the reduced expression of miR-33a-5p in bone metastatic PCa tissues. In turn, miR-33a-5p formed a double negative feedback loop with ZEB1 in target-independent manner, which was dependent on TGF-β signaling. Finally, the clinical negative correlations of miR-33a-5p with ZEB1 expression and TGF-β signaling activity were demonstrated in PCa tissues and primary PCa cells.Conclusion: Our findings elucidated that copy number gains of ZEB1-triggered a TGF-β signaling-dependent miR-33a-5p-mediated negative feedback loop was highly relevant to the bone metastasis of PCa.

Polycomb-Dependent Chromatin Looping Contributes to Gene Silencing during Drosophila Development

Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, we analyze higher order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development. VIDEO ABSTRACT.

Cell Density Dependence of DNA Looping in E. coli Cultures

Sequestering a gene promoter inside a tightly bent DNA loop is a common mechanism of gene repression in bacteria. Many factors contribute to the strength of this type of gene repression, including the mechanical properties of the looped DNA and the concentration and binding strength of the protein that anchors the repression loop. Additionally, E. coli cells undergo numerous highly regulated and dynamic transitions during the well-known phases of a bacterial growth curve (e.g. lag, log, and stationary). We hypothesized that the probability of DNA looping would similarly depend on the growth status of the E. coli culture. To test this idea, we utilized a previously characterized engineered episomal repression loop model containing elements of the well-characterized lac operon with a β-galactosidase reporter gene. We tested multiple episomal constructs, each containing a different operator to operator spacing, grown to three different cell densities (as measured by absorbance at 600 nm) and assessed repression loop formation. We discuss these findings and relate them to DNA supercoiling, which may also influence repression loop formation.

Engineering a Tunable DNA Loop in E. coli

In prokaryotes, genes are typically considered available for expression unless switched off by the binding of a repressor protein at or near the transcriptional start site. The level of gene repression can be further enhanced by the formation of a tunable DNA loop enclosing the transcription start site. One such example of gene control by loop formation occurs within the well-studied lac operon. In E. coli, lac loop formation occurs when LacI, a tetrameric protein, simultaneously binds two operators within close proximity resulting in a DNA loop, repressing transcription of the downstream operon genes. A small molecule (allolactose) causes unlooping. To be useful, engineered loops should be similarly reversible. We have created an artificial dimeric fusion protein capable of binding DNA with designed sequence specificity to form a tunable DNA repression loop. The fusion protein utilizes a transcription activator-like effector (TALE) protein as the DNA binding region fused to FK506 binding protein (FKBP) as the protein-dimerizing domain. We show that the dimeric TALE-FKBP fusion binds DNA in a bidentate manner, similar to LacI, resulting in repression loop formation in living E. coli cells. We assess a TALE-FKBP protein fusion targeted to the Osym operator in the context of our established in vivo lac promoter looping model system. We demonstrate ∼7 fold enhancement of gene repression using the engineered TALE-FKBP fusion when a DNA loop is formed. The dimeric TALE-FKBP fusion can be tuned by addition of the small molecule FK506, which dissociates the FKBP dimer and derepresses the test promoter. We thus demonstrate engineered regulation of bacterial gene expression by DNA looping in living cells.

High-Resolution Characterization of DNA/Protein Complexes in Living Bacteria.

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as the Escherichia coli lactose (lac) operon. Here, we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.

A Prostate Cancer Risk Element Functions as a Repressive Loop that Regulates HOXA13

Prostate cancer (PCa) is the leading cancer among men in the United States, with genetic factors contributing to ∼42% of the susceptibility to PCa. We analyzed a PCa risk region located at 7p15.2 to gain insight into the mechanisms by which this noncoding region may affect gene regulation and contribute to PCa risk. We performed Hi-C analysis and demonstrated that this region has long-range interactions with the HOXA locus, located ∼873 kb away. Using the CRISPR/Cas9 system, we deleted a 4-kb region encompassing several PCa risk-associated SNPs and performed RNA-seq to investigate transcriptomic changes in prostate cells lacking the regulatory element. Our results suggest that the risk element affects the expression of HOXA13 and HOTTIP, but not other genes in the HOXA locus, via a repressive loop. Forced expression of HOXA13 was performed to gain further insight into the mechanisms by which this risk element affects PCa risk.

Engineering DNA Looping in E. Coli

DNA looping plays an important role in many cellular functions including replication, transcription, DNA packaging, and recombination. DNA repression loop formation in prokaryotes acts to regulate transcription in response to a changing environment. One example of gene control by DNA loop formation occurs in the well-studied lac operon. E. coli lac loop formation occurs when LacI, a tetrameric protein, simultaneously binds two DNA operators within close proximity, resulting in a DNA loop. This structure represses transcription of downstream operon genes. We are curious if repression loop formation can be targeted in vivo. Our goal is an engineered protein fusion with both sequence specific DNA binding function and chemically induced dimerization (CID) function. The chemically dimerized protein would have the ability to bind the DNA in a bidentate manner, analogous to LacI, resulting in DNA looping. Targeted DNA loop formation could act in gene repression and might be tunable by the concentration of CID agent. Using designed transcription activator-like effector (TALE) proteins allows creation of DNA binding proteins with arbitrary DNA sequence specificity. TALEs have been used for in vivo genome-engineering applications. FK506 binding protein (FKBP) provides a well-studied protein domain whose ability to undergo CID is exploited in our designs. We utilize our established lac promoter looping system to assess TALE-FKBP protein fusions targeted to lac operators.

Reactive Oxygen Species Function to Mediate the Fe Deficiency Response in an Fe-Efficient Apple Genotype: An Early Response Mechanism for Enhancing Reactive Oxygen Production.

Reactive oxygen species (ROS) are important signaling molecules in plants that contribute to stress acclimation. This study demonstrated that ROS play a critical role in Fe deficiency-induced signaling at an early stage in Malus xiaojinensis. Once ROS production has been initiated, prolonged Fe starvation leads to activation of ROS scavenging mechanisms. Further, we demonstrated that ROS scavengers are involved in maintaining the cellular redox homeostasis during prolonged Fe deficiency treatment. Taken together, our results describe a feedback repression loop for ROS to preserve redox homeostasis and maintain a continuous Fe deficiency response in the Fe-efficient woody plant M. xiaojinensis. More broadly, this study reveals a new mechanism in which ROS mediate both positive and negative regulation of plant responses to Fe deficiency stress.

Supercoiling Effects on Short-Range DNA Looping in E. coli

DNA-protein loops can be essential for gene regulation. The Escherichia coli lactose (lac) operon is controlled by DNA-protein loops that have been studied for decades. Here we adapt this model to test the hypothesis that negative superhelical strain facilitates the formation of short-range (6–8 DNA turns) repression loops in E. coli. The natural negative superhelicity of E. coli DNA is regulated by the interplay of gyrase and topoisomerase enzymes, adding or removing negative supercoils, respectively. Here, we measured quantitatively DNA looping in three different E. coli strains characterized by different levels of global supercoiling: wild type, gyrase mutant (gyrB226), and topoisomerase mutant (ΔtopA10). DNA looping in each strain was measured by assaying repression of the endogenous lac operon, and repression of ten reporter constructs with DNA loop sizes between 70–85 base pairs. Our data are most simply interpreted as supporting the hypothesis that negative supercoiling facilitates gene repression by small DNA-protein loops in living bacteria.

The small chemical enzyme inhibitor 5-phenylnicotinic acid/CD13 inhibits cell migration and invasion of tartrate-resistant acid phosphatase/ACP5-overexpressing MDA-MB-231 breast cancer cells

Tartrate-resistant acid phosphatase (TRAP/ACP5/uteroferrin/purple acid phosphatase/PP5) has received considerable attention as a newly discovered proinvasion metastasis driver associated with different malignancies. This renders TRAP an interesting target for novel anti-cancer therapy approaches. TRAP exists as two isoforms, 5a and 5b, where the 5a isoform represents an enzymatically less active monomeric precursor to the more enzymatically active 5b isoform generated by proteolytic excision of a repressive loop domain. Recently, three novel lead compounds were identified by fragment-based screening and demonstrated to be efficient TRAP enzyme inhibitors in vitro. We conclude that one of the three compounds i.e. 5-phenylnicotinic acid (CD13) was efficient as a TRAP inhibitor with Kic values in the low micromolar range towards the TRAP 5b isoform, but was not able to inhibit the TRAP 5a isoform. Structure-based docking revealed similar interactions of CD13 with the active site in both TRAP isoforms. In stably TRAP-overexpressing MDA-MB-231 breast cancer cells, CD13 inhibited intracellular TRAP activity and showed no cytotoxicity at 200µM. Furthermore, CD13 selectively blocked the TRAP 5b isoform compared to the TRAP 5a in cultured cells, indicating the usefulness of CD13 for assessing the different biological functions of the two TRAP isoforms 5a and 5b in cell systems. Moreover, inhibition of cell migration and invasion of stably TRAP-overexpressing MDA-MB-231 by CD13 was observed. These data establish a proof of principle that a small chemical inhibitor of the TRAP enzyme can block TRAP-dependent functions in cancer cells.

High-resolution mapping of architectural DNA binding protein facilitation of a DNA repression loop in Escherichia coli

Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.

Positive And Negative Feedback Loops Coupled By Common Transcription Activator And Repressor

AbstractDynamical systems consisting of two interlocked loops with negative and positive feedback have been studied using the linear analysis of stability and numerical solutions. Conditions for saddle-node bifurcation were formulated in a general form. Conditions for Hopf bifurcations were found in a few symmetrical cases. Auto-oscillations, when they exist, are generated by the negative feedback repressive loop. This loop determines the frequency and amplitude of oscillations. The positive feedback loop of activation slightly modifies the oscillations. Oscillations are possible when the difference between Hilll’s coefficients of the repression and activation is sufficiently high. The highly cooperative activation loop with a fast turnover slows down or even makes the oscillations impossible. The system under consideration can constitute a component of epigenetic or enzymatic regulation network.

The Role of Transcription Factor Tfii-I/GTF2I in the Response to Cellular Stress in Hematopoietic Cells

The transcription factor ATF3 and the heme regulated eIF2α kinase (HRI) have previously been shown to be involved in the stress response in hematopoietic cells. Many stress-responsive genes are regulated at the level of transcription elongation and are characterized by paused RNA polymerase II (Pol II) at the promoter region. Indeed, analyzing data from the public ENCODE project revealed that the HRI and the ATF3 genes contained paused Pol II at the promoter and at upstream intergenic regulatory elements. In this study we performed a genomic and proteomic analysis of transcription factor TFII-I (also known as GTF2I) in human erythroleukemia K562 cells. TFII-I is a ubiquitously expressed transcription factor previously implicated in regulating genes involved in stress response. TFII-I exhibits both positive and negative effects on transcription. TFII-I is a relatively large protein (about 120 Kda) and contains DNA binding, nuclear localization, and multiple protein/protein interaction domains. Using biotinylation tagging technology and high-throughput sequencing, we determined sites of chromatin interactions for TFII-I. This study revealed that TFII-I binds downstream of Pol II peaks at the ATF3 and HRI gene loci (Fig 1). A proteomic analysis revealed that TFII-I interacts with the elongation factor elongin A, which has been shown to help Pol II overcome the paused state. We demonstrate that Elongin A only associates with the ATF3 gene in response to cellular stress. The stress induction of ATF3 gene transcription requires the function of TFII-I. Furthermore, in the absence of induction, transcription factors E2F6, and E2F4 as well as the histone deacetylase I (HDAC1) bind to the HRI and ATF3 genes in close proximity to the Pol II and TFII-I peaks (Fig. 1). E2F4/6 and HDAC1 have been implicated in the repression of gene expression. The DNA elements that recruit TFII-I, Pol II and the repressor proteins interact over long distances in K562 cells as shown by ChIA-Pet data available from the ENCODE project (Fig.1). Moreover, we demonstrate that TFII-I directly interacts with E2F6 using co-immunoprecipitation assays (Fig. 2). Partial depletion of TFII-I by shRNA led to a decrease in stress mediated transcription without impairing basal transcription levels. The data demonstrate that TFII-I regulates stress-responsive transcription of the HRI and ATF3 genes in hematopoietic cells. In the absence of stress, TFII-I occupies promoter and long distance regulatory elements together with Pol II, E2F6/E2F4, and HDAC1. These proteins may be part of a chromatin loop that keeps Pol II in a repressed configuration. We suggest that upon stress induction, TFII-I recruits Elongin A, which converts Pol II into a transcriptionally active configuration. Transcription elongation disrupts formation of a repressive chromatin loop and leads to transcription activation of the HRI and ATF3 genes. Figure 1 Diagram of the HRI and ATF3 gene loci showing interactions of TFII-I, Pol II, E2F4, E2F6, and HDAC1 as well as RNA seq, and Pol II ChIA-PET data. The ChIA-PET data show interactions of chromatin fragments that have Pol II bound. The arrows indicate the direction of transcription of the HRI gene and from the two promoters of the ATF3 gene (P1 and P2). Figure 1. Diagram of the HRI and ATF3 gene loci showing interactions of TFII-I, Pol II, E2F4, E2F6, and HDAC1 as well as RNA seq, and Pol II ChIA-PET data. The ChIA-PET data show interactions of chromatin fragments that have Pol II bound. The arrows indicate the direction of transcription of the HRI gene and from the two promoters of the ATF3 gene (P1 and P2). Figure 2 Input; TFII-I Figure 2. Input; TFII-I Figure 3 Co-Immunoprecipitation experiment showing the interaction between TFII-I and E2F6. K562 cell nuclear extracts were subjected to precipitation with IgG control or TFII-I specific antibodies. The material was fractionated by SDS-PAGE, transferred to a nylon membrane and probed with an antibody specific for E2F6. Figure 3. Co-Immunoprecipitation experiment showing the interaction between TFII-I and E2F6. K562 cell nuclear extracts were subjected to precipitation with IgG control or TFII-I specific antibodies. The material was fractionated by SDS-PAGE, transferred to a nylon membrane and probed with an antibody specific for E2F6. Disclosures No relevant conflicts of interest to declare.

The ZEB1 Transcription Factor Acts in a Negative Feedback Loop with miR200 Downstream of Ras and Rb1 to Regulate Bmi1 Expression

Ras mutations are frequent in cancer cells where they drive proliferation and resistance to apoptosis. However in primary cells, mutant Ras instead can cause oncogene-induced senescence, a tumor suppressor function linked to repression of the polycomb factor Bmi1, which normally regulates cell cycle inhibitory cyclin-dependent kinase inhibitors (cdki). It is unclear how Ras causes repression of Bmi1 in primary cells to suppress tumor formation while inducing the gene in cancer cells to drive tumor progression. Ras also induces the EMT transcription factor ZEB1 to trigger tumor invasion and metastasis. Beyond its well-documented role in EMT, ZEB1 is important for maintaining repression of cdki. Indeed, heterozygous mutation of ZEB1 is sufficient for elevated cdki expression, leading to premature senescence of primary cells. A similar phenotype is evident with Bmi1 mutation. We show that activation of Rb1 in response to mutant Ras causes dominant repression of ZEB1 in primary cells, but loss of the Rb1 pathway is a hallmark of cancer cells and in the absence of such Rb1 repression Ras induces ZEB1 in cancer cells. ZEB1 represses miR-200 in the context of a mutual repression loop. Because miR-200 represses Bmi1, induction of ZEB1 leads to induction of Bmi1. Rb1 pathway status then dictates the opposing effects of mutant Ras on the ZEB1-miR-200 loop in primary versus cancer cells. This loop not only triggers EMT, surprisingly we show it acts downstream of Ras to regulate Bmi1 expression and thus the critical decision between oncogene-induced senescence and tumor initiation.

Promoter Repression by Strong DNA Bending

In prokaryotes, genes are typically expressed unless switched off by repressor protein binding at or near the transcriptional start site. The lac operon is a classic model of this genetic switch, which also illustrates repression facilitated by DNA looping. In E. coli, lac repressor (LacI) simultaneously binds two distant operator sequences forming a closed loop containing the regulated promoter. Previous looping studies have shown RNA polymerase (RNAP) and LacI compete for overlapping DNA binding sites. Although elements of the lac operon have been extensively studied, fundamental questions remain. Our current work explores the following questions: Is gene repression by DNA looping observed in the absence of direct competition between LacI and RNAP? To what extent is RNAP excluded from a promoter simply by strong DNA bending? To address these questions we created two families of repression loops where a promoter was placed centrally between two operators. Each family of constructs systematically positioned the promoter on different helical faces of the looped DNA to deconvolute the repressive effect of DNA deformation from topological repression due to steric hindrance. RNAP binding to these constructs was assayed in living E. coli cells (-/+ induction) by a standard enzymatic assay of reporter activity, and by ChIP. Our results support several interesting conclusions: i) DNA bending strain dramatically represses the test promoter (∼100-fold relative to non-looping single-operator constructs) even in the absence of binding competition; ii) DNA looping is destabilized ∼10-fold upon gene induction, emphasizing that inducer-saturated LacI is still competent to bind DNA; iii) promoter topology (i.e. outside vs. inside of the DNA loop) has a ∼10-fold effect on RNAP binding and gene expression. These results illustrate and quantitate how gene regulation can be achieved by DNA deformation in the absence of direct protein-protein binding site competition.