The proviral integration site for moloney murine leukemia virus 1 (Pim1) is a serine/threonine kinase and able to promote cell proliferation, survival and drug resistance. Overexpression of Pim1 has been observed in many cancer types and is associated with the poor prognosis of breast cancer. However, it remains unclear whether Pim1 kinase is a potential therapeutic target for breast cancer patients. In this study, we found that Pim1 expression was strongly associated with HER2 expression and that HER2-overexpressing breast cancer cells were more sensitive to Pim1 inhibitor-induced inhibitions of cell viability and metastatic ability. Mechanistically, Pim1 inhibitor suppressed the expression of HER2 at least in part through transcriptional level. More importantly, Pim1 inhibitor overcame the resistance of breast cancer cells to HER2 tyrosine kinase inhibitor lapatinib. In summary, downregulation of HER2 by targeting Pim1 may be a promising and effective therapeutic approach not only for anti-cancer growth but also for circumventing lapatinib resistance in HER2-positive breast cancer patients. Breast cancer is the most common cancer type and ranks second among causes for cancer death in women . AccordiHER2 is a member of human epidermal growth factor receptor (HER/EGFR) tyrosine kinase family, which is frequently overexpressed in many cancer types . HER famThe proviral integration site for moloney murine leukemia virus 1 (Pim1) is a serine/threonine kinase. There are three members in human Pim family, including Pim1, Pim2, and Pim3, which are encoded in chromosome 6, X chromosome, and chromosome 22, respectively. Aberrant elevation of Pim1 has been observed in many cancer types and reported to play a crucial role in tumorigenesis due to the interactions with numerous proteins participating in various signaling pathways involved in cell proliferation, survival, and drug resistance . The onc2. Lapatinib-resistant clones were maintained in the presence of 1 μM lapatinib.Human HER2-positive  and HER2-negative  breast cancer cell lines were obtained from the American Type Culture Collection. HBL-100 cells and HER2-overexpressing clone (HER18) of MCF-7 cells were 5kind gift from Prof. Mien-Chie Hung. Lapatinib-resistant clones (Sk/LR6 and Sk/LR9) were selected from SkBr3 cells by culturing the cells in increasing concentrations of lapatinib . All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium/F12 containing 10% fetal bovine serum (GeneDireX), 100 U/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific) and incubated at 37°C in a humidified atmosphere of 95% air and 5% COCells were washed with 1X phosphate buffered saline (PBS) once and harvested with RIPA buffer (50 mM Tris (pH7.5), 150 mM NaCl, 10 mM EDTA, 1% NP-40, 0.1% SDS, 1 mM PMSF, 10 μg/ml Aprotinin) plus protease inhibitors, followed by homogenization with sonication and centrifugation at 21,500 × g for 15 min. Whole cell lysates were stored at -20°C until used for the experiments .As described previously , the con3 cells/well in a 96-well plate. The next day, cells were cultured with serum-free medium and treated with Pim inhibitors SMI-4a and SGI-1776 at the concentrations of 0, 0.5, 1, 2, 5, 10, 20 μM for 2 days in three independent experiments. Then, the culture medium was refreshed with 100 μl serum-free medium with 5 mg/ml MTT solution (Sigma) for 3 h followed by wash with PBS 3 times. The formazan in the cells was solubilized in 100 μl DMSO per well, followed by the measurement of absorbance at 570 nm.Cells were seeded at a density of 3–4 × 105 cells/well in a 6-well plate overnight. Cells were infected with lentivirus shRNA using a multiplicity of infection of 125 for 24 h. Then, cells were refreshed with the medium containing 2 μg/ml puromycin for 24 h followed by subsequent experiments.Cells were seeded at a density of 2 × 105 cells/well in a 6-well plate. The next day, the cells were transfected with 1 μg plasmid DNA per well for 2 days using TransIT-2020 transfection reagent according to the manufacturer’s instruction as described previously , followed by cell seeding at a density of 2 × 105/well with treatment of SMI-4a at the indicated concentration. After 48 h incubation, cells were washed with 1X PBS once, followed by fixation with 4% formaldehyde for 30 min. Cells were washed with 1X PBS once again, followed by 1% crystal violet staining for 15–30 min at room temperature. Cells remaining on the upper chamber were removed using cotton swab. The number of migrating or invading cells was shown and quantified by counting for five fields/field of view at ×200 magnification.Cell migration and invasion abilities were examined by eviously . For mig3/well in a 24-well plate. The next day, cells were treated with SMI-4a for 14 days. The cells were refreshed with a medium containing SMI-4a every 7 days. 2 weeks later, the cell viability was determined by 1% crystal violet staining (buffered with 30% ethanol).HER2-negative and -positive breast cancer cells were seeded at a density of 1 × 10™ reagent (Roche). 1 μg RNA was subjected to reverse transcription using M-MLV reverse transcriptase according to manufacturer’s instruction (Sigma). The qPCR analysis was performed on Illumina EcoTM system (Bio-genesis Technologies Inc.) using VeriQuest Fast SYBR Green qPCR Master Mix.Total RNA extraction was performed using Trizolp: the sum of the positive reaction rate; Pm: the maximum positive reaction rate; Pn: the minimum positive reaction rate.IC50 of Pim inhibitors was determined by the following equation: lgIC50 = Xm-I (P- (3-Pm-Pn)/4). Xm: lg maximum dose; I: lg (maximum dose/relative dose); p-value is *< 0.05, **<0.01, ***<0.001.Pearson correlation was used to study the correlation between IC50, Pim1, or HER family expression in breast cancer cell lines. All data were displayed as mean ± S.E.M for three independent experiments. The significance of the difference between the experimental and control groups was assessed by Student’s t-test. The difference is significant if R2 score revealed that Pim1 protein expression significantly and positively correlated with HER2 and HER3, but not HER4, protein expressions and that the correlation between Pim1 and EGFR expressions approaches marginal significance (It is known that induction of Pim1 was accompanied by increases in EGFR expression . To addrificance .To further address whether the correlation between Pim1 and HER family expressions relies on Pim1 kinase activity, Pim1 kinase inhibitors SMI-4a and SGI-1776 were employed. First, we determined the sensitivity of various breast cancer cell lines to these Pim1 inhibitors in MTT assays and analyzed the correlation of Pim1 protein expression with the IC50 of these two inhibitors. The IC50 of these Pim1 inhibitors in various breast cancer cell lines were listed in Since we observed that HER2 expression was associated with Pim1 inhibitor sensitivity, we further examine whether HER2-positive breast cancers are more sensitive to Pim1 inhibitors. As shown in We next investigated the mechanism underlying Pim1 inhibitor-mediated anti-cancer activity in HER2-expressing breast cancer cells. As shown in Lapatinib is a HER2 TKI approved for metastatic HER2-positive breast cancer patients. Development of acquired resistance within one year of treatment limited the clinical benefits of this drug . HER2 prThe members of the HER family are well-known oncogenic driver genes in various cancer types. Although targeting the kinase activity by small molecular inhibitors has shown promising clinical benefits, kinase-independent functions have been proposed to contribute to the development of acquired resistance to these drugs . SuppresPim1 is a serine/threonine kinase and promotes cell proliferation, survival, and drug resistance. Overexpression of Pim1 has been observed in many cancer types and reported to play a crucial role in tumorigenesis . In prevIn breast cancer, 20–30% of cases belong to the subgroup of HER2 overexpression, which makes the tumor more aggressive. Therefore, targeted therapy against HER2 activity has been developed and approved for HER2-positive breast cancer . Althoug


In this study, we synthesized PPy@Fe3O4 NPs through a very facile electrostatic absorption method. And we detected reactive oxygen species production, cell apoptosis, migration and protein expression in different groups of A549 cells and established xenograft models to evaluate the effects of PPy@Fe3O4 NPs for inhibiting the growth of NSCLC. The results showed that the PPy@Fe3O4 NPs had negligible cytotoxicity and could efficiently inhibit the cell growth and metastasis of NSCLC in vitro. In addition, the PPy@Fe3O4 NPs decreased tumor volume and growth in vivo and endowed their excellent MRI capability of observing the location and size of tumor. To sum up, our study displayed that the PPy@Fe3O4 NPs had significant synergistic effects of PTT and CDT, and had good biocompatibility and safety in vivo and in vitro. The PPy@Fe3O4 NPs may be an effective drug platform for the treatment of NSCLC.Non-small cell lung cancer (NSCLC) is considered to be a principal cause of cancer death across the world, and nanomedicine has provided promising alternatives for the treatment of NSCLC in recent years. Photothermal therapy (PTT) and chemodynamic therapy (CDT) have represented novel therapeutic modalities for cancer treatment with excellent performance. The purpose of this research was to evaluate the effects of PPy@Fe Lung cancer remains the most common malignant cancer, which has the highest rates of mortality.  Non-smal3+ ions are used as the oxidation agents to produce PPy NPs, many ferric (Fe3+) and ferrous ions (Fe2+) remain in the obtained PPy NPs. And Fe ions in the PPy NPs are selected as precursors for in situ formation of Fe3O4 crystals onto the surface of pre-synthesized PPy NPs. The final products (PPy@Fe3O4 NPs) possess both chemodynamic therapy (CDT) and photothermal therapy (PTT) functions, holding tremendous potential for remarkable efficiency to inhibit tumor growth. PTT, which uses photothermal agents to ablate tumors by converting absorbed light energy into intense localized heat, has attracted widespread attention as a non-invasive therapeutic technology. (2O2) using Fenton/Fenton-like reaction to control tumor progression in the tumor microenvironment (TME). .  As generication.  Thus, PP3O4 NPs could decrease the expression of MMP2, MMP9 and MMP13.Extracellular matrix (ECM) is a complex biopolymer mixture produced by different kinds of cells in the extracellular space. The ECM not only is crucial in cell adhesion and tissue organization, but also modulates cell differentiation, activation and migration.  Matrix m3O4 NPs in vitro and in vivo. Our results displayed that the growth of tumors was considerably inhibited by the PPy@Fe3O4 NPs, and the levels of MMP2, MMP9 and MMP13 decreased. These results demonstrated that PPy@Fe3O4 NPs were excellent MRI-guided synergistic chemodynamic/photothermal cancer therapy agents and might be promising drugs to treat NSCLC because of inhibition of tumor growth and metastasis.In this research, we examined the anti-tumor efficacy of the novel PPy@Fe3; 99.9%) and pyrrole (99%) were purchased from Aladdin chemistry Co., Ltd. . Absolute ethyl alcohol  and ammonium hydroxide solution  were purchased from Sinopharm Chemical Reagent Co., Ltd. . Deionized water (H2O) was prepared by a Milli-Q water purification system . All the chemical reagents were used without further purification.Polyvinyl alcohol 1788 , iron (III) chloride anhydrous  was added in the deionized water (10 ml) and heated to 95°C until the solution was entirely dissolved. Later, FeCl3  was mixed homogeneously with the above mixture under strong magnetic stirring for 1 h. Next, pyrrole monomer  was slowly dropped, and the reaction was maintained for 4 h at 4°C under magnetic stirring. The final solution turned dark green, indicating that PPy NPs were successfully synthesized. Subsequently, the solution (2.5 ml) was taken out, mixed with deionized water (15 ml) and ethanol (2 ml) under stirring at 70°C. Afterwards, 1 ml 1.0 wt% aqueous ammonia liquid was dropped into the solution followed by stirring for 30 min. Then 1 ml of 1.0 wt% aqueous ammonia liquid was added dropwise again, and the mixture lastly maintained for 30 min at 70°C. Finally, after being washed three times by deionized water, the PPy@Fe3O4 NPs were collected by centrifugation (11000rpm) for 50 min.We synthesized the PPy@Fe3O4 NPs were determined using a JEM-200 transmission electron microscopy  at 200kV acceleration voltage. The characteristic functional group and crystal structures of nanomaterials were measured using Fourier-Transformed Infrared (FTIR) spectrometer and X-ray powder diffraction (XRD) respectively.The morphology and size of PPy@Fe3O4 NPs with different concentrations  were separately irradiated by an 808 nm near-infrared (NIR) laser (1.0W/cm2). The temperature profiles were monitored and recorded using a thermal imaging camera  over time. Then the PPy@Fe3O4 NPs (400 μg/ml) solutions were irradiated with the NIR laser for 10 min, and cooled down to room temperature. Last, we calculated the photothermal conversion efficiency (η) by the temperature curves according to published study.  assays. In detail, normal human bronchial epithelial cells (BEAS-2B) and Human lung adenocarcinoma cells (A549) were respectively seeded into 96-well plates at a density of 104 cells per well and cultured in pH7.4. The following day, we replaced the medium with fresh medium (pH7.4) containing the PPy@Fe3O4 NPs of different concentrations  for another 24 h. Then we changed the medium with 100 μl serum-free medium and 10 μl CCK-8 reagent. Following incubation for 2 h, we measured the absorbance value of wells at 450 nm using a microplate reader . The calculated formulas were listed in We evaluated the cytotoxicity of the PPy@Fe3O4 (400 μg/ml); 3) H2O2 (100 μM); 4) PPy@Fe3O4 (400 μg/ml) + H2O2 (100 μM). After 24 h of treatment, we used the probe solution 2, 7-dichlorodihydrofluorescein diacetate  to estimate the intracellular reactive oxygen species (ROS). After DCFH-DA treatment for 20 min, the cells were washed three times by phosphate buffered saline (PBS) buffer and blank medium was added. Finally, we acquired images using Confocal laser scanning Microscopy .The A549 cells were incubated in the culture plates and divided into four groups: 1) control; 2) PPy@Fe3O4 NPs was evaluated by the Calcine-AM/propidium iodide (PI) test  and the Annexin V-FITC/PI apoptosis kit . To investigate the PTT effect of the PPy@Fe3O4 NPs, A549 cells were incubated in the culture plates at pH6.5 and divided into 6 groups; group 1 with cells only; group 2 cultured with NIR only; group 3 cultured with H2O2 (100 μM) only; group 4 cultured with PPy@Fe3O4 NPs (400 μg/ml) + NIR; group 5 cultured with PPy@Fe3O4 NPs (400 μg/ml) + H2O2 (100 μM); group6 cultured with PPy@Fe3O4 NPs (400 μg/ml) + H2O2 (100 μM) + NIR. After incubation with PPy@Fe3O4 NPs (400 μg/ml) for 12 h, the cells of group 2,4 and 6 were exposed to an 808 nm NIR laser (1.0 W/cm2) for 10 min. Afterwards, we stained the A549 cells with Calcein-AM and PI for about 15 min, and images were acquired by CLSM.The anti-cancer effect of PPy@FeAnd we detected cell apoptosis by Annexin V-FITC Apoptosis kit. The A549 cells in different groups were harvested and washed with PBS and Binding Buffer. Then the cells were resuspended in 500 μl Bind Buffer with 5 μl Annexin V-FITC and 10 μl PI solution to mix evenly for 5 min at room temperature in darkness. And we measured cell apoptosis immediately by flow cytometry .In vitro migration assay, 24-well Transwell chambers with a polycarbonate filter membrane of 8 μm pore size  were used to assess the effect of the PPy@Fe3O4 NPs on A549 cell migration. Cells were divided into control group and PPy@Fe3O4 group. After starvation of A549 cells for 24 h, we resuspended cells in serum-free RPMI 1640 medium and serumfree RPMI 1640 medium containing the PPy@Fe3O4 NPs at a concentration of 400 μg/ml respectively. Next, the A549 cells were inoculated into the upper chamber and the RPMI 1640 medium was placed in the lower chamber. After culturing for 24 h, the A549 cells that migrated to the bottom of the membranes were immobilized and stained with 1% crystal violet . Finally, the migrated cells of each groups were counted under a microscope .5) were seeded into 6-well plate and divided into the control group and the PPy@Fe3O4 group (400 μg/ml). After incubation for 24 h, the cells were washed by PBS and lysed in RIPA buffer. Then we removed the cell debris by centrifugation, and the supernatants were harvested and stored at −80°C. Equivalent amounts of protein (25 µg) were separated on 10% SDS-PAGE and transferred onto 0.22 um polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and subsequently incubated with primary antibodies including anti-MMP2/MMP9/MMP13  and anti-β-actin . The following day, we washed the membranes three times for 10 min each time by Tris-buffered saline with Tween20 (TBST), and then incubated them in the corresponding secondary antibody (goat anti-rabbit IgG horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, 1:1,000, Affinity Biosciences, China) at room temperature for 2 h. Following washing three times for 10 min with TBST buffer, the membranes were treated with enhanced chemiluminescence  reagent for exposure.A549 cells . Then we scanned the mice before and 3 h after injection. Thus, we successfully acquired the high-resolution T2-weighted MRI scan images of mice by a 3.0T MRI system . The T2-weighted MRI parameters were as follows: pulse waiting time (TR) = 2,800 ms, echo time (TE) = 60 ms, slice width (SW) = 5.0 mm.For 3O4 NPs (3 mg/ml); 4) PPy@Fe3O4 NPs (3 mg/ml) + NIR. The mice of group3 and 4 were injected intravenously with 200ul PPy@Fe3O4 NPs (3 mg/ml) and the mice of group2 was injected intravenously with 200ul PBS. Then 8 h after injection, the mice of group2 and 4 were irradiated with 808 nm laser (1W/cm2) for 10 min. Meanwhile, we measured the temperature changes of the tumor by a FLIR A300 thermal imaging camera. Simultaneously, tumor volume and body weight were monitored every 2 days. On day 14, the mice were sacrificed. Then the major organs were isolated for photograph or histological analysis (hematoxylin and eosin (H and E) staining), and the tumor was stained subsequently by immunohistochemistry (Ki67) and immunofluorescence (TUNEL).When the tumors reached 7–9 mm in diameter, we divided the mice into four groups (n = 6 per group): 1) control; 2) PBS + NIR; 3) PPy@FeThe tumor tissues specimens of mice were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Then the tissue blocks were cut into sections of 3-5 um thickness. For Ki67 staining, the sections were immunostained overnight at 4°C with an anti-Ki67 antibody (Abcam). After washing in PBS, the sections were subsequently incubated with the second antibody for 1 h. Then the slides were stained with 3, 3-diaminobenzidine (DAB) and hematoxylin separately, dehydrated, and mounted. Images were captured using a microscope . For TUNEL staining, the proportion of apoptotic cells were performed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining kit . We obtained all images using a CLSM.p < 0.05 was considered statistically significant; (**) p < 0.01 and (***) p < 0.001 was considered highly statistically significant; (****) p < 0.0001 was considered extremely statistically significant.All results and measurements were shown as the mean ± SD (standard deviation). Comparison between the mean values of different groups were analyzed by one way analysis of variance (ANOVA) or Student’s t-test: (*) 3O4 NPs. The PPy@Fe3O4 NPs are shown in 3O4 NPs was measured to be 71.0 nm, and many tiny Fe3O4 nano-crystals were surface adsorbed on PPy NPs. The FTIR spectrum showed clearly the characteristic absorption peaks of PPy, indicating the successful formation of PPy . .  The abov3O4 NPs had a broad absorption throughout the visible to the NIR region, which demonstrated that PPy@Fe3O4 NPs were good potential PTT agents. We further evaluate the photothermal properties of PPy@Fe3O4 NPs with NIR light irradiation. With the prolonging of the irradiation time and increased concentration of PPy@Fe3O4 NPs, the temperature rose rapidly  for 24 h to test the cell viability. The results indicated that PPy@Fe3O4 NPs exhibited lower cytotoxicity towards normal and cancer cells as high as 400 μg/ml , which was oxidized to 2, 7′-dichlorofluorescein (DCF) in the presence of ROS. As displayed in 2O2 group and PPy@Fe3O4 NPs group increased weakly. When treated with both H2O2 and PPy@Fe3O4 NPs, the cells displayed strong and extensive green fluorescence, indicating that PPy@Fe3O4 NPs could increase Fenton-induced ROS generation in the presence of H2O2.Chemodynamic therapy is an effective therapeutic treatment that causes damage of tumor cells by producing ROS. To examine the effects of PPy@Fein vitro therapeutic efficacy of PPy@Fe3O4 NPs through Calcine-AM/PI test. Living cells were stained with green fluorescent calcein AM, while dead cells were stained with red fluorescent PI  control; 2) PBS + NIR; 3) PPy@Fe3O4 NPs; 4) PPy@Fe3O4 NPs + NIR. Mice of the group 2 were injected with 200ul PBS via the tail vein, and mice of group 3, 4 were injected with PPy@Fe3O4 NPs (3 mg/ml) via the tail vein. After 8 h, the mice of group 2, 4 were irradiated by an 808 nm NIR laser (1.0W/cm2) for 10 min. Meanwhile, by using an infrared thermal camera, we found that the tumor temperature of mice injected with PPy@Fe3O4 NPs rapidly increased to ∼53°C during irradiation, which was sufficient for ablating tumors. However, the tumor temperature of the group 2 had negligible rise  synthesis. (3O4 NPs significantly increased ROS levels generated from A549 cells under the conditions of H2O2. We also found that the PPy@Fe3O4 NPs promoted apoptosis of the cells of lung cancer induced by H2O2, as revealed by the Annexin V-FITC/PI assay and Calcine-AM/PI test. Therefore, the results of these experiments draw the following conclusion that PPy@Fe3O4 NPs could enhance antitumor effects through ROS damage.On the one hand, excessive production of •OH can oxidize vital cellular constituents, such as DNA, proteins and lipids, which can induce cell apoptosis or necrosis.  On the oategies.  As we alategies.  Thus, thnthesis.  GSH is tnthesis.  Herein, 3O4 NPs on A549 cell migration. The results showed that all PPy@Fe3O4 NPs significantly suppressed A549 cell migration. Moreover, tumor metastasis is a very complicated process that involves the interaction of various genes and proteins, especially MMPs. MMPs have been reported as proteolytic enzymes with the capacity of degrading the extracellular matrix components and other secreted proteins of the lungs. (3O4 NPs. Thus, these findings suggested that PPy@Fe3O4 NPs suppressed human lung cancer cell metastasis.Migration and invasion are two essential features of tumor metastasis. In metastasis, cancer cells invade and migrate from the primary site to the extracellular matrix and surrounding basement membrane, which further colonize the distant site.  Therefore lungs.  And theye lungs.  MMP2, MMe lungs.  Meanwhile lungs.  Here, ou3O4 NPs and demonstrated the safety and effectiveness of the PPy@Fe3O4 NPs in vivo and in vitro. The PPy@Fe3O4 NPs exhibited excellent synergistic effects of PTT and CDT, which could inhibit the growth and metastasis of lung cancer cells efficiently. Additionally, the PPy@Fe3O4 NPs was remarkable MRI contrast agents for tumors. However, the underlying mechanisms of tumor growth and metastasis need to be further discussed. Taken together, our study revealed the potential of PPy@Fe3O4 NPs to develop a new therapeutic strategy for NSCLC.In our study, we synthesized PPy@Fe


Human ARID4A and ARID4B are homologous proteins that are important in controlling gene expression and epigenetic regulation but have distinct functions. Previous studies have shown that the N-terminal domain of ARID4A is an unusual interdigitated double Tudor domain with DNA-binding activity. However, how the Tudor domain of ARID4B differs from that of ARID4A remains unknown. Here, we found that the ARID4B Tudor domain has significantly weaker DNA affinity than the ARID4A Tudor domain despite sharing more than 80% sequence identity. Structure determination and DNA titration analysis indicated that the ARID4B Tudor domain is also an interdigitated double Tudor domain with a DNA-binding surface similar to ARID4A. We identified a residue close to the DNA-binding site of the Tudor domain that differs between ARID4A and ARID4B. The Leu50 in ARID4A is Glu50 in ARID4B, and the latter forms salt bridges with two lysine residues at the DNA-binding surface. This causes a decrease in the strength of positive charge, thus reducing DNA-binding affinity while significantly increasing protein stability. We also found that a C-terminal extension region enhances the DNA-binding affinity of the ARID4B Tudor domain. This C-terminal extension is disordered and contains a positively charged RGR motif, providing an additional DNA-binding site. Finally, sequence and phylogenetic analyses indicated that the residue differences and the presence of the RGR extension region are conserved. These results provide new insight into the functional differences between ARID4A and ARID4B proteins, as well as elucidating the function of the disordered regions in these proteins. Arid4a deficiency were viable and fertile, while Arid4b−/− mice were not born alive (Arid4b knockout (Arid4bSCKO) mice display several unique and more severe phenotypes than Arid4a−/−Arid4b+/− male mice  and RBBP1-like protein 1 (RBBP1L1), are both components of the mSin3A complex, which suppresses gene expression and regulates epigenetic marks , 3, 4, 5rn alive . Furtherale mice . Searchiale mice . BesidesA), three of which are Royal domains, including Tudor, PWWP, and chromobarrel domains. The other two domains are the ARID and R2 domains, which have HDAC-independent and -dependent gene repression activity, respectively. These five domains are highly conserved between the two proteins with sequence identity of 40 to 80%, while disordered regions share much lower sequence identity (<29%) (KD) of 10 to 20 μM mainly via HTD-1  . We havey (<29%) . The Tudy (<29%) . Tudor dy (<29%) , 17. They (<29%) . Howeveria HTD-1 .Figure 1We noticed that the region (residues 122–146) following the construct of the ARID4A Tudor domain is highly conserved between ARID4A and ARID4B with just one residue difference at position 137, and this region was predicted to possibly contain some ordered structure between residues 122 to 138 (B), implying an important function of this region. Therefore, we constructed an extended ARID4B Tudor domain containing residues 1 to 151 (TD151). We also constructed the ARID4B Tudor domain containing residues 1 to 121 (TD121) for comparison.We previously solved the solution structure of the ARID4A Tudor domain including residues 4 to 121, which forms an interdigitated double Tudor domain with DNA-binding activity . NotablyFig. 1C), indicating that the additional C-terminal tail (residues 122–151) enhances DNA-binding affinity of the ARID4B Tudor domain. ARID4B TD151 also shows stronger affinity for the 18-bp dsDNA1 than the 12-bp dsDNA2 (C). Further, DNA-binding affinity of ARID4B TD121 is significantly weaker than ARID4A TD121 (D). Consistent with this, the affinity (KD) of ARID4B TD151 for dsDNA1 was measured as 27 ± 8 μM by ITC (E), whereas the affinity of ARID4B TD121 for DNA is undetectable (F).EMSA results showed that ARID4B TD151 has significantly stronger affinity for the 18-bp dsDNA1 than ARID4B TD121 C, indicap dsDNA2 C. Furthe4A TD121 D. ConsisM by ITC E, whereatectable F.1H-, 15N-, and 13C-NMR resonances of ARID4B TD151. The 1H-15N HSQC spectrum of ARID4B TD151 and the assignments of the amide signals are shown in Fig. S1A. All nonproline backbone amide proton and nitrogen signals of TD151 were assigned, except for His148. The assignment of the backbone resonances  of residues Met1–Glu151 was completed to 99.4%. Assignment of the aliphatic and aromatic side chains was achieved to 94.1% . Based on the assignments, we then determined the solution structure of ARID4B TD151 . HTD-1 contains four β-strands, i.e., β1 (residues 15–20), β2 (residues 23–33), β3' (residues 88–93), and β4' (residues 96–101). Arg103-Ser105 forms a 310 helix (η1') in some conformers. HTD-2 also contains four β-strands, i.e., β3 (residues 36–42), β4 (residues 47–52), β1' (residues 63–69), and β2' (residues 74–85). Asp53-His55 also forms a 310 helix (η1) in some conformers. The root mean square deviation (RMSD) values of secondary backbone atoms of HTD-1, HTD-2, and both HTDs are 0.51 Å, 0.36 Å, and 2.22 Å, respectively .To investigate the structural basis of the DNA-binding affinity of ARID4B TD151, we first assigned the A–C, and . Like thA–C, and A. HTD-1 A–C, and . AlthougA–C, and , and TALD and Fig. S2D). However, compared with the positively charged DNA-binding interface of the ARID4A Tudor domain, the positive charge of the corresponding surface regions of the ARID4B Tudor domain is significantly reduced (E and F). Detailed analysis indicates that the ARID4B Tudor domain contains a negatively charged residue, Glu50, at the interface, corresponding to Leu50 in the ARID4A Tudor domain, while the other charged residues in the interface are conserved between the Tudor domains of ARID4A and ARID4B . The carboxyl group of the Glu50 side chain forms two salt bridges with the side chain amino groups of Lys37 and Lys39. Distances between the Oε atom of Glu50 and the Nξ atoms of the two lysine residues are 3.4 ± 1.0 and 3.5± 1.8 Å, respectively, forming two strong salt bridges .The structure of the ARID4B Tudor domain is highly similar to the ARID4A Tudor domain, with RMSD 0.66, 0.77, and 2.0 Å between secondary structure backbone atoms of the two HTD-1, the two HTD-2, and the two overall structures, respectively . The affinity was obtained by fitting the NMR titration data and the KD obtained was 22 μM (C), in agreement with the ITC result. Besides chemical shift perturbations, addition of dsDNA1 also enhanced NH signal intensities of C-terminal disordered residues of ARID4B TD151, mainly within the sequence 137-GKKTNRGRRS-146  and Gly110-Ile136 (3 > intensity ratio >1.5) (D and E). This suggests that the RGR motif of ARID4B TD151 undergoes intermediate exchange between multiple conformations in the free protein leading to line broadening, but adopts a more rigid conformation with stronger and sharper signals when bound to dsDNA.We then performed NMR titration of ARID4B TD151 by gradually adding dsDNA1, which caused significant chemical shift perturbations , A and Bas 22 μM C, in agrio >1.5) , D and EF) include Glu6, Tyr9, Ala18-Gly22, Glu26, Gln52sc, Trp88, Thr90-Val91, Thr100-Leu101, Ser104, Cys107, Gly110-Glu111, and Ile149; residues showing a moderate CSP  include Val16-Ser17, Cys25, Ala27, Ile29, Thr31, Lys33-Leu35, Thr48, Asp54-His55, Thr84, Val92-Asp95, Asp97-Glu98, Arg102-Arg103, Ser105, Lys109, and Gly143-Arg145. These residues are mainly located within HTD-1, η1 of HTD-2, and the C-terminal RGR motif , indicating important roles of these residues in DNA interaction. Residues showing the largest intensity enhancement (intensity ratio > 3) include Gly137, Lys139, Gly143, Arg145, and Ser146, which are all located in the C-terminal RGR motif . In summary, ARID4B TD151 contains two DNA-binding sites. The first DNA-binding site is mainly located within HTD-1 of the interdigitated double Tudor domain, similar to the ARID4A Tudor domain .We then mapped the results of CSP and intensity enhancement onto the structure of ARID4B TD151. Residues showing the largest CSP  of ∼110 μM for dsDNA1 estimated by fitting the NMR titration data (A and B), about five times weaker than ARID4B TD151, which is consistent with the EMSA result. Similarly, the affinity of ARID4B TD121 for dsDNA2 obtained from titration was 237 μM, about three times weaker than ARID4B TD151 (KD 79 μM) (C–F). Consistent with this, the affinity of ARID4A TD151 with dsDNA1 was measured as 9.7 μM (KD) (G and H), about 2 to 3 times stronger than ARID4A TD121 (KD 27 μM) (KD 22 μM). These results confirm that the C-terminal disordered region can enhance DNA-binding affinity of the Tudor domain.ARID4B TD121 has an affinity  , C–F. Co μM (KD) , G and HA–C), suggesting a very weak sequence preference for AT-rich DNA. To elucidate the role of each residue of the RGR motif in DNA binding, we constructed a series of point mutants in the C-terminal RGR motif of ARID4B TD151, including K139A, R142A, G143A, R144A, and R145A, and measured their binding affinity with dsAT18 and dsGC18 (D). The results showed that these mutations decreased the affinity by up to fivefold. Affinity ratios of dsAT18 versus dsGC18 binding to mutants R142A, G143A and R144A, which are core residues in the RGR motif, are ∼1.1 to 1.3, while the ratios for the mutants K139A and R145A are ∼0.4 to 0.5, which is similar to wild-type ARID4B TD151 (ratio 0.5) (D). These results indicated that the weak preference of ARID4B TD151 for AT-rich DNA is achieved by the core residues Arg142, Gly143, and Arg144, while surrounding residues such as K139 and R145 do not contribute to the preference but contribute to the overall DNA-binding affinity.The C-terminal RGR motif of ARID4B TD151 is similar to the AT-hook motif, which contains a Pro-Arg-Gly-Arg-Pro sequence . The RGRd dsGC18 D. The retio 0.5) D. These KD values for binding of ARID4A TD121 to dsDNA1 and dsDNA2 of 27 μM and 16 μM, respectively . As stated above, the structure determination revealed that most of the charged residues of the DNA-binding site of ARID4A Tudor domain are conserved in the ARID4B Tudor domain except that Leu50 is replaced by the negatively charged Glu50 in the ARID4B Tudor domain (G). Glu50 in the ARID4B Tudor domain forms two salt bridges with Lys37 and Lys39, while the corresponding residues in the ARID4A Tudor domain are indicated to contact with DNA (A), confirming the attenuation effect of Glu50 on DNA binding. Interestingly, this mutation significantly increased the stability of the ARID4A Tudor domain, as the mutant did not precipitate after 2 h incubation at 30 °C, similar to ARID4B TD121, while the wild type underwent significant precipitation under the same conditions, with only ∼30% protein remaining in the supernatant (B). The effect of Glu50/Leu50 on the stability was further investigated by DSC, which indicated that the L50E mutation of ARID4A TD121 increased the Tm value from 40.6 °C to 48.2 °C, while the E50L mutation of ARID4B TD121 decreased the Tm value from 50.8 °C to 44.5 °C (C). Therefore, the salt bridges between Glu50 and Lys37/Lys39 in the ARID4B Tudor domain significantly increase thermostability of the protein and simultaneously decrease its DNA-binding affinity.Previous studies have obtained ectively , which ar domain G. Glu50 with DNA . We therwith DNA A, confirernatant B. The ef 44.5 °C C. TherefBased on the NMR titration results for ARID4B TD151 using dsDNA1, we attempted to construct a structural model for the complex of ARID4B TD151 and dsDNA1 using HADDOCK docking . The che2. The best model in the top cluster is shown in Figure 7A.Analysis of the final 200 HADDOCK models for the ARID4B Tudor domain and dsDNA1 complex resulted in ten clusters and the statistics of the top seven clusters are displayed in A). The axis along HTD-1 and HTD-2 of TD151 is almost parallel to the DNA duplex, with L12 and β1 making contact with the DNA major groove, while β4' and the C-terminal loop of HTD-1 make contact with the DNA minor groove and backbone atoms. The structure model of the complex is quite similar to that of the ARID4A Tudor domain with dsDNA2 (B and C). Detailed analysis of the model indicates that residues Lys19–Arg21 of L12 extend into the DNA major groove, whereas the side chains of Trp88 and Arg102 of the C terminus extend into the minor groove (D). Side chains of Lys37 and Gln52 in HTD-2, and Ser104 and Ser105 in HTD-1 also contact the DNA backbone atoms, while Glu50 has no contact with DNA but still forms a salt bridge with Lys37 (D). These residues form a positively charged DNA-binding surface, which has electrostatic interactions and physical complementarity with DNA (E). These contact residues and the positively charged interacting surface of the ARID4B Tudor domain are basically similar to the structure model of the complex between ARID4A Tudor domain and DNA, although the positive charge of ARID4B Tudor domain is partially neutralized by Glu50 (F and G).In this model of the complex, the DNA duplex mainly binds to the ARID4B HTD-1 at sites containing β1, β4', loop L12, the C-terminal loop of HTD-1, and the linker between HTD-1 and HTD-2 A. The axh dsDNA2 , B and Cr groove D. Side cth Lys37 D. These with DNA E. These by Glu50 , F and GH). In all ten structures, the peptide binds to the DNA minor groove. In the representative structural model, side chains of N141, R144, and R145 penetrate into the groove, and the positively charged side chains of lysine and arginine residues also have electrostatic interaction with DNA (I). Combining the HADDOCK-derived structures of the complexes of the ARID4B Tudor domain and the C-terminal RGR peptide with DNA, Figure 7J shows a model in which the ARID4B Tudor domain and the C-terminal peptide cooperatively bind to the DNA duplex. The Tudor domain binds to both major and minor grooves of the DNA duplex, while the RGR motif only binds to the minor groove. By sliding and rotation along the grooves of the dsDNA1, binding of about three ARID4B TD151 molecules could be accommodated  of ARID4B TD151. As the short segment of peptide is not suitable for cluster analysis using HADDOCK, we chose ten structures with the lowest HADDOCK scores from the final 200 structures to represent the complex in the model H. In allwith DNA I. Combinmmodated , which aC) are located either within HTD-2 (A) or in the N- and C-terminal disordered regions. These residues are generally far away from DNA-binding sites, except that Glu50 of ARID4B forms two salt bridges with DNA-binding lysine residues and decreases the DNA affinity. Besides Glu50, we noticed that a hydrophobic residue Val68 is located in the highly hydrophobic core of HTD-2 of the ARID4B Tudor domain, surrounded by hydrophobic residues Phe42, Val49, and Val51 (B), while in the ARID4A Tudor domain structure, the corresponding residue is a less hydrophobic Thr68 surrounded by Leu42, Gln49, and Val51 (C). Because both Val68 of ARID4A and Thr68 of ARID4B are in the hydrophobic core, the difference in their hydrophobicity may also lead to the observed difference in stability. Consistent with this, we found that wild-type ARID4B TD121 (Tm 50.8 °C) has greater thermostability than the L50E mutant of ARID4A TD121 (Tm 48.2 °C), and the E50L mutant of ARID4B TD121 (Tm 44.5 °C) has greater thermostability than wild-type ARID4A TD121 (Tm 40.6 °C) (C), suggesting an important role of hydrophobic core formation involving Val68 and surrounded residues for stability of the interdigitated Tudor domain.This study reveals that the Tudor domains of ARID4A and ARID4B have different DNA-binding affinities and stability, although the two Tudor domains share ∼80% sequence identity. Our results indicate that both domains bind to DNA using similar structural regions, but the one-residue difference at position 50 is the major reason for the differences in DNA-binding affinity and protein stability. Interestingly, detailed structure-based alignment of the two Tudor domains indicates that most residues that differ between the two Tudor domains  C, suggesBesides the core interdigitated double Tudor domain, we also investigated the C-terminal positively charged disordered RGR motif of ARID4B TD151 and found that the RGR motif can bind to the DNA duplex and enhance the DNA-binding affinity of ARID4B TD151 by about fivefold. The docking results indicate that the C-terminal RGR motif prefers to bind to the DNA minor groove. The RGR motif is similar to the AT-hook motif containing a conserved Arg-Gly-Arg-Pro sequence, which can penetrate into the minor groove through the side chains of the two Arg residues during DNA binding . HoweverDanio rerio to Homo sapiens contain both ARID4A and ARID4B homologues, while lower animals contain only one homologue. In higher animals, all ARID4B Tudor domains contain the Glu residue at the corresponding position to Glu50, while all ARID4A Tudor domains contain a Leu/Val residue at the same position. The corresponding residues in the homologues of lower animals show more variation without charge, which is more similar to ARID4A than ARID4B (A). Therefore, Glu50 of ARID4B has likely evolved for the specific function of ARID4B after it diverged from an ARID4A-like ancestor (B), implying its importance for the function of ARID4B. Our observation of different DNA-binding affinity and stability caused by the difference between Glu50 and Leu50 could be related to the specific functions of the two homologous proteins. The RGR motif is largely conserved in both ARID4A and ARID4B in higher animals, but less conserved in the ARID4A-like homologues of lower animals. Therefore, the function of the RGR motif is likely to be important for both ARID4A and ARID4B in higher animals, but less important in lower animals. The results presented in this study not only provide novel molecular insight into the functional differences between the homologous ARID4A and ARID4B proteins, but also shed light on the role and importance of the RGR intrinsically disordered region in the ARID4A/ARID4B protein family.Sequence alignment and phylogenetic analysis of the Tudor domains and the C-terminal extensions of the ARID4A/ARID4B family proteins revealed different conservation of the Glu50/Leu50 residues and the RGR motif . Higher n ARID4B A. Therefancestor B, implyi6-SMT3 tag and then expressed and purified as described previously for other His-SMT3-tagged proteins , mutant E50L of TD121, TD151 (residues 1–151), and mutants . The ARID4A constructs include TD121 (residues 4–121), TD151 (residues 4–151), and mutant L50E of ARID4A TD121. The proteins were constructed in pET28a with an N-terminal Hisproteins . 15N-13C2HPO4-NaH2PO4, 100 mM NaCl, pH7.0), with addition of 5 mM DTT, 0.02% (w/v) sodium 2,2-dimethylsilapentane-5-sulfonate (DSS), and 10% (v/v) D2O. The two-dimensional 1H-15N and 1H-13C heteronuclear single quantum coherence (HSQC) and three-dimensional CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO, HBHA(CO)NH, HCCH-TOCSY, CCH-TOCSY experiments were performed for backbone and side chain assignments of ARID4B TD151. The three-dimensional 1H-15N and 1H-13C NOESY-HSQC spectra with mixing times of 120 ms were collected to generate distance restraints. All data were processed using NMRPipe  of the protein resonances were calculated using the equation:HN and δN are the changes of 1HN and 15N chemical shifts, respectively. The equilibrium dissociation constants (KD) of protein with DNA were estimated by fitting the CSPs to the equation:max is the CSP value at the theoretical saturated condition obtained from the titration curve fitting; r is the molar ratio of DNA to protein; Cpro is the concentration of initial protein solution; Clig is the stock concentration of DNA. n is the number of equivalent and independent binding sites on the DNA, while the physical meaning of the obtained value of n is complicated as it could also account for any uncertainty in DNA and protein concentrations that were fixed in fitting. To be consistent with fitting for the ARID4A Tudor domain  were mixed with 1 μl dsDNA probe (150 μM), 3 μl glycerol (v/v 40%), and 1 to 9 μl buffer A in a final volume of 13 μl, with 12 μM final dsDNA concentration in each lane. After 60 min incubation at 4 °C, samples were loaded onto 8% native acrylamide gels, run in 1 x TBE buffer  at 4 °C, 150 V for 30 min. Gels were soaked for 5 to 10 min in TBE buffer containing 0.5 μg/ml ethidium bromide and visualized by TANON 1600 Gel Imager.The 12-bp dsDNA2 and 18-bp dsDNA1 were used for EMSA. Free proteins . The titrations were carried out in buffer A. The reactant (0.1 mM protein) was placed in the 200-μl sample cell. Then dsDNA solutions in an injection syringe (0.6 mM) were injected into protein solutions in the cell. The volume of each injection was 2 μl except for the first injection, which was 0.4 μl. A titration experiment consisted of 20 consecutive injections of 4 s duration, with a 120 s interval between injections. Control experiments were performed under identical conditions to determine the heat signals that arise from addition of DNA into the buffer. The resulting data were fitted to a single-site binding model using the Origin software package .g for 30 min. The absorbance of the supernatant at 280 nm was then measured to determine the concentration.Protein samples of ARID4B TD121, ARID4A TD121, and ARID4A L50E mutant, each containing 0.25 mM proteins in 50 μl volume, were incubated for 2 h at 30 °C in the buffer containing 50 mM Tris-HCl (pH 7.5) and 50 mM NaCl. Samples were then centrifuged at 13,000−1 by heating the sample from 15 °C to 75 °C under increased pressure (3 atm). All proteins used in this study were extensively dialyzed against a buffer containing 50 mM NaCl, 50 mM Tris, pH 7.6, and the dialysis buffer was used for instrumental baseline scans and as reference samples. Protein concentrations used were 1.0 mg/ml, corresponding to 75.0 μM for ARID4A/ARID4B TD121 proteins. Data were fitted to a two-state scaled model using NanoAnalyze software.DSC measurements were performed using a Nano DSC system (TA). Prior to scanning, samples were degassed under vacuum for 15 min using a degassing station (TA). DSC thermograms were determined by monitoring the difference in heat capacity in solution upon increasing temperature at a scan rate of 1 °C minStructure modeling of the ARID4B TD151 and DNA complex was performed using HADDOCK . The staDocking of the RGR motif, 138-KKTNRGRRS-146, with DNA was performed using HADDOCK2.2 on a local machine. All peptide residues were considered as active during docking. All DNA bases were considered active in the initial docking, and the active residues were then optimized according to the initial docking result. The final active residues included bases 8 to 12 and 25 to 28. As the short peptide sequence is not suitable for clustering, we chose ten structures with the lowest haddock scores from 200 final water-refined structures to represent the model of the complex between DNA and the peptide.A structure model of the ARID4B TD151 and dsDNA1 complex was constructed by manually combining the models of the Tudor domain-dsDNA1 complex and the RGR motif-dsDNA1 complex. The distance and orientation between the Tudor domain and RGR motif in this model are arbitrarily chosen without further optimization.All atom assignments of ARID4B TD151 have been deposited in BMRB under accession number 50612. The structure and the restraints have been deposited in the Protein Data Bank under accession number 7DM4 for ARID4B TD151. All remaining data are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Interestingly, this NW could be assembled from the stacking of the Li6Si5 aromatic cluster. The 12Si9 solid phase, i.e., metallic character, concerning the perturbation provided by their environment in the Li12Si7 compound.We computationally explore an alternative to stabilize one-dimensional (1D) silicon-lithium nanowires (NWs). The Li The insertion of inorganic materials into single-walled carbon nanotubes (SWCNTs), hereinafter identified simply as CNT, enables the encapsulation of extreme nanowires (NWs) with diameters comparable to a unit cell of the parent material . AlthougnPn clusters (n = 5–9) had previously been reported as global minimum structures  . Note thructures . Some soructures . The stu12Si7, Li7Si3, Li13Si4, and Li22Si5 2 (Li1210+[Si4]10)2, with two well-defined silicon moieties: planar Si5 rings and the Y-shaped Si4 moiety. Such a structural pattern is justified by assigning 26 electrons (20 from 6Si + 6 from 6Li) to the Si56- ring, favoring Hückel’s aromaticity 2 and (Li6Si5)3  contributes decisively to the stabilization of this NW. In mentioned cluster studies, explorations of the potential energy surface have been performed by hybrid methods, including genetic algorithms 3 , which aLi’s see . Howevergorithms .To build new class of materials with desirable properties, using atomic clusters instead of atoms as building blocks, is a remarkable possibility. However, it requires that atomic clusters must retain their identity when assembled, as Khanna and Jena first outlined when they coined the word “cluster-assembled materials” (CAMs) . These ain silico, the stability of the isolated Given the above background, here we evaluated, In the finite models (clusters), geometry optimizations and frequency calculations were performed at the PBE0 /Def2TZVPz0) Å3, considering periodic boundary conditions along the NW direction, where z0 is the periodicity. Within this supercell, the lateral distance between NW images is set to 15 Å. We use a (1 × 1 × 10) Monkhorst-Pack k-point mesh (6Si5)4 structures in the free space, also inside both an armchair and a zigzag single-walled carbon nanotube (SWCNTs). We consider armchair CNTs with chiral indexes , , and , which have diameters of 10.93, 12.27, and 13.63 Å, and zigzag CNTs with chiral indexes , , and  which have diameters of 11.04, 11.80, and 12.59 Å, respectively. For (Li6Si5)4@CNT simulation, (22 × 22 × z0) Å3 volume was used, where z0 is the periodicity chosen for the CNTs. All studied structures were allowed to freely relax without any constraint until forces on each atom were smaller than 25 meV/Å. To gain insights on the stability of the NW models in the free space and inside the SWCNTs, we performed Born-Oppenheimer ab initio molecular dynamics (BO-AIMD) simulations within the NVT ensemble at different temperatures, over a total simulation time of 10 ps, considering a time step of 1 fs.For the solid-state study, we performed first-principles calculations based on DFT  as impleint mesh . We alson = 13–20) in their optimal structure (at the PBE0/Def2TZVP level), covering the diameter range from 10.2 to 15.6 Å. Then the Li7Si5+ cluster was placed, centering it on emulating the growth pattern towards the nanowire .The first question that arises is which is the optimal SWCNT width to favor the finement . To get wire see . We choon = 13–15), Li7Si5+ cluster undergoes noticeable changes in the optimization process due to the confinement effects. In contrast, when [n]cyclocenes with n = 16–20 are used, the Li7Si5+ cluster maintains its structure at the end of the optimization process, leading to the best interaction energy, [Eint = E (Li7Si5+at[n]cyclacene)-(E (Li7Si5)+E ([n]cyclacene))], with [16]cyclacene . Since this analysis is only a reference for estimating the most suitable nanotube diameters to explore in the periodic calculations, we have not included basis set superposition error (BSSE) corrections. The most stable structures, as well as the Eint, are shown in In the case of small [n]cyclacenes (6Si5 units (Li6Si5-NW), which are stacked along the z direction, forming a one-dimensional structure, as shown in We first studied the stability of periodically repeated Li5 rings of 4.04 Å which is the periodicity of the Li6Si5 unit cell. In the equilibrium geometry, the distance between Li atoms of the border is 3.33 Å while the distance with respect to the center one is 2.83 Å. The Si-Si distance between neighboring atoms is 2.37 Å, very close to the ones in Li6Si5 monomer (between 2.30 and 2.35 Å). Our computations of the electronic band structure of the Li6Si5-NW in the primitive unit cell suggest a metallic character  in the free space and encapsulated it inside both armchair and zigzag carbon nanotubes (f-Li6Si5@CNT). For the f-Li6Si5 structure, we consider four Si5 rings surrounded by five Li6 moieties. BO-AIMD simulations provide insights on the stability of the f-Li6Si5 system in the free space at 300 K and 500 K. We find that at 300 K, the f-Li6Si5 structure preserves its stability. Still, at 500 K, it tends to form Si-Si bonds between adjacent Si rings without losing its one-dimensional array, as shown in 6 unit group is added, i.e., [(Li6)5(Si5)4].Next, we studied a finite Li6Si5 structure inside the CNTs, we consider three armchair CNTs with chiral indexes of , , and , and three zigzag CNTs with chiral indexes of , , and . With this choice, we seek to find the CNT size that best accommodates the Li6Si5-NW inside. Note that we selected these CNTs according to our preliminary findings from the Li7Si5+@[n]cyclacene model, suggesting diameters between 12 and 15 Å. The CNTs were simulated with periodic boundary conditions along its axis with a periodicity of z0 = 30 Å. The latter allows a vacuum region for the encapsulated f-Li6Si5 structure of 14 Å, allowing the atomic movement inside the CNT. Next, we calculate the 6Si5 and the CNTs by the equation:6Si5 and the CNT, respectively, and 6Si5 unit inside the CNT. Our results for the 6Si5 inside. In addition, the f-Li6Si5 is better stabilized inside the zigzag  CNT than inside the armchair  CNT by 0.9 eV. Noteworthy, the larger-diameter CNTs are energetically less favorable to encapsulate the f-Li6Si5, as shown in 7Si5+@[n]cyclacene model. This behavior is presumable due to the van der Waals (vdW) interaction between the f-Li6Si5 and the CNT internal walls, stabilizing the system. The non-covalent interaction index (NCI) plots confirm the non-covalent character of f-Li6Si5 with the CNT [f-Li6Si5 inside the zigzag  CNT]. In the NCI method  CNT. It is important to note that the unit cells of the Li6Si5-NW and the CNT have a mismatch of 5.7%, which means that the Li6Si5-NW is not in its equilibrium geometry in the Li6Si5-NW@CNT unit cell, where the distance between the Si5 rings increases by 0.23 Å. However, this mismatch is relatively small and should not affect the electronic properties of the system. For the isolated CNT we find a small bandgap of 0.02 eV as shown in 6Si5-NW@CNT system exhibits metallic properties as shown 6Si5-NW would preserve its electronic properties inside the CNT as can be compared with We also calculate the band structure of the Li6Si5 structure inside both zigzag  and armchair  CNTs by performing BO-AIMD simulations. 5Li6 structure inside the  CNT. We find that the structure remains almost unchanged with respect to f-Si5Li6 in the free space, showing that the CNT would have a small influence in the Li6Si5 NW stability. We only note a small displacement of the Li ions at the extreme of the f-Li6Si5 structure which move toward the CNT wall. The integrity of the f-Li6Si5 structure inside the  and  CNTs was investigated by BO-AIMD simulations at 300 K. We find that the f-Si5Li6 structure preserves its stability where the Li ions move around the Si5 ring without detaching. Similar results are found for the f-Li6Si5 structure inside the armchair  CNT, indicating that the formation and stability of the Li6Si5 NW inside the CNTs is independent of its chirality. This result suggests that Li6 Si5-NW are likely to form inside CNTs in a compact form, which would allow efficient storage of Li ions into CNTs mediated by Si5 rings. 6Si5 inside the  and  CNTs at 300 K. We observe energy fluctuation of around 5 eV in both CNTs, preserving the stability of the f-Li6Si5 structure.Finally, we study the stability of the f-Liab initio molecular dynamic simulations, we have shown that Li6Si5 units can be stacked one above the other, forming a one-dimensional structure linked together by Coulomb interactions. This study complements previous findings, where we demonstrated that Li6Si5, (Li6Si5)2, and (Li6Si5)3 lowest energy structures contain one, two, and three Si56- aromatic rings stabilized by Li+ counterions. Additionally, the 12Si7 compound but coexisting with Y-shaped Si4 moieties. In this case, we support the stability of the isolated Si-Li-nanowire—additionally, the relaxed structure (at room temperature) exhibits metallic characteristics.Using periodic DFT calculations and Born-Oppenheimer 6Si5)4 systems are stable inside both armchair and zigzag carbon nanotubes of around 12 Å in diameter, preserving its stability at room temperature, supporting the viable formation of Li6Si5-NW inside the CNTs. Interestingly, the Li6Si5-NW@CNTs hybrid nanocomposite maintains the metallic character. Finally, in the Li6Si5-NW, the Li6Si5 units are connected by strong electrostatic interactions  in agreement with the Zintl ion concept. In the [Li6Si5-NW]@CNTs, NCI predicts that Li6Si5-NW interacts with the CNT walls by van der Waals interactions We also found that finite (Li


Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in ∼30% of HOXA  •FOXC1 contributes to a monocyte-macrophage lineage differentiation block in AML•FOXC1 and RUNX1 colocalize on chromatin via interaction of their DNA binding domains•A FOXC1/RUNX1/TLE3 repressor complex limits monocyte gene enhancer activity•FOXC1 depletion in AML initiates widespread redistribution of RUNX1/TLE3 on chromatin Simeoni et al. demonstrate that the Forkhead factor FOXC1 recruits a repressor complex of RUNX1, HDAC1, and Groucho family member TLE3 to a discrete set of enhancers distributed close to genes controlling monocyte/macrophage differentiation, thereby contributing to the differentiation block, which is the pathognomonic feature of acute myeloid leukemia. FLT3 internal tandem duplications or NPM1 mutations, which are rarely found in clinical contexts other than AML, yield prominent myeloproliferative phenotypes when modeled in mice  is a blood cancer characterized by a block to normal myeloid lineage differentiation. This results in accumulation of myeloid blast cells in bone marrow (BM) and blood with consequent failure of normal hematopoiesis . Althoug in mice . Even muormation . The preleukemia . In addiFOXC1, which is a critical regulator of normal mesenchymal and mesodermal differentiation, is highly expressed in around 20% of cases of AML, but not expressed in normal hematopoietic lineages  translocation) exhibit high FOXC1 expression. In vitro and in vivo experimental evidence confirm that FOXC1 confers a monocyte/macrophage lineage differentiation block and sustains clonogenic activity in both murine and primary human FOXC1highHOXAhigh AML cells. Co-expression of FOXC1 with Hoxa9 accelerates the onset of AML in murine modeling, with the resulting leukemias exhibiting a higher level of differentiation block by comparison with those initiated by Hoxa9 alone. Further, patients with high FOXC1 expression exhibit inferior survival   translocation indicative of a CALM-AF10 fusion, as well as mutations in NRAS, ETV6, TP53, and EZH2, among others (FOXC1 knockdown (KD) and observed differentiation, as evidenced by morphology, increased expression of the monocyte/macrophage lineage differentiation marker CD86, reduced clonogenic activity, a reduced proportion of cells in the SG2M phase of the cell cycle, as well as an increase in apoptosis (FOXC1 cDNA engineered by site-directed mutagenesis to generate KD-resistant transcripts (FOXC1 SDM3)  A and S1Bg others . To confC1 SDM3) C–1E. We 2 BB475; , with si2 BB475; , misexpr−20), “ATP-dependent chromatin remodelling” (p = 10−12), and “transcription from RNA polymerase II promoter” (p = 10−10).To identify in an unbiased manner for FOXC1 interacting proteins with potential functional roles, we performed rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) . We geneCBFB, which we also identified as a FOXC1 interacting protein, in view of its coding for the obligate heterodimeric binding partner of RUNX1. KD of RUNX1, CBFB, CEBPA, STAT3, and CEBPE using two separate short hairpin RNA (shRNA) hairpins for each gene increased expression of CD86, which we used as a surrogate marker for upregulation of a differentiation program  sequencing (ChIP-seq) for FOXC1, RUNX1, and CEBPA in Fujioka AML cells. In view of its critical role in myeloid development , we alsohigh, H3K4Me1high), active B , primed , or quiet  , Model-based Analysis of ChIP-seq v2 (MACS2)  identifi4Me1low) E, in con4Me1low) D. A simi4Me1low) D. The di4Me1low) D and 3F.FOXC1 KD Fujioka cells MACS identified just 567 peaks, with no evidence of peak redistribution  data D–2H suggFOXC1 KD at sites of strong dual FOXC1 and RUNX1 binding. Given the predominant distribution of FOXC1 and RUNX1 peaks at putative enhancers, we excluded sites located at promoter and 5′ UTR sequences from the analysis in the first instance. There were 581 such genomic locations (which we term “FR-20 enhancer” sites). As comparators, we evaluated strong FOXC1 binding sites without a nearby strong RUNX1 peak  and vice versa for RUNX1 . Most F-20 enhancer sites were at regions of quiet chromatin, whereas the great majority of R-20 and FR-20 enhancer sites were at regions of primed or active chromatin , we observed overall a relative reduction of mean RUNX1 ChIP signal by comparison with R-20 sites ] versus 1,640 ± 73 [FOXC1 KD] reads/600 bp; t test, p = 10−4; R-20: NTC versus KD comparison, p = not significant; FOXC1 KD] reads/600 bp; t test, p = 10−7; FOXC1 KD cells and observed that overall there was an increase in mean relative CEBPA ChIP signal at FR-20 enhancer sites following FOXC1 KD , but no change at F-20 or R20 enhancer sites , we also noted that in control cells there was significantly greater HDAC1 ChIP signal at RF-20 versus R-20 enhancer sites . After FOXC1 KD, 349 genes were upregulated by at least 2-fold and 804 downregulated  to compare the transcriptional changes with, as an example, those observed during phorbol ester-mediated terminal differentiation of THP1 AML cells into macrophages  enhancers exhibited significantly greater baseline RUNX1 binding compared with group 2 (RUNX1down CEBPAdown) and 4 (RUNX1up CEBPAdown) enhancers, and intermediate levels of CEBPA binding compared with group 2 (lower) and group 4 (higher). There was no change in H3K4Me2, SMARCC2, or EP300 ChIP signal, or ATAC-seq signal, for any of the enhancer groupings following FOXC1 KD (data not shown). However, there was a significant reduction in HDAC1 ChIP signal at group 1 and group 2 enhancers  and a significant relative increase in H3K27Ac signal at group 1 sites , FOXC1 serves as a transcription repressor through stabilizing RUNX1 and HDAC1 binding, thus limiting enhancer activity.FOXC1high AML cells, we generated a FOXC1 Forkhead domain-RUNX1 fusion protein (FKD-RUNX1)  A. In thentiation B. In conntiation C, which ntiation F, promotntiation D. We perFOXC1 KD E.FOXC1 KD. MACS2 identified 17,589 RUNX1 binding peaks in FOXC1 KD Fujioka cells. Although the bulk of called peaks in the KD condition overlapped with peaks in the control condition (FOXC1 KD cells only) were 5′ UTR or promoter bound (FOXC1 KD conditions) exhibited an intermediate pattern. There was a significant relative decrease in ChIP signal at group A peaks for RUNX1  and HDAC1 , and a significant increase in CEBPA  and H3K27Ac  ChIP signal (−50) and HDAC1 , and a significant decrease in CEBPA  and H3K27Ac  ChIP signal . Rather, there was enrichment for several short ungapped GC-rich motifs identified by Discriminative Regular Expression Motif Elicitation (DREME), as well as for SP1, a member of the SP1/Kruppel-like family of transcription factors. Interestingly, using ChIP PCR at three exemplar group C promoters of genes whose expression was downregulated, in addition to an increase in RUNX1 ChIP signal, we observed an increase in KLF2 ChIP signal and a concomitant decrease in SP1 ChIP signal   and group 2 (RUNX1down CEBPAdown) FR-20 enhancers  I and 7J.nhancers K; indeednhancers L. In theP signal M and 7N,P signal C. As forKD cells D, and ceKD cells E. Thus, lls  recruits a multitude of co-repressors to its binding sites, whereas somatic mutations of RUNX1, which often target sequences coding for the Runt Homology Domain, are inactivating or confer dominant-negative activity .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Tim Somervaille , HL60 (F) and MonoMac-1 (M), MV (M), Fujioka (M), HEK293 (F), K562 (F) and Kasumi-1 (M). The sex of the cell lines is indicated by either M  or F . Leukaemia cell lines were cultured in 90% RPMI, 10% FBS and 2mM glutamine. HEK293 cells were cultured in 90% DMEM, 10% FBS and 2mM glutamine. Cell line identity was verified where possible by STR analysis.Use of human tissue was in compliance with the ethical and legal framework of the UK’s Human Tissue Act, 2004. Primary human AML samples were from Manchester Cancer Research Centre’s Tissue Biobank and used with the informed consent of donors. The Biobank holds a generic ethics approval (18/NW/0092) which can be conferred to users of banked samples via the MCRC Biobank Access Policy. Samples used in this project were approved for use under application number 08_TISO-02. Details of primary samples used are in 3 cells/ml in methylcellulose medium . Clonogenic assays of primary AML cells were performed in the same methylcellulose medium with addition of IL3, G-CSF and TPO  with puromycin where appropriate (3 μg/ml), as described  . For cloescribed . CytospiFor western blotting (WB), all antibodies were used at a dilution of 1:1000 except anti-Myc tag (1:2000) and anti-ACTB . An anti-RUNX1 antibody from Abcam was used for RUNX1 ChIP-seq, ChIP-qPCR and immunoprecipitation experiments, while an anti-RUNX1 antibody from Cell Signaling was used for WB.To generate a bacterial expression vector for FOXC1, FOXC1 cDNA (NM_001453) was excised from pcDNA3.1-FOXC1 (a gift from Jane Sowden) using EcoR1 and Xho1 restriction sites and sub-cloned into the EcoR1/Xho1 sites of pET-28 (a gift from Iain Hagan). To generate the FOXC1 mutant FOXC1 Δ(69-178), site direct mutagenesis was performed using overlap extension PCR followed by Dpn1 digestion with pET-28-FOXC1 as a template. The vector was then transformed into Rosetta BL21 (DE3) Competent Cells and spread on agar plates containing kanamycin and chloramphenicol, and incubated overnight at 37°C. Cells from a single colony were grown up overnight, diluted to an OD (600nm) of 0.05 and then grown on until the OD reached 0.4. One liter of cells was then supplemented with 1mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 37°C for three hours. Cells were harvested and suspended in bacterial lysis buffer and then subjected to a freeze-thaw cycle. The following day, the sample was defrosted at 37°C for 30 minutes and then supplemented with 4mg of lysozyme and incubated again at 37°C for 30 minutes. Following this second incubation, the sample was supplemented with 10 μg/ml DNase1 and left at room temperature for 30 minutes. Next, 1ml of 10% sodium deoxycholate and 1% Triton were added to the tube and the sample was incubated on ice for 15 minutes. Finally, the lysate was centrifuged at 10000rpm for 15 minutes and the inclusion bodies (pellet) was suspended in 1.5ml PBS supplemented with 10x NuPAGE sample reducing agent and 4x NuPAGE lithium dodecyl sulfate (LDS) sample loading buffer. Recovered proteins were subjected to acrylamide gel electrophoresis and Coomassie gel staining, and the protein band corresponding to FOXC1 was excised. The excised band was then placed into a dialysis membrane and protein eluted overnight at 100mA. The purified protein solution was then transferred into a new dialysis membrane overnight in order to eliminate SDS. Following dialysis, the protein concentration was calculated using a Pierce BCA Protein Assay Kit and 2μg FOXC1 protein was then provided for goat immunisation at Eurogentec  using the Speedy 28-Day program of immunisation.Antibodies were purified from the resulting goat serum using an AminoLink Plus Immobilization Kit according to manufacturer’s instructions. After column elution, the purified antibody was dialysed in PBS, supplemented with an equal volume of glycerol 100% and 0.05% of sodium azide, and stored at −80°C.Flow cytometry analyses were performed using a LSR Model II flow cytometer . Antibodies used for flow cytometry were all used at a dilution of 1/200. Apoptosis was assessed using a BD PharMingen APC Annexin V kit. Propidium iodide cell cycle analyses were performed as described . Through6 cells in 50 μL lysis buffer. Samples were then centrifuged at 20,000xg at 4°C for 15 minutes to pellet cell debris, and the supernatant was then collected. Lysates were stored at −80°C. Equal amounts of protein were loaded and separated by SDS-PAGE.Cells to be lysed were first counted, pelleted by centrifugation and resuspended twice in ice cold PBS in order to wash away media and any debris from cell culture. Cells were lysed in ice-cold high salt lysis buffer (45mM HEPES (pH 7.5), 400mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP40, 6.25mM NaF, 20mM β-glycerophosphate, 1mM DTT, 20mM sodium butyrate and 1x Protease Inhibitor cocktail ), typically at concentrations of 1x10Immunoprecipitation (IP) experiments were performed using lysate generated from Fujioka AML cells and 293 cells. Cells were washed twice with ice-cold PBS and lysed in 1mL (for 10 million cells) of ice-cold TNN buffer (50mM Tris-Cl (pH7.5), 100mM NaCl, 5mM EDTA, 0.5% Nonidet p40) supplemented with 6.25mM NaF, 20mM β-glycerophosphate, 1mM DTT and 1 μL Benzonase® nuclease (Sigma Aldrich), by rotation at 40rpm at 4°C for 15 minutes. Samples were then centrifuged at 20,000xg at 4°C for 15 minutes to pellet cell debris, and 10 μL of the supernatant per sample was taken for input control with the rest being used for IP. For the IP, Protein G Sepharose® Fast Flow beads  were washed three times in TNN buffer before being resuspended in 500 μL TNN buffer with the appropriate antibody or isotype control. Antibodies were used at 10 μg per 100 million cells.The beads were incubated with antibodies overnight at 4°C with constant rotation. Following this incubation, beads were centrifuged at 1700xg at 4°C for 1 minute, combined with the prepared lysates and rotated at 40rpm at 4°C overnight. The following day the antibody-bound beads were centrifuged (1700xg at 4°C for 1 minute) and washed four times in 1mL TNN buffer before being resuspended in 20 μL elution buffer . Proteins bound to the beads were eluted by heating the samples for 10 minutes at 70°C and the beads were subsequently removed by centrifuging the sample through a 0.45 μm Spin-X® centrifuge tube filter within a 2mL DNase/RNase-free polypropylene tube . Immunoprecipitated and co-immunoprecipitated proteins were assayed by western blotting.2O containing 10x NuPAGE® sample reducing agent and 4xNuPAGE® lithium dodecyl sulfate (LDS) sample loading buffer (both from Life Technologies). Samples were then incubated at 95°C for 10 minutes in order to ensure complete unfolding of the protein secondary structure. Lysates were then loaded into pre-cast NuPAGE® 4%–12% Bis-Tris acrylamide gels in a gel tank filled with 1x MOPS® running buffer  to ensure electric conduction. Gels, tanks and MOPS® running buffer were all from Life Technologies. For molecular weight estimation, 5 μL of PageRuler Plus prestained protein ladder (ThermoFisher) was run together with the samples. Empty wells were filled with 4xNuPAGE® LDS loading buffer diluted in ddH2O to ensure an even run of the samples. Gels were electrophoresed for at 150 V for approximately 1 hour.For western blotting, proteins were separated by SDS-PAGE. Equal amounts of lysate were diluted in ddHhttps://www.ge.com). Transfer was performed at 4°C at 70 V for 1 hour 15 minutes in a semi-dry transfer tank (Bio-Rad) filled with transfer buffer. Transfer buffer was prepared by diluting 50mL of transfer buffer 10x solution (30 g Tris and 143 g glycine made up to 1L with deionised water) and 100mL of methanol (Fisher Scientific) with deionised water to a final volume of 500 mL. Following completion of transfer the nitrocellulose membrane was stained with Ponceau Red (Sigma Aldrich) to confirm equal loading of the samples and successful transfer to the nitrocellulose membrane.Following electrophoresis, the pre-cast gel was transferred to a nitrocellulose membrane , prior to blocking with 5% skimmed milk in 1x PBS-Tween for 30 minutes at room temperature. Residual milk was washed away with 1x PBS- Tween and primary antibody incubation was performed on rollers at 4°C overnight. Primary and secondary antibodies were diluted in 5% BSA (Sigma Aldrich) and 2% Western Blocking reagent (Roche) in 1x PBS-Tween. After 3x 10 minutes washes with 1x PBS-Tween, membranes were incubated with secondary Horseradish peroxidase (HRP)-linked secondary antibodies  on rollers for 1 hour at room temperature. After 3x 10 minutes washes with 1x PBS-Tween, membranes were incubated with either ECL  or Supersignal  and the signal generated by the HRP-conjugated immune complexes was exposed using a high performance chemiluminescence film (AmershamTM Hyperfilm - https://www.ge.com) and an X-ray cassette and detected using a Curix 60 film processor  in a dark room.Following Ponceau Red staining, membranes were rinsed with tap water and cut with a sterile scalpel to isolate proteins of the appropriate molecular weight for subsequent staining. Ponceau Red was washed away with 1x PBS-Tween  were grown in RPMI with 10% FBS. The medium was then removed and replaced with PBS containing 2mM di(N-succinimidyl) glutarate (DSG) and crosslinked for 30 minutes. PBS and DSG were then removed and cells were washed twice in PBS. Cell were then further crosslinked in PBS containing 1% formaldehyde for 10 minutes. Crosslinking was quenched by adding glycine to a final concentration of 0.125M. Cells were then washed with ice-cold PBS. The nuclear fraction was extracted by first suspending the pellet in 10ml of LB1 buffer  for 10 min at 4°C. Cells were pelleted and suspended in 10ml of LB2 buffer  and mixed at 4°C for 5 minutes. Cells were pelleted and suspended in 300ml of LB3 buffer  and sonicated using a Bioruptor Pico  for 8 cycles, with 30 s ON, 30 s OFF settings. Triton X-100 was added at 10% concentration and lysate was centrifuged for 10 minutes at 20,000rcf. to purify the debris. The supernatant was then incubated with Dynabeads (Protein G) pre-bound with 10 μg antibody and IP was conducted overnight at 4°C. The beads were washed ten times in 1ml of RIPA buffer and twice in 100mM ammonium hydrogen carbonate (AMBIC) solution. For the second AMBIC wash, beads were transferred to new tubes. RIME samples were prepared and analyzed by mass spectrometry as described (For Rapid Immunoprecipitation Mass spectrometry of Endogenous protein (RIME), Fujioka cells , FOXC1 KD3 pLKO.2 (TRCN0000235693) and a lentiviral vector for expression of FOXC1 cDNA resistant to KD) were from Somerville at al., 2015.To generate lentiviral KD constructs, pLKO.1 puro was digested with Age1 and EcoR1 and ligated with HPLC purified oligonucleotides previously annealed by incubating at 98°C for 5 minutes and slowly cooling to room temperature.To generate doxycycline-inducible Fujioka cells, a lentiviral plasmid expressing the rtTA protein under the control of the EF1α promoter was generated by cloning the rtTA-IRES-neomycin expression cassette from pRetroX-Tet-On Advanced into the BamH1/Sal1 sites of pLentiGS . FujiokaTo generate the tetracycline inducible FOXC1 lentiviral expression construct, human FOXC1 cDNA was PCR amplified from pcDNA3.1-FOXC1 (a gift from Jane Sowden) using oligonucleotides which introduced coding sequences for a C-terminal GSG linker and Myc tag. Oligonucleotide sequences are shown in To generate FOXC1 mutants FOXC1 Δ(1-50) and FOXC1 Δ(436-553), FOXC1 cDNA was amplified from pLentiGS-minCMV-TET-blasticidin vector-FOXC1-Myc using the oligonucleotide sequences shown in To generate FOXC1 mutants FOXC1 Δ(69-178), FOXC1 Δ(215-366), FOXC1 G165R and FOXC1 F112S, site direct mutagenesis was performed using overlap extension PCR followed by Dpn1 digestion with pLentiGS-minCMV-TET-blasticidin vector-FOXC1-Myc as a template. Oligonucleotide primer sequences are shown in RUNX1c was then PCR amplified using the oligonucleotides shown in To generate a doxycycline-inducible FOXC1-DBD RUNX1c lentiviral fusion construct, the DNA binding domain (DBD) of FOXC1 was PCR amplified using the following oligonucleotide primers and full length FOXC1 as template. Oligonucleotide primer sequences are shown in To generate tetracycline inducible KLF2 lentiviral expression construct, human KLF2 cDNA was PCR amplified from Fujioka cells cDNA using the oligonucleotides shown in To generate RUNX1b mutants, pcDNA3.1_Runx1b was created by cloning a PCR amplified FLAG-tagged (DYKDDDDK) murine proximal Runx1 isoform (Runx1b) cDNA  into mam6 cells per dish in 9mL DMEM with 10% FBS (D10). Next day cells were typically at ∼90% confluence. For the transfection, 21 μg polyethylenimine (PEI) was diluted in 500 μL serum-free DMEM at room temperature for each 10cm dish. For the manufacture of lentiviral particles, plasmids containing viral structural genes were combined with lentiviral expression plasmids and diluted as follows: lentiviral vector - 4 μg, pCMVD8.91 - 2 μg, pMDG.2 - 1 μg, serum-free DMEM to 500 μl. Equal volumes of the diluted PEI and plasmid constructs were then combined, mixed by pipetting and left to incubate for 20-30 minutes at room temperature to allow formation of DNA-PEI complexes. The mixture was then added dropwise to 293FT cells. Next day the medium was replaced with 10mL of fresh, pre-warmed D10 medium per dish prior to further overnight incubation and subsequent harvest of viral particle-containing supernatants. All viral supernatants were filtered through a 0.45 μm polyethersulfone filter prior to use. Lentiviral supernatants were either used immediately or stored long-term at −80°C.Lentiviral supernatants were prepared and leukemic human cells were infected with viral particles as previously described . Briefly6 cells or 1.5x106 cells respectively were resuspended in 6ml viral supernatant and centrifuged for 30 minutes at 900xg and 37°C. After centrifugation cell line cells were incubated at standard conditions overnight. The following morning, cells were pelleted and resuspended in 10ml of R10 to reduce Polybrene toxicity. For human cell lines infected with lentiviral shRNA constructs, 24 hours following spinoculation 3 μg/mL puromycin (Sigma-Aldrich) was added for 48 hours to select for successfully transduced cells prior to further manipulation. For primary AML cells, after spinoculation, cells were incubated overnight in AML culture medium with cytokines, without stromal support. The following morning, cells were spinoculated a second time as above. Next day 3 μg/mL puromycin was added for 72 hours to select for successfully transduced cells prior to further manipulation.To increase transduction efficiency of target cells Polybrene was added to all viral supernatants to a final concentration of 8 μg/mL. For lentiviral infection of primary human cells or human cell lines 0.5-1x105 cells or less) or Mini kit (for greater than 5x105 cells) and QIAshredder spin columns. Cells were washed twice in PBS and lysed by vortexing in 350 μL of RLT lysis buffer supplemented with 1% β-mercaptoethanol. Cell lysate was subsequently passed through a QIAshredder spin column for homogenization and homogenized lysates were then passed through a gDNA eliminator spin column to remove genomic DNA contamination. 350 μL of 70% ethanol was added prior to loading the sample onto a MinElute spin column. Following several washes of the column and a 5 minute high speed spin to remove residual ethanol from the column, RNA bound to the column was eluted with RNase-free water. RNA yield was quantified through spectrophotometric analysis using a Nanodrop.RNA was extracted and quantitative PCR performed as described . BrieflyFor reverse transcription, between 1 μg and 100ng of extracted RNA from each cell population was diluted in 10 μL of nuclease-free water and with 10 μL of a reverse transcriptase “mastermix” (High Capacity Reverse Transcription kit) according to the manufacturer’s instructions. The cDNA generated was diluted with nuclease-free water to an appropriate concentration .ACTB) as a housekeeping gene. Primers were designed using the Universal Probe Library Assay Design Center and purchased from Integrated DNA Technologies . For some assays 20xTaqMan primer/probe assays (Life Technologies) were used, as shown in ACTB.qPCR reactions were performed in MicroAmp optical 384-well reaction plates and analyzed using QuantStudio Real Time PCR system. Reactions were performed in triplicate and included primers for β-Actin (http://crispor.tefor.net/). Potential restriction sites were identified in high scoring pairs at alternate ends of the enhancer region. Guides were produced as chemically modified single guide RNA by Synthego. The resulting guides are shown in Guides for KLF2 enhancer  were designed using the CRISPOR tool . Library quality was checked using the Agilent Bioanalyzer. Libraries were quantified by qPCR using the KAPA Library Quantification Kit for Illumina. 1.8 pM pooled libraries were loaded onto the NextSeq 500 and 2x75bp sequencing was carried out using a NextSeq 500/550 High Output v2 kit. Reads were aligned to the human genome (GRCh38 and gene annotated with its corresponding GTF files (GENCODE GRCh38) using STAR version 2.4.2a with the settings–outFilterMultimapNmax 20,–outFilterType BySJout,–alignSJoverhangMin 8,–quantMode GeneCounts . DESeq2 FOXC1 for KD, or a non-targeting control. Next day, cells were drug selected with puromycin 3 μg/mL and incubated for three days. Cells were counted and cross-linked using 1% formaldehyde for 10 minutes , or double cross-linked  with ChIP Cross-link Gold for 30 minutes in PBS with 1mM MgCl2 and then with 1% formaldehyde for 10 minutes. The reaction was stopped by incubation for five minutes with 0.125M glycine. Cell pellets were washed twice with cold PBS containing protease inhibitors (Complete EDTA-free tablets). 108 cells were used per ChIP, as per Fujioka cells were infected with lentiviral particles targeting After washing six times with RIPA buffer , chromatin IP-bound fractions were extracted at 65°C for 30 minutes with elution buffer  vortexing frequently. RNaseA (0.2mg/ml) and proteinase K (0.2 μg/ml) were used to eliminate any RNA or protein from the samples. Finally DNA was extracted using phenol:chloroform:isoamyl alcohol extraction and precipitated with ethanol  and 200mM NaCl) for at least one hour at −80°C. Pellets were washed with 70% ethanol and eluted in 50μl 10mM TrisHCl pH8.0.http://mitra.stanford.edu/kundaje)). Peaks were called with Model-based Analysis of ChIP-Seq version 2 (MACS2) using the following parameters -f BAMPE,–keep-dup 5 to keep only paired-end reads with up to 5 duplicates  using BWA-MEM version 0.7.15 . Reads wplicates . Annotatplicates .For ChIP quantitative PCR, assays were performed in 384-well MicroAmp optical reaction plates using Taqman Fast Universal PCR Mastermix and Universal Probe Library System designed primers and probes. Signal was detected using an ABI PRISM 7900HT Sequence Detection System. Primers and probes used are shown in FOXC1 for KD, or a non-targeting control. Next day, cells were drug selected with puromycin 3 μg/mL and incubated for three days. Next the Assay for Transposase Accessible Chromatin (ATACseq) protocol  all CEBPA peaks in control Fujioka cells , (ii) all RUNX1 peaks in control Fujioka cells , (iii) all SPI1 peaks in control Fujioka cells , (iv) all FOXC1 peaks in control Fujioka cells , (v) all RUNX1 peaks in FOXC1 KD Fujioka cells  and (vi) the coordinates of all MYB binding peaks in THP1 AML cells   . For hisStatistical analyses were performed using Microsoft Excel 2007 or StatsDirect software (v.1.9.7). Details of the statistical tests used for each analysis shown may be found in the figure legends.