Journal: BMC Systems Biology

Article Title: System modeling reveals the molecular mechanisms of HSC cell cycle alteration mediated by Maff and Egr3 under leukemia

doi: 10.1186/s12918-017-0467-4

Figure Lengend Snippet: Differential expression profiles of Maff and Egr3 during leukemia progression. a According to microarray data, Maff and Egr3 are significantly highly expressed under leukemia. Their expressions reach the highest levels at the late stage (≥ Day 14) of leukemia, which are shown herein. The x- and y- axes are the log 2 values of gene expression intensities in the microarray (leukemia and control, respectively); and the two red dashed lines (parallel to the diagonal) represent expression levels that are of 2-fold changes comparing to control, which serve as conventional indicators for significant changes in gene expression. b Relative expression levels of the two genes measured by qRT-PCR are shown. The data validate the high expressions of the two genes in HSCs under leukemia

Article Snippet: The Maff and Egr3 cDNAs were purchased from Origene and HSCs (CD45.1 + LKS + ) were transfected with retroviruses containing plasmids constructed with the Maff and Egr3 cDNAs or an empty segment (control).

Techniques: Quantitative Proteomics, Microarray, Gene Expression, Control, Expressing, Quantitative RT-PCR

Journal: BMC Systems Biology

Article Title: System modeling reveals the molecular mechanisms of HSC cell cycle alteration mediated by Maff and Egr3 under leukemia

doi: 10.1186/s12918-017-0467-4

Figure Lengend Snippet: Simulations of system dynamics with respect to Egr3 expression. a - c Stable equilibriums (steady states) of Cyc D*, Cyc E* and E2F with respect to the expression level of Egr3 are shown in ( a ), ( b ) and ( c ) respectively. Similar to Fig. , data are steady state concentrations under various genetic synthesis rates ( k s _ egr3 , i.e. expression level) of Egr3 . Stable equilibriums of all the three cell cycle checkpoint-determining molecules have shifted downward as k s _ egr3 increases, especially for Cyc E* b and E2F c as their steady states decline sharply in the manner of bistability (i.e. low-level stable equilibriums occur due to dynamical bifurcation; red lines ). The steady states of Cyc D* exhibit very slight upward fluctuations at certain Egr3 expression levels because the decreases in E2F at those k s _ egr3 values result in less amounts of p18 and p19 (inhibitors of Cyc D*), thus causing the transient elevation. However, the Cyc D* level rapidly declines as k s _ egr3 further increases. Noteworthy, when k s _ egr3 exceeds certain values, the three molecules are all suppressed to very low levels, indicating that high Egr3 expression potently suppresses cell cycle. d Proliferation of HSCs over-expressing (i.e. transducted with) Egr3 ; similar to Fig. , “Vector” stands for control data. As shown, the proliferation capability of HSCs highly expressing Egr3 is significantly lowered ( p -value <0.005). e Ratio of HSCs staying in G0 phase before (control) and after Egr3 over-expression. Data show that the ratio of G0 HSCs is significently heightened when Egr3 is highly expressed ( p -value <0.05), indicating that high Egr3 expression arrests HSCs in the G0 phase, reducing the number of HSCs entering the later phase(s) of cell cycle

Article Snippet: The Maff and Egr3 cDNAs were purchased from Origene and HSCs (CD45.1 + LKS + ) were transfected with retroviruses containing plasmids constructed with the Maff and Egr3 cDNAs or an empty segment (control).

Techniques: Expressing, Plasmid Preparation, Control, Over Expression

Journal: BMC Systems Biology

Article Title: System modeling reveals the molecular mechanisms of HSC cell cycle alteration mediated by Maff and Egr3 under leukemia

doi: 10.1186/s12918-017-0467-4

Figure Lengend Snippet: Molecular regulatory system of cell cycle (G0 → G1/G1 → S). Since the HSCs in which Maff and Egr3 are highly expressed (i.e. functional) are mainly G0-phase cells (over 90%), it implicates that if Maff and Egr3 have influence(s) on cell cycle, they must exert influence(s) in G0 → G1/G1 → S in the first place. Therefore, as a preliminary attempt, we summarized all key regulators in G0 → G1/G1 → S (including two cyclins, five CKIs, Rb, E2F and Akt) and examined all possible ways that Maff and Egr3 (highlighted in red) act on the molecular network that result in consistent phenomena with experimental observations. Symbols: “−⊣” - inhibition; “―→” - activation; solid lines - transcriptional regulations; dashed lines - biochemical interactions; red lines : computionally inferred molecular regulatory relationships mediated by Maff and Egr3

Article Snippet: The Maff and Egr3 cDNAs were purchased from Origene and HSCs (CD45.1 + LKS + ) were transfected with retroviruses containing plasmids constructed with the Maff and Egr3 cDNAs or an empty segment (control).

Techniques: Functional Assay, Inhibition, Activation Assay

Journal: BMC Systems Biology

Article Title: System modeling reveals the molecular mechanisms of HSC cell cycle alteration mediated by Maff and Egr3 under leukemia

doi: 10.1186/s12918-017-0467-4

Figure Lengend Snippet: Dynamic simulations of Maff and Egr3 -mediated alterations of cell cycle. a - c Steady states of Cyc D* ( a ), Cyc E* ( b ) and E2F ( c ) with respect to k s _maff under medium Egr3 expression level (low < k s _ egr3 < high) are shown herein. In this circumstance, higher Maff expression is able to uplift the levels of Cyc D*, Cyc E* and E2F, resulting in bistability in their steady states ( red lines ). Dynamics of the three molecules under low Egr3 expression level (with respect to k s _maff ) are equivalent to Fig. , in which high expression of Egr3 is not presumed ( k s _ egr3 = low). d - f Steady states of Cyc D* ( d ), Cyc E* ( e ) and E2F ( f ) with respect to k s _maff under high Egr3 expression level ( k s _ egr3 = high). In this circumstance, increase in Maff expression ( k s _maff ) is unable to effectively uplift the steady states of Cyc D*, Cyc E* and E2F any more. g - i Steady states of Cyc D* ( g ), Cyc E* ( h ) and E2F ( i ) with respect to k s _egr3 when Maff expression level is high ( k s _maff = high). The situation is similar to those of Fig. - c ( k s _maff = low), in which high Egr3 expression potently suppresses the steady-state levels of all three molecules. The results indicate that no matter Maff expression is high or low, cell cycle is suppressed when Egr3 is highly expressed

Article Snippet: The Maff and Egr3 cDNAs were purchased from Origene and HSCs (CD45.1 + LKS + ) were transfected with retroviruses containing plasmids constructed with the Maff and Egr3 cDNAs or an empty segment (control).

Techniques: Expressing

Journal: BMC Systems Biology

Article Title: System modeling reveals the molecular mechanisms of HSC cell cycle alteration mediated by Maff and Egr3 under leukemia

doi: 10.1186/s12918-017-0467-4

Figure Lengend Snippet: Validation of the molecular interactions by sequence analysis. a According to our survey of gene sequences, p18 does not harbor the direct recognition element (i.e. binding motif) of Maff in its transcription promoter region; therefore, we check if there is any intermediate that both contains that motif (in the promoter region) and can negatively regulate p18 . We have found that Blimp1 / Prdm1 , which is also a transcription factor in the regulation of hematopoietic differentiation, harbors the Maff binding motif in its promoter region; and the motif is 156 bp upstream the transcription start site (TSS). Meanwhile, Blimp1 / Prdm1 can transcriptionally repress p18 in regulation of the cycling of hematopoietic cells. Thus the transcription regulatory relation “ Maff ― → Blimp1 − ⊣ p18 ” is indicated, and our computational inference “ Maff − ⊣ p18 ” is well supported. b Our survey has also identified that Anapc11 , which is a core coding gene of the anaphase-promoting complex/cyclosome (APC/C) that inhibits mitotic cyclins and greatly suppresses G0 → G1/G1 → S, links to the cis element that Egr3 recognizes and exerts transcription activation (523 bp upstream the TSS). Therefore, it implies that “ Egr3 ― → Anapc11 ” and “Anapc11 −⊣ Cyclin D/Cdk4/6, Cyclin E/Cdk2”; thus our computational inference that “ Egr3 − ⊣ CyclinD:Cdk4/6, CyclinE:Cdk2” is supported

Article Snippet: The Maff and Egr3 cDNAs were purchased from Origene and HSCs (CD45.1 + LKS + ) were transfected with retroviruses containing plasmids constructed with the Maff and Egr3 cDNAs or an empty segment (control).

Techniques: Biomarker Discovery, Sequencing, Binding Assay, Activation Assay

Journal: BMC Systems Biology

Article Title: System modeling reveals the molecular mechanisms of HSC cell cycle alteration mediated by Maff and Egr3 under leukemia

doi: 10.1186/s12918-017-0467-4

Figure Lengend Snippet: Molecular regulations of cell cycle and hematopoiesis. GATA-1 ( Gata-1 ) activates the cell cycle components as well as p45 NF-E2, the heterodimer partner of Maff. The Maff:p45 NF-E2 functional complex is self-regulated and it activates the transcriptions of various downstream regulators, e.g. Blimp-1 ( Blimp1 ), β1-tubulin ( Tubb1 ), and PF-4 ( Pf4 ). Altogether with our prediction that “ Maff − ⊣ p18 ”, it can be concluded that high expression of Maff eliminates the feedback inhibtion of p18 to Cyclin D:Cdk4/6, as well as up-regulating the downstream TFs. Hence, cell cycle will be accelerated and proliferation/differentiation of hematopoietic cells are enhanced. This is why we hypothesize that high expression of Maff adapts cells to a rapidly proliferative status, i.e. cancerization. On the other side, Egr3 suppresses the checkpoint controllers of G0 → G1/G1 → S and activates CKIs, thus its high expression greatly inhibits cell cycle. Thus it is fairly assumed that it may be a “harsh control” via which HSCs forcibly shut down functionalities when their behaviors exceed the limits of cellularity controlling principles, i.e. self-protection

Article Snippet: The Maff and Egr3 cDNAs were purchased from Origene and HSCs (CD45.1 + LKS + ) were transfected with retroviruses containing plasmids constructed with the Maff and Egr3 cDNAs or an empty segment (control).

Techniques: Functional Assay, Expressing, Control

Journal: The Journal of Neuroscience

Article Title: Opposing Role for Egr3 in Nucleus Accumbens Cell Subtypes in Cocaine Action

doi: 10.1523/JNEUROSCI.0548-15.2015

Figure Lengend Snippet: Egr3 overexpression in MSN subtypes alters cocaine CPP and cocaine-induced locomotor activity. A, Cocaine (7.5 mg/kg) CPP is enhanced with D1-Cre mice that received AAV-DIO-Egr3-EYFP into NAc, whereas cocaine CPP is decreased in D2-Cre mice that received AAV-DIO-Egr3-EYFP into NAc compared with AAV-DIO-EYFP controls. B, Repeated cocaine (10 mg/kg)-induced locomotor activity is increased in D1-Cre mice (days 2–5), whereas it is reduced in D2-Cre mice (day 2–3) expressing AAV-DIO-Egr3-EYFP in NAc compared with controls expressing AAV-DIO-EYFP. Error bars indicate SEM.

Article Snippet: Egr3 shRNA expression vectors were purchased from Origene with four possible sets of Egr3 shRNA and a scrambled sequence cloned into HuSH pGFP-V-RS plasmid vector driven under human U6 promoter (see A ).

Techniques: Over Expression, Activity Assay, Expressing

Journal: The Journal of Neuroscience

Article Title: Opposing Role for Egr3 in Nucleus Accumbens Cell Subtypes in Cocaine Action

doi: 10.1523/JNEUROSCI.0548-15.2015

Figure Lengend Snippet: Egr3 expression after repeated cocaine exposure. A, qRT-PCR and Western blot analysis of Egr3 demonstrate that mRNA and protein levels are decreased in NAc after repeated cocaine exposure (7 d, 20 mg/kg) followed by 24 h of withdrawal in mice. B, Rats that self-administer cocaine display a similar reduction of Egr3 mRNA and protein. All values were normalized to GAPDH. Error bars indicate SEM.

Article Snippet: Egr3 shRNA expression vectors were purchased from Origene with four possible sets of Egr3 shRNA and a scrambled sequence cloned into HuSH pGFP-V-RS plasmid vector driven under human U6 promoter (see A ).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: The Journal of Neuroscience

Article Title: Opposing Role for Egr3 in Nucleus Accumbens Cell Subtypes in Cocaine Action

doi: 10.1523/JNEUROSCI.0548-15.2015

Figure Lengend Snippet: D1-Cre-RT and D2-Cre-RT validation and Egr3 ribosome-associated mRNA levels in MSN subtypes after cocaine exposure. A, Illustration of HA-tagged Rpl22 in D1-MSNs or D2-MSNs by crossing RT mice to D1-Cre or D2-Cre mice. Polyribosomes are immunoprecipitated from D1-MSNs or D2-MSNs with HA-tagged magnetic beads and then cell-specific ribosome-associated mRNA is isolated. B, C, MSN subtypes display enrichment of ribosome-associated mRNA for known MSN subtype genes. D, qRT-PCR analysis of Egr3 ribosome-associated mRNA in D1-Cre-RT and D2-Cre-RT NAc tissue after repeated cocaine (7 d, 20 mg/kg) or saline exposure. Repeated cocaine exposure induces Egr3 ribosome-associated mRNA in D1-MSNs while decreasing Egr3 in D2-MSNs. Error bars indicate SEM.

Article Snippet: Egr3 shRNA expression vectors were purchased from Origene with four possible sets of Egr3 shRNA and a scrambled sequence cloned into HuSH pGFP-V-RS plasmid vector driven under human U6 promoter (see A ).

Techniques: Biomarker Discovery, Immunoprecipitation, Magnetic Beads, Isolation, Quantitative RT-PCR, Saline

Journal: The Journal of Neuroscience

Article Title: Opposing Role for Egr3 in Nucleus Accumbens Cell Subtypes in Cocaine Action

doi: 10.1523/JNEUROSCI.0548-15.2015

Figure Lengend Snippet: Cre-inducible AAV overexpression of Egr3 in D1-MSN and D2-MSN subtypes. A, Schematic of the double-floxed, inverted, open reading frame Cre-dependent AAV vector expressing Egr3-EYFP. B, Image of a D2-Cre NAc demonstrating expression of Egr3-EYFP in NAc but no expression is visible in wild-type (WT) mice. Scale bar, 100 μm. C, D, AAV-DIO-Egr3-EYP overexpression D1-Cre and D2-Cre NAc results in the upregulation of Egr3 mRNA (C) and expression of 70 kDa Egr3-EYFP protein in NAc (D) compared with mice receiving the AAV-DIO-EYFP control virus. Error bars indicate SEM.

Article Snippet: Egr3 shRNA expression vectors were purchased from Origene with four possible sets of Egr3 shRNA and a scrambled sequence cloned into HuSH pGFP-V-RS plasmid vector driven under human U6 promoter (see A ).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Virus

Journal: The Journal of Neuroscience

Article Title: Opposing Role for Egr3 in Nucleus Accumbens Cell Subtypes in Cocaine Action

doi: 10.1523/JNEUROSCI.0548-15.2015

Figure Lengend Snippet: Cre-inducible AAV miRNA knock-down of Egr3 in D1-MSN and D2-MSN subtypes. A, Mouse Egr3 shRNA sequences and their targeting of the Egr3 gene. The position is relative to the ATG start site of Egr3 cDNA. B, qRT-PCR analysis of Egr3 demonstrates Egr3shRNA knock-down in Neuro2a cells. C, Illustration of the miRNA engineering sequence. The first 21 bp of the Egr3 target sequence (red) of shRNA is used for miRNA engineering. D, Schematic of the double-floxed, inverted, open reading frame Cre-dependent AAV vector expressing Egr3miR and mCitrine. E, Image of a D2-Cre NAc demonstrating expression of mCitrine in NAc, but no visible expression is observed in wild-type (WT) NAc. Scale bar, 100 μm. F, G, D1-Cre and D2-Cre mice expressing AAV-DIO-Egr3miR-mCitrine in NAc display reduction of Egr3 mRNA (F) and protein (G) compared with AAV-DIO-Scramble mCitrine controls. Error bars indicate SEM.

Article Snippet: Egr3 shRNA expression vectors were purchased from Origene with four possible sets of Egr3 shRNA and a scrambled sequence cloned into HuSH pGFP-V-RS plasmid vector driven under human U6 promoter (see A ).

Techniques: Knockdown, shRNA, Quantitative RT-PCR, Sequencing, Plasmid Preparation, Expressing

Journal: The Journal of Neuroscience

Article Title: Opposing Role for Egr3 in Nucleus Accumbens Cell Subtypes in Cocaine Action

doi: 10.1523/JNEUROSCI.0548-15.2015

Figure Lengend Snippet: Egr3 transcriptional regulation of cocaine-associated genes after repeated cocaine exposure. ChIP using an Egr3 antibody to immunoprecipitate Egr3 bound to promoters of cocaine-associated genes after 7 d of cocaine (20 mg/kg) or saline exposure followed by 24 h withdrawal. Egr3 binding to the Camk2α, CREB, FosB, Nr4a2, and Sirt1 promoters is increased, whereas binding to the Dnmt3a and G9a promoters is decreased in the cocaine group. Error bars indicate SEM.

Article Snippet: Egr3 shRNA expression vectors were purchased from Origene with four possible sets of Egr3 shRNA and a scrambled sequence cloned into HuSH pGFP-V-RS plasmid vector driven under human U6 promoter (see A ).

Techniques: Saline, Binding Assay

Journal: The Journal of Neuroscience

Article Title: Opposing Role for Egr3 in Nucleus Accumbens Cell Subtypes in Cocaine Action

doi: 10.1523/JNEUROSCI.0548-15.2015

Figure Lengend Snippet: Egr3-regulated genes are differentially expressed in MSN subtypes after repeated cocaine exposure. Cell-type-specific ribosome-associated mRNA analysis from D1-Cre-RT and D2-Cre-RT NAc mice after repeated cocaine (7 d, 20 mg/kg) exposure followed by 24 h withdrawal demonstrates altered induction of Egr3-regulated genes. Ribosome-associated mRNA for Camk2α and CREB is increased in D1-MSNs, whereas it is decreased in D2-MSNs in the cocaine group. FosB, Nr4a2, and Sirt1 are increased in D1-MSNs, with no change in D2-MSNs in the cocaine group. Dnmt3a and G9a are decreased in D1-MSNs, with no change in D2-MSNs in the cocaine group. Error bars indicate SEM.

Article Snippet: Egr3 shRNA expression vectors were purchased from Origene with four possible sets of Egr3 shRNA and a scrambled sequence cloned into HuSH pGFP-V-RS plasmid vector driven under human U6 promoter (see A ).

Techniques: