Journal: Journal of Cell Science

Article Title: Paxillin-dependent regulation of IGF2 and H19 gene cluster expression

doi: 10.1242/jcs.170985

Figure Lengend Snippet: Paxillin affects the expression of H19 and regulates cell proliferation in HepG2 cell line. (A) Quantitative PCR analysis showing that paxillin depletion by shRNA (shPXN) results in upregulation of H19 compared to control (shNON); no effect on IGF2 was observed in HepG2 cells. (B) Paxillin depletion (shPXN) resulted in a decreased number of cells incorporating BrdU, that is, undergoing replication. (C) Expression level of paxillin mRNA and protein level of paxillin in paxillin-depleted (shPXN) and control (shNON) HepG2 cells. qPCR data were normalized to the GAPDH gene and then to the control shNON sample. Actin (ACT) was used as a control of protein amount. Data are shown as mean±s.d. ( n ≥3). *** P <0.001; ns, P >0.05 (Student's t -test).

Article Snippet: Firefly luciferase reporter vectors p H19 P-luc and IGF2 P3-luc were constructed by inserting the H19 promoter (−819 to +13 bp relative to H19 transcription start site) or IGF2 promoter 3 (−499 to +13 bp relative to IGF2 transcript 3 transcription start site; +7858 to +8369 bp relative to the IGF2 exon 1 start) into pGL4.10 (Promega, Madison, WI) using the KpnI and HindIII sites.

Techniques: Expressing, Real-time Polymerase Chain Reaction, shRNA, Control

Journal: Journal of Cell Science

Article Title: Paxillin-dependent regulation of IGF2 and H19 gene cluster expression

doi: 10.1242/jcs.170985

Figure Lengend Snippet: Paxillin depletion does not change allele-specific expression of IGF2 and H19 . (A) A diagram of IGF2 / H19 locus and the amplified regions; arrows show the location of primers for the analyzed polymorphisms ( IGF2 , ApaI; H19 , RsaI). (B) Analysis of single nucleotide polymorphisms (SNPs) in genomic DNA of SAOS2 cells. Restriction analysis showed heterozygosity of H19 SNP rs2839704 and IGF2 SNP rs680 in genomic DNA of SAOS2 cells. IGF2 SNP rs680 (uncut, 220 bp; cut, 150 and 70 bp; the 70 bp fragment migrated out of the gel) and RsaI for H19 SNP rs2839704 (uncut, 720 bp, cut, 540 and 180 bp; the 180 bp fragment is not marked). The restriction site is present in one allele only. The H19 primers are spanning an intron and, therefore, the PCR fragment amplified from genomic DNA is bigger than the cDNA-amplified fragment. (C) IGF2 showed biallelic expression in paxillin-depleted (shPXN) as well as in control SAOS2 cells (shNON). An undigested 217-bp fragment and a digested 155-bp fragment were present after the restriction digest, showing that both alleles are transcribed. H19 retained monoallelic expression in both shPXN and shNON SAOS2 cells. There was no undigested 656-bp fragment, and only digested 488-bp and 168-bp fragments were detected after restriction digest, suggesting that only one allele is transcribed. IGF2 SNP rs680, cut with Apa I (uncut, 220 bp; cut, 150 and 70 bp; the 70 bp fragment migrated out of the gel); H19 SNP rs2839704, cut with RsaI (uncut, 630 bp; cut, 450 and 180 bp). The restriction site is present in one allele only. +RT, reverse transcription included; −RT, reverse transcription omitted to check for possible genomic DNA contamination.

Article Snippet: Firefly luciferase reporter vectors p H19 P-luc and IGF2 P3-luc were constructed by inserting the H19 promoter (−819 to +13 bp relative to H19 transcription start site) or IGF2 promoter 3 (−499 to +13 bp relative to IGF2 transcript 3 transcription start site; +7858 to +8369 bp relative to the IGF2 exon 1 start) into pGL4.10 (Promega, Madison, WI) using the KpnI and HindIII sites.

Techniques: Expressing, Amplification, Control, Reverse Transcription

Journal: Journal of Cell Science

Article Title: Paxillin-dependent regulation of IGF2 and H19 gene cluster expression

doi: 10.1242/jcs.170985

Figure Lengend Snippet: Paxillin regulates the IGF2 P3 and H19 promoters through their shared enhancer. (A) A scheme showing genomic context of the cloned sections; arrows are promoters, boxes are exons. The IGF2 P3 corresponds to the sequence +7858 to +8369 bp relative to the IGF2 exon 1 start. The H19 promoter represents the sequence −819 to +13 bp relative to the H19 transcription start site. The enhancer constitutes the sequence +6036 to +10,970 bp relative to the H19 transcription start site. (B) A scheme of luciferase constructs. H19 P-luc or IGF2 P3-luc consists of Firefly luciferase gene (luc) driven by the H19 promoter ( H19 P) or IGF2 promoter 3 ( IGF2 P3), respectively. H19 P-luc-E or IGF2 P3-luc-E additionally contained an enhancer sequence (E) located downstream of the luciferase gene. (C) Paxillin requires the enhancer to regulate the IGF2 P3 and H19 promoters. Depletion of paxillin from HepG2 cells (shPXN) increases the activity of H19 promoter and decreases the activity of the IGF2 promoter through their shared enhancer compared to control (shNON). Data were normalized to the Renilla luciferase activity. Data are shown as mean±s.d. ( n ≥3). *** P <0.001; ns, P >0.05 (Student's t -test).

Article Snippet: Firefly luciferase reporter vectors p H19 P-luc and IGF2 P3-luc were constructed by inserting the H19 promoter (−819 to +13 bp relative to H19 transcription start site) or IGF2 promoter 3 (−499 to +13 bp relative to IGF2 transcript 3 transcription start site; +7858 to +8369 bp relative to the IGF2 exon 1 start) into pGL4.10 (Promega, Madison, WI) using the KpnI and HindIII sites.

Techniques: Clone Assay, Sequencing, Luciferase, Construct, Activity Assay, Control

Journal: Journal of Cell Science

Article Title: Paxillin-dependent regulation of IGF2 and H19 gene cluster expression

doi: 10.1242/jcs.170985

Figure Lengend Snippet: Paxillin interacts with the IGF2 P3 and H19 promoters and their enhancer. (A) A scheme of IGF2 / H19 cluster with arrows indicating the designed primers. (B) ChIP using anti-paxillin antibody (PXN) or mouse IgG (control IgG) showed a specific enrichment of paxillin at the IGF2 promoter 3 ( IGF2 P3), the H19 promoter and the 5′ end of the enhancer (enhancer A) in HepG2 cells. No binding to the ICR region was detected. For the enhancer sequence (+6033 to +10,972 bp) we used three different primer sets (A, B and C). Set A amplified the region at the 5′ end (+6189 to +6363) of the enhancer, set B the region approximately in the middle (+7814 to +8038) and set C the region close to the 3′ end (+9978 to +10,217). The GAPDH gene was used as a negative control site. Data are shown as mean±s.d. ( n ≥3). *** P <0.001; ** P <0.01 (Student's t -test).

Article Snippet: Firefly luciferase reporter vectors p H19 P-luc and IGF2 P3-luc were constructed by inserting the H19 promoter (−819 to +13 bp relative to H19 transcription start site) or IGF2 promoter 3 (−499 to +13 bp relative to IGF2 transcript 3 transcription start site; +7858 to +8369 bp relative to the IGF2 exon 1 start) into pGL4.10 (Promega, Madison, WI) using the KpnI and HindIII sites.

Techniques: Control, Binding Assay, Sequencing, Amplification, Negative Control

Journal: Journal of Cell Science

Article Title: Paxillin-dependent regulation of IGF2 and H19 gene cluster expression

doi: 10.1242/jcs.170985

Figure Lengend Snippet: Paxillin regulates the interactions between the IGF2 P3 or H19 promoter and the enhancer. (A) Design of 3C analysis. Vertical lines show PstI restriction sites and black arrows represent the designed primers. (B) A chromatin conformation capture (3C) assay was performed using the IGF2 P3 fragment (A1) as an anchor, and the interaction between the IGF2 P3 fragment and fragments of enhancer (A1-B–A1-I) in HepG2 cells was assessed. The interaction frequency between IGF2 P3 and the enhancer was significantly lower after paxillin knockdown (shPXN) compared to control (shNON). (C) A 3C assay was performed in the same way, but using the H19 promoter fragment (A2) as an anchor examining the interaction between the H19 promoter and the fragments of enhancer (A1-B–A1-I). Paxillin depletion increased the interaction frequency between H19 promoter and the enhancer. The interaction frequency corresponds to the intensity of amplified PCR products; analyzed gels are shown in supplementary material Fig. S2 . Data are shown as mean±s.d. ( n ≥3). *** P <0.001; ns, not significant (Student's t -test).

Article Snippet: Firefly luciferase reporter vectors p H19 P-luc and IGF2 P3-luc were constructed by inserting the H19 promoter (−819 to +13 bp relative to H19 transcription start site) or IGF2 promoter 3 (−499 to +13 bp relative to IGF2 transcript 3 transcription start site; +7858 to +8369 bp relative to the IGF2 exon 1 start) into pGL4.10 (Promega, Madison, WI) using the KpnI and HindIII sites.

Techniques: Knockdown, Control, Amplification

Journal: Journal of Cell Science

Article Title: Paxillin-dependent regulation of IGF2 and H19 gene cluster expression

doi: 10.1242/jcs.170985

Figure Lengend Snippet: Paxillin, cohesin and mediator co-occupy the IGF2 / H19 promoters and the enhancer. (A) ChIP using anti-SMC1 or anti-MED23 antibody or rabbit IgG (CON IgG) showed specific enrichment of SMC1A (SMC1) and MED23 on the IGF2 promoter, the H19 promoter and the 5′ end of enhancer. See Fig. 4 A for location of primer sets A, B and C. (B) Depletion of paxillin by shRNA (shPXN) significantly decreased the amount of SMC1 and MED23 on the enhancer compared to control (shNON). We detected less SMC1A and MED23 on the IGF2 promoter (P3), and more SMC1A (but not MED23) on the H19 promoter. Paxillin depletion did not change the binding of SMC1A to the ICR. Data are shown as mean±s.d. ( n ≥3). * P <0.05; ** P <0.01; *** P <0.001; ns, P >0.05 (Student's t -test).

Article Snippet: Firefly luciferase reporter vectors p H19 P-luc and IGF2 P3-luc were constructed by inserting the H19 promoter (−819 to +13 bp relative to H19 transcription start site) or IGF2 promoter 3 (−499 to +13 bp relative to IGF2 transcript 3 transcription start site; +7858 to +8369 bp relative to the IGF2 exon 1 start) into pGL4.10 (Promega, Madison, WI) using the KpnI and HindIII sites.

Techniques: shRNA, Control, Binding Assay

Journal: Journal of Cell Science

Article Title: Paxillin-dependent regulation of IGF2 and H19 gene cluster expression

doi: 10.1242/jcs.170985

Figure Lengend Snippet: Model of IGF2 / H19 regulation by paxillin. Paxillin (PXN) assembles with cohesin and mediator (MED), and engages in the mediation of the long-range chromosomal interactions between the IGF2 or H19 promoter and the shared distal enhancer (E) regulating their transcription. Specifically, paxillin enhances the interaction between the enhancer and the IGF2 promoter, but blocks the H19 -promoter–enhancer interaction, resulting in stimulation of IGF2 and suppression of H19 expression. The model shows the paternal and maternal allele of the genes. Red box, inactive gene; green box, active gene; black lollipops, methylation; blue circle, cohesin complex; dark red arrows, ICR barrier.

Article Snippet: Firefly luciferase reporter vectors p H19 P-luc and IGF2 P3-luc were constructed by inserting the H19 promoter (−819 to +13 bp relative to H19 transcription start site) or IGF2 promoter 3 (−499 to +13 bp relative to IGF2 transcript 3 transcription start site; +7858 to +8369 bp relative to the IGF2 exon 1 start) into pGL4.10 (Promega, Madison, WI) using the KpnI and HindIII sites.

Techniques: Expressing, Methylation

Journal: Cell

Article Title: Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels

doi: 10.1016/j.cell.2015.02.012

Figure Lengend Snippet: (A) Schematic representation of Human ATXN1-3′UTR showing three putative PUM1 binding motifs (gray and red boxes) and their conservation in different species. The numbers indicate positions of PUM1 motifs in the human ATXN1-3′UTR. (B) ATXN1 mRNA quantification by qRT-PCR in HEK293T cells upon overexpression (left panel) or knockdown (siPUM1-81 and -82) (right panel) of PUM1. The destination-cloning vector (Control), scrambled siRNAs (siScr.) and cel-miR-67 were used as negative controls. The housekeeping gene GAPDH was used to normalize the expression of genes in all the qRT-PCR experiments. (C) Luciferase assay in HEK293 cells overexpressing the reporter construct harboring the full length ATXN1-3′UTR. In these conditions, we overexpressed (left panel) or decreased expression (right panel) of PUM1. (D) Luciferase assay in HEK293T cells transfected with single WT and mutant (MUT) putative PUM1 binding site on the ATXN1-3′UTR. The position of cloned regions for each PUM1 binding motif are indicated. The mutagenized nucleotides are highlighted in blue. In panels C and D the destination-vector (control), RNAi scramble (siScramble) and cel-miR-67 were used as negative controls; miR-101a was used as positive control. (RL) Renilla, (FL) Firefly luciferase. All the experiments were performed in triplicate (data represent mean ± S.E.M.). P values were calculated by Student’s t-test. Statistical significance is indicated as follows: *P<0.05, **P<0.01, ***P<0.0001. See also Figure S1.

Article Snippet: HEK293T cells were transfected with 30ng of psiCHECK-2 vectors (Promega) containing ATXN1 -3′UTR WT- or Mut binding sitess using Lipofectamine 2000 (Invitrogen).

Techniques: Binding Assay, Quantitative RT-PCR, Over Expression, Knockdown, Cloning, Plasmid Preparation, Control, Expressing, Luciferase, Construct, Transfection, Mutagenesis, Clone Assay, Positive Control

Journal: Cell

Article Title: Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels

doi: 10.1016/j.cell.2015.02.012

Figure Lengend Snippet: (A) RNA-Clip for the conserved Pum1 binding site in mouse cerebrum and cerebellum. PCRu and PCRd highlight the PCR fragments upstream and downstream of the conserved Atxn1-3′UTR Pum1 binding site (BS). IP with IgG as well Pum1 null mice were used as negative controls. Isolated RNA from a fraction (10%) of pre-cleared lysate was used as input. The experiment was performed in triplicate. Quantification of Atxn1 protein (B) and mRNA levels (C) in WT and Pum1−/− mice in cerebrum and cerebellum (n=8 per genotype). Data represent mean ± S.E.M. and normalized to Gapdh. See Experimental Procedures for more details. P values were calculated by Student’s t-test; *P<0.05, **P<0.01, ***P<0.0001. See also Figure S2.

Article Snippet: HEK293T cells were transfected with 30ng of psiCHECK-2 vectors (Promega) containing ATXN1 -3′UTR WT- or Mut binding sitess using Lipofectamine 2000 (Invitrogen).

Techniques: Binding Assay, Isolation

Journal: Cell

Article Title: Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels

doi: 10.1016/j.cell.2015.02.012

Figure Lengend Snippet: Representative western blot (upper panel) of protein lysates from HEK293T cells upon: (A) Overexpression of both PUM1 and miR-101a; (B) RNAi PUM1 (siPUM1) followed by overexpression of miR-101a; (C) overexpression of PUM1 followed by RNAi AGO2 (siAGO2); and (D) RNAi of both PUM1 and AGO2. The negative controls were destination-cloning vector (Control), RNAi scramble (siScramble) and cel-miR-67. All data were normalized to α-Tubulin (TUBA). (E) mRNA half-life quantification of wild-type (WT) and Mutated (Mut) PUM1 ATXN1-3′UTR binding sites in HEK293T cells at different time points upon DRB treatment (time zero). The numeric values within the panel given the extrapolated half-life for WT and Mut RNA, P=3.6×10−06. Firefly (FL) RNA levels were quantified and normalized to Renilla (RL). Bottom panel: representative western blot of PUM1 in HEK293T cells at different time points. Data were normalized to GAPDH. (F) ATXN1 mRNA half-life quantification in HEK293T cells at different time points, from zero (DRB treatment) to 8 hours total upon RNAi of PUM1 (siPUM1) or RNAi of scramble (siScramble) transfection. The numeric values within the panel given the extrapolated half-life for siPUM1 and siScramble RNA, P=0.012. Bottom panel: representative western blot of PUM1 in HEK293T cells at different time points. Data were normalized to GAPDH mRNA (top panel) or protein (bottom panel). All experiments were performed in triplicate (data represent mean ± S.E.M.); ***P<0.0001. See also Figure S3.

Article Snippet: HEK293T cells were transfected with 30ng of psiCHECK-2 vectors (Promega) containing ATXN1 -3′UTR WT- or Mut binding sitess using Lipofectamine 2000 (Invitrogen).

Techniques: Western Blot, Over Expression, Cloning, Plasmid Preparation, Control, Binding Assay, Transfection

Journal: Cell

Article Title: Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels

doi: 10.1016/j.cell.2015.02.012

Figure Lengend Snippet: (A) Representative western blot showing that haploinsufficiency of Pum1 restores physiological Atxn1 protein levels. All experiments were performed in triplicate in cerebrum and cerebellum from mice at 5 weeks of age (data represent mean ± SD). All data were normalized to Gapdh. Pum1+/−;Atxn1+/− mice showed (B) significant improvement in motor performance on the accelerating rotarod (n=12 per genotype), (C) reduced hind-paw clasping (n>12 per genotype), and (D) reduced kyphosis (curvature of the spine; photo taken at 25 weeks). Purkinje cells loss (E–F) and loss of dendritic arborization (G) were rescued in Pum1+/−;Atxn1+/− mice (n=6 per genotype). Staining was performed with Calbindin/IP3R1 in 3D-deepth-coding images. Data represent mean ± S.E.M. See Experimental Procedures. P values was calculated by Student’s t-test. ns=not significant; *P<0.05, **P<0.01, ***P<0.0001. See also Figure S6.

Article Snippet: HEK293T cells were transfected with 30ng of psiCHECK-2 vectors (Promega) containing ATXN1 -3′UTR WT- or Mut binding sitess using Lipofectamine 2000 (Invitrogen).

Techniques: Western Blot, Staining