Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: AGO1 locates at ERα binding sites, and its binding is enhanced by E2. (A) De novo DNA motif analysis at AGO1-associated genomic regions. (B) Enrichment of TF binding at AGO1-associated regions in serum-maintained MCF-7 cells, divided according to whether they are also bound by ERα (left panel) or not (right panel). ChIP-seq peak overlaps are shown for AGO1(1), PR (2), FOXA1 (2), TEAD4 (1), CEBPB (1), and JUND (1), where the number of replicates is shown between parentheses. (C) TF overlap with ERα-bound regions (in E2-treated cells), expressed as fold enrichment over randomized regions. (D) Heat maps of ChIP-seq signal centered (±1 kb) on ERα binding site midpoints for ERα (+E2), AGO1 (−E2), and AGO1 (+E2) regions. Regions are ordered from high to low ERα signal. (E) AGO1 reads per million mapped reads (RPM) around (±1 kb). AGO1 binding sites are defined by pooling AGO1 peaks from both treatments (−E2 and +E2). (F) UCSC genome browser screenshots showing four loci comprising ERα enhancers and neighboring genes that are up-regulated upon E2 treatment. Tracks correspond to RefSeq genes, AGO1 ChIP-seq, ERα (+E2) ChIP-seq, and GRO-seq plus and minus strands −E2 and +E2, all from MCF-7 cells.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Binding Assay, ChIP-sequencing

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: Relative to : AGO1 locates at ERα binding sites, and its binding is enhanced by E2. (A) Analysis of known DNA binding motifs of TFs expressed in MCF-7 cells at AGO1-associated genomic regions in serum-maintained cells. (B) Enrichment of TF binding at AGO1-associated regions in serum-maintained MCF7 cells. (C) Schematic representation of the hormone treatment experiments time line. (D) Table showing the overlap between the number of genomic sites (peaks) bound by ERα and by AGO1, considering each replicate separately and the common peaks. (E) Correlation heat map between AGO1 ChIP-seq samples and replicates based on read counts data on peak sets. (F) MA plot (gene expression ratios depicting fold changes in AGO1 binding affinity between untreated and treated samples. (G and H) MCF-7 cells were hormone starved and then treated with vehicle or E2 for 1 h. (G) ERα binding to the enhancers was assessed by ChIP-qPCR. Data are represented as mean ± SD ( n = 3). IP, immunoprecipitation. (H) AGO1 binding to the enhancers was assessed by ChIP-qPCR. Data are represented as mean ± SD ( n = 3; *, P < 0.05; **, P < 0.01; two-tailed Student’s t test).

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Binding Assay, ChIP-sequencing, Gene Expression, ChIP-qPCR, Immunoprecipitation, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: AGO1 locates at ERα binding sites, and its binding is enhanced by E2 without changes in its subcellular localization. (A–D) MCF-7 cells were hormone starved and then treated with vehicle or E2 for 1 h. (A) AGO1 levels in starved (−E2) and E2-treated (+E2) cells were tested by Western blot (WB) from whole-cell extracts (WCE; lanes 1–4). AGO1 levels were also assessed under these same conditions but transfected with a specific siRNA against AGO1 (lanes 5–8). (B) AGO1 and levels were assessed by Western blot in nuclear (NE) and cytoplasmic extracts (CE) from starved (−E2) and E2-treated MCF-7 cells. (C) AGO1 subcellular localization was assessed by immunofluorescence followed by confocal microscopy. The box plot corresponds to nucleus/cytoplasm integrated intensity ratios ( n = 100 cells; two-tailed Student’s t test). (D) Panels show representative images from C. Scale bars represent 10 µm.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Binding Assay, Western Blot, Transfection, Immunofluorescence, Confocal Microscopy, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: Relative to and . ERα binding intensity positively correlates with AGO1 binding, and AGO1 participates in the transcriptional activation of E2- responsive genes controlled by ERα enhancers. (A) Enrichment of the indicated chromatin states, obtained from E2-treated MCF-7 cells, over ERα binding sites divided into ERα + /AGO1 + and ERα + /AGO1 − . (B) . Heat maps of ChIP-seq signal centered (±3 kb) on ERα binding site midpoints for ERα (+E2), AGO1 (−E2), and AGO1 (+E2). ERα regions were divided according to whether they overlap with AGO1 (ERα + /AGO1 + ) or not (ERα + /AGO1 − ), and within each group, they were ordered from high to low ERα signal. (C–E) MCF-7 cells were transfected with an siControl or siAGO1. 24 h later, they were hormone starved, and 72 h later, they were treated with E2 or vehicle for the indicated time. (C) E2-dependent AGO1 recruitment to the enhancers upon AGO1 knockdown was controlled by ChIP-qPCR. Data are represented as mean ± SD ( n = 3; *, P < 0.05; **, P < 0.01; two-tailed Student’s t test). (D) RNA was extracted, and the indicated mRNA levels were analyzed by RT-qPCR. (E) Same as D, but a different siRNA against AGO1 was used. Values in D and E represent mean ± SE from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ANOVA and Tukey post-hoc test). In A and B, two different siRNAs against AGO1 were used.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Binding Assay, Activation Assay, ChIP-sequencing, Transfection, Knockdown, ChIP-qPCR, Two Tailed Test, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: ERα binding to chromatin depends on AGO1. (A) ERα (+E2) ChIP-seq normalized read counts over ERα + /AGO1 + and ERα + /AGO1 − regions. (B–D) MCF-7 cells were transfected with an siControl or siAGO1. 24 h later, they were hormone starved, and 72 h later, they were treated with E2 or vehicle for 1 h. (B) AGO1 knockdown efficiency was tested by Western blot (WB) from whole-cell extracts (WCE). (C) E2-dependent ERα recruitment to ERα + /AGO1 + regions was analyzed by ChIP-qPCR. (D) E2-dependent ERα recruitment to ERα + /AGO1 − regions was analyzed by ChIP-qPCR. Values in C and D represent mean ± SE with n = 3 (i.e., three independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ANOVA and Tukey post-hoc test. ns, not significant; IP, immunoprecipitation.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Binding Assay, ChIP-sequencing, Transfection, Knockdown, Western Blot, ChIP-qPCR, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: AGO1 physically interacts with ERα in an E2-dependent and RNA-independent manner. (A) MCF-7 cells were hormone starved and treated with vehicle or E2 for 1 h. Whole-cell extracts (WCE) were treated or not with RNase A, and then they were immunoprecipitated (IP) with control or anti-AGO1 antibody. AGO1 and ERα presence in inputs and immunoprecipitates was analyzed by Western blot (WB). (B) Representative agarose gel showing RNase treatment efficiency.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Immunoprecipitation, Control, Western Blot, Agarose Gel Electrophoresis

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: AGO1 participates in the transcriptional activation of E2-responsive genes controlled by ERα enhancers by promoting three steps of ERα enhancer activation. (A) GRO-seq read count fold change between −E2 and +E2 treatments over ERα + /AGO1 + and ERα + /AGO1 − regions. (B–H) MCF-7 cells were transfected with an siControl or siAGO1. 24 h later, they were hormone starved, and 72 h later, they were treated with E2 or vehicle for 1 h. (B) Total RNA was extracted, and AGO1 knockdown was assessed by RT-qPCR. (C) Nuclear RNA was extracted, and the indicated eRNA levels were analyzed by RT-qPCR. (D) Total RNA was extracted, and the indicated pre-mRNA levels corresponding to E2-regulated genes were analyzed by RT-qPCR. (E) Total RNA was extracted, and the indicated pre-mRNA levels corresponding to non–E2-regulated genes were analyzed by RT-qPCR. (F) Frequency of chromatin loops formation between the indicated restriction fragments was analyzed by 3C. The anchor region, with which all of the interactions were tested, comprises an ERα + /AGO1 + region. AGO1 ChIP-seq and ERα ChIP-seq tracks are shown along the position of the proximal E2-regulated genes. (G) Same as F, but here the anchor region comprises an ERα + /AGO1 − region. (H) Same as G, but here the anchor region comprises a non–E2-regulated enhancer region. Values in B–E represent mean ± SE from three independent experiments (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ANOVA and Tukey post-hoc test). Values in F–H represent mean ± SE from three independent experiments (*, P < 0.05; **, P < 0.01; two-tailed Student’s t test).

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Activation Assay, Transfection, Knockdown, Quantitative RT-PCR, ChIP-sequencing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

doi: 10.1083/jcb.201908097

Figure Lengend Snippet: AGO1 functional role at ERα transcriptional enhancers does not depend on small RNA binding. (A) Schematic representation of AGO1 protein domains showing the position of the point mutation in the MID domain that impairs small RNA binding. (B–D) MCF-7 cells were transfected with the indicated siRNAs, and AGO1 expression was rescued by the indicated constructs before hormone starvation and E2 treatment. (B) AGO1 knockdown and rescue levels were assessed by Western blot. RPB1 corresponds to the housekeeping control. (C) The levels of the indicated pre-mRNA corresponding to E2-regulated genes were assessed by RT-qPCR. (D) The levels of the indicated eRNA corresponding to an E2-regulated eRNA were assessed by RT-qPCR. Values in C and D represent mean ± SE from three independent experiments (*, P < 0.05; two-tailed Student’s t test). ns, not significant; WCE, whole-cell extracts.

Article Snippet: The lentiviral vector driving expression of FLAG/HA-Ago1 (human) from the Syn promoter (pLV-FLAG-HA_Ago1) was generated by amplifying FLAG/HA-Ago1 from pIRESneo-FLAG/HA Ago1 (Addgene plasmid 10822).

Techniques: Functional Assay, RNA Binding Assay, Mutagenesis, Transfection, Expressing, Construct, Knockdown, Western Blot, Control, Quantitative RT-PCR, Two Tailed Test