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

In mammals, argonaute (AGO) proteins have been characterized for their roles in small RNA mediated posttranscriptional and also in transcriptional gene silencing. Here, we report a different role for AGO1 in estradiol-triggered transcriptional activation in human cells. We show that in MCF-7 mammary gland cells, AGO1 associates with transcriptional enhancers of estrogen receptor α (ERα) and that this association is up-regulated by treating the cells with estrogen (E2), displaying a positive correlation with the activation of these enhancers.Moreover, we show that AGO1 interacts with ERα and that this interaction is also increased by E2 treatment, but occurs in the absence of RNA. We show that AGO1 acts positively as a coactivator in estradiol-triggered transcription regulation by promoting ERα binding to its enhancers. Consistently, AGO1 depletion decreases long-range contacts between ERα enhancers and their target promoters. Our results point to a role of AGO1 in transcriptional regulation in human cells that is independent from small RNA binding.Fil: Gómez Acuña, Luciana Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Nazer, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Rodríguez Seguí, Santiago Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Pozzi, María Berta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Buggiano, Valeria Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Marasco, Luciano Edmundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Agirre, Eneritz. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: He, Cody. University of Chicago; Estados UnidosFil: Alló, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Transcriptional signature of islet neogenesis-associated protein peptide-treated rat pancreatic islets reveals induction of novel long non-coding RNAs

BackgroundDiabetes mellitus is characterized by chronic hyperglycemia with loss of β-cell function and mass. An attractive therapeutic approach to treat patients with diabetes in a non-invasive way is to harness the innate regenerative potential of the pancreas. The Islet Neogenesis-Associated Protein pentadecapeptide (INGAP-PP) has been shown to induce β-cell regeneration and improve their function in rodents. To investigate its possible mechanism of action, we report here the global transcriptional effects induced by the short-term INGAP-PP in vitro treatment of adult rat pancreatic islets.Methods and findingsRat pancreatic islets were cultured in vitro in the presence of INGAP-PP for 4 days, and RNA-seq was generated from triplicate treated and control islet samples. We performed a de novo rat gene annotation based on the alignment of RNA-seq reads. The list of INGAP-PP-regulated genes was integrated with epigenomic data. Using the new gene annotation generated in this work, we quantified RNA-seq data profiled in INS-1 cells treated with IL1β, IL1β+Calcipotriol (a vitamin D agonist) or vehicle, and single-cell RNA-seq data profiled in rat pancreatic islets. We found 1,669 differentially expressed genes by INGAP-PP treatment, including dozens of previously unannotated rat transcripts. Genes differentially expressed by the INGAP-PP treatment included a subset of upregulated transcripts that are associated with vitamin D receptor activation. Supported by epigenomic and single-cell RNA-seq data, we identified 9 previously unannotated long noncoding RNAs (lncRNAs) upregulated by INGAP-PP, some of which are also differentially regulated by IL1β and vitamin D in β-cells. These include Ri-lnc1, which is enriched in mature β-cells.ConclusionsOur results reveal the transcriptional program that could explain the enhancement of INGAP-PP-mediated physiological effects on β-cell mass and function. We identified novel lncRNAs that are induced by INGAP-PP in rat islets, some of which are selectively expressed in pancreatic β-cells and downregulated by IL1β treatment of INS-1 cells. Our results suggest a relevant function for Ri-lnc1 in β-cells. These findings are expected to provide the basis for a deeper understanding of islet translational results from rodents to humans, with the ultimate goal of designing new therapies for people with diabetes

Nucleolar Accumulation of RNA Binding Proteins Induced by ActinomycinD Is Functional in Trypanosoma cruzi and Leishmania mexicana but Not in T. brucei

We have recently shown in T. cruzi that a group of RNA Binding Proteins (RBPs), involved in mRNA metabolism, are accumulated into the nucleolus in response to Actinomycin D (ActD) treatment. In this work, we have extended our analysis to other members of the trypanosomatid lineage. In agreement with our previous study, the mechanism seems to be conserved in L. mexicana, since both endogenous RBPs and a transgenic RBP were relocalized to the nucleolus in parasites exposed to ActD. In contrast, in T. brucei, neither endogenous RBPs (TbRRM1 and TbPABP2) nor a transgenic RBP from T. cruzi were accumulated into the nucleolus under such treatment. Interestingly, when a transgenic TbRRM1was expressed in T. cruzi and the parasites exposed to ActD, TbRRM1 relocated to the nucleolus, suggesting that it contains the necessary sequence elements to be targeted to the nucleolus. Together, both experiments demonstrate that the mechanism behind nucleolar localization of RBPs, which is present in T. cruzi and L. mexicana, is not functional in T. brucei, suggesting that it has been lost or retained differentially during the evolution of the trypanosomatid lineage

Severe heat shock induces nucleolar accumulation of mRNAs in Trypanosoma cruzi

Several lines of evidence have shown that, besides its traditional function in ribosome biogenesis, the nucleolus is also involved in regulating other cellular processes such as mRNA metabolism, and that it also plays an important role as a sensor and coordinator of the stress response. We have recently shown that a subset of RNA Binding Proteins and the poly(A)+ RNA are accumulated into the Trypanosoma cruzi nucleolus after inducing transcription inhibition with Actinomycin D. In this study, we investigated the behaviour of the T. cruzi mRNA population in parasites subjected to severe heat shock, an environmental stress that also decreases the rate of RNA synthesis. We found that the bulk of poly(A)+ RNA is reversibly accumulated into the nucleolus when exposing T. cruzi epimastigote forms to severe heat shock. However, the Hsp70 mRNA was able to bypass such nucleolar accumulation. Together, these data reinforce the idea about the involvement of the T. cruzi nucleolus in mRNA metabolism during an environmental stress response. Interestingly, T. brucei procyclic forms did not induce nucleolar accumulation of poly(A)+ RNA under such stress condition, suggesting that different trypanosomatids have adopted different responses to deal with the same stress conditions.Fil: Nazer, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Verdún, Ramiro E.. University of Miami; Estados UnidosFil: Sanchez, Daniel Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

Seeking the truth behind the myth: Argonaute tales from “nuclearland”

Argonaute proteins have been traditionally characterized as a highly evolutionary conserved family engaged in post-transcriptional gene silencing pathways. The Argonaute family is mainly grouped into the AGO and PIWI clades. The canonical role of Argonaute proteins relies on their ability to bind small-RNAs that recognize complementary sequences on target mRNAs to induce either mRNA degradation or translational repression. However, there is an increasing amount of evidence supporting that Argonaute proteins also exert multiple nuclear functions that subsequently regulate gene expression. In this line, genome-wide studies showed that members from the AGO clade regulate transcription, 3D chromatin organization, and splicing of active loci located within euchromatin. Here, we discuss recent work based on high-throughput technologies that have significantly contributed to shed light on the multivariate nuclear functions of AGO proteins in different model organisms. We also analyze data supporting that AGO proteins are able to execute these nuclear functions independently from small RNA pathways. Finally, we integrate these mechanistic insights with recent reports highlighting the clinical importance of AGO in breast and prostate cancer development.Fil: Nazer, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Gómez Acuña, Luciana Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. University of Edinburgh; Reino UnidoFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Argonaute2 attenuates active transcription by limiting RNA Polymerase II elongation in Drosophila melanogaster

Abstract Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. In this study, we show that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, we performed differential ChIP-seq analysis for RNA Polymerase II (Pol II) upon depletion of AGO2. Remarkably, we find specific accumulation of the elongating but not initiating form of Pol II after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (NELF) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology

Code and supplemental data Nazer et al 2018 PLOS Genetics.<br><br><b>nazer_et_al.zip</b>: contains the code and instructions for regenerating the figures.<br><b><br>nazer_et_al_generated_figures.zip</b>: contains the figures and reports created by running the above code.<br

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology

<div><p><i>Drosophila</i> Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, we identified a somatic target of AGO2 transcriptional repression, <i>no hitter</i> (<i>nht</i>), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of <i>AGO2</i> leads to ectopic expression of <i>nht</i> and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the <i>nht</i> TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression.</p></div

AGO2 and LaminB attenuate transcription in active chromatin and additional sites across the genome.

<p>A) Heatmap showing enrichment and depletion of neuRNA-seq up-regulated genes in AGO2-, LaminB, both- or Dcr-2-depleted cells across different chromatin features such as chromatin domains (LADs and DPSs), chromatin colors, transcription start sites (TSS), and transcription polyadenylation site (TPS). The color scale indicates the log₂ odds ratio of the Fisher's exact tests, where negative (blue) indicates depletion and positive (red) indicates enrichment. B) Heatmap showing enrichment and depletion of neuRNA-seq down-regulated genes in AGO2-, LaminB, both- or Dcr-2-depleted cells with respect to chromatin features. C) Heatmap showing enrichment and depletion of AGO2 and Pol II ChIP-seq peaks with respect to chromatin features. Colormap represents the log₂ fold change as reported by the Genomic Association Test (GAT). D) Representative screenshot of co-up-regulated genes in AGO2 KD and LaminB KD embedded within a LAD with TSS located in active RED chromatin. The red and blue bars below the neuRNA-seq signals correspond to significant differences (up and down, respectively) relative to mock-treated cells. Black bars at the bottom of each screenshot correspond to AGO2 peaks. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007276#pgen.1007276.s002" target="_blank">S2 Fig</a>.</p

AGO2 and LaminB attenuate transcription genome-wide.

<p>A) Western blot analysis showing knockdown efficiencies for each protein analyzed. Tubulin is included as loading control. B) Changes in neuRNA levels upon depletion of AGO2. Statistically significant changes include 710 up-regulated genes (red) and 195 down-regulated genes (blue). (For AGO2 rescue experiments see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007276#pgen.1007276.s001" target="_blank">S1 Fig</a>). Genes containing an AGO2 peak at their promoter are additionally colored orange. FET <i>p</i>-values and odds ratios indicate significance of association between affected genes and presence of AGO2 peak at promoters. C) Scatterplot comparing neuRNA-seq profiles from AGO2 and LaminB knockdowns. Pearson’s R corresponds to correlation coefficient of the two profiles. FET <i>p</i>-values and odds ratios indicate significance of the association between coordinately up-regulated (466, red) or down-regulated (76, blue) genes in both knockdowns. D) Scatterplot comparing neuRNA-seq profiles from Dcr-2 and AGO2 knockdowns. Genes with undetermined log2 fold change (due to zero counts in one condition) are fixed to a minimal value of -1.5 so they can be visually represented on the plots. Genes with zero counts in both conditions are not displayed. E) Scatterplot comparing neuRNA-seq profiles from Dcr-2 and LaminB knockdowns. F) Immunofluorescence analysis for AGO2 and LaminB in mock, AGO2- and LaminB-depleted cells. AGO2 (green, Liu antibody), LaminB (red), and DAPI staining (blue) are shown. Scale bar represents 14 μm.</p