scholarly journals ARGONAUTE proteins regulate a specific network of genes through KLF4 in mouse embryonic stem cells

2021 ◽  
Author(s):  
Madlen Mueller ◽  
Moritz Schaefer ◽  
Tara Faeh ◽  
Daniel Spies ◽  
Rodrigo Peña-Hernández ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Mouse Embryonic Stem Cells ◽  
Mirna Biogenesis ◽  
Transcriptional Gene Silencing ◽  
Argonaute Proteins ◽  
Mutant Cells

The Argonaute proteins (AGO) are well-known for their essential role in post-transcriptional gene silencing in the microRNA (miRNA) pathway. Only two AGOs are expressed in mouse embryonic stem cells (mESCs). The transcriptome of Ago mutant mESCs revealed a large and specific set of differentially expressed genes (DEGs), compared to other miRNA biogenesis factor mutant cells, suggesting additional functions for AGOs. Integration of Ago DEGs with ENCODE histone modification data of WT mESCs revealed a correlation with H3K27me3 chromatin mark. We validated experimentally this result and observed a global loss of H3K27me3, which was only partially explaining the DEGs observed in Ago mutant cells. By integrating chromatin accessibility data in conjunction with the prediction of transcription factor (TF) binding sites, we identified differential binding for five TFs, including KLF4 as a key modulator of more than half of the specific DEGs in the absence of AGO proteins. Our findings illustrate that in addition to chromatin state, information about transcription factor binding is more revelatory in understanding the multi-layered mechanism adopted by cells to regulate gene expression.

scholarly journals The pluripotency transcription factor Nanog represses glutathione reductase gene expression in mouse embryonic stem cells

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Claudia Solari ◽  
María Victoria Petrone ◽  
Ayelén Toro ◽  
Camila Vazquez Echegaray ◽  
María Soledad Cosentino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Glutathione Reductase ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Reductase Gene

scholarly journals Foxd3 controls heterochromatin-mediated silencing of repeat elements in mouse embryonic stem cells and represses the 2-cell transcription program

2021 ◽  
Author(s):  
Deepika Puri ◽  
Birgit Koschorz ◽  
Bettina Engist ◽  
Megumi Onishi-Seebacher ◽  
Devon Ryan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Repeat Element ◽  
Negative Regulator ◽  
Major Satellite ◽  
Satellite Repeats

Repeat element transcription plays a vital role in early embryonic development. Expression of repeats such as MERVL characterises mouse embryos at the 2-cell stage, and defines a 2-cell-like cell (2CLC) population in a mouse embryonic stem cell culture. Repeat element sequences contain binding sites for numerous transcription factors. We identify the forkhead domain transcription factor FOXD3 as a regulator of repeat element transcription in mouse embryonic stem cells. FOXD3 binds to and recruits the histone methyltransferase SUV39H1 to MERVL and major satellite repeats, consequentially repressing the transcription of these repeats by the establishment of the H3K9me3 heterochromatin modification. Notably, depletion of FOXD3 leads to the de-repression of MERVL and major satellite repeats as well as a subset of genes expressed in the 2-cell state, shifting the balance between the stem cell and 2 cell-like population in culture. Thus, FOXD3 acts as a negative regulator of repeat transcription, ascribing a novel function to this transcription factor.

scholarly journals Induction of Ventral Spinal V0 Interneurons from Mouse Embryonic Stem Cells

2020 ◽  
Author(s):  
Jennifer Pardieck ◽  
Manwal Harb ◽  
Shelly Sakiyama-Elbert
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Suspension Culture ◽  
Signaling Pathway ◽  
Central Pattern Generators ◽  
Butyl Ester ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Embryoid Bodies

AbstractThe ventral spinal population of V0 interneurons (INs) contribute to the coordinated movements directed by spinal central pattern generators (CPGs), including respiratory circuits and left-right alternation. One challenge in studying V0 INs has been the limited number of cells that can be isolated from primary sources for basic research or therapeutic use. However, derivation from a pluripotent source, such as has been done recently for other IN populations could reduce this issue. However, there is currently no protocol to specifically derive V0 interneurons from embryonic stem cells or induced pluripotent stem cells. To generate an induction protocol, mouse embryonic stem cells (mESCs) were grown in suspension culture and then exposed to retinoic acid (RA) and collected at different time points to measure mRNA expression of the V0 progenitor transcription factor marker, Dbx1, and post-mitotic transcription factor marker, Evx1. The cultures were also exposed to the sonic hedgehog signaling pathway agonist purmorphamine (purm) and the Notch signaling pathway inhibitor N-{N-(3,5-difluorophenacetyl-L-alanyl)}-(S)-phenylglycine-t-butyl-ester (DAPT) to determine if either of these pathways contribute to V0 IN induction, specifically the ventral (V0V) subpopulation. From the various parameters tested, the final protocol that generated the greatest percentage of cells expressing V0V IN markers was an 8 day protocol using 4 days of suspension culture to form embryoid bodies followed by addition of 1 μM RA from days 4 to 8, 100 nM purm from days 4 to 6, and 5 μM DAPT from days 6 to 8. This protocol will allow investigators to obtain V0 IN cultures for use in in vitro studies, such as those examining CPG microcircuits, electrophysiological characterization, or even for transplantation studies in injury or disease models.

scholarly journals The role of MORC3 in silencing transposable elements in mouse embryonic stem cells

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Varsha P. Desai ◽  
Jihed Chouaref ◽  
Haoyu Wu ◽  
William A. Pastor ◽  
Ryan L. Kan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Transposable Elements ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Mouse Embryonic Stem Cells ◽  
Endogenous Retroviruses ◽  
Direct Role ◽  
Developmental Abnormalities

Abstract Background Microrchidia proteins (MORCs) are involved in epigenetic gene silencing in a variety of eukaryotic organisms. Deletion of MORCs result in several developmental abnormalities and their dysregulation has been implicated in developmental disease and multiple cancers. Specifically, mammalian MORC3 mutations are associated with immune system defects and human cancers such as bladder, uterine, stomach, lung, and diffuse large B cell lymphomas. While previous studies have shown that MORC3 binds to H3K4me3 in vitro and overlaps with H3K4me3 ChIP-seq peaks in mouse embryonic stem cells, the mechanism by which MORC3 regulates gene expression is unknown. Results In this study, we identified that mutation in Morc3 results in a suppressor of variegation phenotype in a Modifiers of murine metastable epialleles Dominant (MommeD) screen. We also find that MORC3 functions as an epigenetic silencer of transposable elements (TEs) in mouse embryonic stem cells (mESCs). Loss of Morc3 results in upregulation of TEs, specifically those belonging to the LTR class of retrotransposons also referred to as endogenous retroviruses (ERVs). Using ChIP-seq we found that MORC3, in addition to its known localization at H3K4me3 sites, also binds to ERVs, suggesting a direct role in regulating their expression. Previous studies have shown that these ERVs are marked by the repressive histone mark H3K9me3 which plays a key role in their silencing. However, we found that levels of H3K9me3 showed only minor losses in Morc3 mutant mES cells. Instead, we found that loss of Morc3 resulted in increased chromatin accessibility at ERVs as measured by ATAC-seq. Conclusions Our results reveal MORC3 as a novel regulator of ERV silencing in mouse embryonic stem cells. The relatively minor changes of H3K9me3 in the Morc3 mutant suggests that MORC3 acts mainly downstream of, or in a parallel pathway with, the TRIM28/SETDB1 complex that deposits H3K9me3 at these loci. The increased chromatin accessibility of ERVs in the Morc3 mutant suggests that MORC3 may act at the level of chromatin compaction to effect TE silencing.

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