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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 01, 2016 |
| Title |
Geminin promoter occupancy and Geminin-dependent histone acetylation during neuroectodermal cell specification |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by genome tiling array
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| Summary |
The nucleoprotein Geminin (Gmnn) promotes neural cell fate acquisition of embryonic stem cells, while knockdown reduces the efficiency of neural gene activation. This occurs, at least in part, through Geminin’s ability to promote histone hyperacetylation at neural genes, to activate their expression. In mouse models in vivo, Geminin deficiency in the embryonic neural tube between embryonic days 8.5-10.5 also reduces the expression of genes controlling neural specification and/or differentiation, contributing to neural tube defects. To determine where Geminin binding and Gmnn-dependent acetylation occurs throughout the genome, we performed ChIP-chip analysis (these data) and ChIP-seq analysis (GSE77246) to define Geminin-bound chromatin locations in mouse embryonic stem cells (ESCs) and in ESC-derived neuroectoderm. We also performed ChIP-chip analysis of H3K9 acetylation in ESC-derived neuroectoderm, with or without Doxycycline-dependent Gmnn knockdown, to define the requirements for Geminin activity for histone acetylation of promoters during neural fate specification.
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| Overall design |
For ChIP sample preparation for promoter occupancy assays, three independent biological replicates each were generated for the following sample types: (1) Geminin ChIP in ESCs (2) Geminin ChIP in ESC-derived neuroectoderm after 2.5 days of N2B27 monolayer culture, using the N18 Gmnn antibody (Santa Cruz sc-8449). (3-4) A clonal A2lox embryonic stem cell line engineered for Doxycycline-dependent Gmnn knockdown (KD) was differentiated in N2B27 medium for 2.5 days with or without 500ng Doxcycline and these samples were used for H3K9ac ChIP (antibody Abcam; Ab10812) to assess Geminin-dependent histone acetylation (H3K9ac in GemKD cells on day 2.5, three biological replicates each were generated with Gmnn knockdown (plus Dox) or under control conditions (no Dox). The A2lox mouse ES cell line (Iacovino et al., Stem Cells 2011, 29(10): 1580-88) and a clonal derivative engineered for Doxycycline-dependent Gmnn shRNA.expression (Yellajoshyula et al., PNAS 2011, 108(8): 3294-99) were routinely propagated on feeder cells (mouse embryonic fibroblast cells, Chemicon) in DMEM (Invitrogen) supplemented with 15% fetal calf serum (Hyclone), 1 mM sodium pyruvate, 1 mM Non-Essential amino acids, 1mM L-Glutamine (Invitrogen), 10−4 M 2-mercaptoethanol (Sigma) and 103 units per ml of leukemia inhibitory factor (ESGRO, Chemicon). Monolayer neural differentiation of ES cells was done as previously described (Ying and Smith, Methods in Enzymology 2003, 365: 327-41). Briefly, ES cells cultured on feeder cells were dissociated and plated at 1x105/cm2 for one day on 0.1% gelatin-coated tissue culture dishes. After 24 hours, cells were plated onto 0.1% gelatin-coated tissue culture dishes at low density (1x104 cells/cm2) in N2B27 medium and cultured for 2.5 days.
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| Contributor(s) |
Yellajoshyula D, Sankar S, Zhang B, Kroll KL |
| Citation(s) |
27881878 |
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| Submission date |
May 15, 2016 |
| Last update date |
Dec 01, 2016 |
| Contact name |
Kristen L Kroll |
| E-mail(s) |
[email protected]
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| Organization name |
Washington University School of Medicine
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| Department |
Developmental Biology
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| Lab |
Kristen Kroll
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| Street address |
320 McDonnell Sciences/660 S. Euclid Ave.
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| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platforms (1) |
| GPL16158 |
NimbleGen Mus musculus 100718_MM8_Deluxe_Promoter_HX1 array |
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| Samples (12)
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| This SubSeries is part of SuperSeries: |
| GSE81595 |
Genome-wide association of Geminin and Zic1 during neuroectodermal cell specification |
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| Relations |
| BioProject |
PRJNA321631 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE81450_RAW.tar |
1.2 Gb |
(http)(custom) |
TAR (of GFF, PAIR, TXT) |
| Processed data provided as supplementary file |
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