1. Volume 64, Issue 5, Pages 967-981 (December 2016)
LncPRESS1 Is a p53-Regulated LncRNA that Safeguards Pluripotency by Disrupting SIRT6-Mediated De-acetylation of Histone H3K56 
Abhinav K. Jain, Yuanxin Xi, Ryan McCarthy, Kendra Allton, Kadir C. Akdemir, Lalit R. Patel, Bruce Aronow, Chunru Lin, Wei Li, Liuqing Yang, Michelle C. Barton 
Molecular Cell 
Volume 64, Issue 5, Pages (December 2016)
DOI: /j.molcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2016 64, 967-981DOI: (10.1016/j.molcel.2016.10.039)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Genome-wide Analysis of p53-Dependent Transcriptome of Human ESCs
(A) Process to identify p53-regulated lncRNAs.
(B) The biotype distribution of all (left) versus differentially expressed transcripts (right) by RNA-seq analysis of human ESCs transfected with siControl or siTP53 ± RA.
(C) Heatmap showing clustering of 275 lncRNAs differentially expressed in human ESCs depleted of p53, untreated or treated with RA.
(D) Heatmap showing expression data for p53-dependent lncRNAs in human ESCs treated with RA.
(E) Subtype distribution of p53-dependent lncRNAs differentially expressed during hESC differentiation.
(F) Aggregate plots showing profiles of histone modifications around ±3.5 kb from TSS of lncRNAs directly regulated by p53.
See also Figure S1 and Tables S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 LncRNAs Directly Regulated by p53 during hESC Differentiation
(A and B) Expression levels of selected lncRNAs during hESC differentiation with RA (A) and BMP4 (B).
(C and D) Expression levels of selected genes during RA (C) or BMP4 (D) induced differentiation in shControl or shTP53 hESCs.
(E and F) Protein (E) and mRNA (F) expression levels in shControl and shTP53 hESCs treated with Nutlin-3a. The error bars represent SEM from three different experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).
See also Figure S2.
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5. Figure 3 p53 Binds at the Promoters of LncRNAs
(A) ChIP-seq tracks of normalized sequence tags show enrichment for p53, H3K4me3, and H3K27me3 along the lncRNA loci in pluripotent (−RA) and hESCs treated with RA (+RA) visualized on UCSC genome browser (
(B and C) H3K27me3 and H3K4me3 status at gene promoters (B) and H3K79me2 along lncPRESS1 gene (C) normalized to H2B.
(D) Summary table of location and status of p53REs on selected lncRNAs.
(E) ChIP-qPCR analysis of p53 occupancy at lncRNA promoters during differentiation (+RA for 1 day). The error bars represent SEM from three different experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < compared to untreated).
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Single-Cell Analyses Reveal Lineage-Specific Expression of p53-Regulated LncRNAs
(A) Heatmap showing unsupervised clustering of 300 individual hESCs undergoing ectoderm-, endoderm-, or mesoderm-specific differentiation analyzed by single-cell qRT-PCR for p53-regulated lncRNAs and 28 marker genes for pluripotency and lineage-specific differentiation.
(B) Principal component analysis visualization of FCM-generated clusters (cluster identity indicated by color). The lines link each gene to its two nearest neighbors from the full dimensional parameter space.
(C) Expression levels of selected genes during lineage-specific differentiation to dEC, dEN, and dME. The error bars represent SEM from three different experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).
See also Figure S4 and Table S3.
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7. Figure 5 Depletion of LncPRESS1 Affects Pluripotency
(A) Expression levels of selected pluripotency-specific genes in shControl or shLncPRESS1 hESCs. The error bars represent SEM from three different experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).
(B) Alkaline phosphatase staining and quantitation of hESCs, scored as undifferentiated, partially differentiated, or fully differentiated.
(C) Immunofluorescence images of hESCs stained with StainAlive TRA 1-81 antibody. The percent of TRA 1-81 positive or negative cell populations sorted by FACS are displayed.
(D) CyTOF analysis and quantification of pluripotent and differentiated cell populations were performed based upon twelve markers. The significant changes in the percent of pluripotent cells (∗∗p = ) and ectodermal cells (∗p < 5 × 10−4) were observed in the shLncPRESS1 hESCs.
(E) Heatmap showing expression data for lncPRESS1-dependent genes in hESCs.
(F) Volcano plot of gene expression data; each point corresponds to a RefSeq gene (q value cutoff <0.1 is marked). The consensus pluripotency- (red dots) and differentiation-specific (blue dots) genes are highlighted.
(G) The top ten gene ontology terms of the genes dependent on lncPRESS1 in hESCs.
See also Figure S5 and Table S4.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 LncPRESS1 Interacts with SIRT6
(A) Scheme of RNA pull-down assay.
(B) Top lncPRESS1-interacting proteins identified by mass spectrometry analysis.
(C) RIP of lncPRESS1 using anti-SIRT6 antibody. The percent of input lncPRESS1 or H19 in SIRT6-RIP during differentiation (+RA for 2 days) compared to negative control IgG is shown. The error bars represent SEM from three different experiments.
(D) Chromatin fractions and total lysates were analyzed for SIRT6, H3, and GAPDH. The relative SIRT6 levels normalized to loading controls are shown.
(E) Protein lysates were analyzed for H3K56ac, OCT4, H3, and GAPDH. The protein levels are reported as fold change relative to shControl.
(F) Purified histone extracts were analyzed for histone PTMs and fold changes normalized to total histones are reported.
See also Figure S6 and Table S5.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 LncPRESS1 Regulates SIRT6-Mediated H3K56/K9 Acetylation
(A) SIRT6 occupancy at pluripotency-specific gene promoters during hESC differentiation.
(B) H3K56ac and H3K9ac status normalized to H2B at gene promoters.
(C) CyTOF analysis of shControl and shSIRT6 hESCs based upon twelve markers. The dimensional reduction using viSNE shows no apparent changes in pluripotency marker OCT4 upon SIRT6 knockdown. The SIRT6 depletion had no effect on the percent of pluripotent or ectodermal cells.
(D) H3K56ac and H3K9ac status normalized to H2B at gene promoters. All of the error bars represent SEM from three different experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < compared to shControl untreated).
(E) Model of lncPRESS1-mediated regulation of SIRT6 activity.
See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions