NUP98 and RAE1 sustain progenitor function through HDAC-dependent chromatin targeting to escape from nucleolar localization

Abstract Self-renewing somatic tissues rely on progenitors to support the continuous tissue regeneration. The gene regulatory network maintaining progenitor function remains incompletely understood. Here we show that NUP98 and RAE1 are highly expressed in epidermal progenitors, forming a separate complex in the nucleoplasm. Reduction of NUP98 or RAE1 abolishes progenitors’ regenerative capacity, inhibiting proliferation and inducing premature terminal differentiation. Mechanistically, NUP98 binds on chromatin near the transcription start sites of key epigenetic regulators (such as DNMT1, UHRF1 and EZH2) and sustains their expression in progenitors. NUP98’s chromatin binding sites are co-occupied by HDAC1. HDAC inhibition diminishes NUP98’s chromatin binding and dysregulates NUP98 and RAE1’s target gene expression. Interestingly, HDAC inhibition further induces NUP98 and RAE1 to localize interdependently to the nucleolus. These findings identified a pathway in progenitor maintenance, where HDAC activity directs the high levels of NUP98 and RAE1 to directly control key epigenetic regulators, escaping from nucleolar aggregation

VEGF/Flk1 Signaling Cascade Transactivates <em>Etv2</em> Gene Expression

<div><p>Previous reports regarding the genetic hierarchy between Ets related protein 71 (Er71/Etv2) and Flk1 is unclear. In the present study, we pursued a genetic approach to define the molecular cascade between Etv2 and Flk1. Using a transgenic Etv2-EYFP reporter mouse, we examined the expression pattern of Etv2 relative to Flk1 in the early conceptus. Etv2-EYFP was expressed in subset of Flk1 positive cells during primitive streak stages, suggesting that Flk1 is upstream of Etv2 during gastrulation. Analysis of reporter gene expression in Flk1 and Etv2 mutant mice further supports the hypothesis that Flk1 is necessary for Etv2 expression. The frequency of cells expressing Flk1 in Etv2 mutants is only modestly altered (21% decrease), whereas expression of the Etv2-EYFP transgenic reporter was severely reduced in the Flk1 null background. We further demonstrate using transcriptional assays that, in the presence of Flk1, the Etv2 promoter is activated by VEGF, the Flk1 ligand. Pharmacological inhibition studies demonstrate that VEGF mediated activation is dependent on p38 MAPK, which activates Creb. We identify the VEGF response element in the Etv2 promoter and demonstrate that Creb binds to this motif by EMSA and ChIP assays. In summary, we provide new evidence that VEGF activates Etv2 by signaling through Flk1, which activates Creb through the p38 MAPK signaling cascade.</p> </div

VEGF activates the Etv2 promoter.

<p>(A–B) Transcriptional assays using an empty luciferase vector or the 3.9 kb Etv2 luciferase reporter standardized to the CMV-Renilla Luciferase reporter. Flk1 cDNA or a balancer DNA plasmid is co-transfected and the cells are treated with or without VEGF (50 ng/ml) for 2–18 hours as indicated in the panel (A). Flk1 or a balancer DNA is cotransfected and cells are treated for 6 hours with varying amounts of VEGF (0–50 ng/ml) as indicated in the panel (B). (C–D) Transcriptional inhibition assays in which Flk1 cDNA and the 3.9 kb Etv2 Luciferase promoter are co-transfected followed by pretreatment of various doses of SB202190 (C), or GF109203X (D) and a 6 hour treatment of 50 ng/ml VEGF. Data were analyzed by two-way anova (A,C,D) or one-way anova (B). n.s.: not significant; *: p<0.05; ***: p<0.001; ****: p<0.0001.</p

VEGF activation of the Etv2 promoter is dependent on a Creb binding site.

<p>(A) A schematic of the 3.9 kb Etv2 luciferase reporter showing 3 evolutionary conserved Creb binding motifs (CRE1-3) and the mutations schematized in panel B. Numbers reflect genomic position relative to the translational start site of Etv2. (B) A schematic of the truncation and mutation strategy. × indicates a CG to AA mutation of either CRE1 or CRE2. Numbers indicate genomic position relative to the translational start site of Etv2. (C) Transcriptional assays using the constructs shown in B co-transfected with Flk1 cDNA and treated with or without VEGF. (D) Transcriptional assay using the 1 kb Etv2 luciferase cotransfected with increasing amounts of expression vector encoding constitutively phosphorylated Creb cDNA. Data were analyzed using two-way anova (C) or Kruskal-Wallis nonparametric t-test with Dunn’s post test comparison (D). n.s.: not significant; *: p<0.05; **: p<0.01; ****: p<0.0001.</p

Flk1 expression precedes Etv2 expression during embryogenesis.

<p>(A) Diagram of the transgenic construct used in this study. Numbers shown identify the genomic location relative to the translation start site of Etv2. CR-1 represents conserved region one and CR-2 represents conserved region two. (B) Schematic diagram of the embryonic axes and germ layers of a late streak stage embryo. Black lines indicate approximate levels of sections in C. (C) A series of transverse sections of a late streak stage embryo. Representative sections were stained with antibodies to Brachyury (Bry), Flk1 and EYFP (scale bar: 100 microns).</p

Creb1 binds to the CRE2 motif in the Etv2 promoter.

<p>(A–B) Creb1 specifically interacts with the CRE2 motif in the Etv2 promoter as shown by ChIP assay. EBs were collected 3.5 days after initiating mesoderm differentiation. A region in the Gapdh gene was used as a negative control (Control) and 1% of the total chromatin DNA before the immunoprecipitation was used as a positive control (Input). The PCR products were run on a gel for direct visualization (A) and qPCR was performed for quantitation (B). (C) Creb1 binds CRE2 directly <i>in vitro</i>. Creb1 was synthesized <i>in vitro</i>. The oligonucleotides harboring CRE2 motif is labeled with 32P. Synthesized Creb1, non-radioactive oligonucleotides, and the antibodies were incubated with radioactive oligonucleotide probes as indicated in the figure. The interaction between Creb1 and the CRE2 motif was analyzed on a 4% TBE gel.</p

Etv2 is coexpressed with Flk1.

<p>(A–Q) Expression analysis of Etv2-EYFP and Flk1 in E7.0 to E9.5 embryos. Sections were stained with the GFP antibody (A, B, D, E, G–I, K, L, N, P; shown in green) or the Flk1 antibody (A, C, D, F–H, J, K, M, O, Q; shown in red). DAPI nuclear staining is shown in blue (N–Q). Yellow indicates overlap of green and red channels (A, D, G, H, K). A–C: No bud stage. D–F: Early-bud stage. G–M: Early head-fold stage. N–Q: E9.5. The boxed area in A is enlarged in B–C; the boxed area in D is enlarged in E–F; the boxed area in G is enlarged in H–J, and the boxed area in K is enlarged in L–M. Arrowheads in B, C point to progenitors of blood islands coexpressing Flk1 and EYFP. Arrows and arrowheads in H–M indicate endothelial lineages co-expressing Flk1 and EYFP. Asterisks in A, H–M indicate regions in which either gene is expressed alone. Arrowheads in P indicate developing intersomitic vessels. Structures are designated as follows (al: allantois, bi: blood island, da: dorsal aorta, h: heart, ec: endocardium, hf: head fold, nt: neural tube, ph: pharynx, s: somite). Bars in A, D, G, K, N, and P indicate 200 µm. Bars in C, F, J, and M indicate 50 µm.</p

ETV2 primes hematoendothelial gene enhancers prior to hematoendothelial fate commitment

Summary: Mechanisms underlying distinct specification, commitment, and differentiation phases of cell fate determination remain undefined due to difficulties capturing these processes. Here, we interrogate the activity of ETV2, a transcription factor necessary and sufficient for hematoendothelial differentiation, within isolated fate intermediates. We observe transcriptional upregulation of Etv2 and opening of ETV2-binding sites, indicating new ETV2 binding, in a common cardiac-hematoendothelial progenitor population. Accessible ETV2-binding sites are active at the Etv2 locus but not at other hematoendothelial regulator genes. Hematoendothelial commitment coincides with the activation of a small repertoire of previously accessible ETV2-binding sites at hematoendothelial regulators. Hematoendothelial differentiation accompanies activation of a large repertoire of new ETV2-binding sites and upregulation of hematopoietic and endothelial gene regulatory networks. This work distinguishes specification, commitment, and sublineage differentiation phases of ETV2-dependent transcription and suggests that the shift from ETV2 binding to ETV2-bound enhancer activation, not ETV2 binding to target enhancers, drives hematoendothelial fate commitment