OCT4/POU5F1 is indispensable for the lineage differentiation of the inner cell mass in bovine embryos

The mammalian blastocyst undergoes two lineage segregations, that is, formation of the trophectoderm and subsequently differentiation of the hypoblast (HB) from the inner cell mass, leaving the epiblast (EPI) as the remaining pluripotent lineage. To clarify the expression patterns of markers specific for these lineages in bovine embryos, we analyzed day 7, 9, and 12 blastocysts completely produced in vivo by staining for OCT4, NANOG, SOX2 (EPI), and GATA6, SOX17 (HB) and identified genes specific for these developmental stages in a global transcriptomics approach. To study the role of OCT4, we generated OCT4-deficient (OCT4 KO) embryos via somatic cell nuclear transfer or in vitro fertilization. OCT4 KO embryos reached the expanded blastocyst stage by day 8 but lost NANOG and SOX17 expression, while SOX2 and GATA6 were unaffected. Blastocysts transferred to recipient cows from day 6 to 9 expanded, but the OCT4 KO phenotype was not rescued by the uterine environment. Exposure of OCT4 KO embryos to exogenous FGF4 or chimeric complementation with OCT4 intact embryos did not restore NANOG or SOX17 in OCT4-deficient cells. Our data show that OCT4 is required cell autonomously for the maintenance of pluripotency of the EPI and differentiation of the HB in bovine embryos

Allelic Imbalance of Recurrently Mutated Genes in Acute Myeloid Leukaemia

The patho-mechanism of somatic driver mutations in cancer usually involves transcription, but the proportion of mutations and wild-type alleles transcribed from DNA to RNA is largely unknown. We systematically compared the variant allele frequencies of recurrently mutated genes in DNA and RNA sequencing data of 246 acute myeloid leukaemia (AML) patients. We observed that 95% of all detected variants were transcribed while the rest were not detectable in RNA sequencing with a minimum read-depth cut-off (10x). Our analysis focusing on 11 genes harbouring recurring mutations demonstrated allelic imbalance (AI) in most patients. GATA2, RUNX1, TET2, SRSF2, IDH2, PTPN11, WT1, NPM1 and CEBPA showed significant AIs. While the effect size was small in general, GATA2 exhibited the largest allelic imbalance. By pooling heterogeneous data from three independent AML cohorts with paired DNA and RNA sequencing (N = 253), we could validate the preferential transcription of GATA2-mutated alleles. Differential expression analysis of the genes with significant AI showed no significant differential gene and isoform expression for the mutated genes, between mutated and wild-type patients. In conclusion, our analyses identified AI in nine out of eleven recurrently mutated genes. AI might be a common phenomenon in AML which potentially contributes to leukaemogenesis.Peer reviewe

Monoubiquitination of Ancient Ubiquitous Protein 1 Promotes Lipid Droplet Clustering

<div><p>Lipid droplets, the intracellular storage organelles for neutral lipids, exist in a wide range of sizes and of morphologically distinct organization, from loosely dispersed lipid droplets to tightly packed lipid droplet clusters. We show that the lipid droplet protein AUP1 induces cluster formation. A fraction of AUP1 is monoubiquitinated at various lysine residues. This process depends on its internal CUE domain, which is a known ubiquitin-binding domain. AUP1 with a deleted or point mutagenized CUE domain, as well as a lysine-free mutant, are not ubiquitinated and do not induce lipid droplet clustering. When such ubiquitination deficient mutants are fused to ubiquitin, clustering is restored. AUP1 mutants with defective droplet targeting fail to induce clustering. Also, another lipid droplet protein, NSDHL, with a fused ubiquitin does not induce clustering. The data indicate that monoubiquitinated AUP1 on the lipid droplet surface specifically induces clustering, and suggest a homophilic interaction with a second AUP1 molecule or a heterophilic interaction with another ubiquitin-binding protein.</p></div

Knockdown of AUP1 causes declustering of LDs.

<p>A) A431 cells were either mock transfected or transfected with one of three different siRNAs against AUP1. Cells were lysed and proteins separated by SDS-PAGE and immunoblotted with anti-AUP1 antibody. GAPDH served as loading control. B) Fluorescence micrographs of mock transfected (control) or siRNA treated (siRNA3) A431 cells, both grown in medium supplemented with 50 µM oleate. Cells were immunostained with anti-AUP1 antibody (AUP1, left), and LD540 (LDs, middle panels). Merged images (right) show nuclei stained by DAPI in blue, AUP1 in red and LDs in green. Bars, 10 µm. C) Quantification of LD clustering in mock- (control) or siRNA- (as indicated) treated A431 cells. Results are displayed as average ± standard deviation of three independent experiments. For each individual experiment at least 25 cells were analyzed.</p

The AUP1 CUE domain is important for LD clustering.

<p>A–C) COS7 cells were transfected with different HA-tagged AUP1 domain deletion or mutation constructs as indicated and grown in medium supplemented with 50 µM oleate. Cells were immunostained with anti-HA antibody (left), and LD540 (LDs, middle panels). Merged images (right) show nuclei stained by DAPI in blue, AUP1 in red and LDs in green. Bars, 10 µm. D) Quantification of LD clustering in COS7 cells overexpressing HA-tagged AUP1 constructs as indicated. Empty vector was used as control. Results are displayed as average ± standard deviation of three independent experiments. For each individual experiment at least 25 cells were analyzed. F) Expression levels of HA-tagged AUP1 constructs. Proteins from COS7 cells overexpressing HA-tagged AUP1 constructs as indicated were separated by SDS-PAGE and immunoblotted with anti-HA antibody. GAPDH served as loading control. Note: AUP1-ΔCUE-HA and AUP1-mutCUE3-HA migrated at an apparent molecular weight around five kDa higher than expected. E) Amino acid sequence of the AUP1 CUE domain and predicted relative position of the three α-helices after Prag <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072453#pone.0072453-Prag1" target="_blank">[34]</a>. Mutated amino acid residues of the three AUP1-mutCUE constructs used in this work are highlighted in grey.</p

AUP1 monoubiquitination is sufficient to promote LD clustering.

<p>A) COS7 cells were transfected with HA-tagged AUP1 mutation constructs (as indicated), fused to monoubiquitin and grown in medium supplemented with 50 µM oleate. Cells were immunostained with anti-HA antibody (left), and LD540 (LDs, middle panels). Merged images (right) show nuclei stained by DAPI in blue, AUP1 in red and LDs in green. Bars, 10 µm. B) COS7 cells were transfected with HA-tagged NSDHL with or without a fused monoubiquitin as indicated and grown in medium supplemented with 50 µM oleate. Cells were immunostained with anti-HA antibody (left), and LD540 (LDs, middle panels). Merged images (right) show nuclei stained by DAPI in blue, AUP1 in red and LDs in green. Bars, 10 µm. C) Quantification of LD clustering in COS7 cells overexpressing HA-tagged AUP1 fusion constructs (as indicated). Results are displayed as average ± standard deviation of three independent experiments. For each individual experiment at least 25 cells were analyzed.</p

Possible mechanisms for AUP1 induced LD clustering.

<p>From left to right: Type 1: monoubiquitinated AUP1 dimerizes <i>in trans</i> with another (1A) ubiquitinated or (1B) non-ubiquitinated AUP1 by binding between the ubiquitin moieties and the CUE domains. Type 2: Monoubiquitinated AUP1 interacts <i>in trans</i> with another LD protein containing a ubiquitin-binding domain (UBD). Type 3: Interaction of type 1 or 2, but mediated by a soluble adaptor protein.</p

AUP1 is ubiquitinated.

<p>A) COS7 cells were transfected with His-ubiquitin and HA-tagged AUP1 constructs and controls (as indicated). His-ubiquitin and His-ubiquitin modified proteins were isolated from cell lysates using Ni-NTA agarose. Proteins from lysates (10% input, left panel) and His-purifications (right panel) were separated by SDS-PAGE and immunoblotted with anti-HA antibody. B, C) COS7 cells were transfected with His-ubiquitin and HA-tagged AUP1 constructs (as indicated). Samples were processed as under A). D) Quantification of LD clustering in COS7 cells overexpressing HA-tagged Lys to Arg mutation AUP1 constructs (as indicated). Results are displayed as average ± standard deviation of three independent experiments. For each individual experiment at least 25 cells were analyzed. E) COS7 cells were transfected with a construct expressing AUP1-10KR-HA and grown in medium supplemented with 50 µM oleate. Cells were immunostained with anti-HA antibody (left), and LD540 (LDs, middle panel). The merged image (right) shows nuclei stained by DAPI in blue, AUP1 in red and LDs in green. Bars, 10 µm.</p

AUP1 overexpression causes LD clustering.

<p>A–C) COS7 cells were transfected with empty control vector (control) or different HA-tagged AUP1 constructs as indicated and grown in medium supplemented with 50 µM oleate. Cells were immunostained with anti-HA antibody (left), and LD540 (LDs, middle panels). Merged images (right) show nuclei stained by DAPI in blue, AUP1 in red and LDs in green. Bars, 10 µm. COS7 cells not expressing AUP1-HA do not show LD clustering (marked by asterisk (*)).</p