Understanding mechanisms of epigenetic regulation in embryonic stem cells (ESCs) is of fundamental importance for stem cell and developmental biology. Here, we identify
, a member of the ETS family ...of transcription factors (TFs), as a marker of ground state pluripotency. We show that
is rapidly induced in ground state ESCs and in response to extracellular signal-regulated kinase (ERK) inhibition. We find that SPIC binds to enhancer elements and stabilizes NANOG binding to chromatin, particularly at genes involved in choline/one-carbon (1C) metabolism such as
,
, and
. Gain-of-function and loss-of-function experiments revealed that
controls 1C metabolism and the flux of
-adenosyl methionine to
-adenosyl-L-homocysteine (SAM-to-SAH), thereby, modulating the levels of H3R17me2 and H3K4me3 histone marks in ESCs. Our findings highlight betaine-dependent 1C metabolism as a hallmark of ground state pluripotency primarily activated by SPIC. These findings underscore the role of uncharacterized auxiliary TFs in linking cellular metabolism to epigenetic regulation in ESCs.
Ubiquitin-specific protease 7 (USP7) has been implicated in cancer progression and neurodevelopment. However, its molecular targets remain poorly characterized. We combined quantitative proteomics, ...transcriptomics, and epigenomics to define the core USP7 network. Our multi-omics analysis reveals USP7 as a control hub that links genome regulation, tumor suppression, and histone H2A ubiquitylation (H2AK119ub1) by noncanonical Polycomb-repressive complexes (ncPRC1s). USP7 strongly stabilizes ncPRC1.6 and, to a lesser extent, ncPRC1.1. Moreover, USP7 represses expression of AUTS2, which suppresses H2A ubiquitylation by ncPRC1.3/5. Collectively, these USP7 activities promote the genomic deposition of H2AK119ub1 by ncPRC1, especially at transcriptionally repressed loci. Notably, USP7-dependent changes in H2AK119ub1 levels are uncoupled from H3K27me3. Even complete loss of the PRC1 catalytic core and H2AK119ub1 has only a limited effect on H3K27me3. Besides defining the USP7 regulome, our results reveal that H2AK119ub1 dosage is largely disconnected from H3K27me3.
Developmental biology. A Me6Age for pluripotency Stunnenberg, Hendrik G; Vermeulen, Michiel; Atlasi, Yaser
Science (American Association for the Advancement of Science),
2015-Feb-06, 20150206, Letnik:
347, Številka:
6222
Journal Article
BACKGROUND: EGFR is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumor types have different mutation spectra. It is possible ...that functional differences underlie this tumor-type specific mutation spectrum. METHODS: We have determined whether specific mutations in EGFR (EGFR, EGFRvIII and EGFR-L858R) have differences in binding partners, differences in downstream pathway activation (gene expression and phosphoproteins), and have functional consequences on cellular growth and migration. RESULTS: Using biotin pulldown and subsequent mass spectrometry we were able to detect mutation specific binding partners for EGFR. Differential binding was confirmed using a proximilty ligation assay and/or Western Blot for the dedicator of cytokinesis 4 (DOCK4), UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), MYC binding protein 2 (MYCBP2) and Smoothelin (SMTN). We also demonstrate that each mutation induces the expression of a specific set of genes, and that each mutation is associated with specific phosphorylation patterns. Finally, we demonstrate using stably expressing cell lines that EGFRvIII and EGFL858R display reduced growth and migration compared to EGFR wildtype expressing cells. CONCLUSION: Our results indicate that there are distinct functional differences between different EGFR mutations. The functional differences between different mutations argue for the development of mutation specific targeted therapies.
Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous ...polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.
BACKGROUND: EGFR is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumor types have different mutation spectra. It is possible ...that functional differences underlie this tumor-type specific mutation spectrum. METHODS: We have determined whether specific mutations in EGFR (EGFR, EGFRvIII and EGFR-L858R) have differences in binding partners, differences in downstream pathway activation (gene expression and phosphoproteins), and have functional consequences on cellular growth and migration. RESULTS: Using biotin pulldown and subsequent mass spectrometry we were able to detect mutation specific binding partners for EGFR. Differential binding was confirmed using a proximilty ligation assay and/or Western Blot for the dedicator of cytokinesis 4 (DOCK4), UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), MYC binding protein 2 (MYCBP2) and Smoothelin (SMTN). We also demonstrate that each mutation induces the expression of a specific set of genes, and that each mutation is associated with specific phosphorylation patterns. Finally, we demonstrate using stably expressing cell lines that EGFRvIII and EGFL858R display reduced growth and migration compared to EGFR wildtype expressing cells. CONCLUSION: Our results indicate that there are distinct functional differences between different EGFR mutations. The functional differences between different mutations argue for the development of mutation specific targeted therapies.
Survivin, an inhibitor of apoptosis protein (IAP), has been regarded as a valuable tumor marker for diagnosis and prognosis of bladder cancer. Recently, three splice variants of the gene with ...different anti-apoptotic activities have been reported. However, there is no data on the expression and potential causative roles of these transcripts in bladder cancer. Here, we have investigated the expression pattern of survivin and two of its splice variants, survivin-DeltaEx3 and survivin-2B, in malignant versus non-malignant bladder tissues.
We used semi-quantitative RT-PCR analysis to examine the expression of survivin variants in 30 transitional cell carcinoma, 19 matched non-tumor, and 9 apparently healthy control tissue samples of the bladder. DNA sequencing was used to confirm the identity of amplified fragments.
For all examined samples, survivin was the predominant transcript, being present in 83% of tumor and 25% of non-tumor bladder tissue samples, and survivin-2B was the least detected isoform. The expression levels of survivin and survivin-DeltaEx3 was significantly higher in neoplastic compared to non-neoplastic tissues (P<0.001) and both the sensitivity and specificity of survivin were superior to survivin-DeltaEx3 (83% and 75% for survivin and 76% and 64% for survivin-DeltaEx3, respectively). Also, the expression levels of survivin and survivin-DeltaEx3 showed a significant correlation (P=0.02) with tumor invasiveness (pT1/pTa).
Our data revealed for the first time a differential expression pattern of survivin splice variants in bladder tissues, which potentially could have a practical usefulness in diagnosis and/or therapy of the tumor.
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