scholarly journals OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development

2017 ◽  
Author(s):  
Joel Hrit ◽  
Cheng Li ◽  
Elizabeth Allene Martin ◽  
Mary Goll ◽  
Barbara Panning
Keyword(s):  
Stem Cell ◽  
Cell Formation ◽  
Nutrient Status ◽  
Hematopoetic Stem Cell ◽  
Intracellular Proteins ◽  
Tet Enzymes ◽  
And Function ◽  
First Time

AbstractMammalian TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs are targets of the enzyme OGT, which post-translationally modifies intracellular proteins in response to cellular nutrient status. The biological implications of the OGT-TET interaction have not been thoroughly explored. Here, we show for the first time that modification of TET1 by OGT enhances its activity in vitro. We identify a previously uncharacterized domain of TET1 responsible for binding to OGT and report a point mutation that disrupts the OGT-TET1 interaction. Finally, we show that the interaction between TET1 and OGT is necessary for TET1 to rescue tet mutant zebrafish hematopoetic stem cell formation, suggesting that OGT promotes TET1’s function in development. Our results demonstrate regulation of TET activity by OGT in vitro and in vivo. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

scholarly journals OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Joel Hrit ◽  
Leeanne Goodrich ◽  
Cheng Li ◽  
Bang-An Wang ◽  
Ji Nie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Stem ◽  
Nutrient Sensing ◽  
Zebrafish Embryos ◽  
Hematopoetic Stem Cell ◽  
Tet Enzymes ◽  
And Function ◽  
Necessary And Sufficient ◽  
First Time

TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a TET1 domain that is necessary and sufficient for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that this interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1’s function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET2 and 5-methylcytosine, which is accompanied by alterations in gene expression. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

scholarly journals OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development

2018 ◽  
Author(s):  
Joel Hrit ◽  
Cheng Li ◽  
Elizabeth Allene Martin ◽  
Eric Simental ◽  
Mary Goll ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Weight ◽  
Embryonic Stem ◽  
Nutrient Sensing ◽  
Zebrafish Embryos ◽  
Hematopoetic Stem Cell ◽  
Tet Enzymes ◽  
And Function ◽  
First Time

AbstractTET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a domain of TET1 responsible for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that the TET1-OGT interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1’s function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET-containing high molecular weight complexes and causes widespread gene expression changes. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

scholarly journals Dickkopf-1 Inhibition Reactivates Wnt/β-Catenin Signaling in Rhabdomyosarcoma, Induces Myogenic Markers In Vitro and Impairs Tumor Cell Survival In Vivo

2021 ◽  
Vol 22 (23) ◽  
pp. 12921
Author(s):  
Irina Giralt ◽  
Gabriel Gallo-Oller ◽  
Natalia Navarro ◽  
Patricia Zarzosa ◽  
Guillem Pons ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Positive Effects ◽  
Novel Therapeutic Strategies ◽  
And Function ◽  
Myogenic Markers ◽  
First Time ◽  
Dickkopf 1 ◽  
Proliferation And Invasion

The Wnt/β-catenin signaling pathway plays a pivotal role during embryogenesis and its deregulation is a key mechanism in the origin and progression of several tumors. Wnt antagonists have been described as key modulators of Wnt/β-catenin signaling in cancer, with Dickkopf-1 (DKK-1) being the most studied member of the DKK family. Although the therapeutic potential of DKK-1 inhibition has been evaluated in several diseases and malignancies, little is known in pediatric tumors. Only a few works have studied the genetic inhibition and function of DKK-1 in rhabdomyosarcoma. Here, for the first time, we report the analysis of the therapeutic potential of DKK-1 pharmaceutical inhibition in rhabdomyosarcoma, the most common soft tissue sarcoma in children. We performed DKK-1 inhibition via shRNA technology and via the chemical inhibitor WAY-2626211. Its inhibition led to β-catenin activation and the modulation of focal adhesion kinase (FAK), with positive effects on in vitro expression of myogenic markers and a reduction in proliferation and invasion. In addition, WAY-262611 was able to impair survival of tumor cells in vivo. Therefore, DKK-1 could constitute a molecular target, which could lead to novel therapeutic strategies in RMS, especially in those patients with high DKK-1 expression.

Constructing stem cell microenvironments using bioengineering approaches

2013 ◽  
Vol 45 (23) ◽  
pp. 1123-1135 ◽  
Author(s):  
David A. Brafman
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Disease Modeling ◽  
Mechanical Stimuli ◽  
Culture Methods ◽  
Stem Cell Fate ◽  
And Function

Within the adult organism, stem cells reside in defined anatomical microenvironments called niches. These architecturally diverse microenvironments serve to balance stem cell self-renewal and differentiation. Proper regulation of this balance is instrumental to tissue repair and homeostasis, and any imbalance can potentially lead to diseases such as cancer. Within each of these microenvironments, a myriad of chemical and physical stimuli interact in a complex (synergistic or antagonistic) manner to tightly regulate stem cell fate. The in vitro replication of these in vivo microenvironments will be necessary for the application of stem cells for disease modeling, drug discovery, and regenerative medicine purposes. However, traditional reductionist approaches have only led to the generation of cell culture methods that poorly recapitulate the in vivo microenvironment. To that end, novel engineering and systems biology approaches have allowed for the investigation of the biological and mechanical stimuli that govern stem cell fate. In this review, the application of these technologies for the dissection of stem cell microenvironments will be analyzed. Moreover, the use of these engineering approaches to construct in vitro stem cell microenvironments that precisely control stem cell fate and function will be reviewed. Finally, the emerging trend of using high-throughput, combinatorial methods for the stepwise engineering of stem cell microenvironments will be explored.

scholarly journals Formation and Function of the Rbl2p–β-Tubulin Complex

1998 ◽  
Vol 18 (3) ◽  
pp. 1757-1762 ◽  
Author(s):  
Julie E. Archer ◽  
Margaret Magendantz ◽  
Leticia R. Vega ◽  
Frank Solomon
Keyword(s):  
Half Life ◽  
Cell Formation ◽  
Cellular Mechanism ◽  
Wild Type ◽  
Yeast Protein ◽  
And Function ◽  
Β Tubulin

ABSTRACT The yeast protein Rbl2p suppresses the deleterious effects of excess β-tubulin as efficiently as does α-tubulin. Both in vivo and in vitro, Rbl2p forms a complex with β-tubulin that does not contain α-tubulin, thus defining a second pool of β-tubulin in the cell. Formation of the complex depends upon the conformation of β-tubulin. Newly synthesized β-tubulin can bind to Rbl2p before it binds to α-tubulin. Rbl2p can also bind β-tubulin from the α/β-tubulin heterodimer, apparently by competing with α-tubulin. The Rbl2p–β-tubulin complex has a half-life of ∼2.5 h and is less stable than the α/β-tubulin heterodimer. The results of our experiments explain both how excess Rbl2p can rescue cells overexpressing β-tubulin and how it can be deleterious in a wild-type background. They also suggest that the Rbl2p–β-tubulin complex is part of a cellular mechanism for regulating the levels and dimerization of tubulin chains.

scholarly journals Expression of Stem Cell Markers in Primo Vessel of Rat

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Eun Seok Park ◽  
Jeong Hoon Lee ◽  
Won Jin Kim ◽  
Jinbeom Heo ◽  
Dong Myung Shin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pcr Analysis ◽  
Acupuncture Points ◽  
Stem Cell Markers ◽  
Molecular Features ◽  
Detection Frequency ◽  
Liver Surface ◽  
First Time

Accumulating line of evidence support that adult tissues contain a rare population of pluripotent stem cells (PSCs), which differentiate into all types of cells in our body. Bonghan microcell (primo microcells (PMCs)) discovered in 1960s was reported to have a pluripotency like a stem cellin vivoas well asin vitrocondition. Here, we describe the detailed morphology and molecular features of PMCs. PMCs reside in Bonghan duct (primo vessel (PV)) reported as a corresponding structure of acupuncture points and meridian system. We found that PMCs were frequently observed in the liver surface of the rat between 300 g and 400 g from April to June, suggesting that the their detection frequency depends on the weight, the season, and the organ of rat. As reported, PMCs freshly isolated from PVs were spherical ~1-2 μm microsized cells. In contrast, a unique bithread or budding-shaped PMCs emerged during tissue culture around 8 days. RT-PCR analysis demonstrated that PVs-derived cells express theOct4, the most important PSCs gene, in addition to several PSCs markers (Sox2,Stella,Rex1, andKlf4). Thus, we for the first time provide the evidence about Oct4-expressing stem-like characteristics for cells resident in PVs, a possible novel stem cell enriched niche.

scholarly journals USP7 as part of non-canonical PRC1.1 is a druggable target in leukemia

2017 ◽  
Author(s):  
Henny Maat ◽  
Jennifer Jaques ◽  
Aida Rodríguez López ◽  
Shanna M. Hogeling ◽  
Marcel P. de Vries ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Therapeutic Approach ◽  
Human Leukemia ◽  
List Type ◽  
Essential Component ◽  
Poor Risk ◽  
And Function

ABSTRACTAcute myeloid leukemia (AML) is a highly heterogeneous disease in which genetic and epigenetic changes disturb regulatory mechanisms controlling stem cell fate and maintenance. AML still remains difficult to treat, in particular in poor risk AML patients carrying TP53 mutations. Here, we identify the deubiquitinase USP7 as an integral member of non-canonical PRC1.1 and show that targeting of USP7 provides an alternative therapeutic approach for AML. USP7 inhibitors effectively induced apoptosis in (primary) AML cells, also independent of the USP7-MDM2-TP53 axis, whereby survival of both the cycling as well as quiescent populations was affected. MLL-AF9-induced leukemia was significantly delayed in vivo in human leukemia xenografts. We previously showed that non-canonical PRC1.1 is critically important for leukemic stem cell self-renewal, and that genetic knockdown of the PRC1.1 chromatin binding component KDM2B abrogated leukemia development in vitro and in vivo [1]. Here, by performing KDM2B interactome studies in TP53mut cells we identify that USP7 is an essential component of PRC1.1 and is required for its stability and function. USP7 inhibition results in disassembly of the PRC1.1 complex and consequently loss of binding to its target loci. Loss of PRC1.1 binding coincided with reduced H2AK119ub and H3K27ac levels and diminished gene transcription, whereas H3K4me3 levels remained unaffected. Our studies highlight the diverse functions of USP7 and link it to Polycomb-mediated epigenetic control. USP7 inhibition provides an efficient therapeutic approach for AML, also in the most aggressive subtypes with mutations in TP53.Key pointsUSP7 is a therapeutic target in leukemia, including poor risk TP53mut AML.USP7 is an essential component of non-canonical PRC1.1 and is required for its stability and function.

scholarly journals Aurora B Kinase Regulates the Postmitotic Endoreduplication Checkpoint via Phosphorylation of the Retinoblastoma Protein at Serine 780

2009 ◽  
Vol 20 (8) ◽  
pp. 2218-2228 ◽  
Author(s):  
Jayasree S. Nair ◽  
Alan L. Ho ◽  
Archie N. Tse ◽  
Jesse Coward ◽  
Haider Cheema ◽  
...  
Keyword(s):  
Critical Role ◽  
Cell Formation ◽  
Polyploid Cell ◽  
Phenotypic Change ◽  
Aurora B ◽  
Cyclin Dependent Kinase ◽  
Molecule Inhibitor ◽  
First Time

The phenotypic change characteristic of Aurora B inhibition is the induction of polyploidy. Utilizing specific siRNA duplexes and a selective small molecule inhibitor (AZD1152) to inhibit Aurora B activity in tumor cells, we sought to elucidate the mechanism by which Aurora B inhibition results in polyploidy. Cells treated with AZD1152 progressed through mitosis with misaligned chromosomes and exited without cytokinesis and subsequently underwent endoreduplication of DNA despite activation of a p53-dependent pseudo G1 checkpoint. Concomitant with polyploid cell formation, we observed the appearance of Rb hypophosphorylation, an event that occurred independently of cyclin-dependent kinase inhibition. We went on to discover that Aurora B directly phosphorylates Rb at serine 780 both in vitro and in vivo. This novel interaction plays a critical role in regulating the postmitotic checkpoint to prevent endoreduplication after an aberrant mitosis. Thus, we propose for the first time that Aurora B determines cellular fate after an aberrant mitosis by directly regulating the Rb tumor suppressor protein.

scholarly journals TAp63α targeting of Lgr5 mediates colorectal cancer stem cell properties and sulforaphane inhibition

Oncogenesis ◽  
2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Yue Chen ◽  
Meng-huan Wang ◽  
Jian-yun Zhu ◽  
Chun-feng Xie ◽  
Xiao-ting Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Stem Cell ◽  
Cancer Progression ◽  
Inhibitory Effect ◽  
P53 Family ◽  
First Time ◽  
Established Role

Abstract Cancer stem cells (CSCs) have an established role in cancer progression and therapeutic resistance. The p63 proteins are important transcription factors which belong to the p53 family, but their function and mechanism in CSCs remain elusive. Here, we investigated the role of TAp63α in colorectal CSCs and the effects of sulforaphane on TAp63α. We found that TAp63α was upregulated in spheres with stem cell properties compared to the parental cells. Overexpression of TAp63α promoted self-renewal capacity and enhanced CSC markers expression in colorectal sphere-forming cells. Furthermore, we showed that TAp63α directly bound to the promoter region of Lgr5 to enhance its expression and activate its downstream β-catenin pathway. Functional experiments revealed that sulforaphane suppressed the stemness of colorectal CSCs both in vitro and in vivo. Upregulation of TAp63α attenuated the inhibitory effect of sulforaphane on colorectal CSCs, indicating the role of TAp63α in sulforaphane suppression of the stemness in colorectal cancer. The present study elucidated for the first time that TAp63α promoted CSCs through targeting Lgr5/β-catenin axis and participated in sulforaphane inhibition of the stem cell properties in colorectal cancer.

CD8+/TCR− Graft Facilitating Cells Enhance HSC Engraftment and Survival Via TNF-α Mediated Prevention of Apoptosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1352-1352
Author(s):  
Suzanne T. Ildstad ◽  
Francine Rezzoug ◽  
Yiming Huang ◽  
Marcin Wysoczynski ◽  
Carrie L. Schanie ◽  
...  
Keyword(s):  
Stem Cell ◽  
Regulatory Effect ◽  
Hematopoietic Stem ◽  
Accessory Cells ◽  
Tnf Α ◽  
Apoptotic Effect ◽  
Low Levels ◽  
First Time

Abstract The use of accessory cells to enhance hematopoietic stem cell (HSC) engraftment could have a significant therapeutic impact, especially when stem cell numbers are limited. The bone marrow (BM) microenvironment is involved in regulation of HSC, allowing production of mature blood cells while maintaining HSC self renewal. To date, the precise identity of specific cells in the microenvironment that exert this regulatory effect on HSC has not been defined. We recently reported that CD8+/TCR− facilitating cells (FC), a subpopulation of BM cells containing predominantly B220+/CD11c+/CD11b− tolerogenic precursor-plasmacytoid dendritic cells, enhance HSC engraftment in allogeneic recipients. Additionally, FC significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. In the present studies, we investigated the mechanism of FC-mediated enhancement of HSC engraftment. We show for the first time that FC significantly increase HSC survival in vitro and exert an anti-apoptotic effect on HSC via TNF-α. Co-culture of FC with HSC induces production of physiologically relevant low levels of TNF-α by FC. FC from TNF-α−/− mice are impaired in function in vitro and in facilitating HSC engraftment in vivo. Furthermore, neutralization of TNF-α on FC using anti-TNF antibody results in loss of FC function in vitro, confirming a major role for TNF-α in FC function. Notably, co-culture of FC with HSC prevents HSC apoptosis and is associated with significant upregulation of the anti-apoptotic I-κB family member Bcl-3 in HSC. Blocking of TNF-α on FC abrogates the anti-apoptotic effect of FC on HSC and prevents upregulation of Bc1-3 in HSC. Taken together, these findings demonstrate that TNF-α-induced in FC affects highly primitive HSC and identify Bcl-3 as a possible pathway for TNF-α in regulating HSC survival.

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