scholarly journals In Vitro Histone Methyltransferase Assay

2008 ◽  
Vol 2008 (3) ◽  
pp. pdb.prot4939-pdb.prot4939 ◽  
Author(s):  
I. M. Fingerman ◽  
H.-N. Du ◽  
S. D. Briggs
Keyword(s):  
Histone Methyltransferase

scholarly journals Histone methyltransferase WHSC1 inhibits colorectal cancer cell apoptosis via targeting anti-apoptotic BCL2

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yu Wang ◽  
Liming Zhu ◽  
Mei Guo ◽  
Gang Sun ◽  
Kun Zhou ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Cell ◽  
Cell Apoptosis ◽  
B Cell Lymphoma ◽  
Histone Methyltransferase ◽  
Chemotherapeutic Agents ◽  
Cancer Cell Apoptosis ◽  
Active Transcription

AbstractWHSC1 is a histone methyltransferase that facilitates histone H3 lysine 36 dimethylation (H3K36me2), which is a permissive mark associated with active transcription. In this study, we revealed how WHSC1 regulates tumorigenesis and chemosensitivity of colorectal cancer (CRC). Our data showed that WHSC1 as well as H3K36me2 were highly expressed in clinical CRC samples, and high WHSC1 expression is associated with poorer prognosis in CRC patients. WHSC1 reduction promoted colon cancer cell apoptosis both in vivo and in vitro. We found that B cell lymphoma-2 (BCL2) expression, an anti-apoptotic protein, is markedly decreased in after WHSC1 depletion. Mechanistic characterization indicated that WHSC1 directly binds to the promoter region of BCL2 gene and regulate its H3K36 dimethylation level. What’s more, our study indicated that WHSC1 depletion promotes chemosensitivity in CRC cells. Together, our results suggested that WHSC1 and H3K36me2 modification might be optimal therapeutic targets to disrupt CRC progression and WHSC1-targeted therapy might potentially overcome the resistance of chemotherapeutic agents.

scholarly journals Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

2016 ◽  
Vol 37 (3) ◽  
pp. 301-307 ◽  
Author(s):  
Ana S.A. Cohen ◽  
Damian B. Yap ◽  
M.E. Suzanne Lewis ◽  
Chieko Chijiwa ◽  
Maria A. Ramos‐Arroyo ◽  
...  
Keyword(s):  
Histone Methyltransferase ◽  
Weaver Syndrome ◽  
Protein Variants

The Histone Methyltransferase EZH2 Adopts a Nuclear Localization During Cleavage in Porcine Embryos Fertilized In Vitro.

2010 ◽  
Vol 83 (Suppl_1) ◽  
pp. 304-304
Author(s):  
Celeste E. Morrison ◽  
K. Park ◽  
X. Wang ◽  
R. A. Cabot
Keyword(s):  
Nuclear Localization ◽  
Histone Methyltransferase

scholarly journals Transcriptional Repression by the Retinoblastoma Protein through the Recruitment of a Histone Methyltransferase

2001 ◽  
Vol 21 (19) ◽  
pp. 6484-6494 ◽  
Author(s):  
Laurence Vandel ◽  
Estelle Nicolas ◽  
Olivier Vaute ◽  
Roger Ferreira ◽  
Slimane Ait-Si-Ali ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Histone H3 ◽  
Histone Methyltransferase ◽  
S Phase ◽  
E2f Transcription Factor ◽  
Cycle Dependent

ABSTRACT The E2F transcription factor controls the cell cycle-dependent expression of many S-phase-specific genes. Transcriptional repression of these genes in G0 and at the beginning of G1by the retinoblasma protein Rb is crucial for the proper control of cell proliferation. Rb has been proposed to function, at least in part, through the recruitment of histone deacetylases. However, recent results indicate that other chromatin-modifying enzymes are likely to be involved. Here, we show that Rb also interacts with a histone methyltransferase, which specifically methylates K9 of histone H3. The results of coimmunoprecipitation experiments of endogenous or transfected proteins indicate that this histone methyltransferase is the recently described heterochromatin-associated protein Suv39H1. Interestingly, phosphorylation of Rb in vitro as well as in vivo abolished the Rb-Suv39H1 interaction. We also found that Suv39H1 and Rb cooperate to repress E2F activity and that Suv39H1 could be recruited to E2F1 through its interaction with Rb. Taken together, these data indicate that Suv39H1 is involved in transcriptional repression by Rb and suggest an unexpected link between E2F regulation and heterochromatin.

scholarly journals Coordinated Recruitment of Histone Methyltransferase G9a and Other Chromatin-Modifying Enzymes in SHP-Mediated Regulation of Hepatic Bile Acid Metabolism

2006 ◽  
Vol 27 (4) ◽  
pp. 1407-1424 ◽  
Author(s):  
Sungsoon Fang ◽  
Ji Miao ◽  
Lingjin Xiang ◽  
Bhaskar Ponugoti ◽  
Eckardt Treuter ◽  
...  
Keyword(s):  
Bile Acid ◽  
Histone Deacetylases ◽  
Trichostatin A ◽  
Critical Role ◽  
Histone Methyltransferase ◽  
Bile Acid Metabolism ◽  
Dependent Manner ◽  
H3k9 Methylation ◽  
Hepatic Bile

ABSTRACT SHP has been implicated as a pleiotropic regulator of diverse biological functions by its ability to inhibit numerous nuclear receptors. Recently, we reported that SHP inhibits transcription of CYP7A1, a key gene in bile acid biosynthesis, by recruiting histone deacetylases (HDACs) and a Swi/Snf-Brm complex. To further delineate the mechanism of this inhibition, we have examined whether methylation of histones is also involved and whether a functional interplay between chromatin-modifying enzymes occurs. The histone methyltransferase G9a, but not SUV39, was colocalized with SHP in the nucleus and directly interacted with SHP in vitro. G9a, which was coimmunoprecipitated with hepatic SHP, methylated Lys-9 of histone 3 (H3K9) in vitro. Expression of G9a enhanced inhibition of CYP7A1 transcription by SHP, while a catalytically inactive G9a dominant negative (DN) mutant reversed the SHP inhibition. G9a was recruited to and H3K9 was methylated at the CYP7A1 promoter in a SHP-dependent manner in bile acid-treated HepG2 cells. Expression of the G9a-DN mutant inhibited H3K9 methylation, blocked the recruitment of the Brm complex, and partially reversed CYP7A1 inhibition by bile acids. Inhibition of HDAC activity with trichostatin A blocked deacetylation and methylation of H3K9 at the promoter, and, conversely, inhibition of H3K9 methylation by G9a-DN partially blocked deacetylation. Hepatic expression of G9a-DN in mice fed cholic acid disrupted bile acid homeostasis, resulting in increased bile acid pools and partial de-repression of Cyp7a1 and Cyp8b1. Our studies establish a critical role for G9a methyltransferase, histone deacetylases, and the Swi/Snf-Brm complex in the SHP-mediated inhibition of hepatic bile acid synthesis via coordinated chromatin modification at target genes.

scholarly journals DZNep, an inhibitor of the histone methyltransferase EZH2, suppresses hepatic fibrosis through regulating miR-199a-5p/SOCS7 pathway

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11374
Author(s):  
Rongrong Ding ◽  
Jianming Zheng ◽  
Ning Li ◽  
Qi Cheng ◽  
Mengqi Zhu ◽  
...  
Keyword(s):  
Hepatic Fibrosis ◽  
Histone Methyltransferase ◽  
Potential Effect ◽  
Collagen I ◽  
Luciferase Reporter ◽  
Regulatory Relationship ◽  
Ezh2 Expression ◽  
Relationship Of

Background Hepatic fibrosis is a common response to chronic liver injury. Recently, the role of DZNep (a histone methyltransferase EZH2 inhibitor) in repressing pulmonary and renal fibrosis was verified. However, the potential effect of DZNep on hepatic fibrosis has not been elucidated. Methods The hepatic fibrosis model was established in rats treated with CCl4 and in hepatic stellate cells (HSCs) treated with TGF-β1. The liver tissues were stained with H&E and Masson’s trichrome. The expression of EZH2, SOCS7, collagen I, αSMA mRNA and miR-199-5p was assessed using qPCR, immunohistochemical or western blot analysis. A dual-luciferase reporter assay was carried out to validate the regulatory relationship of miR-199a-5p with SOCS7. Results The EZH2 level was increased in CCl4-treated rats and in TGF-β1-treated HSCs, whereas DZNep treatment significantly inhibited EZH2 expression. DZNep repressed hepatic fibrosis in vivo and in vitro, as evidenced by the decrease of hepatic fibrosis markers (α-SMA and Collagen I). Moreover, miR-199a-5p expression was repressed by DZNep in TGF-β1-activated HSCs. Notably, downregulation of miR-199a-5p decreased TGF-β1-induced expression of fibrosis markers. SOCS7 was identified as a direct target of miR-199a-5p. The expression of SOCS7 was decreased in TGF-β1-activated HSCs, but DZNep treatment restore d SOCS7 expression. More importantly, SOCS7 knockdown decreased the effect of DZNep on collagen I and α SMA expression in TGF-β1-activated HSCs. Conclusions DZNep suppresses hepatic fibrosis through regulating miR-199a-5p/SOCS7 axis, suggesting that DZNep may represent a novel treatment for fibrosis.

High Throughput Screening Identifies Potential Inhibitors of WHSC1/MMSET, a Histone Methyltransferase Oncoprotein in Multiple Myeloma and Acute Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3251-3251 ◽  
Author(s):  
Christine M Will ◽  
Michael Scholle ◽  
Roodolph St. Pierre ◽  
Gary Schiltz ◽  
James E. Bradner ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Enzymatic Activity ◽  
Small Molecule ◽  
High Throughput ◽  
Conflicts Of Interest ◽  
Histone Methyltransferase ◽  
Response To Therapy ◽  
Open Chromatin ◽  
Activating Mutations

Abstract Aberrant epigenetic gene regulation is now considered a hallmark of many diseases, including cancer. Mutations and disruptions of epigenetic modifier enzymes are among the commonest mutation in human cancer. MMSET/NSD2/WHSC1 is a histone methyltransferase overexpressed in many cancers, including a subset of multiple myeloma (MM) harboring the t(4;14) translocation. This increased expression of MMSET is linked to a poorer prognosis and response to therapy than other MM subtypes. More recently activating mutations of MMSET were identified in relapsed pediatric ALL. The major substrate for MMSET in vivo is lysine 36 on histone H3 (H3K36). The ability of MMSET to methylate H3 depends on the enzymatic activity of its SET domain, found in most histone methyltransferases. Overexpression or activating mutations of MMSET lead to a global increase in dimethylation of H3K36 (H3K36me2) and a concomitant global loss of trimethylated H3K27 (H3K27me3), which leads to a more open chromatin confirmation. We, and others, have shown that MMSET has a role in a number of pathways including cell cycle, apoptosis, cell adhesion, and DNA repair. The loss of MMSET in myeloma cells decreases cell proliferation and leads to a reduced clonogenic capacity. These data highlight MMSET as a promising therapeutic target for t(4;14)+ MM. A small molecule inhibitor could also prove advantageous in our efforts to understand the various mechanisms of MMSET's enzymatic activity. Here we performed two high throughput screens on two different small molecule libraries. The screens were conducted by using the recombinant SET methyltransferase domain of MMSET (aa980-1214), generated in vitro, in combination with H4 (aa36-50) as the peptide substrate and S-adenosylmethionine (SAM) as the methyl donor. These screens were designed to avoid SAM competitors by screening under high SAM concentrations (50μM). The first screen used SAMDI technology, which combines mass spectrometry with high-density arrays. This method allows us to identify compounds within the libraries that inhibit MMSET in a rapid, label-free format. The second screen used alphalisa technology, a luminescent proximity assay that provides a homogeneous, sensitive method to screen for molecules that reduce MMSET activity. A total of 88 compounds from both libraries were identified as potential MMSET inhibitors (>3-standard deviation) and were counter-screened against the histone demethylase LSD1 to confirm their specificity. Numerous candidate compounds promiscuously inhibited the enzymatic activity of LSD1, yielding eleven putative MMSET inhibitors, with IC50 values ranging from 6-42μM. These eleven compounds were then screened against full-length MMSET enzyme using HeLa-derived mono/di-nucleosomes as the substrate. Eight of the compounds demonstrated a 50% or greater inhibition of MMSET at 10 μM and three compounds exhibited a dose-dependent inhibition upon dilution. Future in vitro, cellular, and animal based assays will determine the potency and selectivity of these compounds against MMSET and gauge their effectiveness as targeted therapies in cancers characterized by MMSET overexpression and activating mutations. Disclosures No relevant conflicts of interest to declare.

In Acute Myeloid Leukemia (AML), Targeting the Histone Methyltransferase EZH2 Promotes Differentiation, Impairs Clonogenic Survival and Augments the Anti-Leukemic Effects of the Retinoid, All-Trans-Retinoic Acid (ATRA)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3786-3786 ◽  
Author(s):  
Ronan T. Swords ◽  
Aymee Perez ◽  
Ana Rodriguez ◽  
Justin M. Watts ◽  
Tino Schenk ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Retinoic Acid ◽  
Myeloid Leukemia ◽  
Clonogenic Survival ◽  
Histone Methyltransferase ◽  
Self Renewal ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Acute Myeloid

Abstract The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the polycomb group complex, is critical for normal hematopoietic stem cell development. EZH2 mediates transcriptional repression through histone tri-methylation (H3K27me3). The activity of EZH2 influences cell fate regulation, namely the balance between self-renewal and differentiation. The contribution of aberrant EZH2 expression to tumorigenesis is becoming increasingly recognized. Its role in hematological malignancies however, is complex. Both gain-of-function and loss-of-function mutations have been respectively reported in lymphoma and leukemia, suggesting that EZH2 may serve a dual purpose as an oncogene and tumor-suppressor gene. Impaired self-renewal via EZH2 inhibition has been observed and offers a potentially attractive therapeutic approach in acute myeloid leukemia. Indeed, overexpression of EZH2 has been reported in patients with AML, particularly in those with complex karyotypes. In the present study, we show that deletion of EZH2 compromises the growth potential of AML cells by promoting their differentiation. To understand the role of EZH2 in vitro, we first examined the cell growth and colony-forming ability of EZH2 knockdown vs WT HL-60 cells. We found that proliferation of HL-60 cells was severely compromised following deletion of EZH2. Additionally, EZH2 deletion resulted in retarded cell-cycle entry and resulted in increased apoptotic cell death Similarly, the number of total colonies generated by EZH2 deleted cells in the secondary and tertiary re-plating assays was considerably less than that of controls. EZH2 deleted cells tended to form dispersed colonies that were mainly composed of differentiated myeloid cells, whereas control cells mostly formed compact colonies composed of myeloblasts. The proportion of dispersed colonies in the EZH2deleted cell culture increased with serial replatings. Deletion of EZH2 affects the growth and replating capacity of AML cell in vitro. When EZH2 deleted HL-60 cells were treated with the retinoid all-trans-retinoic acid (ATRA), we observed a marked induction of differentiation (as measured by the myeloid maturation marker CD11b) compared to the effects of ATRA on differentiation in wild type (WT) cells. Similarly, impaired clonogenic survival was more pronounced following ATRA treatment in EZH2 deleted vs WT HL-60 cells (see figure). We then profiled a number of small molecule inhibitors of EZH2 alone (EPZ005687, EPZ-6438, GSK126, El1, DZNeP, UNC1999 and GSK343) and in combination with ATRA, confirming these phenotypic changes. To elucidate the mechanism for how EZH2 regulates the balance of self-renewal vs differentiation in AML, we examined the genome-wide distribution of H3K27me3 by ChIP-seq analysis. First, western blot analysis revealed a marked decrease in the levels of H3K27me3 in EZH2 deleted AML cells. Next, we examined the presence of H3K27me3 marks in leukemia cells purified by ChIP-seq analysis. We focused on the region from 5.0 kb upstream to 3.0 kb downstream of transcription start sites (TSSs) of reference sequence (RefSeq) genes (http://www.ncbi.nlm.nih.gov/RefSeq/) because H3K27me3 marks are usually enriched near TSSs or across the body of genes. As expected, the deletion of EZH2 caused a drastic reduction in these H3K27me3 marks. Targeting EZH2 presents and interesting dichotomy as a novel drug target since inhibition of this protein could potentially be beneficial or detrimental depending on the context of the disease. In the case of AML, EZH2 mutations likely impede differentiation and block retinoic acid led differentiation programs. Updated studies outlining the interaction between the retinoic acid signaling pathway and EZH2 will be presented. These studies justify clinical investigation of EZH2 inhibitors combined with ATRA for patients with AML. Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Disclosures No relevant conflicts of interest to declare.

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