scholarly journals SAT-726 Estrogen Receptor Alpha as a Potential Target for Bisphenol A-Mediated Epigenetic Reprogramming: An in Vitro Analysis

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Morgan Gallo ◽  
Lindsey S Treviño ◽  
Tiffany A Katz
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Target Genes ◽  
Hepatic Cell ◽  
Time Points ◽  
Target Gene Expression ◽  
Er Alpha ◽  
Hepatic Cell Lines ◽  
Gene Expression Levels

Abstract Perinatal exposure to bisphenol A (BPA) has been shown to reprogram the hepatic epigenome of rodents and may promote the development of various metabolic diseases later in life, such as nonalcoholic fatty liver disease (NAFLD). This developmental reprogramming is characterized by the creation of “super promoters” at target genes implicated in metabolic pathways. While it is unclear how these “super promoters” are created, their creation is potentially mediated through BPA and estrogen receptor (ER) interaction. In order to test this potential mechanism, in vitro methods were used to examine ER target gene expression via RT-qPCR in 2 human hepatic cell lines transiently transfected with the ER isoform, ER alpha, prior to BPA exposure for various lengths of time. Within individual time points, there were no significant differences in target gene expression levels between cells that had been transfected with ER alpha and the vector control. Gene expression levels in the target genes were visibly increased at the 24-hour exposure mark in both transfection groups in comparison to the 0- and 6-hour time points, however only a fraction of these increases were found to be statistically significant. These gene expression patterns are not only consistent with previous studies examining target gene expression in BPA-treated hepatic cell lines, but more importantly, suggest BPA does not act via ER alpha to orchestrate the epigenetic changes seen in vitro. BPA may interact with a different ER isoform or an unknown target to create the observed “super promoters” at target genes, reinforcing the promiscuity of BPA and other xenoestrogens in facilitating epigenetic modifications, and ultimately, disease phenotypes.

scholarly journals Relationship of DUX4 and target gene expression in FSHD myocytes

2020 ◽  
Author(s):  
Jonathan Chau ◽  
Xiangduo Kong ◽  
Nam Nguyen ◽  
Katherine Williams ◽  
Rabi Tawil ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Target Gene ◽  
Target Genes ◽  
Myoblast Differentiation ◽  
Target Gene Expression ◽  
Gene Transcripts ◽  
Relationship Of

AbstractFacioscapulohumeral dystrophy (FSHD) is linked to misexpression of the transcription factor, DUX4. Although DUX4 target gene expression is often readily detectable, analysis of DUX4 expression has been limited due to its low expression in patient samples. Recently, single cell/nucleus RNA-sequencing was used to detect the native expression of DUX4 for the first time, but important spatial relationships with its target gene expression was missing. Furthermore, dynamics of DUX4 expression during myoblast differentiation has not been fully explored. In order to study the spatiotemporal relationship of DUX4 and key target genes, we performed RNA FISH on immortalized FSHD2 patient skeletal muscle cells. Using two probe sets, DUX4 transcripts were detected in 1-4% of myotubes after 3-day differentiation in vitro. We found that DUX4 transcripts mainly localize as foci in one or two nuclei in a myotube compared to abundant accumulation of the target gene transcripts in the cytoplasm. Over a 13-day differentiation timecourse, DUX4 expression without target gene expression significantly increased and peaked at day 7. Target gene expression correlates better with DUX4 expression early in differentiation while the expression of target genes without detectable DUX4 transcripts increases later. Consistently, shRNA depletion of DUX4-activated transcription factors, DUXA and LEUTX, specifically repressed a DUX4-target gene, KDM4E, later in differentiation, suggesting that following the initial activation by DUX4, target genes themselves contribute to the maintenance of downstream gene expression. Together, in situ detection of the DUX4 and target gene transcripts provided new insight into dynamics of DUX4 transcriptional network in FSHD patient myocytes.Significance StatementFSHD is the third most common muscular dystrophy and is associated with upregulation of DUX4, a transcription factor, and its target genes. Although target genes are easily detectable in FSHD, low frequency DUX4 upregulation in patient myocytes is difficult to detect, and examining the relationship and dynamics of DUX4 and target gene expression without artificial overexpression of DUX4 has been challenging. Using RNAScope with highly specific probes, we detect the endogenous DUX4 and target gene transcripts in situ in patient skeletal myotubes during differentiation in vitro. Our study reveals a unique DUX4 expression pattern and its relationship to the expression of target genes, and evidence for self-sustainability of the target gene network. The study provides important new insights into the FSHD disease mechanism.

scholarly journals Hottip Lncrna Reinforces CTCF Defined Chromatin Boundaries and Drives Wnt Target Gene Expression in AML Leukemogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 277-277
Author(s):  
Huacheng Luo ◽  
Ganqian Zhu ◽  
Tsz Kan Fung ◽  
Yi Qiu ◽  
Mingjiang Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Triple Helix ◽  
Target Gene ◽  
Target Genes ◽  
Ctcf Binding ◽  
Target Gene Expression ◽  
Rna Immunoprecipitation

We reported recently that HOXA locus associated lncRNA, HOTTIP, is highly expressed in AML patients carrying MLL rearrangement and NPM1c+ mutations. The expression of HOTTIP positively correlates with posterior HOXA gene expression and poor patient survival. We further demonstrated that HOTTIP acts as an epigenetic regulator to define oncogenic HOXA topologically associated domain (TAD) and drive HOXA associated leukemic transcription program. However, it remains unclear whether and how HOTTIP lncRNA is involved in remodeling leukemic genome to facilitate AML leukemogenesis. Here, we showed that HOTTIP regulates a fraction of CTCF binding sites (CBSs) in the AML genome by directly interacting with CTCF and its binding motifs. We carried out CTCF ChIP-seq and HOTTIP ChIRP (chromatin isolation by RNA purification)-seq comparing WT and HOTTIP knockout (KO) MOLM13 cells. KO of HOTTIP in MLL-rearranged MOLM13 AML cells specifically impaired CTCF binding sites that were co-occupied by HOTTIP lncRNA, whereas loss of HOTTIP did not affect global CTCF binding. These target genes include posterior HOXA genes and Wnt target genes such as C-MYC, EVI1, AXIN, and TWIST1. Furthermore, we found that HOTTIP interacts with its putative target sites by formation of DNA: RNA hybridization structure triple helix and R-loop in vivo and in vitro. We then carried out DRIP (DNA-RNA immunoprecipitation)-seq and DRIPc(DNA-RNA immunoprecipitation followed by cDNA conversion)-Seq, which utilize a sequence independent but structure-specific S9.6 antibody for DRIP to capture global R-loops, by comparing WT and HOTTIP KO MOLM13 cells. The obtained DRIP-seq and DRIPc-seq data were then incorporated and integrated with the HOTTIP ChIRP-seq and CTCF ChIP-seq data to explore global collaboration between R-loop and HOTTIP associated CTCF binding sites. We found that HOTTIP interacts with CTCF binding motif that defines the TADs and the promoters of the HOTTIP target genes by formation of R-loop or triple helix structure. Loss of HOTTIP disrupted the R-loop formation at promoters and enhancers of the HOTTIP target genes to inhibit their expression. In MLL-rearranged AML genome, in addition to the HOXA locus, CTCF forms leukemic specific TADs that protect aberrant Wnt target genes. Depletion of HOTTIP lncRNA impaired CTCF defined TADs in the Wnt target gene loci and reduced Wnt target gene expression. In contrast, overexpression of Hottip lncRNA (Hottip-Tg) in the mice bone marrow hematopoietic compartment perturbs hematopoietic stem cell (HSC) self-renewal and differentiation leading to AML like disease by reinforcing CTCF defined TADs, enhancing chromatin accessibility within TADs, and upregulating gene transcription in the Wnt target loci. Finally, when we treated HOTTIP expressed primary patient AML cells carrying MLL-rearrangement and their derived PDX mouse model with a canonical Wnt inhibitor, ICG-001, ICG-001 inhibited AML LSC self-renewal in in vitro by LTC-IC assays and in vivo leukemogenesis in the PDX mouse models with an aberrant HOTTIP lncRNA expression, but not in HOTTIP negative/low non-MLL AML samples. Thus, HOTTIP lncRNA and CTCF cooperate to specifically reinforce CTCF defined WNT target locus TADs and drive Wnt target gene expression in the HOTTIP expressed AML. Disclosures No relevant conflicts of interest to declare.

2049-P: The Chd4 Helicase of the Nucleosome Remodeling and Deacetylase Complex Modulates Pdx1 Target Gene Expression In Vitro

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 2049-P
Author(s):  
REBECCA K. DAVIDSON ◽  
NOLAN CASEY ◽  
JASON SPAETH
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Nucleosome Remodeling ◽  
Target Gene Expression

scholarly journals On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

2021 ◽  
Author(s):  
Philipp Moritz Fricke ◽  
Angelika Klemm ◽  
Michael Bott ◽  
Tino Polen
Keyword(s):  
Gene Expression ◽  
Acetic Acid ◽  
Literature Search ◽  
Target Gene ◽  
Target Genes ◽  
Recombinant Dna ◽  
Acetic Acid Bacteria ◽  
Homoserine Lactone ◽  
Expression Systems ◽  
Target Gene Expression

Abstract Acetic acid bacteria (AAB) are valuable biocatalysts for which there is growing interest in understanding their basics including physiology and biochemistry. This is accompanied by growing demands for metabolic engineering of AAB to take advantage of their properties and to improve their biomanufacturing efficiencies. Controlled expression of target genes is key to fundamental and applied microbiological research. In order to get an overview of expression systems and their applications in AAB, we carried out a comprehensive literature search using the Web of Science Core Collection database. The Acetobacteraceae family currently comprises 49 genera. We found overall 6097 publications related to one or more AAB genera since 1973, when the first successful recombinant DNA experiments in Escherichia coli have been published. The use of plasmids in AAB began in 1985 and till today was reported for only nine out of the 49 AAB genera currently described. We found at least five major expression plasmid lineages and a multitude of further expression plasmids, almost all enabling only constitutive target gene expression. Only recently, two regulatable expression systems became available for AAB, an N-acyl homoserine lactone (AHL)-inducible system for Komagataeibacter rhaeticus and an l-arabinose-inducible system for Gluconobacter oxydans. Thus, after 35 years of constitutive target gene expression in AAB, we now have the first regulatable expression systems for AAB in hand and further regulatable expression systems for AAB can be expected. Key points • Literature search revealed developments and usage of expression systems in AAB. • Only recently 2 regulatable plasmid systems became available for only 2 AAB genera. • Further regulatable expression systems for AAB are in sight.

In vitro chemosensitivity of freshly explanted tumor cells to pemetrexed is correlated with target gene expression

2007 ◽  
Vol 25 (5) ◽  
pp. 417-423 ◽  
Author(s):  
Axel-Rainer Hanauske ◽  
Ulrike Eismann ◽  
Olaf Oberschmidt ◽  
Heike Pospisil ◽  
Steve Hoffmann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Cells ◽  
Target Gene ◽  
Target Gene Expression ◽  
In Vitro Chemosensitivity

Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling

Development ◽  
1999 ◽  
Vol 126 (16) ◽  
pp. 3607-3616 ◽  
Author(s):  
Y. Chen ◽  
J.R. Cardinaux ◽  
R.H. Goodman ◽  
S.M. Smolik
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Ectopic Expression ◽  
Proteolytic Processing ◽  
Dominant Negative ◽  
Cubitus Interruptus ◽  
Target Gene Expression ◽  
Exogenous Expression

Hedgehog (HH) is an important morphogen involved in pattern formation during Drosophila embryogenesis and disc development. cubitus interruptus (ci) encodes a transcription factor responsible for transducing the hh signal in the nucleus and activating hh target gene expression. Previous studies have shown that CI exists in two forms: a 75 kDa proteolytic repressor form and a 155 kDa activator form. The ratio of these forms, which is regulated positively by hh signaling and negatively by PKA activity, determines the on/off status of hh target gene expression. In this paper, we demonstrate that the exogenous expression of CI that is mutant for four consensus PKA sites [CI(m1-4)], causes ectopic expression of wingless (wg) in vivo and a phenotype consistent with wg overexpression. Expression of CI(m1-4), but not CI(wt), can rescue the hh mutant phenotype and restore wg expression in hh mutant embryos. When PKA activity is suppressed by expressing a dominant negative PKA mutant, the exogenous expression of CI(wt) results in overexpression of wg and lethality in embryogenesis, defects that are similar to those caused by the exogenous expression of CI(m1-4). In addition, we demonstrate that, in cell culture, the mutation of any one of the three serine-containing PKA sites abolishes the proteolytic processing of CI. We also show that PKA directly phosphorylates the four consensus phosphorylation sites in vitro. Taken together, our results suggest that positive hh and negative PKA regulation of wg gene expression converge on the regulation of CI phosphorylation.

487 Correlations of in vitro chemosensitivity of solid tumors to Pemetrexed (P, ALIMTA®) and target gene expression

2004 ◽  
Vol 2 (8) ◽  
pp. 148-149
Author(s):  
O. Oberschmidt ◽  
U. Eismann ◽  
M. Ehnert ◽  
S. Struck ◽  
J. Blatter ◽  
...  
Keyword(s):  
Gene Expression ◽  
Solid Tumors ◽  
Target Gene ◽  
Target Gene Expression ◽  
In Vitro Chemosensitivity

The Interaction Between DOT1L and AF10 Is Required for H3K79 Dimethylation and MLL-AF9 Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 401-401
Author(s):  
Aniruddha J Deshpande ◽  
Liying Chen ◽  
Kathrin M Bernt ◽  
Stuart Dias ◽  
Deepti Banka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Target Gene ◽  
Leucine Zipper ◽  
Target Genes ◽  
Fusion Gene ◽  
Transformed Cells ◽  
Target Gene Expression ◽  
Transforming Activity ◽  
H3k79 Methylation

Abstract Abstract 401 MLL-fusion proteins induce changes in histone modifications that result in the abnormal and sustained expression of downstream oncogenic target genes. A number of recent studies have identified aberrant histone 3 lysine 79 (H3K79) methylation by the chromatin modifying enzyme DOT1L as an important epigenetic modification that sustains MLL-target gene expression. Aberrant H3K79 methylation has been shown to be necessary for oncogenic transformation mediated by a number of MLL-fusions. These recent findings have generated tremendous interest in H3K79 methylation as a therapeutic target in the MLL rearranged leukemias. The plant-homeodomain (PHD) and leucine zipper-containing protein AF10 biochemically interacts with DOT1L and is believed to influence H3K79 methylation. We generated conditional knockout mice in which the Dot1l-interacting octapeptide-motif leucine zipper (OM-LZ) domain of Af10 was flanked by LoxP sites. Deletion of the Af10OM-LZ domain with the Cre recombinase is predicted to abrogate the Af10-Dot1l interaction. Deletion of the Af10OM-LZ domain greatly reduced global H3K79 dimethylation as assessed by immunoblotting as well as mass spectrometry in Af10OM-LZ deleted HoxA9/Meis1a transformed cells. Given the importance of H3K79 methylation in MLL-rearranged leukemias, we sought to assess whether the transforming activity of the MLL-AF9 fusion gene was dependent on the Af10-Dot1l interaction. Using an MLL-AF9-IRES-GFP encoding retrovirus, we established immortalized blast-colony forming cultures from mouse lineage negative Sca-1 positive/Kit positive (LSK) bone marrow cells bearing floxed Af10OM-LZ alleles. Deletion of the Af10OM-LZ domain with Cre-recombinase dramatically reduced H3K79me2 on the MLL-target genes Hoxa5-10 and Meis1, leading to downregulation of these transcripts. We performed colony-forming cell (CFC) assays from MLL-AF9 transformed cells in the presence or absence of the Af10OM-LZ allele. In the first week, Af10OM-LZ deletion profoundly impaired the blast-colony forming potential of MLL-AF9 transformed LSKs and the only clones that could serially replate in subsequent passages had escaped Af10OM-LZ excision. Af10OM-LZ deleted colonies were very small and spread-out and showed morphological features of terminal myeloid differentiation. In contrast, HoxA9/Meis1 transformed LSK cells expanded normally in the absence of the Af10OM-LZ domain. These results demonstrate that the Af10OM-LZ, much like Dot1l, is critical for the in vitro transforming activity of the MLL-AF9 fusion gene, but does not non-specifically inhibit cellular proliferation. We then sought to investigate the potential role of the Af10OM-LZ domain in the in vivo leukemogenic activity of MLL-AF9. We generated primary MLL-AF9 leukemias from LSKs harboring floxed Af10OM-LZ alleles. Deletion of the Af10OM-LZ domain in cells explanted from the MLL-AF9 primary leukemias led to a significant increase in the disease latency in secondary recipient mice. Moreover, limiting dilution analysis of MLL-AF9 leukemias with or without the Af10OM-LZ domain demonstrated a >100 fold decrease in the frequency of leukemia initiating cells in the absence of the Af10OM-LZ domain. Microarray analysis showed that a vast majority of MLL-AF9 target genes were significantly downregulated in Af10OM-LZ deleted as compared to Af10OM-LZ wildtype MLL-AF9 leukemias. However, the Af10OM-LZ deleted cells could still eventually cause leukemia. This is intriguing given that Af10OM-LZ deletion, similar to Dot1l deletion, leads to a significant reduction in H3K79 dimethylation as well as MLL-target gene expression. A more detailed analysis of H3K79 methylation using mass spectrometry revealed that in contrast to H3K79 dimethylation, global levels of H3K79 mono-methylation were largely unchanged in Af10OM-LZ deleted cells. This suggests the residual MLL-AF9 target gene expression seen in Af10OM-LZ deleted cells is maintained by H3K79 monomethylation. Our results demonstrate a surprising role for Af10 in the conversion of H3K79 monomethylation to dimethylation and reveal the AF10-DOT1L interaction as an attractive therapeutic target in MLL-rearranged leukemias. Disclosures: Armstrong: Epizyme: Consultancy.

scholarly journals E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop

2007 ◽  
Vol 27 (1) ◽  
pp. 65-78 ◽  
Author(s):  
Cynthia Timmers ◽  
Nidhi Sharma ◽  
Rene Opavsky ◽  
Baidehi Maiti ◽  
Lizhao Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Target Genes ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Control Of Gene Expression ◽  
Target Gene Expression ◽  
Rb Phosphorylation ◽  
P53 Target Genes

ABSTRACT E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21 CIP1 . Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21 CIP1 and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

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