scholarly journals HCF-2 inhibits cell proliferation and activates differentiation-gene expression programs

2019 ◽  
Author(s):  
Daria Gudkova ◽  
Oleksandr Dergai ◽  
Viviane Praz ◽  
Winship Herr
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Protein Family ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Cell Proliferation And Differentiation ◽  
293 Cells ◽  
Proliferation And Differentiation ◽  
Localization Signal ◽  
Parallel Activation

ABSTRACTHCF-2 is a member of thehost-cell-factor protein family, which arose in early vertebrate evolution as a result of gene duplication. Whereas its paralog, HCF-1, is known to act as a versatile chromatin-associated protein required for cell proliferation and differentiation, much less is known about HCF-2. Here, we show that HCF-2 is broadly present in human and mouse cells, and possesses activities distinct from HCF-1. Unlike HCF-1, which is excluded from nucleoli, HCF-2 is nucleolar — an activity conferred by one and a half C-terminal Fibronectin type 3 repeats and inhibited by the HCF-1 nuclear localization signal. Elevated HCF-2 synthesis in HEK-293 cells results in phenotypes reminiscent of HCF-1-depleted cells, including inhibition of cell proliferation and mitotic defects. Furthermore, increased HCF-2 levels in HEK-293 cells lead to inhibition of cell proliferation and metabolism gene-expression programs with parallel activation of differentiation and morphogenesis gene-expression programs. Thus, the HCF ancestor appears to have evolved into a small two-member protein family possessing contrasting nuclear vs. nucleolar localization, and cell proliferation and differentiation functions.

scholarly journals THAP11F80L cobalamin disorder-associated mutation reveals normal and pathogenic THAP11 functions in gene expression and cell proliferation

2019 ◽  
Author(s):  
Harmonie Dehaene ◽  
Viviane Praz ◽  
Philippe Lhôte ◽  
Maykel Lopes ◽  
Winship Herr
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Gene Transcription ◽  
Vitamin B ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Key Enzyme ◽  
Neurodevelopmental Abnormalities ◽  
Biallelic Mutations

AbstractTwelve human THAP proteins share the THAP domain, an evolutionary conserved zinc-finger DNA-binding domain. Studies of different THAP proteins have indicated roles in gene transcription, cell proliferation and development. We have analyzed this protein family, focusing on THAP7 and THAP11. We show that human THAP proteins possess differing homo- and heterodimer formation properties and interaction abilities with the transcriptional co-regulator HCF-1. HEK-293 cells lacking THAP7 were viable but proliferated more slowly. In contrast, HEK-293 cells were very sensitive to THAP11 alteration. Nevertheless, HEK-293 cells bearing a THAP11 mutation identified in a patient suffering from cobalamin disorder (THAP11F80L) were viable although proliferated more slowly. Cobalamin disorder is an inborn vitamin deficiency characterized by neurodevelopmental abnormalities, most often owing to biallelic mutations in the MMACHC gene, whose gene product MMACHC is a key enzyme in the cobalamin (vitamin B12) metabolic pathway. We show that THAP11F80L selectively affected promoter binding by THAP11, having more deleterious effects on a subset of THAP11 targets, and resulting in altered patterns of gene expression. In particular, THAP11F80L exhibited a strong effect on association with the MMACHC promoter and led to a decrease in MMACHC gene transcription, suggesting that the THAP11F80L mutation is directly responsible for the observed cobalamin disorder.

scholarly journals The influence of direct and indirect fibroblast cell contact on human myogenic cell behavior and gene expression in vitro

2019 ◽  
Vol 127 (2) ◽  
pp. 342-355 ◽  
Author(s):  
Cecilie J. L. Bechshøft ◽  
Peter Schjerling ◽  
Michael Kjaer ◽  
Abigail L. Mackey
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Cell Proliferation ◽  
Myogenic Cell ◽  
Mrna Levels ◽  
Indirect Contact ◽  
Permeable Membrane ◽  
Primary Myoblasts ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation

Underpinning skeletal muscle plasticity is the interplay between many cell types, of which fibroblasts are emerging as potent players, both negatively in the development of fibrosis but also positively in stimulating muscle repair through enhancing myogenesis. The mechanisms behind this interaction however remain unknown. To investigate this, waste hamstring muscle tissue was obtained from eight healthy young men undergoing reconstructive anterior cruciate ligament surgery and primary myoblasts and fibroblasts were isolated. Myoblasts were cultured alone or with fibroblasts, either in direct or indirect contact (separated by an insert with a permeable membrane). The myogenesis parameters proliferation, differentiation, and fusion were determined from immunostained cells, while, in replicate samples, gene expression levels of GAPDH, Ki67, Pax7, MyoD, myogenin, myomaker, MHC-Iβ, TCF7L2, COL1A1, and p16 were determined by RT-PCR. We found only trends for an influence of skeletal muscle fibroblasts on myogenic cell proliferation and differentiation. While greater mRNA levels of GAPDH, Pax7, MyoD, myogenin, and MHC-Iβ were observed in myogenic cells in indirect contact with fibroblasts (insert) when compared with cells cultured alone, a similar effect of an empty insert was also observed. In conclusion we find very little influence of skeletal muscle fibroblasts on myoblasts derived from the same tissue, although it cannot be excluded that a different outcome would be seen under less optimal myogenic growth conditions. NEW & NOTEWORTHY Using passage one primary myoblasts and fibroblasts isolated from human skeletal muscle, we found only a trend for an effect of skeletal muscle fibroblasts on myogenic cell proliferation and differentiation. This is contrary to previous reports and raises the possibility that fibroblasts of different tissue origins exert distinct roles.

Effects of Scutellariae Radix on gene expression in HEK 293 cells using cDNA microarray

2006 ◽  
Vol 105 (3) ◽  
pp. 346-351 ◽  
Author(s):  
Chia-Sheng Chen ◽  
Nae-Jing Chen ◽  
Li-Wei Lin ◽  
Chia-Chang Hsieh ◽  
Guang-Wei Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cdna Microarray ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Scutellariae Radix ◽  
293 Cells

scholarly journals Dysregulation of ANKRD11 Influenced Hematopoisis By Histone Acetylation-Mediated Gene Expression in Myelodysplastic Syndrome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4292-4292
Author(s):  
Youshan Zhao ◽  
Feng Xu ◽  
Juan Guo ◽  
Sida Zhao ◽  
Chunkang Chang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Histone Acetylation ◽  
Mrna Expression ◽  
Cell Line ◽  
Expression Levels ◽  
Target Sequencing ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Mrna Expression Levels

Abstract Background and Object In addition to histone deacetylation, the importance of histone over-acetylation induced oncogene transcription in initiation and progression of myelodysplastic syndrome (MDS) has been proposed recently. Our previous whole-exome sequencing identified a new somatic mutation, ANKRD11, an important factor in histone acetylation regulation. Its roles in MDS pathophysiology need to be clarified. Methods The next generation target sequencing (Including ANKRD11) was carried out in 320 patients with MDS using the MiSeq Benchtop Sequencer. ANKRD11 mRNA expression in bone marrow of MDS was measured by real-time PCR. Loss and gain of function assay were carried out in myeloid cell lines K562, MEG-01£¬or SKM-1 to observe the influence on cell proliferation and differentiation . The levels of histone acetylation at H3 and H4 were detected by Western blot. Results Target sequencing in a cohort of 320 MDS patients identified 14 of ANKRD11 mutations (4.38%, Fig.1), which were confirmed by Sanger sequencing. Meanwhile, no ANKRD11 mutations in 100 normal controls were defined. ANKRD11 mutations occurred frequently in exons 10 and 9. The mRNA expression levels of ANKRD11 were significantly decreased in MDS patients, especially in ANKRD11mutant patients (Fig.2). ANKRD11 knockdown in K562 and MEG-1 resulted in growth inhibition, cell cycle arrest and erythroid/megakaryocytic differentiation retardant. In MDS cell line SKM-1, the arrested differentiation was rescued by over-expression of ANKRD11. Consistent with a role for ANKRD11 in histone acetylation, ANKRD11 KD increased acetylation of histones H3 and H4 at H3K14 and H4K5 and resulted in the upregulation of genes involved in differentiation inhibilation (SOX6, P21, et al). Finally, the ANKRD11 KD-mediated influence on cell proliferation and differentiation were reversed by inhibiting histone acetyltransferase activity. Conclusion Our assay defined that ANKRD11 was a crucial chromatin regulator that suppress histone acetylation and then decrease gene expression during myeloid differentiation, providing a likely explanation for its role in MDS pathogenesis. This study further support histone acetylase inhibitor as a potential treatment in MDS. Figure ANKRD11mutation distribution (a) and coexist with other mutations (b). Figure. ANKRD11mutation distribution (a) and coexist with other mutations (b). Figure The mRNA expression levels of ANKRD11in our MDS (A, C) subset and GEO data (B). Figure. The mRNA expression levels of ANKRD11in our MDS (A, C) subset and GEO data (B). Changes of histone acetylation in ANKRD11-KD cell line (MEG-01). ANKRD11 KD significantly increased acetylation of histones H3 and H4 at H3K14 and H4K5. Changes of histone acetylation in ANKRD11-KD cell line (MEG-01). ANKRD11 KD significantly increased acetylation of histones H3 and H4 at H3K14 and H4K5. Disclosures No relevant conflicts of interest to declare.

A Lymphoma-Associated Mutation in BAFF-R Drives Constitutive PI3K Signaling and Increased Expression of Pro-Survival Genes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2642-2642
Author(s):  
Frank J Secreto ◽  
Michelle K Manske ◽  
Tammy Price-Troska ◽  
Steven C. Ziesmer ◽  
Stephen M Ansell ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Pi3k Signaling ◽  
Hek 293 ◽  
Akt Phosphorylation ◽  
Hek 293 Cells ◽  
Pi3k Activity ◽  
293 Cells ◽  
The Impact ◽  
In Cells

Abstract Abstract 2642 BAFF is essential for B cell maturation, and a lack of either BAFF or its primary receptor, BAFF-R, results in a severe depletion of T2 marginal zone and follicular B cells. Elevated serum BAFF levels have been correlated with an increased risk of developing non-Hodgkin's lymphoma (NHL), along with a more aggressive phenotype. A growing body of genetic evidence points toward an association between the development of human disease and variation in genes encoding BAFF and its receptors. Recently, we characterized a novel lymphoma-associated mutation in TNFRSF13C, the gene encoding BAFF-R. This mutation (BAFF-R H159Y) encodes a His159Tyr substitution in the C-terminus of BAFF-R adjacent to the TRAF3 binding motif. Signaling through BAFF-R H159Y results in increased NF-κB activity, elevated immunoglobulin production and increased association with TRAF2, TRAF3 and TRAF6 compared to wild type (WT) BAFF-R. We have detected this mutation in 6% of total NHL cases (n=129), and in 10% of follicular lymphoma (FL) cases (n=41) evaluated thus far. We previously reported that BAFF-R H159Y expressing mouse B cells exhibited significantly more resistance to Fas ligand (FasL) induced apoptosis compared to their cells expressing BAFF-R WT, and we propose here that BAFF-R H159Y mediated increases in PI3K activity may explain such an enhanced anti-apoptotic response. In this study we now show that BAFF stimulated HEK 293 cells stably expressing BAFF-R H159Y not only display significantly increased Akt phosphorylation when compared to BAFF-R WT expressing cells, but also demonstrate robust Akt phosphorylation in the absence of BAFF. BAFF-R H159Y-dependent Akt activation also led to activation of the downstream Akt targets mTOR and GSK3β and their phosphorylation was inhibited following treatment with the PI3- kinase inhibitor wortmannin. We next examined the impact of the BAFF-R H159Y mutation on expression of BAFF-target genes. Quantitative PCR analyses revealed that BAFF-R H159Y cells exhibited a pattern of gene expression indicative of promoting cell survival, displaying significantly higher levels of BCL2, BCL2L1 and PIN1, while down-regulating expression of the pro-apoptotic gene BIM. We recently reported that TRAF6 associates with BAFF-R, and that such binding is more pronounced in cells expressing BAFF-R H159Y. In order to investigate the role TRAF6 plays in mediating BAFF-R-dependent PI3K activity, we silenced TRAF6 expression in HEK 293 and Karpas 422 lymphoma cells using TRAF6 shRNA. Reduced TRAF6 protein expression resulted in a parallel decrease in BAFF-R WT mediated phosphorylation of mTOR in Karpas 422 cells and phosphorylation of both Akt and GSK3β was markedly reduced in BAFF-R H159Y expressing HEK 293 cells. Interestingly, TRAF6 knock-down did not affect NF-kB2 activation in either Karpas 422 or HEK BAFF-R expressing cells suggesting that Akt does not play a role in BAFF-R mediated activation of non-canonical NF-kB. Finally, preliminary co-precipitation studies indicate that Akt can be recruited to BAFF-R itself, and our initial observations suggest that such an association is significantly reduced in cells expressing BAFF-R H159Y. Taken together, these studies suggest that the BAFF-R H159Y mutation confers enhanced BAFF-R-dependent PI3K signaling and pro-survival gene expression independent of BAFF. Moreover, such enhanced P13K activation is partly dependent upon TRAF6, and decreased recruitment of Akt to BAFF-R H159Y may function to increase the amount of this PI3K target for activation. Thus, BAFF-R H159Y likely contributes to BAFF signaling irregularities in NHL patients harboring this mutation, and may predispose individuals to developing lymphoma regardless of their serum BAFF concentration. Disclosures: No relevant conflicts of interest to declare.

Gene expression profiles in HEK-293 cells with low or high store-operated calcium entry: can regulatory as well as regulated genes be identified?

2005 ◽  
Vol 21 (1) ◽  
pp. 14-33 ◽  
Author(s):  
Tatiana K. Zagranichnaya ◽  
Xiaoyan Wu ◽  
Arpad M. Danos ◽  
Mitchel L. Villereal
Keyword(s):  
Gene Expression ◽  
Transient Receptor Potential ◽  
Expression Profiles ◽  
Physiological Role ◽  
Gene Expression Profiles ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Gene expression profiles were generated using cDNA microarray technology for clones of human embryonic kidney (HEK)-293 cells selected to have either high or low levels of store-operated Ca2+ entry (SOCE). For five high clones, three low clones, and control HEK-293 cells, duplicate Affymetrix U133A human gene arrays were run after extraction of total RNA from cells growing in the presence of serum. Of the ∼22,000 genes represented on the microarray, 58 genes had readings at least twofold higher, while 32 genes had readings at least twofold lower, in all five high SOCE clones compared with control HEK-293 cells. In the low SOCE clones, 92 genes had readings at least twofold higher, while 58 genes had readings at least twofold lower, than in HEK-293 cells. Microarray results were confirmed for 18 selected genes by real-time RT-PCR analysis; for six of those genes, predicted changes in the low SOCE clone were confirmed by an alternative method, monitoring mRNA levels in HEK-293 with SOCE decreased by expression of small interfering (si)RNA to canonical transient receptor potential protein-1. Genes regulated by SOCE are involved in signal transduction, transcription, apoptosis, metabolism, and membrane transport. These data provide insight into the physiological role of SOCE. In addition, a potential regulator of SOCE, insulin receptor substrate (IRS)-2, has been identified. A reduction of IRS-2 levels by siRNA methods in two high clones dramatically reduced SOCE, whereas overexpression of IRS-2 in a low SOCE clone elevated SOCE.

Unfolded protein response (UPR) gene expression during antibody-dependent enhanced infection of cultured monocytes correlates with dengue disease severity

2011 ◽  
Vol 31 (3) ◽  
pp. 221-230 ◽  
Author(s):  
Prasad N. Paradkar ◽  
Eng Eong Ooi ◽  
Brendon J. Hanson ◽  
Duane J. Gubler ◽  
Subhash G. Vasudevan
Keyword(s):  
Gene Expression ◽  
Unfolded Protein Response ◽  
Disease Severity ◽  
Clinical Isolates ◽  
Hek 293 ◽  
Dengue Disease ◽  
Hek 293 Cells ◽  
Unfolded Protein ◽  
293 Cells ◽  
Protein Response

DENV (dengue virus) induces UPR (unfolded protein response) in the host cell, which strikes a balance between pro-survival and pro-apoptotic signals. We previously showed that Salubrinal, a drug that targets the UPR, inhibits DENV replication. Here, we examine the impact on UPR after direct or ADE (antibody-dependent enhanced) infection of cells with DENV clinical isolates. THP-1 cells in the presence of subneutralizing concentration of humanized antibody 4G2 (cross-reactive with flavivirus envelope protein) or HEK-293 cells (human embryonic kidney 293 cells) were infected with DENV-1–4 serotypes. UPR gene expression was monitored under these infection conditions using real-time RT–PCR (reverse transcription–PCR) and Western blots to analyse serotype-dependent variations. Subsequently, in a blinded study, strain-specific differences were compared between DENV-2 clinical isolates obtained from a single epidemic. Results showed that THP-1 cells were infected efficiently and equally by DENV-1–4 in the ADE mode. At 48 hpi (h post infection), DENV-1 and -3 showed a higher replication rate and induced higher expression of several UPR genes such as BiP (immunoglobulin heavy-chain-binding protein), GADD34 (growth arrest DNA damage-inducible protein 34) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein]. The ADE infection of THP-1 cells with epidemic DENV-2 high-UPR-gene-expressing strains appears to correlate with severe disease; however, no such correlation could be made when the same viruses were used to infect HEK-293 cells. Our finding that UPR gene expression in THP-1 cells during ADE infection correlates with dengue disease severity is consistent with a previous study [Morens, Marchette, Chu and Halstead (1991) Am. J. Trop. Med. Hyg. 45, 644–651] that showed that the growth of DENV 2 isolates in human peripheral blood leucocytes correlated with severe and mild dengue diseases.

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