scholarly journals FOXL2 Interacts with Steroidogenic Factor-1 (SF-1) and Represses SF-1-Induced CYP17 Transcription in Granulosa Cells

2010 ◽  
Vol 24 (5) ◽  
pp. 1024-1036 ◽  
Author(s):  
Mira Park ◽  
Eunkyoung Shin ◽  
Miae Won ◽  
Jae-Hong Kim ◽  
Hayoung Go ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Ovarian Follicle ◽  
Type I ◽  
Yeast Two Hybrid ◽  
Steroidogenic Enzyme ◽  
Steroidogenic Factor 1 ◽  
Ovarian Follicle Development ◽  
Two Hybrid

Abstract Mutations in FOXL2 are responsible for blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) type I, in which affected women exhibit premature ovarian failure. FOXL2-null mice showed defects in granulosa cell development during folliculogenesis. We screened a rat ovarian yeast two-hybrid cDNA library to identify FOXL2-interacting proteins and found steroidogenic factor-1 (SF-1). Here, we show that human FOXL2 and SF-1 proteins interact in human granulosa cells and that FOXL2 negatively regulates the transcriptional activation of a steroidogenic enzyme, CYP17, by SF-1. Furthermore, FOXL2 mutants found in blepharophimosis-ptosis-epicanthus inversus syndrome type I patients lost the ability to repress CYP17 induction mediated by SF-1. Chromatin immunoprecipitation and EMSA results further revealed that FOXL2 inhibited the binding of SF-1 to the CYP17 promoter, whereas the FOXL2 mutants failed to block this interaction. Therefore, this study identifies a novel regulatory role for FOXL2 on a key steroidogenic enzyme and provides a possible mechanism by which mutations in FOXL2 disrupt normal ovarian follicle development.

scholarly journals Loose Plant Architecture 1-Interacting Kinesin-like Protein KLP Promotes Rice Resistance to Sheath Blight Disease

2021 ◽  
Author(s):  
Jin Chu ◽  
Han Xu ◽  
Hai Dong ◽  
Yuanhu Xuan
Keyword(s):  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Plant Architecture ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Sheath Blight Disease ◽  
Blight Disease ◽  
Two Hybrid ◽  
Wild Type Control

Abstract BackgroundSheath blight disease (ShB) is a destructive disease affecting rice production. Previously, we have reported that Loose Plant Architecture 1 (LPA1) promotes resistance to ShB. However, the mechanisms by which LPA1 confers resistance against this disease have not been extensively investigated. Notably, interactors that regulate LPA-1 activity remain elusive.FindingsIn this study, we identified the interaction of kinesin-like protein (KLP) with LPA1 in the nucleus of rice cells by yeast two-hybrid, bimolecular fluorescent complimentary (BiFC), and co-immunoprecipitation (co-IP) assays. To investigate the role of KLP in promoting resistance to ShB, wild-type, klp mutant, and KLP overexpressor (KLP OX) rice plants were inoculated with Rhizoctonia solani AG1-IA. The results indicated that, compared with the wild-type control, klp mutants were more susceptible while KLP OX plants were less susceptible to ShB. Since LPA1 transcriptionally activates PIN-FORMED 1a (PIN1a), we examined the expression of 8 related PIN genes. The results showed that only the expression of PIN1a and PIN3b coincided with KLP expression levels. In addition, a chromatin immunoprecipitation (ChIP) assay showed that KLP bound directly to the promoter region of PIN1a but not of PIN3b. Transient expression assays confirmed that LPA1 and KLP transcriptionally activate PIN1a, and that coexpression of KLP and LPA1 had an additive effect on the activation of PIN1a, suggesting that KLP enhances LPA1 transcriptional activation activity. ConclusionsTaken together, our results show that KLP is a novel LPA1 interactor that promotes resistance of rice to ShB.

scholarly journals Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

1996 ◽  
Vol 16 (2) ◽  
pp. 593-602 ◽  
Author(s):  
R Candau ◽  
P A Moore ◽  
L Wang ◽  
N Barlev ◽  
C Y Ying ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Sequence Similarity ◽  
Adaptor Proteins ◽  
Yeast Two Hybrid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Evolutionarily Conserved ◽  
Human Proteins ◽  
Two Hybrid

Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation.

scholarly journals Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development

2019 ◽  
Vol 33 (9) ◽  
pp. 10049-10064 ◽  
Author(s):  
Xiangmin Lv ◽  
Chunbo He ◽  
Cong Huang ◽  
Hongbo Wang ◽  
Guohua Hua ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Follicle Development ◽  
Ovarian Follicle Development

scholarly journals Involvement of miRNAs in equine follicle development

Reproduction ◽  
2013 ◽  
Vol 146 (3) ◽  
pp. 273-282 ◽  
Author(s):  
S N Schauer ◽  
S D Sontakke ◽  
E D Watson ◽  
C L Esteves ◽  
F X Donadeu
Keyword(s):  
Cell Survival ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Ovarian Follicle ◽  
Follicle Development ◽  
Previous Evidence ◽  
Ovarian Follicle Development ◽  
Fluid Levels ◽  
Follicle Selection

Previous evidence fromin vitrostudies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle anovulatory wave (n=6 mares). Follicular fluid levels of progesterone and estradiol were lower (P<0.01) in S follicles than in DO follicles, whereas mean levels of IGF1 were lower (P<0.01) in S and DA follicles than in DO follicles. Relative to DO and DA follicles, S follicles had higher (P≤0.01) follicular fluid levels of miR-145 and miR-378. In Experiment 2, follicular fluid and granulosa cells were aspirated from dominant follicles before (DO) and 24 h after (L) administration of an ovulatory dose of hCG (n=5 mares/group). Relative to DO follicles, L follicles had higher follicular fluid levels of progesterone (P=0.05) and lower granulosa cell levels ofCYP19A1andLHCGR(P<0.005). Levels of miR-21, miR-132, miR-212, and miR-224 were increased (P<0.05) in L follicles; this was associated with reduced expression of the putative miRNA targets,PTEN,RASA1, andSMAD4. These novel results may indicate a physiological involvement of miR-21, miR-145, miR-224, miR-378, miR-132, and miR-212 in the regulation of cell survival, steroidogenesis, and differentiation during follicle selection and ovulation in the monovular ovary.

Localization of ovine follistatin and α and βA inhibin mRNA in the sheep ovary during the oestrous cycle

1994 ◽  
Vol 12 (2) ◽  
pp. 181-193 ◽  
Author(s):  
D J Tisdall ◽  
N Hudson ◽  
P Smith ◽  
K P McNatty
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Indirect Evidence ◽  
Oestrous Cycle ◽  
Corpora Lutea ◽  
Α Subunit ◽  
Ovarian Follicle Development ◽  
Antral Follicles

ABSTRACT The sites of follistatin and α and βA inhibin gene expression were examined by in situ hybridization in sheep ovaries during the early and mid-luteal phases (days 3 and 10) of the oestrous cycle and a prostaglandin F2α (PGF2α)-induced follicular phase. Follistatin mRNA was detected in the granulosa cells of preantral, antral and early atretic follicles at all stages of the oestrous cycle, and in the corpora lutea at the early and mid-luteal stages of the cycle. However, only low levels of expression of follistatin were observed in the presumptive preovulatory follicle at 56 h after treatment with PGF2α. Both α and βA inhibin were shown to be expressed in ovaries at all stages of the oestrous cycle. In situ hybridization localized α subunit mRNA to the granulosa cells of most, but not all, healthy antral follicles, and to no other ovarian cell type. In contrast, expression of the βA subunit was confined to a few medium-to-large healthy antral follicles. In antral follicles expressing βA inhibin, mRNAs for α inhibin and follistatin were always detected, but the converse was not true. Unlike follistatin, no α and βA inhibin expression was seen in preantral follicles, developing corpora lutea, or follicles undergoing atresia. These results show that, in the adult sheep ovary, follistatin gene expression is a constitutive event in all growing follicles from the early preantral stage, and also provide indirect evidence of the involvement of follistatin, but not inhibin or activin, in the early stages of ovarian follicle development in sheep.

scholarly journals SMAD3 regulates the diverse functions of rat granulosa cells relating to the FSHR/PKA signaling pathway

Reproduction ◽  
2013 ◽  
Vol 146 (2) ◽  
pp. 169-179 ◽  
Author(s):  
Yexia Li ◽  
Yujie Jin ◽  
Yuxia Liu ◽  
Chunyan Shen ◽  
Jingxia Dong ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Transforming Growth Factor ◽  
Ovarian Follicle ◽  
Transforming Growth Factor Β ◽  
Female Rats ◽  
Nuclear Antigen ◽  
Follicle Development ◽  
Sprague Dawley ◽  
Ovarian Follicle Development ◽  
Estrogen Production

The function of Smad3, a downstream signaling protein of the transforming growth factor β (TGFβ) pathway, in ovarian follicle development remains to be elucidated. The effects of Smad3 on ovarian granulosa cells (GCs) in rat were studied. Female rats (21 days of age Sprague–Dawley) received i.p. injections of pregnant mare serum gonadotropin, and GCs were harvested for primary culture 48 h later. These cells were engineered to overexpress or knockdown Smad3, which were validated by immunohistochemistry and western blot. The expression of proliferating cell nuclear antigen (PCNA), cyclin D2, TGFβ receptor II (TGFβRII), protein kinase A (PKA), and FSH receptor (FSHR) was also detected by western blotting. Cell cycle and apoptosis of GCs were assayed by flow cytometry. The level of estrogen secreted by GCs was detected by ELISA. Smad3 overexpression promoted estrogen production and proliferation while inhibiting apoptosis of GCs. Reduction in Smad3 by RNAi resulted in reduced estrogen production and proliferation and increased apoptosis of GCs. Manipulation of Smad3 expression also resulted in changes in FSHR and PKA expression, suggesting that the effects of Smad3 on follicle development are related to FSHR-mediated cAMP signaling.

scholarly journals Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 239-254 ◽  
Author(s):  
Claire Glister ◽  
C Fred Kemp ◽  
Philip G Knight
Keyword(s):  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Viable Cell ◽  
Receptor Activation ◽  
Cell Number ◽  
Activin A ◽  
Follicle Cells ◽  
Viable Cell Number ◽  
Type I ◽  
Antral Follicles

Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed BMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced ‘basal’ and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed ‘basal’ and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under ‘basal’ (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (Kd 0.28 ± 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.

scholarly journals Ebolavirus VP35 Interacts with the Cytoplasmic Dynein Light Chain 8

2009 ◽  
Vol 83 (13) ◽  
pp. 6952-6956 ◽  
Author(s):  
Toru Kubota ◽  
Mayumi Matsuoka ◽  
Tsung-Hsien Chang ◽  
Mike Bray ◽  
Steven Jones ◽  
...  
Keyword(s):  
Life Cycle ◽  
Light Chain ◽  
Viral Protein ◽  
Cytoplasmic Dynein ◽  
Type I ◽  
Dynein Light Chain ◽  
Yeast Two Hybrid ◽  
Binding Partners ◽  
Mapping Analysis ◽  
Two Hybrid

ABSTRACT The viral protein VP35 of ebolavirus (EBOV) is implicated to have diverse roles in the viral life cycle. We employed a yeast two-hybrid screen to search for VP35 binding partners and identified the cytoplasmic dynein light chain (DLC8) as a protein that interacts with VP35. Mapping analysis unraveled a consensus motif, SQTQT, within VP35 through which VP35 binds to DLC8. The disruption of DLC8 binding does not affect the ability of VP35 to inhibit type I IFN production. Given that VP35 from various EBOV species interacts with DLC8, this interaction may have a role in regulating the EBOV life cycle.

scholarly journals Dax1 Up-Regulates Oct4 Expression in Mouse Embryonic Stem Cells via LRH-1 and SRA

2010 ◽  
Vol 24 (12) ◽  
pp. 2281-2291 ◽  
Author(s):  
Victoria R. Kelly ◽  
Bin Xu ◽  
Rork Kuick ◽  
Ronald J. Koenig ◽  
Gary D. Hammer
Keyword(s):  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Embryonic Stem ◽  
Steroid Receptor ◽  
Orphan Nuclear Receptor ◽  
Steroidogenic Factor 1 ◽  
Oct4 Expression ◽  
Mes Cells

Abstract Dax1 (Nr0b1) is an atypical orphan nuclear receptor that has recently been shown to play a role in mouse embryonic stem (mES) cell pluripotency. Here we describe a mechanism by which Dax1 maintains pluripotency. In steroidogenic cells, Dax1 protein interacts with the NR5A nuclear receptor steroidogenic factor 1 (Nr5a1) to inhibit transcription of target genes. In mES cells, liver receptor homolog 1 (LRH-1, Nr5a2), the other NR5A family member, is expressed, and LRH-1 has been shown to interact with Dax1. We demonstrate by coimmunoprecipitation that Dax1 is, indeed, able to form a complex with LRH-1 in mES cells. Because Dax1 was historically characterized as an inhibitor of steroidogenic factor 1-mediated transcriptional activation, we hypothesized that Dax1 would inhibit LRH-1 action in mES cells. Therefore, we examined the effect of Dax1 on the LRH-1-mediated activation of the critical ES cell factor Oct4 (Pou5f1). Chromatin immunoprecipitation localized Dax1 to the Oct4 promoter at the LRH-1 binding site, and luciferase assays together with Dax1 overexpression and knockdown experiments revealed that, rather than repress, Dax1 accentuated LRH-1-mediated activation of the Oct4 gene. Similar to our previously published studies that defined the RNA coactivator steroid receptor RNA activator as the critical mediator of Dax1 coactivation function, Dax1 augmentation of LRH-1-mediated Oct4 activation is dependent upon steroid receptor RNA activator. Finally, utilizing published chromatin immunoprecipitation data of whole-genome binding sites of LRH-1 and Dax1, we show that LRH-1 and Dax1 commonly colocalize at 288 genes (43% of LRH-1 target genes), many of which are involved in mES cell pluripotency. Thus, our results indicate that Dax1 plays an important role in the maintenance of pluripotency in mES cells through interaction with LRH-1 and transcriptional activation of Oct4 and other genes.

Screening and Verification of Host Proteins Interacting with Neospora Caninum AMA1 Using a Yeast Two-hybrid System

2020 ◽  
Author(s):  
Baoyi Wu ◽  
Chunmei Jin ◽  
Yinan Wang ◽  
Yan Zhang ◽  
Tianyu Zheng ◽  
...  
Keyword(s):  
Neospora Caninum ◽  
Transmembrane Protein ◽  
Type I ◽  
Filamin A ◽  
Biological Functions ◽  
Yeast Two Hybrid ◽  
Apical Membrane Antigen 1 ◽  
Invasion Mechanism ◽  
First Time ◽  
Two Hybrid

Abstract Background: Neospora caninum apical membrane antigen 1 (NcAMA1) is a conservative type I transmembrane protein that is secreted by the microneme to the surface of the parasite, and is a key component of the invasion mechanism. In order to explore further the biological functions of NcAMA1 in the process of parasite invasion, we conducted research on NcAMA1 and its interacting partners. Methods: In this study, Vero cell yeast two-hybrid (Y2H) cDNA library was constructed. Using the constructed recombinant vector pGBKT7-NcAMA1 as bait, the Y2H system was used to screen the proteins that interact with NcAMA1. In addition, the interaction between NcAMA1 and the screened transmembrane emp24 domain trafficking protein 2 （Tmed2）was further verified by one-to-one Y2H experiments and pull-down, and the role of Tmed2 protein in the process of N. caninum invasion was initially verified by RNA silencing and antibody blocking experiments.Results: Our results show that, through the Y2H experiment, we have identified two proteins that interact with NcAMA1, which are the Chlorocebus sabaeus filamin A, alpha and Chlorocebus sabaeus Tmed2. When the expression of Tmed2 protein decreased or blocked, the invasion rate of N. caninum was increased.Conclusions: These findings give us a deeper understanding of the biological functions of NcAMA1, and for the first time suggest that Tmed2 may be involved in the process invasion by of N. caninum, inhibiting the invasion of parasites by interacting with the protein secreted by N. caninum.

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