scholarly journals Target Gene Specificity of E2F and Pocket Protein Family Members in Living Cells

2000 ◽  
Vol 20 (16) ◽  
pp. 5797-5807 ◽  
Author(s):  
Julie Wells ◽  
Kathryn E. Boyd ◽  
Christopher J. Fry ◽  
Stephanie M. Bartley ◽  
Peggy J. Farnham
Keyword(s):  
Cell Cycle ◽  
Target Gene ◽  
Target Genes ◽  
Protein Complexes ◽  
Current Model ◽  
Family Members ◽  
Living Cells ◽  
Gene Promoters ◽  
Pocket Protein ◽  
Protein Dna Interactions

ABSTRACT E2F-mediated transcription is thought to involve binding of an E2F-pocket protein complex to promoters in the G0 phase of the cell cycle and release of the pocket protein in late G1, followed by release of E2F in S phase. We have tested this model by monitoring protein-DNA interactions in living cells using a formaldehyde cross-linking and immunoprecipitation assay. We find that E2F target genes are bound by distinct E2F-pocket protein complexes which change as cells progress through the cell cycle. We also find that certain E2F target gene promoters are bound by pocket proteins when such promoters are transcriptionally active. Our data indicate that the current model applies only to certain E2F target genes and suggest that Rb family members may regulate transcription in both G0 and S phases. Finally, we find that a given promoter can be bound by one of several different E2F-pocket protein complexes at a given time in the cell cycle, suggesting that cell cycle-regulated transcription is a stochastic, not a predetermined, process.

scholarly journals Regulation of Poly(ADP-ribose) Polymerase-1-dependent Gene Expression through Promoter-directed Recruitment of a Nuclear NAD+ Synthase

2012 ◽  
Vol 287 (15) ◽  
pp. 12405-12416 ◽  
Author(s):  
Tong Zhang ◽  
Jhoanna G. Berrocal ◽  
Jie Yao ◽  
Michelle E. DuMond ◽  
Raga Krishnakumar ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Target Gene ◽  
Target Genes ◽  
Enzymatic Activities ◽  
Gene Promoters ◽  
Protein Coding ◽  
Photon Excitation ◽  
Gene Sets ◽  
Cellular Compartments ◽  
Parp 1

NMNAT-1 and PARP-1, two key enzymes in the NAD+ metabolic pathway, localize to the nucleus where integration of their enzymatic activities has the potential to control a variety of nuclear processes. Using a variety of biochemical, molecular, cell-based, and genomic assays, we show that NMNAT-1 and PARP-1 physically and functionally interact at target gene promoters in MCF-7 cells. Specifically, we show that PARP-1 recruits NMNAT-1 to promoters where it produces NAD+ to support PARP-1 catalytic activity, but also enhances the enzymatic activity of PARP-1 independently of NAD+ production. Furthermore, using two-photon excitation microscopy, we show that NMNAT-1 catalyzes the production of NAD+ in a nuclear pool that may be distinct from other cellular compartments. In expression microarray experiments, depletion of NMNAT-1 or PARP-1 alters the expression of about 200 protein-coding genes each, with about 10% overlap between the two gene sets. NMNAT-1 enzymatic activity is required for PARP-1-dependent poly(ADP-ribosyl)ation at the promoters of commonly regulated target genes, as well as the expression of those target genes. Collectively, our studies link the enzymatic activities of NMNAT-1 and PARP-1 to the regulation of a set of common target genes through functional interactions at target gene promoters.

scholarly journals Nkx3.1 binds and negatively regulates the transcriptional activity of Sp-family members in prostate-derived cells

2005 ◽  
Vol 393 (1) ◽  
pp. 397-409 ◽  
Author(s):  
Steven O. Simmons ◽  
Jonathan M. Horowitz
Keyword(s):  
Dna Binding ◽  
Histone Deacetylases ◽  
Target Genes ◽  
Protein Complexes ◽  
Trichostatin A ◽  
Prostate Gland ◽  
Specific Antigen ◽  
Family Members ◽  
Early Event ◽  
Prostate Tumorigenesis

Nkx3.1 is a homeodomain-containing transcription factor that is expressed early in the development of the prostate gland and is believed to play an important role in the differentiation of prostatic epithelia. Loss of Nkx3.1 protein expression is often an early event in prostate tumorigenesis, and the abundance of Nkx3.1-negative epithelial cells increases with disease progression. In a number of systems, homeodomain proteins collaborate with zinc-finger-containing transcription factors to bind and regulate target genes. In the present paper, we report that Nkx3.1 collaborates with Sp-family members in the regulation of PSA (prostate-specific antigen) in prostate-derived cells. Nkx3.1 forms protein complexes with Sp proteins that are dependent on their respective DNA-binding domains and an N-terminal segment of Nkx3.1, and Nkx3.1 negatively regulates Sp-mediated transcription via Trichostatin A-sensitive and -insensitive mechanisms. A distal 1000 bp portion of the PSA promoter is required for transrepression by Nkx3.1, although Nkx3.1 DNA-binding activity is itself not required. We conclude that Nkx3.1 negatively regulates Sp-mediated transcription via the tethering of histone deacetylases and/or by inhibiting the association of Sp proteins with co-activators.

scholarly journals G1 arrest and differentiation can occur independently of Rb family function

2010 ◽  
Vol 191 (4) ◽  
pp. 809-825 ◽  
Author(s):  
Stacey E. Wirt ◽  
Adam S. Adler ◽  
Véronique Gebala ◽  
James M. Weimann ◽  
Bethany E. Schaffer ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Target Genes ◽  
Family Members ◽  
Mouse Embryos ◽  
G1 Arrest ◽  
Cell Cycle Exit ◽  
Family Function ◽  
Rb Pathway

The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9–11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.

scholarly journals Coactivation of Estrogen Receptor β by Gonadotropin-Induced Cofactor GIOT-4

2008 ◽  
Vol 29 (1) ◽  
pp. 83-92 ◽  
Author(s):  
Madoka Kouzu-Fujita ◽  
Yoshihiro Mezaki ◽  
Shun Sawatsubashi ◽  
Takahiro Matsumoto ◽  
Ikuko Yamaoka ◽  
...  
Keyword(s):  
Target Gene ◽  
Target Genes ◽  
Gene Promoters ◽  
Ovulatory Cycle ◽  
Independent Manner ◽  
Ovarian Granulosa Cell ◽  
Transcriptional Regulatory Mechanism ◽  
Transcriptional Regulatory ◽  
Specific Stage ◽  
Regulatory Convergence

ABSTRACT Estrogen exerts its diverse effects through two subtypes of estrogen receptors (ER), ERα and ERβ. Each subtype has its own distinct function and expression pattern in its target tissues. Little, however, is known about the transcriptional regulatory mechanism of ERβ in the major ERβ-expressing tissues. Using biochemical methods, we identified and described a novel ERβ coactivator. This protein, designated GIOT-4, was biochemically purified from 293F cells. It coactivated ERβ in ovarian granulosa cells. GIOT-4 expression was induced by stimulation with follicle-stimulating hormone (FSH). GIOT-4 recruited an SWI/SNF-type complex in a ligand-independent manner to ERβ as an ER subtype-specific physical bridging factor and induced subsequent histone modifications in the ERβ target gene promoters in a human ovarian granulosa cell line (KGN). Indeed, two ERβ-specific target genes were upregulated by FSH at a specific stage of a normal ovulatory cycle in intact mice. These findings imply the presence of a novel regulatory convergence between the gonadotropin signaling cascade and ERβ-mediated transcription in the ovary.

Repression of Mir-106b Family Members Does Not Alter Cell Cycle Progression in Hodgkin Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4722-4722
Author(s):  
Johan H Gibcus ◽  
Lu Ping Tan ◽  
Rikst Nynke Schakel ◽  
Geert Harms ◽  
Peter Moeller ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Cycle Arrest ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Target Genes ◽  
Family Members ◽  
Luciferase Reporter ◽  
P21 Protein ◽  
Cycle Arrest

Abstract MicroRNAs (miRNAs) are 19–25 nucleotide long RNA molecules derived from precursor genes that inhibit the expression of target genes by binding to their 3′ UTR region. Expression of miRNAs is often tissue specific and miRNA profiling has shown specific miRNA expression patterns in both B-cell development and lymphomagenesis. Hodgkin lymphoma is derived from pre-apoptotic germinal center B-cells, although a general loss of B cell phenotype is noted. Using quantitative RT-PCR and miRNA microarray, we determined the miRNA profile of HL and compared this with the profile of a panel of B-cell non-Hodgkin lymphomas (NHL). The two methods showed a very good correlation for the expression levels of the individual miRNAs. Using a large panel of cell lines, we confirmed differential expression between HL and other B-cell lymphoma derived cell lines for 27 miRNAs. The HL specific miRNAs included miR-155, miR-21 and miR-106b seed family members miR-17-5p, miR-20a, miR-93, miR-106a and miR- 106b. Next, we performed target gene validation of predicted target genes for miR-17-5p, which is highly expressed in HL. Using luciferase reporter assays with stabilized anti-sense miR17-5p oligonucleotides, we showed that GPR137B, RAB12 and RBJ are likely miR-17-5p target genes in two different HL cell lines. Previous publications indicated that miR-106b seed family members negatively regulate the cyclin-dependent kinase inhibitor 1A (p21/CIP1) resulting in cell cycle arrest at G1. Consistent with these findings, we show that the miR-106b family members are highly expressed in L428, whereas p21 is not. However, inhibition of the miR-106b seed family members in L428 does not result in elevated p21 protein expression. Furthermore, there is no cell cycle arrest, growth reduction or increase in cell death and apoptosis after inhibition of the miR-106b seed family members. Thus, we conclude that blocking of the miR-106b seed family members does not necessarily lead to indiction of p21 protein. This suggests an additional regulatory layer of p21 expression in L428 cells.

scholarly journals Enhancer-bound LDB1 regulates a corticotrope promoter-pausing repression program

2015 ◽  
Vol 112 (5) ◽  
pp. 1380-1385 ◽  
Author(s):  
Feng Zhang ◽  
Bogdan Tanasa ◽  
Daria Merkurjev ◽  
Chijen Lin ◽  
Xiaoyuan Song ◽  
...  
Keyword(s):  
Target Gene ◽  
Target Genes ◽  
Binding Protein ◽  
Homeodomain Protein ◽  
Gene Promoters ◽  
Cell Type ◽  
Lim Domain ◽  
Transcriptional Initiation ◽  
Nucleosome Remodeling ◽  
Enhancer Binding Protein

Substantial evidence supports the hypothesis that enhancers are critical regulators of cell-type determination, orchestrating both positive and negative transcriptional programs; however, the basic mechanisms by which enhancers orchestrate interactions with cognate promoters during activation and repression events remain incompletely understood. Here we report the required actions of LIM domain-binding protein 1 (LDB1)/cofactor of LIM homeodomain protein 2/nuclear LIM interactor, interacting with the enhancer-binding protein achaete-scute complex homolog 1, to mediate looping to target gene promoters and target gene regulation in corticotrope cells. LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes. Although LDB1-dependent activated genes are regulated at the level of transcriptional initiation, the LDB1-dependent repressed transcription units appear to be regulated primarily at the level of promoter pausing, with LDB1 regulating recruitment of metastasis-associated 1 family, member 2, a component of the nucleosome remodeling deacetylase complex, on these negative enhancers, required for the repressive enhancer function. These results indicate that LDB1-dependent looping events can deliver repressive cargo to cognate promoters to mediate promoter pausing events in a pituitary cell type.

scholarly journals Overlapping and CpG methylation-sensitive protein-DNA interactions at the histone H4 transcriptional cell cycle domain: distinctions between two human H4 gene promoters.

1992 ◽  
Vol 12 (7) ◽  
pp. 3273-3287 ◽  
Author(s):  
A J van Wijnen ◽  
F M van den Ent ◽  
J B Lian ◽  
J L Stein ◽  
G S Stein
Keyword(s):  
Cell Cycle ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Consensus Sequence ◽  
Proximal Promoter ◽  
Gene Promoters ◽  
Histone H4 ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Diploid Cells

Transcriptional regulation of vertebrate histone genes during the cell cycle is mediated by several factors interacting with a series of cis-acting elements located in the 5' regions of these genes. The arrangement of these promoter elements is different for each gene. However, most histone H4 gene promoters contain a highly conserved sequence immediately upstream of the TATA box (H4 subtype consensus sequence), and this region in the human H4 gene FO108 is involved in cell cycle control. The sequence-specific interaction of nuclear factor HiNF-D with this key proximal promoter element of the H4-FO108 gene is cell cycle regulated in normal diploid cells (J. Holthuis, T.A. Owen, A.J. van Wijnen, K.L. Wright, A. Ramsey-Ewing, M.B. Kennedy, R. Carter, S.C. Cosenza, K.J. Soprano, J.B. Lian, J.L. Stein, and G.S. Stein, Science, 247:1454-1457, 1990). Here, we show that this region of the H4-FO108 gene represents a composite protein-DNA interaction domain for several distinct sequence-specific DNA-binding activities, including HiNF-D, HiNF-M, and HiNF-P. Factor HiNF-P is similar to H4TF-2, a DNA-binding activity that is not cell cycle regulated and that interacts with the analogous region of the H4 gene H4.A (F. LaBella and N. Heintz, Mol. Cell. Biol. 11:5825-5831, 1991). The H4.A gene fails to interact with factors HiNF-M and HiNF-D owing to two independent sets of specific nucleotide variants, indicating differences in protein-DNA interactions between these H4 genes. Cytosine methylation of a highly conserved CpG dinucleotide interferes with binding of HiNF-P/H4TF-2 to both the H4-FO108 and H4.A promoters, but no effect is observed for either HiNF-M or HiNF-D binding to the H4-FO108 gene. Thus, strong evolutionary conservation of the H4 consensus sequence may be related to combinatorial interactions involving overlapping and interdigitated recognition nucleotides for several proteins, whose activities are regulated independently. Our results also suggest molecular complexity in the transcriptional regulation of distinct human H4 genes.

scholarly journals Overlapping and CpG methylation-sensitive protein-DNA interactions at the histone H4 transcriptional cell cycle domain: distinctions between two human H4 gene promoters

1992 ◽  
Vol 12 (7) ◽  
pp. 3273-3287
Author(s):  
A J van Wijnen ◽  
F M van den Ent ◽  
J B Lian ◽  
J L Stein ◽  
G S Stein
Keyword(s):  
Cell Cycle ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Consensus Sequence ◽  
Proximal Promoter ◽  
Gene Promoters ◽  
Histone H4 ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Diploid Cells

Transcriptional regulation of vertebrate histone genes during the cell cycle is mediated by several factors interacting with a series of cis-acting elements located in the 5' regions of these genes. The arrangement of these promoter elements is different for each gene. However, most histone H4 gene promoters contain a highly conserved sequence immediately upstream of the TATA box (H4 subtype consensus sequence), and this region in the human H4 gene FO108 is involved in cell cycle control. The sequence-specific interaction of nuclear factor HiNF-D with this key proximal promoter element of the H4-FO108 gene is cell cycle regulated in normal diploid cells (J. Holthuis, T.A. Owen, A.J. van Wijnen, K.L. Wright, A. Ramsey-Ewing, M.B. Kennedy, R. Carter, S.C. Cosenza, K.J. Soprano, J.B. Lian, J.L. Stein, and G.S. Stein, Science, 247:1454-1457, 1990). Here, we show that this region of the H4-FO108 gene represents a composite protein-DNA interaction domain for several distinct sequence-specific DNA-binding activities, including HiNF-D, HiNF-M, and HiNF-P. Factor HiNF-P is similar to H4TF-2, a DNA-binding activity that is not cell cycle regulated and that interacts with the analogous region of the H4 gene H4.A (F. LaBella and N. Heintz, Mol. Cell. Biol. 11:5825-5831, 1991). The H4.A gene fails to interact with factors HiNF-M and HiNF-D owing to two independent sets of specific nucleotide variants, indicating differences in protein-DNA interactions between these H4 genes. Cytosine methylation of a highly conserved CpG dinucleotide interferes with binding of HiNF-P/H4TF-2 to both the H4-FO108 and H4.A promoters, but no effect is observed for either HiNF-M or HiNF-D binding to the H4-FO108 gene. Thus, strong evolutionary conservation of the H4 consensus sequence may be related to combinatorial interactions involving overlapping and interdigitated recognition nucleotides for several proteins, whose activities are regulated independently. Our results also suggest molecular complexity in the transcriptional regulation of distinct human H4 genes.

scholarly journals Pocket Protein Complexes Are Recruited to Distinct Targets in Quiescent and Proliferating Cells

2005 ◽  
Vol 25 (18) ◽  
pp. 8166-8178 ◽  
Author(s):  
Egle Balciunaite ◽  
Alexander Spektor ◽  
Nathan H. Lents ◽  
Hugh Cam ◽  
Hein te Riele ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Regulatory Networks ◽  
Protein Complexes ◽  
Proliferating Cells ◽  
Quiescent Cells ◽  
Pocket Protein ◽  
Genome Wide ◽  
E2f Transcription Factor ◽  
A Cell ◽  
Cycle Dependent

ABSTRACT Biochemical and genetic studies have determined that retinoblastoma protein (pRB) tumor suppressor family members have overlapping functions. However, these studies have largely failed to distinguish functional differences between the highly related p107 and p130 proteins. Moreover, most studies pertaining to the pRB family and its principal target, the E2F transcription factor, have focused on cells that have reinitiated a cell cycle from quiescence, although recent studies suggest that cycling cells exhibit layers of regulation distinct from mitogenically stimulated cells. Using genome-wide chromatin immunoprecipitation, we show that there are distinct classes of genes directly regulated by unique combinations of E2F4, p107, and p130, including a group of genes specifically regulated in cycling cells. These groups exhibit both distinct histone acetylation signatures and patterns of mammalian Sin3B corepressor recruitment. Our findings suggest that cell cycle-dependent repression results from recruitment of an unexpected array of diverse complexes and reveals specific differences between transcriptional regulation in cycling and quiescent cells. In addition, factor location analyses have, for the first time, allowed the identification of novel and specific targets of the highly related transcriptional regulators p107 and p130, suggesting new and distinct regulatory networks engaged by each protein in continuously cycling cells.

scholarly journals Liver X Receptor Ligands Suppress Ubiquitination and Degradation of LXRα by Displacing BARD1/BRCA1

2009 ◽  
Vol 23 (4) ◽  
pp. 466-474 ◽  
Author(s):  
Kang Ho Kim ◽  
Jeong Min Yoon ◽  
A Hyun Choi ◽  
Woo Sik Kim ◽  
Gha Young Lee ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Target Gene ◽  
Target Genes ◽  
Cancer Susceptibility ◽  
Liver X Receptor ◽  
Lipid Homeostasis ◽  
Gene Promoters ◽  
Receptor Ligands ◽  
Ubiquitin E3 Ligase ◽  
Ligand Free

Abstract Liver X receptor (LXR) is a ligand-activated transcription factor that plays important roles in cholesterol and lipid homeostasis. However, ligand-induced posttranslational modification of LXR is largely unknown. Here, we show that ligand-free LXRα is rapidly degraded by ubiquitination. Without ligand, LXRα interacts with an ubiquitin E3-ligase protein complex containing breast and ovarian cancer susceptibility 1 (BRCA1)-associated RING domain 1 (BARD1). Interestingly, LXR ligand represses ubiquitination and degradation of LXRα, and the interaction between LXRα and BARD1 is inhibited by LXR ligand. Consistently, T0901317, a synthetic LXR ligand, increased the level of LXRα protein in liver. Moreover, overexpression of BARD1/BRCA1 promoted the ubiquitination of LXRα and reduced the recruitment of LXRα to the target gene promoters, whereas BARD1 knockdown reversed such effects. Taken together, these data suggest that LXR ligand prevents LXRα from ubiquitination and degradation by detaching BARD1/BRCA1, which might be critical for the early step of transcriptional activation of ligand-stimulated LXRα through a stable binding of LXRα to the promoters of target genes.

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