Studying the recruitment of Sp1 to the β-globin promoter with  an
            <i>in vivo</i>
            method: Protein position identification  with nuclease tail (PIN*POINT)

Lee, Jong-Soo; Lee, Chang-Hun; Chung, Jay H.

doi:10.1073/pnas.95.3.969

Cited by 24 publications

(13 citation statements)

References 40 publications

(35 reference statements)

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“…Antibodies may no longer be a significant issue for E2A, since ChIP assays for E2A have been recently reported using monoclonal antibodies against E2A or directed against an epitope-tagged E2A (17,20,56). The Pinpoint technique, which utilizes fusion constructs between a DNA endonuclease and a transcription factor (27), introduces DNA nicks that are likely to generate a DNA damage-and-repair response. In contrast, Dam in transgenic Drosophila had little effect on development (62).…”

Section: Vol 24 2004 E2a Binds Cyclin D3 In Vivo 8797mentioning

confidence: 99%

Identification of Cyclin D3 as a Direct Target of E2A Using DamID

Song

Cooperman²,

Letting

et al. 2004

Molecular and Cellular Biology

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The transcription factor E2A can promote precursor B cell expansion, promote G 1 cell cycle progression, and induce the expressions of multiple G 1 -phase cyclins. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated the 1-kb promoter of cyclin D3, which contains two E boxes. However, deletion of the E boxes did not disrupt the transactivation by E2A, raising the possibility of indirect activation via another transcription factor or binding of E2A to non-E-box DNA elements. To distinguish between these two possibilities, promoter occupancy was examined using the DamID approach. A fusion construct composed of E2A and the Escherichia coli DNA adenosine methyltransferase (E47Dam) was subcloned in lentivirus vectors and used to transduce precursor B-cell and myeloid progenitor cell lines. In both cell types, specific adenosine methylation was identified at the cyclin D3 promoter. Chromatin immunoprecipitation analysis confirmed the DamID findings and localized the binding to within 1 kb of the two E boxes. The methylation by E47Dam was not disrupted by mutations in the E2A portion that block DNA binding. We conclude that E2A can be recruited to the cyclin D3 promoter independently of E boxes or E2A DNA binding activity.E2A is essential to normal B-cell development. B cells, even at the earliest detectable precursor-B-cell developmental stage, do not develop in mice that lack E2A (2, 68). Heterozygote murine fetuses that express only one copy of E2A have half the number of B cells, suggesting that the level of E2A may dictate the number of B cells (68). E2A function is intrinsic to B cells, since transfer of E2A null bone marrow cells into wild-type mice fails to rescue B-cell development while ectopic expression of E2A in E2A null mice rescues B-cell development (67).E2A is frequently mutated in precursor-B-cell lymphoblastic leukemia. Acute lymphoblastic leukemia (ALL) is the most common neoplasm in childhood (65). Approximately 6% of pediatric ALLs have chromosomal translocations involving the E2A gene (29). These translocations represent the secondmost-frequent translocation in ALL and are almost always associated with ALL of precursor-B-cell origin. The most common E2A translocation, t(1;19), is associated with poor response to older standard risk ALL therapies. Even with the current ALL therapies, the t(1:19) translocation is sufficient to decrease the prognosis from low risk (Ͼ90% 4-year event-free survival) to standard risk (approximately 80% 4-year eventfree survival).Normal E2A regulates cell cycle progression, an important contributor of cell accumulation that is frequently disrupted during carcinogenesis. Hence, mutations in E2A may contribute to abnormal B-cell accumulation by disrupting normal regulation of cell cycle progression by E2A. The nature of this regulation is unclear. Numerous studies support the current model that E2A inhibits cell cycle progression (19,40,41).However, other st...

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Section: Vol 24 2004 E2a Binds Cyclin D3 In Vivo 8797mentioning

confidence: 99%

Identification of Cyclin D3 as a Direct Target of E2A Using DamID

Song

Cooperman²,

Letting

et al. 2004

Molecular and Cellular Biology

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“…The Pur␣ expression construct, pHAPur1, was kindly provided by E. Johnson (Mount Sinai School of Medicine, New York, NY) (11), and the empty vector equivalent, pHA, was produced by religation following liberation of the Pur␣ sequence by RsrII and EcoRI digestion. The Sp1 expression construct, pCMV-Sp, was produced from the parent plasmid p588 (12). This parent plasmid contains the Sp1 coding region cloned downstream of the CMV promoter and upstream of a region encoding the nuclease domain of the restriction endonuclease FokI.…”

Section: Plasmid Constructionmentioning

confidence: 99%

During Differentiation of the Monocytic Cell Line U937, Purα Mediates Induction of theCD11cβ2 Integrin Gene Promoter

Shelley

Teodoridis

Park

et al. 2002

The Journal of Immunology

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CD11c is a member of the β2 integrin family of adhesion molecules that, together with CD18, forms a heterodimeric receptor on the surface of myeloid, NK, dendritic, and certain leukemic, lymphoma, and activated lymphoid cells. Monocytic differentiation is associated with an induction of both CD11c and CD18 gene expression. The resulting CD11c/CD18 receptor mediates firm adhesion to the vascular endothelium, transendothelial migration, chemotaxis, and phagocytosis. Monocytic differentiation can be mimicked in vitro by treatment of the promonocytic cell line U937 with PMA. Recently, we reported that in U937 cells, expression of the CD11c gene is controlled by an unidentified transcription factor that binds ssDNA. This finding suggested that DNA secondary structure plays an important role in controlling the CD11c gene and prompted us to search for additional ssDNA-binding activities with which this gene interacts. In this study, we report that in U937 cells, expression of the CD11c gene is mediated by the ssDNA-binding protein Purα. During PMA-induced differentiation, the ability of Purα to activate the CD11c promoter in U937 cells increases, as does that of Sp1. Together, these increases in the functional activity of both Purα and Sp1 combine to induce CD11c expression.

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“…Cells were harvested 48 h after transfection, and cell lysates were then used in CAT assays. A Hirt extraction was performed to recover plasmid DNA from transfected cells (Lee et al 1998). The recovered plasmid was subjected to primer extension using the CAT primer, and the products were resolved by denaturing electrophoresis and autoradiographed.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

Activator-mediated disruption of sequence-specific DNA contacts by the general transcription factor TFIIB

Evans¹,

Fairley²,

Roberts³

2001

Genes Dev.

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The transcription factor TFIIB plays a central role in preinitiation complex assembly, providing a bridge between promoter-bound TFIID and RNA Polymerase II. TFIIB possesses sequence-specific DNA-binding ability and interacts with the TFIIB-recognition element (BRE), present in many promoters. Here we show that the BRE suppresses the basal level of transcription elicited by a core promoter, which increases the amplitude of transcriptional stimulation in the presence of an activator protein. Further, we find that an activator can disrupt the TFIIB–BRE interaction within a promoter-bound complex. Our results reveal a novel function for activators in the modulation of core promoter recognition by TFIIB

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Studying the recruitment of Sp1 to the β-globin promoter with an in vivo method: Protein position identification with nuclease tail (PIN*POINT)

Cited by 24 publications

References 40 publications

Identification of Cyclin D3 as a Direct Target of E2A Using DamID

Identification of Cyclin D3 as a Direct Target of E2A Using DamID

During Differentiation of the Monocytic Cell Line U937, Purα Mediates Induction of theCD11cβ2 Integrin Gene Promoter

Activator-mediated disruption of sequence-specific DNA contacts by the general transcription factor TFIIB

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