Reconstitution and Transcriptional Analysis of Chromatin In Vitro

An, Woojin; Roeder, Robert G.

doi:10.1016/s0076-6879(03)77030-x

Cited by 52 publications

(69 citation statements)

References 25 publications

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“…FLAG-tagged human DNA topoisomerase I for relaxation of supercoiled DNA was expressed in Sf9 cells and purified using M2 agarose (64). Expression and purification of recombinant ACF and NAP-1 and procedures for chromatin assembly, supercoiling assay, and micrococcal nuclease (MNase) analysis were essentially as described previously (3). Reaction mixtures (70 l) contained 0.35 g relaxed plasmid DNA tRNA Met (34) and 0.6 g core histones.…”

Section: Methodsmentioning

confidence: 99%

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Different Functional Modes of p300 in Activation of RNA Polymerase III Transcription from Chromatin Templates

Mertens

Roeder

2008

Molecular and Cellular Biology

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Transcriptional coactivators that regulate the activity of human RNA polymerase III (Pol III) in the context of chromatin have not been reported. Here, we describe a completely defined in vitro system for transcription of a human tRNA gene assembled into a chromatin template. Transcriptional activation and histone acetylation in this system depend on recruitment of p300 by general initiation factor TFIIIC, thus providing a new paradigm for recruitment of histone-modifying coactivators. Beyond its role as a chromatin-modifying factor, p300 displays an acetyltransferase-independent function at the level of preinitiation complex assembly. Thus, direct interaction of p300 with TFIIIC stabilizes binding of TFIIIC to core promoter elements and results in enhanced transcriptional activity on histone-free templates. Additional studies show that p300 is recruited to the promoters of actively transcribed tRNA and U6 snRNA genes in vivo. These studies identify TFIIIC as a recruitment factor for p300 and thus may have important implications for the emerging concept that tRNA genes or TFIIIC binding sites act as chromatin barriers to prohibit spreading of silenced heterochromatin domains.It is well established that assembly of regulatory DNA sequences into nucleosomes represses transcription by restricting access of the transcriptional machinery to DNA (reviewed in reference 28). Thus, overcoming nucleosomal repression through alteration of chromatin structure is essential for gene activation. Eukaryotic cells contain at least two general classes of chromatin-modifying factors, the ATP-dependent chromatin-remodeling complexes (43) and histone-modifying enzymes that effect histone acetylation, methylation, phosphorylation, and ubiquitination (63). Whereas the function and mechanism of action of both types of factors have been extensively studied on genes transcribed by RNA polymerase II (Pol II), much less is known about chromatin-modifying factors involved in the regulation of Pol III-dependent genes.Pol III synthesizes small structural RNAs such as 5S RNA, tRNA, adenovirus VA1 RNA, and U6 RNA. Accurate and specific transcription of DNA templates by Pol III requires the assistance of the general initiation transcription factors TFIIIB and TFIIIC and, in some cases, various gene-specific factors such as the 5S gene-specific factor TFIIIA (for review, see references 15 and 53). In the case of tRNA and VA1 genes (subclass 2 genes), gene-internal core promoter elements (A and B boxes) are recognized directly by TFIIIC, which in turn directs the sequential binding of TFIIIB and Pol III. Human TFIIIC has been chromatographically separated into two distinct activities, TFIIIC1 and TFIIIC2 (74). TFIIIC2 is a stable complex of six subunits (220, 110, 102, 90,63, and 35 kDa) that binds directly to the B box (10a, 30, 75). TFIIIC1 displays no strong DNA-binding activity on its own but stabilizes TFIIIC2 binding to A and B boxes (66, 74). Other factors derived from TFIIIC preparations, such as coactivator PC4, also stabilize TFIIIC bind...

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Section: Methodsmentioning

confidence: 99%

“…Reaction mixtures (70 l) contained 0.35 g relaxed plasmid DNA tRNA Met (34) and 0.6 g core histones. Chromatin acetylation assays were performed as described previously (3). Reaction mixtures contained 650 ng histones (as assembled chromatin), 100 ng TFIIIC, and 20 ng p300.…”

Section: Methodsmentioning

confidence: 99%

Different Functional Modes of p300 in Activation of RNA Polymerase III Transcription from Chromatin Templates

Mertens

Roeder

2008

Molecular and Cellular Biology

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“…(Fig. 5B,C ;Luger et al 1999;An and Roeder 2004;Vary et al 2004). The DNA template contained six LexA binding sites, which can be bound by a LexA protein fused to an RNAbinding protein (Fig.…”

Section: In Vitro Chromatin Reconstitution Of A2/b1-hotair Interactionmentioning

confidence: 99%

“…PCR with a biotinylated reverse primer was used to add a biotin tag to the 3 ′ end of the 1.1-kb DNA substrate. Chromatin was reconstituted through a previously developed enzymatic assembly method (An and Roeder 2004;Vary et al 2004). Biotinylated DNA, human histone octamers, human histone chaperone Nap1, nucleosome positioning factor Isw1a, and an ATPregeneration system (final concentrations: 30 mM creatine phosphate, 3 mM ATP, 4.1 mM MgCl 2 , and 6.4 μg/mL creatine kinase) were incubated at 30°C for 5 h in R+ Buffer (10 mM Hepes pH hnRNPA2/B1 regulates the HOTAIR lncRNA www.rnajournal.org 1007 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 500 µM EGTA, 10% Glycerol, 2.5 mM β-glyerophosphate, 200 µM PMSF, 1 mM DTT).…”

Section: Chromatin Reconstitutionmentioning

confidence: 99%

An RNA matchmaker protein regulates the activity of the long noncoding RNA HOTAIR

Meredith

Balas

Sindy

et al. 2016

RNA

110

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The human long noncoding RNA (lncRNA) HOTAIR acts in trans to recruit the Polycomb repressive complex 2 (PRC2) to the HOXD gene cluster and to promote gene silencing during development. In breast cancers, overexpression of HOTAIR increases metastatic potential via the repression of many additional genes. It has remained unclear what factors determine HOTAIR-dependent PRC2 activity at specific genomic loci, particularly when high levels of HOTAIR result in aberrant gene silencing. To identify additional proteins that contribute to the specific action of HOTAIR, we performed a quantitative proteomic analysis of the HOTAIR interactome. We found that the most specific interaction was between HOTAIR and the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, a member of a family of proteins involved in nascent mRNA processing and RNA matchmaking. Our data suggest that A2/B1 are key contributors to HOTAIR-mediated chromatin regulation in breast cancer cells: A2/B1 knockdown reduces HOTAIR-dependent breast cancer cell invasion and decreases PRC2 activity at the majority of HOTAIR-dependent loci. We found that the B1 isoform, which differs from A2 by 12 additional amino acids, binds with highest specificity to HOTAIR. B1 also binds chromatin and associates preferentially with RNA transcripts of HOTAIR gene targets. We furthermore demonstrate a direct RNA-RNA interaction between HOTAIR and a target transcript that is enhanced by B1 binding. Together, these results suggest a model in which B1 matches HOTAIR with transcripts of target genes on chromatin, leading to repression by PRC2.

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“…HeLa core histones were purified as described previously (45). In vitro chromatin assembly was done as described previously (2). HAT reactions using HeLa core histones (1 g) or in vitro-assembled nucleosomes (0.3 g) were carried out as described previously (39).…”

Section: Expression Vectors Antibodies and Pcr Primersmentioning

confidence: 99%

GAS41 Is Required for Repression of the p53 Tumor Suppressor Pathway during Normal Cellular Proliferation

Park

Roeder

2006

Molecular and Cellular Biology

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GAS41 is a common subunit of the TIP60 and SRCAP complexes and is essential for cell growth and viability. Here, we report that GAS41 is required for repression of the p53 tumor suppressor pathway during normal cellular proliferation. Either GAS41 small interfering RNA-mediated knockdown of GAS41 expression or specific interruptions of the carboxy-terminal coiled-coil motif of the GAS41 protein activate the p53 tumor suppressor pathway, as evidenced by p53 up-regulation, p53 serine-15 phosphorylation, and p21 transcriptional activation. Activation of the p53 pathway does not result from changes in TIP60 complex assembly or TIP60 coactivator functions for p53, since a TIP60 complex containing a coiled-coil mutant of GAS41 retains the same composition and histone acetyltransferase activity as its wild-type counterpart and since mutant GAS41 does not compromise ectopic p53-dependent transcriptional activation in a reporter gene assay. Finally, we demonstrate that GAS41 is prebound to the promoters of two p53 tumor suppressor pathway genes (p21 and p14 ARF ) in normal unstressed cells but is dissociated from both promoters in response to stress signals that activate p53. Our data suggest that GAS41 plays a role in repressing the p53 tumor suppressor pathway during the normal cell cycle by a TIP60-independent mechanism.Epigenetic mechanisms are now widely accepted as important regulators of gene expression (9, 19). Eucaryotic DNA is packaged into a chromatin structure that consists of arrays of core histone-containing nucleosomes organized, along with linker histone H1, into higher-order structures. Tightly packed nucleosomes generate an inherently repressive structure for gene expression by restricting access of various transcription factors and the general transcription machinery. Moreover, DNA modifications, such as CpG island methylation, and histone tail modifications, such as acetylation, methylation, phosphorylation, and ubiquitylation, allow complex epigenetic controls of nuclear activities that include DNA replication, transcription, DNA repair, and recombination (41).The increasingly large group of chromatin-modifying complexes contains enzymatic activities that covalently modify histone tails. The enzymatic subunits of complexes with histone acetyltransferase (HAT) activity include the H3 HAT GCN5 in Saccharomyces cerevisiae SAGA and human STAGA complexes and the H4 HAT Esa1 in the yeast NuA4 complex (1,22,35). In humans, a NuA4-like complex with TIP60 as the H4 HAT has been described, although there is also a similar complex that lacks HAT activity but shares the TRRAP, p400, Epc1, TIP49, TIP48, BAF53, and ␤-actin components (21, 24). It is not clear whether the last complex has a distinct function or whether it represents an intermediate in the assembly of the TIP60-containing complex. The TIP60 complex was proposed to play a role in DNA repair and apoptosis, and its largest subunit, TRRAP, has been implicated in p53-dependent mdm2 transcriptional activation (3,24,27). TIP60 also has been proposed as an ...

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Reconstitution and Transcriptional Analysis of Chromatin In Vitro

Cited by 52 publications

References 25 publications

Different Functional Modes of p300 in Activation of RNA Polymerase III Transcription from Chromatin Templates

Different Functional Modes of p300 in Activation of RNA Polymerase III Transcription from Chromatin Templates

An RNA matchmaker protein regulates the activity of the long noncoding RNA HOTAIR

GAS41 Is Required for Repression of the p53 Tumor Suppressor Pathway during Normal Cellular Proliferation

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