Reconstitution of Chromatin Complexes from High-Performance Liquid Chromatography-Purified Histones

Ausió, Juan; Moore, Susan C.

doi:10.1006/meth.1998.0637

Cited by 40 publications

(45 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Reconstitution of Nucleosome and Oligonucleosome Arrays-Recombinant human H2A.1 and H2A.Z proteins were mixed with chicken native H2B, H3, and H4 histones in stoichiometric amounts, and histone octamers were reconstituted onto random 146-bp 1 and positioning 208-12-mer templates (28) by a 2 to 0 M stepwise salt gradient dialysis (29) in 10 mM Tris-HCl (7.5), 0.1 mM EDTA (8.0) as described elsewhere (28). The 208-12 DNA template consisting of 12 tandem repeats of a 208-bp sequence derived from the sea urchin Lytechinus variegatus 5 S rRNA gene was amplified and purified from plasmid p5S-208-12 kindly provided to us by Dr. R. T. Simpson (30).…”

Section: Methodsmentioning

confidence: 99%

“…The 208-12 DNA template consisting of 12 tandem repeats of a 208-bp sequence derived from the sea urchin Lytechinus variegatus 5 S rRNA gene was amplified and purified from plasmid p5S-208-12 kindly provided to us by Dr. R. T. Simpson (30). The stoichiometry of the histone octamer to the DNA in the reconstituted 208-12 oligonucleosome complexes was determined as described previously (28). The reconstituted chromatin particles thus obtained (at a concentration of ϳ40 -50 g/ml) were dialyzed against the appropriate buffers (25, 31) and used for subsequent analytical ultracentrifuge analysis (32,33).…”

Section: Methodsmentioning

confidence: 99%

“…DNase I Footprinting-DNase I digestion of 32 P-labeled mononucleosomes and autoradiography were performed according to Ausió and Moore (28).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Characterization of the Stability and Folding of H2A.Z Chromatin Particles

Abbott

Ivanova

Wang

et al. 2001

Journal of Biological Chemistry

Self Cite

147

110

View full text Add to dashboard Cite

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of the Stability and Folding of H2A.Z Chromatin Particles

Abbott

Ivanova

Wang

et al. 2001

Journal of Biological Chemistry

Self Cite

147

110

View full text Add to dashboard Cite

“…Purification of Defined Sequence DNA Templates-p5S 208-12 plasmid containing 12 tandem repeats of a 208-bp sequence derived from the Lytechinus 5 S rRNA gene (44) was prepared as described elsewhere (45). The 208-12 DNA and 208 DNA were excised from the plasmid by HhaI and AvaI digestion, respectively.…”

Section: Sds-page-electrophoreticmentioning

confidence: 99%

“…Reconstitution of Nucleosomes-Nucleosome cores were reconstituted onto purified 167-bp random sequence or sequence-defined 208-bp 5 S rDNA fragments using the reconstituted hybrid chicken erythrocyte octamers as a histone source containing either calf H2A (control) or calf uH2A and following the procedures already described (45).…”

Section: Sds-page-electrophoreticmentioning

confidence: 99%

Histone H2A Ubiquitination Does Not Preclude Histone H1 Binding, but It Facilitates Its Association with the Nucleosome

Jason

Finn

Lindsey

et al. 2005

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.Despite the renewed interest in histone H2A/H2B ubiquitination (1-3), the functional role of uH2A 1 still remains controversial. This is in contrast to ubiquitinated H2B where a strong correlation with transcriptional activation has long been established (2), and even a molecular mechanism involving a transhistone regulatory pathway has been shown to be involved in this process (4, 5). With uH2A, there is almost as much experimental evidence for its association to actively transcribing chromatin as there is to the opposite. For instance, it was shown that nucleosomes from the transcriptionally poised hsp70 and copia genes from Drosophila contain 50% uH2A (6). Also nucleosomes containing mono-and diubiquitinated H2A were found to preferentially occur near the 5Ј-end of the transcribed mouse dihydrofolate reductase gene (7). On the other hand, early studies with rat liver nuclei linked the disappearance of uH2A to increased transcription (8), and it was shown that Drosophila nucleosomes consisting of non-transcribed 1.705 satellite DNA showed an enrichment in uH2A (9). In support of these early data, a role for histone uH2A in Polycomb silencing has been demonstrated recently (10). Furthermore uH2A has been shown to be concentrated in the heterochromatic inactive sex body in pachytene spermatocytes (11). Nevertheless the molecular mechanism(s) involved remains completely unknown.Despite the controversy, ubiquitinated histones have been associated with chromatin partially depleted of linker histones (12) and transcriptionally active or poised sequences (1, 2, 6, 7, 13) that are thought to be have a reduced linker histone content or have altered association with linker histones (for a review, see Ref. 14). However, there is evidence that suggests that histone H2A ubiquitination does not interfere with linker histone binding. Two independent studies have shown that H1 can be cross-linked to uH2A in vitro (15) and in vivo (16). Moreover, in the cross-linking studies performed in mouse cells, the molar ratio of H1-uH2A to H1-H2A was the same as the molar ratio of uH2A to H2A (16).Linker histones are required for the stabilization of well defined chromatin fibers (17-19), and interference with the binding of lin...

show abstract

Analysis of Protein Lysine Acetylation In Vitro and In Vivo

2008

View full text Add to dashboard Cite

Protein lysine acetylation, referring to acetylation of the epsilon-amino group of a lysine residue, has recently emerged as an important post-translational modification for regulating protein functions in various organisms. Like phosphorylation, lysine acetylation is a rapidly reversible and precisely controlled covalent modification that serves as a simple on/off switch or participates in a codified manner with other post-translational modifications to regulate protein functions in different cellular and developmental processes. This unit describes and discusses methods used for in vitro and in vivo determination of lysine acetylation.

show abstract

Reconstitution of Chromatin Complexes from High-Performance Liquid Chromatography-Purified Histones

Cited by 40 publications

References 54 publications

Characterization of the Stability and Folding of H2A.Z Chromatin Particles

Characterization of the Stability and Folding of H2A.Z Chromatin Particles

Histone H2A Ubiquitination Does Not Preclude Histone H1 Binding, but It Facilitates Its Association with the Nucleosome

Analysis of Protein Lysine Acetylation In Vitro and In Vivo

Contact Info

Product

Resources

About