DNA Methylation Increases Nucleosome Compaction and Rigidity

Choy, John S.; Wei, Sijie; Lee, Ju‐Yeon; Tan, Song; Chu, Steven; Lee, Tae Hee

doi:10.1021/ja910264z

Cited by 243 publications

(218 citation statements)

References 15 publications

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“…Perhaps because the sensor somewhat resembles the structure of actin dimers, the actin probe AcpA expresses well and labels f-actin efficiently, presumably at random locations. For imaging purposes, we found that, as previously published, polarized FRET (16)(17)(18) reduces bleed-through corrections and provides increased contrast.…”

supporting

confidence: 81%

Actin stress in cell reprogramming

Guo

Wang

Sachs

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

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Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin-cpstFRET-actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFPtagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals.actin | force probe | stem cell | reprogramming | cell mechanics C ells have only three sources of free energy-chemical, electrical, and mechanical potential (1)-and life is driven by the flow of energy between them. The flow of mechanical energy has not been well studied in living cells outside of muscle, yet all cells are subject to endogenous and exogenous mechanical forces. The lack of progress in measuring these forces (stress) has primarily been due to a lack of suitable probes, but that has now been remedied with the development of genetically coded FRETbased force sensors (2). The literature shows many macroscopic effects of mechanical stress on cellular processes including cell motility, embryogenesis, stem cell replication and differentiation (3, 4), bone and muscle homeostasis, gene expression, protein folding (5), and membrane potential (6). However, these studies lacked the ability to measure stress in specific structural proteins, and the analyses have tended to treat cytoskeletal stresses as uniform, which we now know is not true (2, 7). We, and other groups (8), have shown that the distribution of forces is nonuniform in both time and space and protein specificity (9).The force sensors consist of a FRET pair (typically CFP/ YFP) connected by a protein linker, a biological analog of a mechanic...

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supporting

confidence: 81%

Actin stress in cell reprogramming

Guo

Wang

Sachs

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

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“…We then a global genomic scale (particularly at repetitive sequences) and hypermethylation at CGIs. 27 CpG methylation may induce a more compact and rigid nucleosome structure 67 that would significantly affect the CGIs, which normally are relatively nucleosome-deficient and permissive for TF binding. 62 Perhaps more significantly, hypomethylation of the bulk of the genome might open access to many potential binding sites that are not accessible in normal cells.…”

Section: Resultsmentioning

confidence: 99%

Distinct p53 genomic binding patterns in normal and cancer-derived human cells

Botcheva¹,

McCorkle²,

McCombie³

et al. 2011

Cell Cycle

118

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“…3) (36). Nucleosomes containing the 147-bp DNA displayed one major band, some minor amounts of higher molecular weight species, and free DNAs.…”

Section: Dna Fragment Preparation and Reconstitution Of Nucleosomes Umentioning

confidence: 99%

Effects of Histone Acetylation by Piccolo NuA4 on the Structure of a Nucleosome and the Interactions between Two Nucleosomes

Lee

Wei

Lee

2011

Journal of Biological Chemistry

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We characterized the effect of histone acetylation on the structure of a nucleosome and the interactions between two nucleosomes. In this study, nucleosomes reconstituted with the Selex "Widom 601" sequence were acetylated with the Piccolo NuA4 complex, which acetylates mainly H4 N-terminal tail lysine residues and some H2A/H3 N-terminal tail lysine residues. Upon the acetylation, we observed directional unwrapping of nucleosomal DNA that accompanies topology change of the DNA. Interactions between two nucleosomes in solution were also monitored to discover multiple transient dinucleosomal states that can be categorized to short-lived and long-lived (ϳ1 s) states. The formation of dinucleosomes is strongly Mg 2؉ -dependent, and unacetylated nucleosomes favor the formation of long-lived dinucleosomes 4-fold as much as the acetylated ones. These results suggest that the acetylation of histones by Piccolo NuA4 disturbs not only the structure of a nucleosome but also the interactions between two nucleosomes. Lastly, we suggest a structural model for a stable dinucleosomal state where the two nucleosomes are separated by ϳ2 nm face-to-face and rotated by 34°with respect to each other.

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DNA Methylation Increases Nucleosome Compaction and Rigidity

Cited by 243 publications

References 15 publications

Actin stress in cell reprogramming

Actin stress in cell reprogramming

Distinct p53 genomic binding patterns in normal and cancer-derived human cells

Effects of Histone Acetylation by Piccolo NuA4 on the Structure of a Nucleosome and the Interactions between Two Nucleosomes

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