Phosphorylation and an ATP-dependent process increase the dynamic exchange of H1 in chromatin - PubMed (original) (raw)

Phosphorylation and an ATP-dependent process increase the dynamic exchange of H1 in chromatin

Yali Dou et al. J Cell Biol. 2002.

Abstract

In Tetrahymena cells, phosphorylation of linker histone H1 regulates transcription of specific genes. Phosphorylation acts by creating a localized negative charge patch and phenocopies the loss of H1 from chromatin, suggesting that it affects transcription by regulating the dissociation of H1 from chromatin. To test this hypothesis, we used FRAP of GFP-tagged H1 to analyze the effects of mutations that either eliminate or mimic phosphorylation on the binding of H1 to chromatin both in vivo and in vitro. We demonstrate that phosphorylation can increase the rate of dissociation of H1 from chromatin, providing a mechanism by which it can affect H1 function in vivo. We also demonstrate a previously undescribed ATP-dependent process that has a global effect on the dynamic binding of linker histone to chromatin.

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Figures

Figure 1.

Figure 1.

GFP-tagged H1s replace endogenous H1 by homologous recombination. (A) Maps of the macronuclear HHO1 locus before and after gene replacement. The WT HHO1 gene is shown as a box in a 4.15-kb Hind III fragment. In WT and A5 or E5, _Eco_RI will cleave the original 4.15-kb fragment into 2.3- and 1.85-kb fragments. The position of the probe used in the Southern blot is shown at the bottom. (B) Southern blot analysis shows complete gene replacement in WT-GFP, A5-GFP, and E5-GFP strains. Genomic DNAs were isolated from WT, WT-GFP, A5-GFP, and E5-GFP cells and digested with _Hin_dIII and _Eco_RI. The 2.3-kb band observed in WT cells has been completely replaced by a 3.0-kb band in the GFP-tagged cells.

Figure 2.

Figure 2.

GFP-tagged H1 is correctly targeted to macronuclei and does not affect the transcription regulation of CyP1 by H1 phosphorylation. (A) Nuclear localization of GFP-tagged H1 in A5 cells. A DIC image is shown on the left, GFP fluorescence in the middle and the merged image is shown on the right. WT and E5 cells gave the same results (not depicted). (B) The function of H1 in the regulation of CyP1 expression is not disturbed by GFP tagging. Whole-cell RNAs isolated from WT, A5, E5, WT-GFP, A5-GFP, and E5-GFP strains after 0, 6, and 12 h of starvation were analyzed on a Northern blot probed with a _CyP1_-specific probe and a _ngoA_-specific probe. A 26S rRNA probe was used as a loading control. The expression patterns of CyP1 and ngoA were similar to the results obtained from WT, A5, and E5 cells without GFP tag.

Figure 3.

Figure 3.

Phosphorylation affects the dynamics of H1 binding in vivo. (A) Time-lapse images were taken before and during recovery after bleaching macronuclei in Tetrahymena. An A5 cell expressing A5-GFP is used as an example. Images were taken immediately before photobleaching, immediately after photobleaching, and at indicated intervals during recovery. The bleached region is shown within the square. (B) Quantitative analysis of FRAP experiments after bleaching macronuclei in WT-GFP, A5-GFP, or E5-GFP cells. The number of cells analyzed for each curve is indicated.

Figure 4.

Figure 4.

The dynamic binding of H1 to chromatin is an ATP-dependent process. FRAP analysis on A5-GFP or E5-GFP in macronuclei of living cells after treatment with the ATP depleting drug, Rotenone. Cells were treated with 180 μM Rotenone until they stopped swimming, an indicator of ATP depletion.

Figure 5.

Figure 5.

ATP-dependent H1 binding to chromatin in two in vitro systems. (A) ATP-dependent binding of H1 in isolated nuclei. Quantitative analysis of FRAP experiments after bleaching A5-GFP and E5-GFP nuclei incubated with or without energy mix. Nuclei were isolated immediately before the analysis, and images were taken within 2 h of isolation. (B) ATP-dependent binding of H1 in Tetrahymena cell ghosts. The fluorescent recovery of bleached A5-GFP cell ghosts and E5-GFP cell ghosts was determined in the presence or absence of ATP (100 μM) mix.

Figure 6.

Figure 6.

ATP titration of A5 and E5 cell ghosts. The 50% recovery times were calculated for A5 and E5 cell ghosts at 0, 10, 25, 50, and 100 μM ATP.

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