Global modulation of chromatin dynamics mediated by dephosphorylation of linker histone H1 is necessary for erythroid differentiation

Abstract

Differentiation of metazoan cells involves dramatic changes in gene expression patterns and proliferative capacity driven primarily by epigenetic mechanisms. Here we used in vivo photobleaching techniques and biochemical assays to investigate the contribution of alterations in chromatin dynamics to the differentiation of murine erythroleukemia (MEL) cells, a model system for erythroid development. As MEL cells differentiate the binding affinity of all linker histone variants increases, indicative of an overall decrease in chromatin flexibility. Changes in H1(0) binding properties depend on phosphorylation at one or more of three cyclin-dependent kinase sites. The presence of constructs mimicking constitutively phosphorylated H1 results in a significant inhibition in the acquisition of commitment to terminal cell division and the expression of erythroid-specific properties. These data indicate that the progressive loss of cdk activity associated with MEL cell differentiation leads to the accumulation of dephosphorylated linker histones and restricted chromatin flexibility and that these are necessary events in the progression of erythroid differentiation. We present additional data indicating that the presence of phosphorylated H1 has a dominant effect on the binding behavior of other linker histones and propose a model for the role of linker histone phosphorylation in which these modifications act within the context of assembled chromatin.

Fig. 1.

Chromatin binding properties of H1-GFP variants in untreated and differentiated MEL cells. (A–D) FRAP recovery curves of the indicated constructs from control MEL cultures (Untreated) and parallel cultures treated with 5 mM HMBA for 4 days (Differentiated). The FRAP recovery curve is a plot of the normalized fluorescence recovery versus time and represents the average from at least five individual cells. (E) The average T₅₀ ± SD derived from FRAP assays of cells expressing the indicated H1-GFP variants.

Fig. 2.

Changes in H1⁰–chromatin interactions are linked to commitment. (A) Time course study of H1⁰-GFP recovery in HMBA-induced MEL cells. T₅₀ ± SD values of H1⁰-GFP recovery (○) in HMBA-induced MEL cells at different time points is represented along with the percentage of committed cells (●) and cells staining positive for Hb with the benzidine reagent (■). (B) Average T₅₀ ± SD values of H1⁰-GFP recovery in DEX-sensitive cultures treated for 60 h with HMBA, DEX, or HMBA and DEX compared with those of untreated cells.

Fig. 3.

The state of H1 phosphorylation affects linker histone–chromatin interactions. (A and B) FRAP recovery curves of H1⁰Ala-GFP and H1⁰Glu-GFP from at least 10 individual untreated or differentiated MEL cells. (C) T₅₀ ± SD values derived from FRAP assays. (D–F) Elution profiles of the chromatin-bound H1⁰-GFP, H1⁰Ala-GFP, or H1⁰Glu-GFP at different KCl concentrations as a percentage of the values obtained from unwashed nuclei from proliferating or differentiated MEL cells. The amount of chromatin-bound H1⁰-GFP, H1⁰Ala-GFP, or H1⁰Glu-GFP was determined by fluorimetry as described in Materials and Methods.

Fig. 4.

Dephosphorylation of H1 is necessary for the differentiation program. (A and B) Wild-type MEL cells and cell lines expressing exogenous H1⁰, H1⁰Ala, or H1⁰Glu were treated with 5 mM HMBA. The percentage of hemoglobin-positive cells (A) and committed cells (B) was determined. (C and D) Comparison of the relative nuclear volume and amount of condensed chromatin of HMBA-induced wild-type MEL cells and those overexpressing H1⁰Glu.

Fig. 5.

Expression of H1⁰Glu affects the mobility of other linker histones. Shown are FRAP recovery curves of H1⁰Ala-GFP (A) and H1⁰cc-GFP (B) in the wild-type MEL cells and those overexpressing H1⁰Glu. Undifferentiated MEL cell lines overexpressing H1⁰Glu were transiently transfected with vectors expressing H1⁰Ala-GFP or H1⁰cc-GFP using Lipofectamine as per the manufacturer's instructions. FRAP assays were conducted 24 h after transfection.