Competition between histone H1 and HMGN proteins for chromatin binding sites

Abstract

The ability of regulatory factors to access their nucleosomal targets is modulated by nuclear proteins such as histone H1 and HMGN (previously named HMG-14/-17 family) that bind to nucleosomes and either stabilize or destabilize the higher-order chromatin structure. We tested whether HMGN proteins affect the interaction of histone H1 with chromatin. Using microinjection into living cells expressing H1-GFP and photobleaching techniques, we found that wild-type HMGN, but not HMGN point mutants that do not bind to nucleosomes, inhibits the binding of H1 to nucleosomes. HMGN proteins compete with H1 for nucleosome sites but do not displace statically bound H1 from chromatin. Our results provide evidence for in vivo competition among chromosomal proteins for binding sites on chromatin and suggest that the local structure of the chromatin fiber is modulated by a dynamic interplay between nucleosomal binding proteins.

Fig. 1. FRAP analysis of competition between proteins for binding sites in living cells. (A) Experimental approach. (1) A nuclear protein is injected into the cytoplasm of cells expressing a GFP-fusion protein. (2) Either fluorescent protein alone or a mixture of fluorescent and non-fluorescent proteins is microinjected into the cytoplasm. After the microinjected proteins enters the nucleus, the mobility of the fluorescent proteins (either as GFP-fusion products or chemically labeled) is analyzed by FRAP. (B–D) Quantitative analysis of FRAP experiments after bleaching cells containing either fluorescent protein alone (blue) or a mixture of fluorescent and non-fluorescent protein (red). The proteins analyzed are indicated in each panel. The values are the mean ± SD from at least eight cells of a typical experiment. Arrows on the abscissa point to the time required to reach either 40% (B) or 80% (C and D) of the pre-bleach fluorescence intensity. The percentages on the right of the curves indicate the difference in the percentage recovery between the curves within 23 s after photobleaching.

Fig. 2. Competition between H1⁰–GFP and HMGN proteins for chromatin binding sites. (A) Colocalization of HMGN1 and HMGN2 with H1⁰–GFP. Confocal images of living cells expressing H1⁰–GFP and microinjected with fluorescently labeled HMGN proteins. Scale bar = 5 µm. (B) SDS–PAGE analysis of competitors microinjected into the H1⁰–GFP expressing cells. (C–K) Quantitative analysis of FRAP experiments of cells expressing H1⁰–GFP that either were not (blue) or were (red) injected with the proteins indicated in each panel. The values are the mean ± SD from at least eight cells of a typical experiment. Arrows on the abscissa point to the time required to reach 40% of the pre-bleach fluorescence intensity. The percentages on the right of the curves indicate the difference in the percentage recovery between the curves within 23 s after photobleaching. Note the differences between euchromatin and heterochromatin and that the controls in (E) and (H) did not affect the mobility of H1⁰–GFP.

Fig. 3. Loss of competition between HMGN1 and H1⁰–GFP in cells treated with compounds that affect the interaction of these proteins with chromatin. (A) FRAP analysis of the effect of HMGN1 on the mobility of H1⁰–GFP in heterochromatin. (B) FRAP analysis of the effect of HMGN1 on the mobility of H1⁰–GFP in euchromatin. Note that HMGN does not affect the immobile fraction of H1⁰–GFP. (C) FRAP analysis of HMGN1 in euchromatin and heterochromatin. (D) FRAP analysis indicating that Act-D treatment decreases the residence time of HMGN proteins on chromatin. (E) FRAP analysis indicating that Act-D treatment abolishes the ability of HMGN proteins to reduce the residence time of H1⁰–GFP on chromatin. (F) FRAP analysis indicating that DRB abolishes the ability of HMGN proteins to reduce the residence time of H1⁰–GFP on chromatin.

Fig. 4. Dynamic interplay between HMGN proteins and histone H1 on the surface of the nucleosome. The interaction of H1 with nucleosomes stabilizes the higher-order chromatin structure and promotes the formation of a compacted, folded chromatin fiber. An increase in the local concentration of HMGN proteins, which compete with H1 for shared binding sites on the nucleosome (Alfonso et al., 1994), decreases the H1 residence time at selected chromatin loci and promotes the unfolding of the chromatin fiber.