Mammalian linker-histone subtypes differentially affect gene expression in vivo

Abstract

Posttranslational modifications and remodeling of nucleosomes are critical factors in the regulation of transcription. Higher-order folding of chromatin also is likely to contribute to the control of gene expression, but the absence of a detailed description of the structure of the chromatin fiber has impaired progress in this area. Mammalian somatic cells contain a set of H1 linker-histone subtypes, H1 (0) and H1a to H1e, that bind to nucleosome core particles and to the linker DNA between nucleosomes. To determine whether the H1 histone subtypes play differential roles in the regulation of gene expression, we combined mice lacking specific H1 histone subtypes with mice carrying transgenes subject to position effects. Because position effects result from the unique chromatin structure created by the juxtaposition of regulatory elements in the transgene and at the site of integration, transgenes can serve as exquisitely sensitive indicators of chromatin structure. We report that some, but not all, linker histones can attenuate or accentuate position effects. The results suggest that the linker-histone subtypes play differential roles in the control of gene expression and that the sequential arrangement of the linker histones on the chromatin fiber might regulate higher-order chromatin structure and fine-tune expression levels.

Figure 1

Age-dependent silencing is modulated by specific linker-histone deletions. (a) A 4.4-kb human β-globin gene PstI fragment (position 59859–64301 of GenBank HUMHBB) is subject to an age-dependent variegating position effect. Expression of the human β-globin gene was quantified by HPLC on RBC hemolysates and was found to decrease with time. % h β-globin = 100 × human β-globin/(mouse β-like chains + human β-globin). (b) Permeabilized RBCs were stained with FITC-labeled anti-human β-globin antibodies and periodically analyzed by flow cytometry. The proportion of RBCs expressing the human transgene decreases with age. (c) Metaphases from splenocytes were hybridized with a FITC-labeled human β-globin probe and a CY-3 labeled major satellite probe. In this mouse line, the human β-globin transgene is located far from pericentromeric heterochromatin. (d) Linker-histone subtypes differentially affect transgene expression. Silencing of the h-β-globin transgene in mice homozygous or heterozygous for deletions of linker histones was monitored over time by flow cytometry and compared with controls. Deletion of H1d, H1e, or H1e and H1 (0) dramatically attenuates the rate of silencing. Deletion of H1a, H1c, or H1 (0) has no effect on the rate of silencing. n = number of mice analyzed.

Figure 2

Analysis of DNA methylation of the transgene during silencing. (a Left) Splenic cells from phenylhydrazine-treated mice were analyzed by flow cytometry with a Ter-119 antibody. About 80% of the nucleated cells are erythroid [nonnucleated RBCs were gated out by using the forward scatter (FSC) and side scatter (SSC) parameters]. A cell population >90% erythroid was obtained by further purifying the spleen cells on a magnetic column by using Ter-119 antibodies attached to magnetic beads. (Right) Permeabilized RBCs from 12- and 90-week-old animals were labeled with anti-human β-globin antibodies. About 75% of the younger animals and <10% of the older animals were expressing the transgene in their RBCs. (b) Southern blot analysis with methylation-sensitive restriction nucleases HgaI (H) and SnaBI (S). Lanes 1–6: Genomic DNA from spleen cells was digested with BamH1 (B) BamHI/SnaBI or BamHI/HgaI and hybridized with a probe hybridizing in the 5′ flanking region of the β-globin gene (at position −1356 to −817 from the cap site of the β-globin gene). Lanes 1, 2, 3, 7, and 9: 12-week-old animals; lanes 4, 5, 6, 8, 10: 90-week-old animals. The human β-globin promoter in both the young and old animals is ≈70% methylated. Lanes 7 and 8: DNA from 90% pure erythroid cell population was digested with BamHI/SnaBI and hybridized as above. Again ≈70% of the transgene DNA is methylated regardless of age. Lanes 9 and 10: Same as lane 7 and 8 but using brain DNA. In this tissue, the transgene is almost completely methylated. (c) Southern blots were quantified by phosphor-imaging, and the mean of the results from two or three independent determinations performed on at least two animals was plotted.

Figure 3

Variegated expression of the human β-globin gene in a 150-kb YAC transgene is modulated by linker-histone deletions. (a) Representative FACS histograms obtained by analysis of permeabilized RBCs from littermates transgenic for YAC_277w and wt, and heterozygous or homozygous for deletion of the H1e linker histone were stained with a monoclonal FITC-labeled anti-human β-globin antibody. x axis, intensity of FITC labeling; y axis, cell numbers. The peak on the left represents nonexpressing cells; the peak on the right represents expressing cells. (b) Quantitation of the results for the H1a, H1c, and H1e series (see text). All three linker histones influence this position effect but H1e and has the strongest effect. +/+, +/−, and −/− mice were derived from the same cross.

Figure 4

Expression of the human β-globin gene in a 150-kb nonvariegating YAC transgene is modulated by H1a. (a) Typical reverse-phase HPLC chromatograms obtained by analysis of RBC hemolysates from mice transgenic for YAC_264-inv and wt or null (−/−) at the H1a locus. (b) Quantitative analysis of the results: Percent human β-globin expression (% h β-globin) = 100 × hβ/(hβ + mβ) determined by HPLC in mice heterozygous for YAC_264-inv and null for H1a, H1c, or H1e. Controls were littermates of the null mice that were either wt, H1a+/−, H1c+/−, or H1e+/−. Differences between H1a−/− mice and all of the other types of mice are statistically significant (t test P value < 0.001 in all cases). Absence of H1a is therefore associated with a decrease in expression of the human β-globin gene in YAC_264-inv.

Figure 5

Linker-histone composition in the murine erythroid lineage. (Left) Typical HPLC profile of total histone extract from bone marrow erythroid cells. The ratio of total H1 to core histones was determined by using the amount of H2b as a reference (13). (Center) Relative amounts of H1d and H1e (which cannot be resolved by HPLC) were determined by time-of-flight mass spectrometry on eluted HPLC peaks. Peaks of 21969 and 22089 are the phosphorylated forms of H1e and H1d, respectively. (Right) Summary of triplicate determinations of the distribution of the linker-histone subtypes in purified bone marrow erythroid cells.