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- Fig. 1.
Sequence comparison of Mus musculus replication-dependent somatic linker histone subtypes. The following sequences were aligned to H1.4 (Genbank accession no. NM_015787) using BLOSUM62: H1.1 (Genbank accession no. NM_030609), H1.5 (Genbank accession no. NM_020034), H1.2 (Genbank accession no. NM_015786), H1.3 (Genbank accession no. NM_145713). Subtypes are designated according to the recently proposed unified nomenclature (Talbert et al., 2012). The previously used mouse subtype designations are shown in parentheses. The sequence span representing the globular domain is marked with a black bar.
- Fig. 2.
FRAP analysis of individual H1 subtypes. (A) Representative FRAP analyses demonstrating the kinetic properties of each of the somatic linker histone subtypes. BALB/c 3T3 cells stably expressing H1-GFP proteins were imaged before and during recovery after bleaching of a nucleoplasmic area 2 µm in diameter. Images were taken before (pre-bleach, left column) and at indicated times after the bleach pulse. (B-D) Quantitative analysis of FRAP recovery of cells expressing the indicated H1-GFP subtypes. Error bars have been removed for clarity. (F) Histogram of t50 values. Plotted values represent means±s.d. from at least 12 measurements (see Table 1); **P<0.0001; *P<0.001.
- Fig. 3.
Quantitative analysis of FRAP recovery of domain switch constructs. (A) Sequence alignment of H1.1 and H1.5. The sequence span representing the globular domain is marked with a black bar. Arrows represent the location of the complimentary Sac1 and Hind3 restriction sites used to swap domains between the two variants. (B) Schematic of domain switch mutants. Construct 551 consists of residues 1-99 of H1.5 and residues 102-213 of H1.1; Construct 115 consists of residues 1-101 of H1.1 and residues 100-223 of H1.5. Construct 511 consists of residues 1-42 of H1.5 and residues 45-213 of H1.1. Construct 155 consists of residues 1-44 of H1.1 and residues 43-223 of H1.5. Construct 151 consists of residues 1-44 of H1.1, residues 43-99 of H1.5 and residues 102-213 of H11. Construct 515 consists of residues 1-42 of H1.5, residues 45-101 of H1.1 and residues 100-223 of H1.5. (C-E) Quantitative analysis of FRAP recovery of cells expressing the indicated domain switch constructs as GFP fusions. (F) Plotted t50 values represent means±s.d. from at least 12 cells (see Table 2).
- Fig. 4.
Quantitative analysis of FRAP recovery of mutant H1 constructs. (A) Quantitative analysis of FRAP recovery of cells expressing the indicated constructs as GFP fusions. (B) Plotted t50 values represent means±s.d. from at least 16 cells (see Table 2). *P<0.0001.
- Fig. 5.
Effects of overexpression of H1.1 or H1.5 on cell cycle progression. (A) Separation of H1 subtypes by HPLC. Total histones were isolated from density arrested cell cultures from the indicated lines and separated by HPLC as previously described (Brown et al., 1996). The subtypes present in each peak are indicated based on prior studies (Yellajoshyula and Brown, 2006). Estimates of the relative amounts of each subtype were determined by quantitation of the absorbance at 210 nm via integration of the area in each peak (see Table 3). (B) Cell cycle distribution after release from density arrest as determined by FACS. Results are the average of three independent experiments. (C) Individual FACS profiles from a representative experiment.
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