Single-molecule study reveals Hmo1, not Hho1, promotes chromatin assembly in budding yeast

Abstract

Linker histone H1 plays a crucial role in various biological processes, including nucleosome stabilization, high-order chromatin structure organization, gene expression, and epigenetic regulation in eukaryotic cells. Unlike higher eukaryotes, little about the linker histone in Saccharomyces cerevisiae is known. Hho1 and Hmo1 are two long-standing controversial histone H1 candidates in budding yeast. In this study, we directly observed at the single-molecule level that Hmo1, but not Hho1, is involved in chromatin assembly in the yeast nucleoplasmic extracts (YNPE), which can replicate the physiological condition of the yeast nucleus. The presence of Hmo1 facilitates the assembly of nucleosomes on DNA in YNPE, as revealed by single-molecule force spectroscopy. Further single-molecule analysis showed that the lysine-rich C-terminal domain (CTD) of Hmo1 is essential for the function of chromatin compaction, while the second globular domain at the C-terminus of Hho1 impairs its ability. In addition, Hmo1, but not Hho1, forms condensates with double-stranded DNA via reversible phase separation. The phosphorylation fluctuation of Hmo1 coincides with metazoan H1 during the cell cycle. Our data suggest that Hmo1, but not Hho1, possesses some functionality similar to that of linker histone in Saccharomyces cerevisiae, even though some properties of Hmo1 differ from those of a canonical linker histone H1. Our study provides clues for the linker histone H1 in budding yeast and provides insights into the evolution and diversity of histone H1 across eukaryotes. IMPORTANCE There has been a long-standing debate regarding the identity of linker histone H1 in budding yeast. To address this issue, we utilized YNPE, which accurately replicate the physiological conditions in yeast nuclei, in combination with total internal reflection fluorescence microscopy and magnetic tweezers. Our findings demonstrated that Hmo1, rather than Hho1, is responsible for chromatin assembly in budding yeast. Additionally, we found that Hmo1 shares certain characteristics with histone H1, including phase separation and phosphorylation fluctuations throughout the cell cycle. Furthermore, we discovered that the lysine-rich domain of Hho1 is buried by its second globular domain at the C-terminus, resulting in the loss of function that is similar to histone H1. Our study provides compelling evidence to suggest that Hmo1 shares linker histone H1 function in budding yeast and contributes to our understanding of the evolution of linker histone H1 across eukaryotes.

Keywords: Hmo1; chromatin assembly; linker histone H1; magnetic tweezers; total internal reflection fluorescence microscopy.

Fig 1

The role of Hmo1 and Hho1 in chromatin assembly. (A) Experimental setup using single-molecule total internal reflection fluorescence microscopy (TIRFM). λDNA molecules were immobilized on the surface of the flow cell via streptavidin-biotin interactions. Yeast nucleoplasmic extracts (YNPE), containing all soluble proteins in the yeast nuclei, were diluted with blocking buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM EDTA, 0.2 mg/mL BSA) and injected into the flow cell at 50 µL/min to assembly chromatin at room temperature. (B) The DNA compaction induced by YNPE_wt, YNPE_hho1Δ , YNPE_hmo1Δ , and YNPE_{hho1Δ&hmo1Δ} . Snapshots represent representative individual DNA molecules at specified time points in the time-lapse movie recorded for each type of YNPE. (C) Kinetic curves of λDNA compaction in YNPE_wt (dark blue), YNPE_hho1Δ (blue), YNPE_hmo1Δ (red), and YNPE_{hho1Δ&hmo1Δ} (orange). The curves are plotted based on the average length of more than 30 individual DNA molecules measured in experiments.

Fig 2

Hmo1 acts directly on chromatin assembly and facilitates further compaction of nucleosomes. (A) Kinetic analysis of supplementing four different purified proteins into YNPE_hmo1Δ [YNPE_hmo1Δ + H1.4 (gray), YNPE_hmo1Δ + Hmo1 (light blue), YNPE_hmo1Δ + Hho1 (red), YNPE_hmo1Δ + LmHU (orange)]. The wild-type YNPE curve (dark blue) was used as a control. More than 20 DNA molecules were measured in each experiment. (B) Kinetic analysis of four different purified proteins without YNPE [H1.4 (gray), LmHU (orange), Hmo1 (blue), and Hho1 (red)]. More than 20 DNA molecules were measured in each experiment. (C) DNA compaction time under various conditions (n > 3, **** and *** means P value < 0.0001 and P value < 0.001, respectively). (D) Top: fluorescence imaging of nucleosomes on λDNA molecules in YNPE_{hmo1Δ- H2B-3FLAG}. A total of 50 DNA molecules were counted, and the percentage of each representative image is shown in the parentheses. Bottom: fluorescence imaging of nucleosomes on λDNA molecule in YNPE_{hmo1Δ- H2B-3FLAG}+ Hmo1. A total of 30 DNA molecules were counted, and the percentage of the representative image is shown in the parentheses.

Fig 3

Single-molecule force spectroscopy demonstrated that Hmo1 facilitates the assembly of nucleosomes in YNPE. (A) Top: constructs of the DNA template: a 1,491-bp double-stranded DNA labeled with three digoxigenin (3Dig-) and biotin (Bio-) at each end. Bottom: schematic setup of the single-molecule magnetic tweezers used in our experiments. (B) The representative force-extension curves of chromatin assembled in different types of YNPE [YNPE_wt (red), YNPE_hho1Δ (orange), YNPE_hmo1Δ + Hmo1 (olive), YNPE_hmo1Δ (blue)]. The control force-extension curve of 1,500-bp dsDNA was plotted in violet. All measurements were repeated more than five times. The WLC model fits the gray lines with different DNA contour lengths. The interval between two adjacent gray lines is 25 nm, representing half of the nucleosome dissociation length. Different colored triangles indicate the region where the jumps occurred in YNPE_wt (gray triangles), YNPE_hho1Δ (black triangles), and YNPE_hmo1Δ + Hmo1 (brown triangles), respectively. The right inset model depicts the nucleosome disruption process in YNPE_wt (red), YNPE_hho1Δ (orange), and YNPE_hmo1Δ + Hmo1 (olive). (C) Plots show the details of the representative jump during chromatin stretching in YNPE_wt (red) or YNPE_hho1Δ (orange), indicating the dissociation of the nucleosomal wrap. The gray lines show the changes in force during the experiment.

Fig 4

Lysine-rich extension is critical for the chromatin assembly function of Hmo1. (A) Top: schematic diagrams of Hmo1, Hmo1-AB, Hho1, Hho1(1-176), and H1.4(Homo sapiens). Hmo1-AB was derived by removing the portion of Hmo1 after the 190th amino acid, while Hho1(1-176) was derived by removing the portion of Hho1 after the 176th amino acid. Bottom: protein structure simulation of Hmo1, Hmo1-AB, Hho1, Hho1(1-176), and H1.4(Homo sapiens) by AlphaFold2 (Alphafold protein structure database, https://alphafold.ebi.ac.uk/). (B) Kinetic analysis of DNA compaction after supplementing the same concentration (8.2 nM) of purified Hmo1 (light blue), Hmo1-AB (orange), Hho1 (red), Hho1(1-176) (olive), and H1.4 (gray) in YNPE_hmo1Δ . The curves of wild-type YNPE (dark blue) and the YNPE_hmo1Δ (purple) were used as controls. More than 20 DNA molecules were measured in each experiment.

Fig 5

Hmo1 possesses phase separation property when mixed with dsDNA. (A) Bright-field images of five kinds of purified proteins mixed with 300-bp DNA. The images with red borders represent the occurrence of phase separation. (B) Snapshot images of phase separation condensate before and after photobleaching and recovery (left). Purified proteins [H1.4, Hmo1, Hho1(1-176)] were labeled with red dyes carrying the NHS-ester group (RED-NHS). 60-bp dsDNA was labeled with fluorescein amidite (FAM). The percentage of recovery of phase separation condensates versus time (right; mean ± SD, n = 3).

Fig 6

The degree of phosphorylation of Hmo1 changes during the cell cycle in a manner consistent with that of H1. (A) The representative western blotting image of Hmo1 phosphorylation. The experiments were performed three times. (B) Relatively quantification of western blotting data (mean ± SD, n = 3). The degree of Hmo1 phosphorylation varies across the cell cycle. The y-axis represents the intensity ratio of phosphorylated Hmo1 to GAPDH.