Analysis of histone chaperone antisilencing function 1 interactions

Abstract

The assembly and disassembly of chromatin impacts all DNA-dependent processes in eukaryotes. These processes are intricately regulated through stepwise mechanisms, requiring multiple proteins, posttranslational modifications, and remodeling enzymes, as well as specific proteins to chaperone the highly basic and aggregation-prone histone proteins. The histone chaperones are acidic proteins that perform the latter function by maintaining the stability of the histones when they are not associated with DNA and guiding the deposition and removal of histones from DNA. Understanding the thermodynamics of these processes provides deeper insights into the mechanisms of chromatin assembly and disassembly. Here we describe complementary thermodynamic and biochemical approaches for analysis of the interactions of a major chaperone of the H3/H4 dimer, anti-silencing function 1 (Asf1) with histones H3/H4, and DNA. Fluorescence quenching approaches are useful for measuring the binding affinity of Asf1 for histones H3/H4 under equilibrium conditions. Electrophoretic mobility shift analyses are useful for examining Asf1-mediated tetrasome (H3/H4-DNA) assembly and disassembly processes. These approaches potentially can be used more generally for the study of other histone chaperone-histone interactions and provide a means to dissect the role of posttranslational modifications and other factors that participate in chromatin dynamics.

Figure 2.1. Asf1 protein

(A) Schematic diagram showing the globular core of Asf1 in grey and the C-terminal intrinsically disordered tail in white. (B) Schematic diagram showing Asf1 in black with the positions of the cysteines in the globular core of Asf1 and the cysteine introduced at −1 position shown as white spheres. (C) 15% SDS-PAGE analysis of proteins. Coomassie Blue stain (lanes: molecular weight ladder, yAsf1^*532, and yAsf1) and fluorescent image showing Alexa Fluor 532 labeling of Asf1 (Lane yAsf1^*532, and yAsf1).

Figure 3.1. Histone Proteins

(A) 15% SDS-PAGE analysis of histone proteins and fluorophore labeling. Coomassie Blue stain (left hand side) and fluorescent image (right hand side) showing H3/H4*^FM, H3/H4^Qsy9, and H3/H4 samples. (B) Schematic diagram of Asf1-H3/H4 complex with the positions used for fluorophore labeling indicated. Asf1, shown in grey, is labeled at cysteine -1 with Alexa Fluor 532 shown in a black space filling representation. Histone H3 is shown in black. Histone H4, shown in white, is labeled at Cysteine 71 with Qsy9 or FM shown in a black space filling representation.

Figure 4.1. K_D determination using a fluorescence quenching approach

Fluorescence quenching of the yAsf1^*532 fluorescence signal by H3/H4^Qsy9 and unlabeled H3/H4 was observed and quantitated to determine the K_D of the yAsf1-H3/H4 interaction. H3/H4 or H3/H4*^Qsy9 was titrated into 1.0 nM yAsf1^*532. The dilution corrected and background subtracted fluorescence data were fitted with a ligand-depleted binding model (Equation 1; GraphPad Prism) because the concentration of yAsf1^*532 was within 10-fold of the K_D value.

Figure 5.1. Non-denaturing gel electrophoretic analysis of histones, Asf1, and tetrasomes

7% non-denaturing PAGE illustrates the migration of DNA, proteins and complexes in electrophoretic assays. The images were obtained using a Typhoon 9400 Variable mode imager (GE Healthcare) in two modes. First the fluorescence of the labeled proteins was imaged using two excitation and emission filter sets to detect the signal of the histone H4 FM label (488 nm excitation and 526SP emission filter) and the Alexa Fluor 532 signal of yAsf1 (532 nm excitation and 555BP20 emission filter). The gel was then stained with SYBR Green I (Invitrogen) nucleic acid stain and imaged again (488 nm excitation and 526SP emission filter). Typically, 80 ng of DNA was loaded and the PMT was set to 300 V. Finally, the sequential scans were merged with FluorSep, and band intensity was quantitated with ImageQuant. Migration of yAsf1^*532 (top), Tetrasomes (FM) (middle), and SYBR Green I stained DNA (bottom) are shown with a DNA ladder. While the tetrasomes migrate at approximately the same position as 200 bp linear DNA in this system, H3/H4^*FM and the Asf1-H3/H4 complex do not enter the gel.

Figure 5.2. Activities of Asf1 in the assembly and disassembly of H3/H4-DNA complexes

(A) Effect of prior addition of Asf1 on tetrasome and disome formation. H3/H4*^FM histones at 0.8 μM were incubated in the absence and presence of increasing concentrations of unlabeled yAsf1 (0, 0.4, 0.8, 2.0, or 4.0 μM) for 30 minutes at 20 °C prior to addition of 0.4 μM concentration 80 bp DNA fragments of 5SDNA. After further incubation for 60 minutes at 20 °C the products were analyzed by non-denaturing PAGE. The gel was scanned to obtain the H3/H4*^FM fluorescence before the gel was stained with SYBR Green I nucleic acid stain and then rescanned. The position of each species is indicated with an arrow. (B) The data from at least three independent experiments were quantitated and are presented in graphical form (bottom panel). (C) Effect of the addition of Asf1 on preformed tetrasomes and disomes. H3/H4*^FM histones at 0.8 μM were incubated with 0.4 μM concentration 80 bp DNA fragments of 5SDNA prior to the addition of buffer or increasing concentrations of unlabeled yAsf1 (0, 0.4 0.8. 2, and 4 μM) for 90 minutes at 20 °C. The products were analyzed by non-denaturing PAGE and scanned for FM fluorescence, and the data from three independent experiments are presented in graphical form (D).