Semisynthesis of site-specifically succinylated histone reveals that succinylation regulates nucleosome unwrapping rate and DNA accessibility

Abstract Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.


Figure S3
LC-MS and deconvolution results for the ligated product before and after desulfurization.

Figure S4
LC-MS and deconvolution results of purified Alexa 488-labeled histone H2B at T112 position.

Figure S5
LC-MS and deconvolution results of purified Cy5-labeled histone H2A at K119 position.

Figure S6
The succinylation on H4K77 effects nucleosome stability.

Figure S8
FRET-based approach to study the effect of histone H4K77succ on nucleosome stability.

Figure S9
FRET-based approach to study the effect of histone H4K77succ on nucleosome reconstituted from MMTV-B, native positioning sequence.

Figure S10
The results of 'one-pot' assay.

Figure S11
FRET-based approach to study the effect of histone H4K77E on nucleosome

Figure S12
Monitor the amount of H2A-H2B remains on beads by Western blot.

Table S1
Summary of the nucleosome outer rip hopping data.

Table S2
Yeast strains used in this study.      H4K77succ (red trace) nucleosomes that labeled by FRET pair at internal DNA (DNA +42 -DNA -52 ).

Experimental Methods
The salt concentration at which the FRET has decreased by 50% is denoted as C1/2. For visualization, all curves were normalized between 100% at the maximum FRET value and 0 at high salt. Salt titration -5 -was repeated 3 times on each type of nucleosomes (n=3, C1/2 = mean ± s.e.m).  which the FRET has decreased by 50% is denoted as S1/2. For visualization, all curves were normalized -8 -between 100% at the maximum FRET value and 0 at high concentration of LexA. Titration was repeated 3 times on nucleosomes from one preparation (n=3, S1/2 = mean ± s.e.m). Middle panel, the S1/2 values of independent experiments on H4 K77E nucleosomes were showed. The average of S1/2 was calculated from 2 biological replicates (independent nucleosome preparations). Right panel, histogram showed the change in nucleosomal DNA accessibility associated with K-to-E mutation. The mean of S1/2 values of unmodified nucleosomes were set to 100%. The beads were then heated in SDS loading buffer for SDS-PAGE and Western blot analysis. The histone H2B was detected by Western blot using anti-H2B. The representative result from two repeats.

Experimental Methods
Site-directed mutagenesis on histone H2A and H2B.

Western blotting
The samples were separated by 15% SDS-PAGE and then were transferred onto a polyvinylidene fluoride (PVDF) membrane followed by blocking in TBST buffer (0.1% Tween-20 in TBS buffer) containing 5% nonfatdried milk for 1 h at room temperature. The membrane was then incubated with primary antibody diluted in TBST buffer with 5% nonfat-dried milk for overnight at 4 o C, followed by washing with TBST buffer for twice, each time 15 min. After incubating with secondary antibody diluted in TBST buffer with 5% BSA for 1 h at room temperature, the membranes were washed with TBST for 4 times, 10 min each, and visualized with SuperSignal West Pico or Dura Chemiluminescent Substrate by a MyECL Imager system (Thermo Fisher Scientific).