XB-ART-56790Nat Commun January 1, 2020; 11 (1): 1345.
SSRP1-mediated histone H1 eviction promotes replication origin assembly and accelerated development.
In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.
PubMed ID: 32165637
PMC ID: PMC7067836
Article link: Nat Commun
Genes referenced: ctrl h2bc21 mcm7 mmut myc orc1 ssrp1
GO keywords: cell cycle
Article Images: [+] show captions
|Fig. 3. SSRP1 counteracts histone H1-dependent inhibition of origin assembly.a Western blot (WB) of the indicated proteins bound to chromatin isolated from SN incubated with mitotic or interphase extracts for the indicated times. b WB of the indicated proteins bound to chromatin isolated from SN pre-incubated with 200 ng/μl recombinant SSRP1 or control buffer and transferred to interphase extracts for the indicated times. SSRP1 input is 10% of the total. c WB of chromatin isolated from interphase egg extracts supplemented with sperm nuclei pre-incubated with buffer (−) or purified histone H1 (+) at a final concentration of 2 μM for 30 min in the presence of 200 ng/μl recombinant SSRP1 or control buffer and probed with the indicated antibodies. d Representative autoradiography of replication time course assay. Sperm nuclei were pre-incubated for 30 min with 200 ng/μl recombinant SSRP1, 2 μM histone H1, or both and then transferred to interphase extracts. Samples were taken at the indicated times after nuclei and α-32P-dCTP addition to egg extracts. OD for each lane is indicated. Images in all figures show a typical result of experiments repeated at least three times.|
|Fig. 4. SSRP1 interaction with histone H1 through its N terminal domain.a WB of Flag pull-down performed with recombinant human SPT16, Flag-SSRP1 and purified histone H1 using the indicated antibodies. b Schematic representation of Flag-SSRP1 mutated recombinant proteins used. Structural domains of wild-type SSRP1 protein are marked in color: N-terminal domain, NTD (Yellow); intrinsically disordered domain, IDD (green); C-terminal domain, CID (orange); Middle domain, MD (blue); HMG-box (red) and positively charged domains (black). Numbers indicate amino acid residues. PH indicates pleckstrin homology domain. *Indicates mutated residue. c) WB of in vitro pull-downs using the anti-Flag antibodies bound to agarose beads incubated with the indicated combinations of recombinant wild-type and mutant proteins. The mutated Flag-SSRP1 proteins used for the pull-downs are indicated under each panel. The inputs are shown in the panel below (Input).|
|Fig. 6. SSRP1 induced delay of MBT onset.a Time lapse video frames taken from movie S1 at the indicated times from the 4th cleavage (set as time zero) of embryos injected at the one-cell stage. Top row: control buffer (CTRL) injected embryos; Bottom row: embryos injected with Myc-SSRP1 mRNA. Size bar = 500 μm. b Graph showing the number of synchronous divisions-only of 24 embryos injected with Myc-SSRP1 mRNA or buffer and monitored up to 450 min from fertilization. Only cleavages after the 10th are shown to simplify graph layout. Each dot represents one embryo. Bars show mean ± SEM. c WB using the indicated antibodies of whole embryos injected with Myc-SSRP1 mRNA or buffer and taken at the indicated stages. Stage 8 was sampled every 30 min. d Images taken as in (a) from time-lapse movie S2. Embryos injected with control buffer or Myc-∆NTD mRNA. Size bar = 500 μm. e Graph showing the number of synchronous divisions monitored as in (b) of embryos injected with buffer or Myc-∆NTD mRNA. f WB of whole embryos injected with Myc-∆NTD mRNA or buffer and taken as in (c). g Images taken as in (a) from time-lapse movie S3 of embryos injected with control buffer or NTD mRNA. Size bar = 500 μm. h Graph showing the number of synchronous divisions monitored as in (b) of embryos injected with control buffer or Myc-NTD mRNA. i WB of whole embryos injected with control buffer or Myc-NTD mRNA taken as in (c).|
|Fig. 8. Proposed model.In vitro SSRP1 suppresses histone H1 chromatin association and histone H1-mediated inhibition of DNA replication origins assembly. In vivo SSRP1 levels decrease around MBT when somatic forms of histone H1 start to be expressed. Overexpression of SSRP1 delays MBT onset. Persistent expression of SSRP1 in post-MBT embryos promotes faster somatic cell cycles and accelerated development. See text for more details.|