Falbo L , Raspelli E , Romeo F , Fiorani S , Pezzimenti F , Casagrande F , Costa I , Parazzoli D , Costanzo V .

Cancer Research UK

	Fig. 1. Isolation of SSRP1 as somatic nuclei replication stimulating factor.a Graph showing DNA replication kinetics of sperm (8000 n/μl) or somatic nuclei (SN) (4000 n/μl). DNA was directly incubated in interphase egg extract (Interphase) or exposed for 30 min to CSF-arrested mitotic extract, which was then driven into interphase by 0.4 mM CaCl2 addition (Mitosis + Interphase). DNA synthesis was quantified by measuring the percentage of α-32P-dCTP incorporation relative to the input DNA for each condition. Each point represents the mean value ± standard error of the mean (SEM). n = 3 independent experiments; p < 0.0001 when comparing mean values for all points; Two-way anova. b Graph showing DNA replication of the indicated amounts of sperm and SN directly incubated in interphase egg extract for 120 min. DNA synthesis was quantified as in (a). Bars represent mean ± SEM. n = 3 independent experiments; **p < 0.01, ***p < 0.001 or ns when comparing mean values for the indicated samples; unpaired t test. c Schematic representation of Xenopus egg extract fractionation. Extract was precipitated by centrifugation following incubation with increasing amount of polyethylene glycol (PEG). Either pellet (P) or supernatant (S) fractions collected at different PEG concentrations were assayed for their ability to stimulate replication when incubated with SN then transferred to interphase extracts in the presence of radiolabelled nucleotide. d Graph showing α-32P-dCTP incorporation (counts per minute, cpms) in SN pre-incubated with the indicated PEG fractions for 30 min and then transferred to interphase egg extract for 120 min. e Graph showing α-32P-dCTP incorporation in somatic nuclei pre-incubated with the fractions eluted from the column as described in (c) loaded with the active PEG fraction (9%P) and then transferred to interphase egg extract for 120 min.
	Fig. 2. SSRP1-dependent stimulation of replication origin assembly.a Graph showing replication time course of SN (4000 n/μl) replication. SN were pre-incubated for 30 min with buffer, 200 ng/μl recombinant SSRP1, SPT16 or both, as indicated, and transferred to interphase extracts (Interphase) or pre-incubated in CSF-arrested mitotic egg extract for 30 min (Mitotic) then driven into interphase by CaCl2 addition. Each point represents the mean ± SEM. n = 3 independent experiments; p < 0.0001 when comparing mean values for all samples; Two-way anova. b Representative autoradiography of a DNA replication time course assay in interphase egg extract showing α-32P-dCTP incorporation in SN. Optical density (OD) for each lane is indicated. c Graph showing replication time course of sperm nuclei (8000 n/μl) treated as indicated. Each point represents mean ± SEM. n = 3 independent experiments; p = ns when comparing mean values for all samples; Two-way anova. d DNA fiber assay showing digoxigenin-dUTP (dig-dUTP) incorporation in SN incubated directly in interphase egg extract together with buffer (left), pre-incubated with 200 ng/μl recombinant SSRP1 (center) or pre-incubated for 30 min in CSF-arrested mitotic egg extract then driven into interphase by CaCl2 addition (right). DNA fibers are in red. Dig-dUTP labeled tracts are in green. Bar = 10 Kb. e Graphs showing IODs indicated in Kb for each sample. The center-to-center distances between adjacent Dig-dUTP tracks were measured and plotted analyzing 50 fibers pooled from three independent experiments; IODs and mean values ± SEM are shown; n = 100 individual IODs; p < 0.0001; Two-way anova. f Chromatin binding showing somatic nuclei incubated in interphase extract in the presence of buffer or recombinant SSRP1. Chromatin was isolated at the indicated times after addition to egg extract and blotted using the indicated antibodies. No DNA indicates absence of nuclei. SN indicates somatic nuclei alone. Image shows typical result. g Graph showing relative quantification of signal intensity of ORC1 and MCM7 proteins loaded on chromatin at the indicated times normalized to histone H2B. Bars represent mean intensity ± SEM. n = 3 independent experiments; p < 0.0005 when comparing mean values for ORC1 and MCM7; t-test.
	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. 5. SSRP1 N-Terminal domain dependent stimulation of DNA replication through histone H1 chromatin eviction.a Representative autoradiography of a DNA replication assay showing α-32P-dCTP incorporation for 120 min in SN pre-incubated with 200 ng/μl recombinant SSRP1 or equimolar amounts of its mutated versions, as indicated. b Graph indicating fold change in somatic DNA replication efficiency relative to buffer considered as 1. SN were pre-incubated with 200 ng/μl recombinant SSRP1 or equimolar amounts of its mutated versions as indicated. Bars represent mean ± SEM. n = 3 independent experiments; p < 0.005 when comparing all values. Two-way anova. c WB of chromatin isolated at 60 min after addition to interphase egg extracts of SN pre-incubated with 200 ng/μl recombinant SSRP1 or equimolar amounts of its mutant versions and probed with the indicated antibodies. d WB of chromatin isolated at the indicate times from interphase egg extracts supplemented with SN pre-incubated with Flag-∆NTD or control buffer. e WB of chromatin isolated from interphase egg extracts incubated for 60 min with SN exposed to Flag-NTD or control buffer in the presence or absence of purified histone H1. f WB of chromatin isolated at the indicate times from interphase egg extracts incubated with somatic nuclei exposed to Flag-NTD or control buffer.
	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. 7. SSRP1 induced development acceleration through cell cycle shortening.a Representative images of control buffer (CTRL) and Myc-SSRP1 (SSRP1) injected Xenopus laevis embryos at 24 and 48 hours pf. Size bar = 1000 µm. b Graph showing lengths of CTRL and SSRP1- injected embryos measured 24 and 48 h pf. Each point represents one embryo. Bars show mean ± SEM; ****p < 0.0001; ns = not significant; n = 120 embryos; unpaired two-tailed t test. c Graph showing average length of cell cycle post-MBT per embryo measured for 30 cells starting at the 12th cell division using four embryos for each indicated condition. Cell cycle duration times between two sequential divisions were manually annotated from movies; n = 120 cell cycles; *p < 0.01; unpaired two-tailed t-test. d Representative images of CTRL and Myc-∆NTD—injected embryos at 24 and 48 h post-fertilization. Size bar = 1000 µm. e Graph showing length of CTRL and Myc-∆NTD mRNA injected embryos measured 24 and 48 h pf. Each point represents one animal. Bars show mean ± SEM; n = 100 embryos; **p < 0.01; unpaired two-tailed t test. f Graph showing average duration of cell cycle post-MBT measured as in (c); n = 120 cells; *p < 0.01; unpaired two-tailed t test. g Representative images of CTRL and NTD- injected embryos 24 and 48 h pf. All images are at identical scale. Size bar = 1000 μm. h Graph showing length of CTRL and NTD mRNA injected embryos measured 24 and 48 h pf. Each point represents one embryo. Bars show mean ± SEM; n = 70 embryos; ****p < 0.0001; unpaired two-tailed t test. i Graph showing average duration of cell cycle post-MBT measured as in (c); n = 120 cell cycles; *p < 0.01; unpaired two-tailed t test. For graphs in (c), (f), (i), box height represents the 25th to 75th percentiles and the centerline represents the median. The whiskers extend to the farthest data point.
	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.

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