Intensification of p-coumaric acid heterologous production using extractive biphasic fermentation

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Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage factory, enabling tight coupling between DSB introduction and repair during PGR

Models for the assembly of an active DNA cleavage complex.

<p>(A) Ku associates with DNA-bound Pgm and activates DNA cleavage. Pgm and its putative partners (in grey) would recognize and bind the boundaries of eliminated sequences. The binding of Ku (in blue) activates Pgm for DNA cleavage (symbolized by the switch from a rectangular box to an oval), perhaps by assisting the formation of a synapse between both IES ends, in a transpososome-like intermediate. The DNA-PKcs catalytic subunit (in peach) may also be part of the active DNA cleavage complex. (B) Ku forms a complex with Pgm in the absence of DNA, activating the DNA binding and/or cleavage activities of Pgm. Following DNA cleavage, conformational remodeling of the complex would position Ku on broken DNA ends and allow it to perform its classical role in C-NHEJ-mediated DSB repair. MAC DNA is represented in black, IES DNA in red.</p

Physical interaction between Pgm and Ku.

<p>(A) Co-precipitation of HA-Ku80c and Ku70a-HA with MBP-Pgm from insect cell extracts (top panel), revealed on western blots using an anti-HA antibody. (B) Co-immunoprecipitation of Pgm with HA-Ku80c and/or Ku70a-HA from insect cell extracts, revealed on western blots using an anti-Pgm antibody. In A and B, the input proteins from each extract are displayed in the bottom panel. (C) The interaction between HA-Ku80c and Pgm is resistant to DNaseI treatment. The co-precipitation experiment was performed as described in A, using MBP-Pgm, HA-Ku80c and 6His-Ku70a recombinant proteins produced from baculovirus vectors. EDTA was removed from the lysis buffer and replaced by 10 mM MgCl₂. Half of the sample was treated with 40 µg/mL of Dnase I during the 2-hr incubation with amylose-coupled magnetic beads. The presence of HA-Ku80c in the purified complexes was revealed on western blots using an anti-HA antibody.</p

Molecular analysis of genome rearrangements after Ku80c depletion.

<p>(A) Detection of DNA fragments with excised or non-excised IES 51G4404 from the surface antigen <i>G⁵¹</i> gene. Total genomic DNA was extracted during autogamy of 51ΔA cells subjected to RNAi against <i>ICL7</i> (control) or <i>KU80c</i>. To compare the two time-courses, similar autogamy stages were numbered from 1 to 6, based on the observation of DAPI-stained cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen.1004552.s001" target="_blank">Figure S1C</a>). <i>Pst</i>I-hydrolyzed total genomic DNA was run on 1% agarose gels. Southern blots were hybridized with the Gmac probe (in grey), which hybridizes to the flanking MAC DNA downstream of the IES. (B) Detection of fragmented or non-fragmented DNA downstream of the <i>G⁵¹</i> gene by Southern blot hybridization of <i>Pst</i>I-digested total genomic DNA (same samples as in A) run on 0.8% agarose gels. The subtelomeric tel51G probe is shown in grey. The white box in the bottom right diagram represents telomeric repeats. (C) PCR detection of <i>de novo</i> IES excision junctions during autogamy of 51ΔA cells, in an <i>ICL7</i> (control) or a <i>KU80c</i> RNAi. Note that a few autogamous cells were present at time-point 1 in the control RNAi (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen.1004552.s001" target="_blank">Figure S1C</a>). (D) PCR detection of IES circle junctions during autogamy in an <i>ICL7</i> (control) or a <i>KU80c</i> RNAi. Triangles in C and D represent PCR primers (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen.1004552.s006" target="_blank">Table S1</a>).</p

Detection of programmed DSBs at IES boundaries during autogamy.

<p>(A) LMPCR detection of DSBs at MAC or IES ends during autogamy of 51ΔA cells subjected to RNAi against <i>ICL7</i> (control) or <i>KU80c</i> (same samples as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen-1004552-g004" target="_blank">Figure 4</a>). A Sanger DNA sequencing ladder provides size markers. On the diagram, the LMPCR linker is drawn as grey boxes and <i>Paramecium</i> DNA as black lines, with a black dot representing the 3′ end generated by Pgm-dependent cleavage. In the <i>KU80c</i> RNAi, the LMPCR signals at 51G4404 MAC ends are likely due to background DNA breaks generated at the MAC <i>G⁵¹</i> locus during DNA extraction. This background is not detected for IESs of the <i>A⁵¹</i> gene, because this locus is absent from the old MAC. (B) TdT tailing of free 3′OH ends during autogamy of 51ΔA cells subjected to RNAi against <i>ICL7</i> (control) or <i>KU80c</i> (same samples as in (A)). On the diagram, the potentially resected 5′ end is represented by a dotted line.</p

RNAi screen for essential <i>KU</i> genes during autogamy.

<p>(A) Survival of the post-autogamous progeny of cells submitted to different combinations of RNAi. Kp: autogamy in standard <i>K. pneumoniae</i> medium; <i>ICL7</i>: RNAi against <i>ICL7</i>, a nonessential gene that encodes an infraciliary lattice centrin <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen.1004552-Gogendeau1" target="_blank">[47]</a>; <i>XR</i>: RNAi against <i>XRCC4</i>. RNAi experiments against <i>KU80</i> genes were performed using gene-specific inserts KU80-a2, KU80-b2 and KU80-c2 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen.1004552.s002" target="_blank">Figure S2</a>). For each condition, 30 to ∼140 post-autogamous cells were analyzed. Each bar represents the percentage of viable post-autogamous cells carrying a functional new MAC, for each condition. Error bars represent the Wilson score intervals (95% confidence level), which are appropriate for a small number of trials or for values close to an extreme probability. (B) DAPI-staining of developing MACs during RNAi against <i>ND7</i> (control), <i>KU70</i>, <i>KU80c</i>, <i>XRCC4/LIG4</i> and <i>LIG4</i> + <i>KU80c</i>. Developing MACs are indicated by yellow arrowheads. Upon <i>LIG4</i> or <i>XRCC4</i> RNAi, developing MACs exhibit faint DAPI staining, which correlates with a defect in DNA amplification <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen.1004552-Kapusta1" target="_blank">[16]</a>. (C) Northern blot hybridization of total RNA during a control time course experiment (<i>ND7</i> RNAi) and in a <i>KU80c</i> RNAi. V: vegetative cells; T0: 60% of cells with fragmented MAC; other time-points refer to hours following T0 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen.1004552.s001" target="_blank">Figure S1B</a>).</p

Nuclear localization of GFP-Ku fusions during autogamy.

<p>Cells were microinjected with fusion transgenes expressing GFP-Ku70a (panels a, b, c) and GFP-Ku80c (panels d, e, f) under the control of their respective transcription signals. In autogamous cells, developing MACs are indicated by yellow arrowheads, the other DAPI-stained nuclei are fragments from the old vegetative MAC. For each protein, the GFP fluorescence sometimes concentrated in nuclear foci of unclear biological significance (see cells in panels c and f, and enlarged inserts on the right). In this particular experiment, expression of the GFP fusions had no significant effect on the recovery of viable post-autogamous progeny (87% progeny with functional new MACs for GFP-Ku70a, 90% for GFP-Ku80c).</p

Nuclear accumulation of a Pgm-GFP fusion in Ku80c-depleted cells.

<p>Cells were microinjected with a <i>PGM-GFP</i> fusion transgene and one transformant was submitted to RNAi against <i>ICL7</i> (control: panel A) or <i>KU80c</i> (panel B). The progression of autogamy was monitored over a four-day starvation period (a and e: day 1; b and f: day 2; c and g: day 3; d and h: day 4). To compare the intensities of GFP fluorescence, signals were acquired with the same exposure time, and identical window settings were applied to the image display using the ImageJ software (National Institute of Health). Developing MACs are indicated by yellow arrowheads. In the enlarged inserts shown on the right of each panel, the display settings were modified to highlight the Pgm-GFP nuclear foci. The white arrowheads in panel B point to the DAPI-free regions, in which overproduced Pgm-GFP accumulates following <i>KU80c</i> RNAi. In the control RNAi, 93% of post-autogamous progeny had a functional new MAC, while the <i>KU80c</i> RNAi yielded no progeny with a functional new MAC.</p

<i>KU70</i> and <i>KU80</i> genes in <i>P. tetraurelia</i>.

<p>(A) Diagram of the WGD relationships between <i>KU70</i> or <i>KU80</i> genes in <i>P. tetraurelia</i>. The % of identity between genes (nt) and proteins (aa) are indicated along the arrows connecting two genes. (B) Transcription profiles of <i>KU70</i> and <i>KU80</i> genes during an autogamy time-course of strain 51, as determined by high-throughput RNA-seq. V: vegetative cells; S: starved or meiotic cells with intact parental MAC; T0: 50% of cells with fragmented MAC; the following time-points refer to hours after T0 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen.1004552-Nowak1" target="_blank">[51]</a>. On the vertical axis, FPKM represents the number of fragments per gene kb per million of fragments that were uniquely mapped on the genome. (C) Detection of <i>KU70</i> and <i>KU80c</i> mRNA during autogamy, through northern blot hybridization. Control RNAi: RNAi against the nonessential <i>ND7</i> gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen.1004552-Skouri1" target="_blank">[43]</a>, which encodes an exocytosis protein. V: vegetative cells. The times refer to hours after T0, the time at which 50% of cells have a fragmented MAC (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004552#pgen.1004552.s001" target="_blank">Figure S1A</a>).</p