Multivalent interaction of ESCO2 with the replication machinery is required for sister chromatid cohesion in vertebrates

Abstract

The tethering together of sister chromatids by the cohesin complex ensures their accurate alignment and segregation during cell division. In vertebrates, sister chromatid cohesion requires the activity of the ESCO2 acetyltransferase, which modifies the Smc3 subunit of cohesin. It was shown recently that ESCO2 promotes cohesion through interaction with the MCM replicative helicase. However, ESCO2 does not significantly colocalize with the MCM complex, suggesting there are additional interactions important for ESCO2 function. Here we show that ESCO2 is recruited to replication factories, sites of DNA replication, through interaction with PCNA. We show that ESCO2 contains multiple PCNA-interaction motifs in its N terminus, each of which is essential to its ability to establish cohesion. We propose that multiple PCNA-interaction motifs embedded in a largely flexible and disordered region of the protein underlie the unique ability of ESCO2 to establish cohesion between sister chromatids precisely as they are born during DNA replication.

Keywords: DNA replication; chromosome biology; cohesin; sister chromatid cohesion.

Fig. 1.

ESCO2 associates with sites of active DNA replication. (A) The Eco acetyltransferases. Shown are the S. cerevisiae Eco1 protein and the vertebrate homologs ESCO1 and ESCO2. All 3 proteins contain a highly conserved domain containing a PIP box (P; black), a zinc finger motif (Z; yellow), and the catalytic acetyltransferase domain (A; blue). The vertebrate proteins ESCO1 and ESCO2 have unique N-terminal extensions, with no apparent sequence homology to each other (light green and light blue). (B) ESCO2 colocalizes with PCNA in replication foci. Confocal micrographs of nuclei of U2OS cells cotransfected with mCherry-ESCO2 and GFP-PCNA. ESCO2 colocalized with PCNA in replication foci in patterns of early, mid, and late DNA replication. Sites of colocalization appear yellow in the merged images. (C) The PIP box in ESCO2 is dispensable for localization to replication foci. Confocal micrographs of U2OS cells cotransfected with GFP-PCNA and mCherry-ESCO2 in which the PIP box is deleted. Pearson’s correlation coefficient for red and green signal intensities R = 0.837, 0.754, and 0.591 for early, mid, and late images, respectively (B). R = 0.189 for lacI, and 0.837 for ΔPIP (C). Shown is a representative example of 3 independent experiments. (Scale bars, 10 μm.)

Fig. 2.

ESCO2 interacts with PCNA outside of replication foci. (A) Tethered ESCO2 recruits PCNA. An mCherry-lacI-ESCO2 fusion protein, expressed in U2OS cells containing a stably integrated tandem array of lac operator sequences, is recruited to nuclear loci where it can be seen as 1 or 2 red foci in confocal micrographs. Cotransfected GFP-PCNA was recruited to the ESCO2 focus (Top), and this was unaffected by deletion of the PIP box in ESCO2 (Middle). PCNA was not enriched in the foci in cells containing mCherry-lacI (without ESCO2) (Bottom). (Scale bars, 10 μm.) (B) Normalized fluorescence intensity. The fluorescence intensity of lines drawn across the lac operator arrays from a number of cells treated as in A was averaged. Fluorescence of mCherry-lacI fusion is indicated by a red line, DAPI in blue, and GFP in green. n > 25 cells in each sample. Shown is a representative example of 3 independent experiments.

Fig. 3.

Sequences in the N terminus of ESCO2 are required for cohesion establishment. (A) The N terminus of ESCO2 contains several short, conserved motifs. Clustal Omega alignment of ESCO2 proteins from the indicated species is shown. The overall consensus is shown at Top, in which the grayscale indicates the degree of conservation (black is 100% and the lightest gray is <60%), and gaps are shown with a gray line. Mean hydrophobicity is also shown, with red indicating hydrophobic patches. Accession nos. are in SI Appendix, Table S1. (B) Enlargement of conserved motifs in the ESCO2 N terminus. The alignment of the 3 motifs, box A, box B, and box C, as well as the conserved PIP box, are shown. Sequence logos are colored according to the RasMol scheme (52). (C) Representative chromosome spreads as analyzed to score sister chromatid cohesion. Categories i and ii, in which sister chromatids were clearly tethered together, were considered normal cohesion, while chromatids that were well separated, as in categories iii (separated) and iv (scattered), were scored as loss of cohesion. (D) Cohesion assay. HeLa cells expressing siRNA-resistant FLAG-tagged derivatives of ESCO2 with the indicated mutations were treated with siRNA against ESCO2 to deplete endogenous transcripts and were scored for cohesion as shown in C (n ≥ 100/sample). ***P < 0.005 compared to wild-type (WT) control indicated by the black circle (Fisher’s exact test with Bonferonni’s correction for multiple comparisons; ns, not significant). Shown is a representative experiment; each mutant was tested at least 4 times independently (data from additional experiments are summarized in SI Appendix, Table S2). (E) Immunoblot showing expression of ESCO2 transgenes and SMC3 acetylation. Cell lysates from samples in D were probed with antibodies for the indicated proteins. SMC3^Ac, NCK, and ESCO2 came from the same gel. NCK was used as a loading control. SMC3 and ESCO2 were analyzed separately. Tg, transgene; Dox, doxycyline (used to activate expression of transgenes).

Fig. 4.

PCNA interacts with ESCO2 motifs in vivo and in vitro. (A) ESCO2N-GFP fusions. A cartoon depicting constructs in which the N-terminal 375 amino acids (a.a.) of ESCO2 are fused directly to eGFP. Motifs A, B, and C were deleted independently, as shown; numbers indicate the number of amino acids deleted (not drawn to scale). (B) Localization to replication foci. Confocal images of U2OS cells cotransfected with Ruby-PCNA and the GFP-fusion constructs. Colocalization is indicated in yellow in the merge image, as in Fig. 2. (C) Box C is critical for PCNA recruitment by tethered ESCO2. mCherry-lacI-ESCO2 (full-length) fusions with the deletions indicated in A were coexpressed with GFP-tagged PCNA as in Fig. 2, and the colocalization at nuclear foci was scored by fluorescence intensity profile analysis as in Fig. 2. (D) MCM4 recruitment to tethered ESCO2 is dependent upon box A. The experiment in C was repeated, only in this case the ESCO2 constructs were coexpressed with mEmerald-MCM4. Recruitment to tethered ESCO2 was scored as in Fig. 2. (E) Pull-down assay using GST-fusion proteins. Short peptide sequences including box A, box B, box C, or the ESCO2 PIP box motifs were expressed as GST-fusion proteins. A parallel set was made in which alanine substitutions were made at the invariant amino acids (shown in red). The PIP box from p21 was used as a positive control. (F) Coprecipitation from cell-free extracts. GST-fusion proteins shown in A were mixed with Xenopus egg extract and incubated with glutathione sepharose beads. The beads were washed and bound proteins were eluted and probed for PCNA by immunoblot. A duplicate gel was stained with Coomassie dye to detect the GST-fusion proteins. (G) Coprecipitation of purified proteins. The indicated GST-fusion proteins (E) were mixed with purified recombinant PCNA, pulled down with glutathione agarose beads, and analyzed as in F for PCNA.

Fig. 5.

Contributions of the canonical PIP motif to PCNA binding. (A) Generation of combined fusions. GST was fused to a peptide spanning amino acids 320 to 388 of human ESCO2 containing both box C and the downstream canonical PIP-like motif at amino acids 374 to 377. A derivative in which the PIP-like motif was mutated (ESCO2 Q374A, I377A) was also purified. (B) Surface plasmon resonance analysis. The GST-fusion peptides in A were used to test PCNA binding using SPR, in which GST fusions were bound by anti-GST antibody and recombinant trimeric PCNA was used as an analyte. Shown are the background-subtracted sensorgrams (Left) and binding curves (Right) for p21 (positive control), GST (negative control), and the indicated fusions. Binding constants are shown at Right. The χ² residuals are represented as RU², an indication of curve fitting. Vertical blue lines indicate K_D. (C) Contribution of the Eco1 PIP box to PCNA interaction in S. cerevisiae. GST was fused to the first 33 amino acids of S. cerevisiae Eco1p (GST-Eco1-PIP) for use in a pull-down assay (as in Fig. 4). Disruption of the PIP motif by 2 amino acid substitutions (Q18A, L21A = GST-Eco1-PIP-AA) disrupted the ability to pull down PCNA from whole cell yeast extract, as indicated by immunoblot analysis. A parallel gel was Coomassie stained, showing the bead-associated GST fusions. WCE, whole cell yeast extract.

Fig. 6.

Structural disorder is an intrinsic property of the ESCO2 N terminus. (A) Functional motifs are embedded in the disordered region. The protein sequences of ESCO2 from multiple species were analyzed to detect regions of predicted disorder using the PrDOS algorithm. Sequences above the dotted horizontal line are predicted to be disordered. Conserved motifs, including box A, PIP1, PIP2, and PIP3, are predicted to be structured and are indicated by the gray bars. Numbers indicate the number of amino acids between each conserved motif. Accession nos. are shown in SI Appendix, Table S3. (B) Spacing between motifs. The number of amino acids between each of the conserved motifs in the ESCO2 N terminus are indicated in blue. Species included are the same as in A, except that Xenopus tropicalis ESCO2 was also included. (C) Models. ESCO2 is initially recruited to chromatin through interaction with the loaded MCM helicase (gray) through box A (blue). Subsequently, ESCO2 associates with sites of active DNA replication through multiple PCNA-interaction motifs (orange, green, and black lozenges). ESCO2 may interact with a single PCNA trimer, as shown at Top, or may interact simultaneously with more than one PCNA trimer (Middle). The interaction of ESCO2 with MCMs and PCNA may occur simultaneously, or ESCO2 may be released from the MCM complex to associate with PCNA. Multiple, flexible low-affinity interactions may ensure association of ESCO2 with the dynamic replisome.