To find out what proportion, if any, of cohesin complexes topologically entrap DNA molecules during the G1 phase in mammalian cells, we analyzed the possibility of extracting chromatin-bound cohesin with a high-salt solution. We synchronized HeLa cells in the G1 phase, lysed them in isotonic buffer, and incubated permeabilized cells on ice in either isotonic buffer or in a buffer containing 0.5M NaCl. This relatively high concentration of salt should cause the extraction of most non-histone DNA binding proteins whereas topologically bound cohesin rings should remain associated with long chromosomal DNA molecules [12–14].
We separated extracted proteins from the insoluble material by centrifugation and assessed with western blotting the distribution of cohesin subunits Rad21 and Smc3 as well as CTCF between the fractions in different conditions. As expected, CTCF remained associated with chromatin in isotonic conditions but was completely extracted from nuclei in a high-salt buffer (Fig. 1b). On the other hand, approximately one half of the cohesin molecules (50–55%) were solubilized during cellular lysis in the isotonic conditions, a result that is in general agreement with previous publications [13, 36]. It is assumed that this easily solubilized fraction of cohesin roughly corresponds to a subpopulation of unbound free-diffusing cohesin molecules revealed by FRAP experiments [16, 36]. In contrast to CTCF, approximately 25–30% of cohesin complexes remain associated with chromatin even after incubation in high-ionic-strength conditions (Fig. 1b). Recently, a salt-resistant form of cohesin DNA binding was described that does not necessarily involve true topological entrapment; this mode of cohesin-DNA interactions was referred to as a “gripping state” [37–39]. Although gripping state is salt resistant at 4°C, it was shown that it could potentially be disrupted in high-salt buffers at higher temperatures [38]. Albeit that the gripping state seems to be short lived in vivo and could only be captured in special in vitro conditions (such as the usage of non-hydrolysable ATP analogues or ATPase-deficient cohesin complexes), we checked whether salt-resistant cohesin complexes observed in G1 cells are represented at some level by “gripping” cohesin complexes. To achieve that aim, we incubated permeabilized cells in a high-salt buffer at 37°C and assessed the redistribution of cohesin subunits between supernatant and chromatin-associated fraction. We found that in these conditions, the proportion of solubilized cohesin increased, but a substantial fraction (10–20%) still remained associated with chromatin (Fig. 1b). It is, therefore, likely that this portion is represented by cohesin that topologically entraps chromosomal DNA during the G1 stage of the cell cycle (i.e. before the onset of DNA replication). This notion is supported by observations made in a yeast model, where topologically engaged cohesin rings could be biochemically detected even in replication-deficient cells [9].
In the next set of experiments, we investigated whether chromatin loops generated by LE are resistant to salt extraction. With this aim, we generated 3C-seq libraries from permeabilized G1 cells incubated for 30 minutes in either isotonic or high-salt buffer. We chose the ~ 1Mb region on chromosome 21 that contains several well-defined CTCF-anchored cohesin loops in HeLa cells and enriched 3C-seq libraries with ligation products from this region using the C-TALE protocol [40]. Examination of heatmaps showed that high-salt treatment caused the complete disappearance of bright spots located away from the diagonal that are believed to reflect the presence of chromatin loops (Fig. 1c). It is, therefore, likely that LE-generated loops in vivo are sensitive to high concentrations of salt; this behavior is similar to that of cohesin loops generated in vitro [28, 29]. The latter are disrupted along with a complete dissociation of cohesin from DNA molecules when the salt concentration increases [29]. These in vitro results were interpreted in favor of a non-topological mode of cohesin LE [29].
However, our in vivo results can be explained otherwise because the C-TALE data alone, in contrast to the results of the above-mentioned in vitro experiments, do not show whether loop-maintaining cohesin molecules remained associated with chromatin after high-salt treatment. Theoretically, loops in which cohesin molecules topologically entrap DNA can be, nonetheless, sensitive to increased ionic strength. There are several possible structures of such loops (Fig. 1d). First, the cohesin molecule can associate with CTCF loop anchors asymmetrically, with one DNA anchor entrapped in a topological manner, whereas the other is not (Fig. 1d-(ii)) (hereinafter, we will refer to loops of such structure as being semi-topological). Alternatively, each cohesin molecule of a dimer, maintaining one loop, can interact with DNA in a semi-topological manner (Fig. 1d-(iii–iv)). Two principally different configurations actually correspond to such a dimer, with either both CTCF anchors occupied by topologically bound cohesin or the other way round, with both CTCF anchors associated with the non-topologically engaged salt-sensitive pole of cohesin. Finally, our experimental settings involve comparatively prolonged incubation of nuclei in a high-salt buffer. It is possible that in such a time interval, even topologically engaged cohesin molecules can diffuse from their original CTCF anchors along DNA molecules. In this scenario, even loops that do not rely on electrostatic cohesin–DNA interactions can produce blurred and, thus, indiscernible spots in C-TALE heatmaps (Fig. 1d-(v)).
To determine which of the above-presented configurations better describes real cohesin-CTCF loops, we performed ChIP-seq to identify profiles of cohesin association with the genomic region under study (1Mb region on chromosome 21) in control and salt-treated nuclei. We found that high-salt treatment caused the displacement of cohesin from CTCF-defined loop anchors sites, which were originally enriched in it (Fig. 1e).
Although the extraction of CTCF by a high-salt solution should release cohesin from anchorage sites, the topologically bound cohesin is expected to reside in proximity to these sites because it has limited capacity to passively diffuse along nucleosome-bound DNA [41]. However, the possibility that topologically bound cohesin rings can passively diffuse along DNA under conditions of increased salt concentration cannot be ruled out. In particular, such diffusion can occur during the 30-min incubation in a high-salt solution performed in our experiments. To exclude this possibility we repeated the ChIP-seq experiments using a significantly shorter time of incubation in the high-salt buffer (1 min instead of 30 min). We expected to observe the preservation or, perhaps, partial flattening of cohesin peaks at the original locations after this short treatment if, indeed, cohesin remained topologically bound to DNA but started to diffuse along the chromatin fiber. However, we again registered a complete disappearance of cohesin peaks (Fig. 1f). Thus, we concluded that, at least around CTCF-bound sites, cohesin likely does not interact with DNA topologically. Overall, the ChIP-seq data support either a non-topological or semi-topological structure of CTCF-anchored chromatin loops (Fig. 1d-(i) and Fig. 1d-(iv)).
Our results can be accommodated by a wide range of hypothetical models of LE, in which the cohesin ring either does not physically entrap DNA at all (non-topological LE) or entraps it during only some stages of the ATP hydrolysis cycle. Below we present a model (Fig. 2a) that provides reasonable explanations for most of the apparently controversial observations. First, we suggest that LE is performed by Scc2-bound cohesin complexes in a non-topological manner. This proposal is consistent with the in vitro data on cohesin LE [28, 29] and is corroborated by our results. Additionally, topological entrapment was shown to be dispensable for cohesin translocation from the loading sites in yeast [9]. Further, we postulate that Pds5 blocks in two ways the Scc2 activity in the LE process, namely (i) Pds5 competes with Scc2 for a binding surface on Rad21 and (ii) Pds5 participates in LE termination by recruiting Wapl, which causes the temporary opening of the Smc3-kleisin gate, leading to topological DNA entrapment. This process apparently leads to the termination of loop extrusion. The suggested mechanism explains how both Pds5 and Wapl negatively regulate the processivity of LE [26, 35]. In the proposed scenario, the Pds5-Wapl complex, rather than Scc2, serves as an actual cohesin loader in vivo. Such activity has been indeed demonstrated in vitro [44]. The Scc2 in vitro loading activity shown in several studies is likely to rely on the same process of transient Smc3-kleisin gate opening. Taking into account several circumstantial pieces of evidence [14, 37, 38], it is reasonable, however, to assume that Scc2, in contrast to Pds5-Wapl, poorly catalyzes DNA passage through the Smc3-kleisin gate; such a reaction apparently requires multiple rounds of ATP hydrolysis cycle and specifically tailored conditions.
According to the proposed model (Fig. 2a), topologically loaded cohesin complexes are not able to resume loop extrusion and are, therefore, subsequently released from chromatin through an additional round of Pds5-Wapl-catalyzed Smc3-kleisin gate opening. Accordingly, the Pds5-Wapl complex mediates both the engagement of cohesin in topological interactions with DNA and disengagement from it, as previously suggested [44].
We propose that CTCF inhibits both LE progression and termination by selectively recruiting Pds5 to cohesin while preventing Wapl and Scc2 binding (Fig. 2b). Thus, CTCF sites are, in fact, locations for a temporal pausing of LE. It was, indeed, reported that CTCF N-terminal binding to cohesin inhibits LE termination by blocking Wapl binding to the ‘‘conserved essential surface’’ (CES) of SA protein [33]. Furthermore, various cohesin regulators, including Wapl, Shugoshin, Sororin and Scc2 (but not Pds5), contain the amino acid motif F/YXF involved in CTCF-CES interactions. Hence, it is reasonable to assume that CTCF binding may also interfere with Scc2 recruitment to cohesin. Pds5A was recently shown to interact with CTCF through its N-terminal domain [45]. Additionally, Pds5 knockdown data suggest a contribution of Pds5 in CTCF-dependent LE blockage [26]. Thus, it is possible that CTCF inhibits the processivity of cohesin by selectively recruiting Pds5 in place of Scc2 to the complex and also prevents loop dissociation by inhibiting Wapl activity.
Overall, the presented model implies that active cohesin LE does not involve topological DNA entrapment and that LE termination is catalyzed by the Pds5-Wapl complex and is associated with cohesin topological loading and subsequent release. Such a hypothetical framework reconciles non-topological LE with the fact that both Pds5 and Wapl, primarily recognized as unloading factors, negatively regulate the processivity of LE. Released cohesin rings can be involved in new rounds of LE. However, a time gap exists between LE termination and the release of cohesin from chromatin. This gap explains the existence of topologically loaded cohesin rings during the G1 phase reported in our study and in previous publications [9, 13]. This subpopulation of engaged rings can be stabilized on chromosomes during the S phase by Smc3 K112/113 acetylation and Sororin recruitment, which block Wapl-dependent cohesin release (Fig. 2a) [46, 47].