Materials and Methods
3.3 DNMT1, Usp7 and SCML2 cross-talk on the H3 ubiquitylated chromatinubiquitylated chromatin
3.3.5 SCML2 positions Usp7 at the N-terminus of histone H3
To better understand how SCML2 activates Usp7 on chromatin, crosslinking mass spec-trometry analysis was performed (Figure3.32). Recombinant FL His.Usp7 and recombinant GST.RBR-DUF were incubated with unmodified chromatin on streptavidin beads. Excess His.Usp7 and RBR-DUF were removed during the chromatin pull-down. The GST.RBR-DUF construct was prefered over FL SCML2 to avoid SPM-mediated multimerisation of SCML2 during protein-protein crosslinking (Figure3.26B) [106]. GST.RBR-DUF was shown to be necessary and sufficient for SCML2’s interaction with Usp7, for SCML2 and Usp7 chro-matin targeting and for SCML2 stimulation of Usp7 deubiquitylation activity (Figure3.21E, Figure 3.30C, Figure 3.28D). Stable as well as transient interactions between GST.RBR-DUF, FL His.Usp7 and the histones making up the unmodified chromatin were covalently linked at the end of the affinity purification experiment. The crosslinker used was BS3, a homobyfunctional crosslinker with a spacer of 11.4 ˚A that links lysine residues found in close proximity to each other.
Besides the known interaction with the TRAF domain of Usp7 (Figure 3.28F) [98],
RBR-and UBL5 (Figure 3.32A). Most crosslinks originated from the RBR region and contacted the UBL2-3 domains. The RBR region also contacted the nucleosome at the N-termini of histones H2B, H3 and H4. The Usp7 catalytic domain contacted the nucleosome on the N-terminal tail of histone H3 (residues K15 and K37) and the C-terminal tail of histone H4. Additional contacts were detected between UBL4-5 domains and the N-terminus of histone H2B. The extreme C-terminus of Usp7 formed intramolecular crosslinks with Usp7’s catalytic domain and the UBL1 domain, in what was previously shown to constitute an ac-tivating conformational change [70]. GST.RBR-DUF recruited Usp7 to the nucleosome and positioned it in the right orientation to mount a subsequent deubiquitylation attack onto the N-terminal tail of histone H3 (Figure 3.32B).
Previously, it was shown that ubiquitin specific protease adaptors bind the nucleosome at its acidic patch [72]. In the crosslinking mass spectrometry experiment, GST.RBR-DUF bound the nucleosome around the DNA gyres proximal to the N-terminus of histone H3. Only two crosslinking sites (H2BK112; H3K56) were found proximal to the nucleosomal acidic patch.
As suggested before, SCML2 may bind the nucleosome on two distinct surfaces. (Figure 3.17, Figure 3.24A, Figure 3.27A, Figure 3.28D).
The known interation between SCML2 and the TRAF domain of Usp7 was only indicated by two crosslinks (Figure 3.32A), one to the RBR domain, one to the DUF domain. Arguably, this was a consequence of the tight interaction between RBR-DUF and TRAF, which created a solvent-inaccesible interface. GST.RBR-DUF’s crosslinking sites on Usp7 mapped primar-ily to the UBL1-5 domains (Figure3.32C). Surprisingly, the crosslinking sites coincided with known interaction surfaces between Usp7 and the polybasic region (PBR) of UHRF1 and between Usp7 and the bromo-adjacent homology (BAH) domains of DNMT1 (Figure3.32D, Figure3.32E) [91], [92].
The crosslinking mass spectrometry experiments suggested the formation of a cross-talk or competition between DNMT1, SCML2 (and UHRF1) for interaction with the UBL1-2-3 do-mains of Usp7. Previously, we showed that DNMT1 inhibits SCML2 stimulation of Usp7 (Figure 3.30, Figure 3.31). During the deubiquitylation experiment, Usp7 likely switched between the DNMT1-bound state and the SCML2-bound state which caused conformational changes with functional implications in the enzyme. SCML2 may be needed on the H3ub chromatin to lower the activation energy for the Usp7-catalysed deubiquitylation reaction, to foster the recycling of the H3ub-bound DNMT1. To test this hypothesis, di-ubiquitylated chromatin arrays were preincubated to saturation with recombinant DNMT1 and then sub-jected to deubiquitylation in the presence or absence of GST.RBR-DUF over a time course of four hours (Figure 3.33). After chromatin afinity purification, Usp7 partially converted the H3ub2 mark to H3ub1 and decreased marginally the DNMT1 concentration on the chromatin array. In the presence of excess GST.RBR-DUF, more Usp7 was recruited to chromatin which resulted in more conversion of H3ub2 to H3ub1 and free H3 and less recov-ery of DNMT1 on the biotinylated arrays. This finding is in agreement with the hypothesis that SCML2 is needed for recycling of DNMT1 by facilitating the removal of the H3ub mark from chromatin arrays.
Figure 3.32(preceding page): SCML2 adapts Usp7 to H3ub chromatin. (A) Crosslinking mass spectrometry analysis of Usp7 and GST.RBR-DUF on unmodified chromatin arrays. Inter-molecular crosslinks are depicted with straight lines. IntraInter-molecular crosslinks are depicted as curves. SCML2 crosslinks to the nuclesome or to Usp7 are colored in cyan. Usp7 crosslinks to the nucleosome are colored in green. (B) Representation of the observed crosslinks on the nucleosome and Usp7 crystal structures. SCML2 crosslinked sites on Usp7 and on the nucleosome are high-lighted as cyan spheres. The SCML2 crosslinks on the nuclesome are displayed for each of the two copies of H2B, H3 and H4. Usp7 crosslinks on the nucleosome and nucleosome crosslinks on Usp7 are highlighted as green spheres. The interaction between the catalytic domain and the H3 tail is represented by straight green lines. Usp7 catalytic centre is depicted in yellow. (C) Represen-tation of SCML2 crosslinks on the UBL1-2-3-4-5 crystal structure. (D) SCML2 crosslinks on the UBL1-2-3 domains coincide with the interaction surface occupied by the UHRF1’s PBR peptide.
(E) SCML2 crosslinks on the UBL1-2-3-4-5 coincide with both of DNMT1’s binding surfaces onto Usp7. Structural information was obtained from crystal structures with PDB accession numbers 1KX5 (mononucleosome), 5FWI (Usp7 CD and UBL1-2-3), 5C6D (Usp7 UBL1-2-3 with UHRF1 PBR) and 4YOC (Usp7 UBL1-2-3-4-5 with DNMT1) [191], [192], [91], [92].
Figure 3.33: SCML2 controls DNMT1 residence time on H3 ubiquitylated chromatin.
Coomassie-stained SDS-PAGE gel of a time-course chromatin pull-down experiment with pre-incubated FL DNMT1 in the presence of equimolar FL Usp7 or equimolar FL Usp7 and over-stoichiometric GST.RBR-DUF. Samples were collected 1, 2 and 4 h after addition of Usp7 or Usp7 and GST.RBR-DUF.