RNA is required for the interaction between H1.4 and CAPD

Unpublished results from this laboratory have shown that the C-terminus of H1.4 can mediate the interaction with other proteins through RNA. To establish if the interaction between the C-terminus of H1.4 and the CTD of CAPD2 requires RNA, which may have been copurified with GST-CTD, RNase A was added to the pull-down reaction and the bound protein was analysed by western blotting for FLAG (Section 2.3.12). As shown in Figure 5.9, the interaction between H1.4 and the CTD of CAPD2 was lost upon degradation of RNA. The interaction was only maintained when low concentrations of RNase A were used (0.01 μg/mL or less). Below this concentration some of the bacterial RNA copurified with the proteins may have remained intact, and was therefore able to mediate the interaction. This result suggests that H1.4 and CAPD2 require an RNA component to interact in vitro, although, further investigation will be required to establish if this interaction occurs through a specific RNA species in vivo.

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Figure 5.9. RNA is required to mediate the interaction between H1.4 and

CAPD2.

GST pull-down assay of H1.4-FLAG (0.5 μg) and GST-CTD (CTD) at 150

mM sodium chloride with RNase A. RNase A was added to each reaction at the following concentrations from lane 1 through 6; 0, 0.001, 0.01, 0.1, 1 and

10 μg/mL. Bound H1.4-FLAG was analysed by western blotting for FLAG.

RNase A

α-FLAG

-

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5.3 Discussion

The hyperphosphorylation of H1.4 by the Cyclin dependent kinases that occurs during mitosis abolished the interaction with HP1β, whereas the interaction with the Condensin I subunit CAPD2 was maintained. While phosphorylation of the CDK sites in the C-terminus of H1.4 did affect the ability of HP1β to interact with methylated H1.4K26, phosphorylation of H1.4T18 also contributed. H1.4T18 is in the immediate vicinity of the methylated ‘ARKS’ motif to which HP1β binds, and H1.4T18 has been shown to be spatially orientated form hydrogen bonds with HP1γ bound to methylated H1.4 (Ruan et al., 2012). Thus phosphorylation of H1.4T18 may disrupt weak interactions important for facilitating the interaction between the HP1β chromo domain and H1.4 methylated at lysine 26. As the H1.4T18 phosphorylation site contributes to the displacement of HP1β as much as H1.4S27 phosphorylation does, then it will be interesting to establish if simultaneous phosphorylation of both sites together is sufficient to dislodge HP1β from methylated H1.4

CDK phosphorylation itself has been shown to induce conformational changes in H1 that may contribute to changes in the wider chromatin landscape (Lopez et al., 2015; Roque et al., 2008), although, the contribution of other protein factors acting together with H1 has not been well investigated. The report by Ball et al. (2002) linking the Condensin I regulatory subunit, CAPD2, and histone H1, provided an additional mechanism through which H1 could be implicated in chromosome condensation. While the localisation and histone interaction domains of CAPD2 were explored, the interaction with H1 was shown only via far-western blotting and in a GST pull-down assay with bovine H1 (Ball et al., 2002). As shown here, the H1.4 subtype could interact with CAPD2, regardless of its CDK phosphorylation status, through its C-terminal tail. However, this interaction was also mediated by the equivalent region in H1.2, which has a lesser condensation capacity relative to H1.4 (Clausell et al., 2009; Th'ng et al., 2005). This indicates that these two ubiquitous subtypes of H1 can mediate the interaction with CAPD2 through their C-terminal tail. Although the C-terminal tail is the most divergent region within H1, the other subtypes are yet to be tested for their ability to interact with CAPD2.

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Not only is H1.4 phosphorylated by CDK (Langan et al., 1989; Swank et al., 1997), but CAPD2 has also been reported to be a substrate for CDK (K. Kimura et al., 1998). This phosphorylation has been reported to activate the supercoiling activity in Xenopus laevis 13S condensin, leading to chromosome condensation (K. Kimura et al., 1998). Both H1.4 and CAPD2 can interact regardless of their phosphorylation status, suggesting that phosphorylation does not regulate this interaction. Regulation of the interaction by phosphorylation may not be necessary given that the Condensin I complex is excluded from the nucleus until the nuclear envelope disassembles (Hirota

et al., 2004). The observation that H1 and CAPD2 can interact when Condensin I is in its active state, suggests that H1 could aid in the targeting and maintenance of Condensin I to the chromatin to perform its condensation function indicating that phosphorylated H1 plays a role in chromosome condensation.

Most intriguing is the potential requirement for RNA to mediate the interaction between CAPD2 and H1.4, as shown here. The interaction through the C-terminal tail of H1.4 and the dependence on RNA bears similarity to the interaction between H1.4 and the hinge domain of HP1α (T.K. Hale, personnal communication; Hale et al., 2006). In addition, as for the hinge in HP1α, there are three adjacent lysine residues in the region of CAPD2 that mediates the interaction with H1 (Ball et al., 2002); these lysines may facilitate an interaction through RNA. Although mutation of these residues in CAPD2 was not attempted here, the dependence on RNA shown here, together with the failure of the lysine to alanine mutant tested by Ball et al. (2002) to localise to the nucleus suggests that these residues may mediate their nuclear localisation effect through an interaction between RNA and H1.4, or even H3. Establishing if CAPD2 and H1.4 do in fact interact in an RNA-dependent manner in vivo, and whether this is important for the correct localisation of the Condensin I complex requires further investigation. While attempts were made to study the in vivo interaction between CAPD2 and H1.4 using fluorescently tagged proteins, this was hindered by the inability to obtain cotransfected cells that were in mitosis. Future exploration could establish if this RNA mediated interaction occurs in vivo and if there is specificity in the type of RNA required to facilitate the interaction between CAPD2 and H1.4.

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Together, this in vitro data indicates that further exploration is required to elucidate whether these mechanisms occur in the context of chromatin in the nucleus during mitosis.

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Chapter Six