This study has provided the first demonstration that two members of the human SR protein family, SRp20 and ASF/SF2, associate with interphase chromatin, dissociate from mitotic chromosomes following histone H3S10P, and reassociate with post-mitotic chromatin. In the future it will be important to determine whether this is a common property of all members of the SR family of proteins. It appears that the release of SR proteins from chromatin is determined by SR protein phosphorylation by SRPKs and histone H3 serine 10 phosphorylation by Aurora B kinase. Consistent with this view, SRPK1 has been shown to translocate to the nucleus at the onset of M-phase, and some SR proteins have been shown to be hyperphosphorylated in M-phase (Ding et al., 2006
; Gui et al., 1994a
). We propose that a dual phosphorylation event involving SRPK-mediated phosphorylation of SR proteins and Aurora B kinase mediated H3 phosphorylation probably regulates the dynamics of SR protein interactions with chromatin. Although primarily nuclear, shuttling of SRp20 and ASF/SF2 to the cytoplasm when transcription is blocked or Clk/Sty kinase is exogenously expressed, has been observed (Caceres et al., 1998
). It remains to be seen whether these events are linked and thus establish a regulatory circuit.
Another interesting feature of the SR protein interaction with chromatin is that while a dual modification of histone H3 appears necessary for HP1 release (Fischle et al., 2005
; Hirota et al., 2005
; Mateescu et al., 2004
), H3S10P alone is sufficient for release of SRp20 and ASF/SF2 from mitotic chromatin. While the interaction of SRp20 and ASF/SF2 with chromatin might appear unexpected, previous work established that inactivation of ASF/SF2 causes transcription-related and cell-cycle progression defects, suggesting a role for ASF/SF2 in modulation of chromosome dynamics. Li et al. (2005)
suggested that a mechanism other than an alteration in mRNA splicing, specifically the generation of DNA double strand breaks resulting from cotranscriptional R-loop formation (Li and Manley, 2005
; Li et al., 2005
), could be responsible for the G2/M transition defects in cells depleted of ASF/SF2. Similarly, roles for another splicing factor, SC35, beyond its function in RNA processing have recently been reported in chromatin based processes: SC35 regulates transcription elongation of specific genes and also plays a critical role in regulating genomic stability and cell cycle progression (Lin et al., 2008
; Xiao et al., 2007
). Based on these and our current findings, we propose that chromatin association/dissociation properties of the SR proteins also contributes to proper cell cycle progression, and that they may contribute to the G2/M defect, as well as explain the delayed G0/G1 entry we observed upon ASF/SF2 depletion.
SRp20 and ASF/SF2’s chromatin association/dissociation properties provide insight into how these proteins may work to regulate proper chromatin function. We hypothesize that the release of SR proteins from hyperphosphorylated chromatin and the subsequent reassociation of SR proteins with chromatin once histone H3S10P has diminished are both important events for proper chromatin function. siRNA-mediated knockdown of ASF/SF2 caused retention of HP1 proteins on histone H3 despite serine 10 phosphorylation, suggesting that dissociation of ASF/SF2 from hyperphosphorylated histone H3 also influences HP1 dissociation from mitotic chromatin. Indeed, Fischle et al. (2005)
suggested that in addition to H3S10P, other mechanisms may be involved in mitotic release of HP1 from chromatin, such as further modification of HP1 proteins and/or their interaction partners (Fischle et al., 2005
). Our findings implicate ASF/SF2 as one such interacting partner responsible for HP1 release from mitotic chromatin. HP1 and ASF/SF2 are both associated with interphase chromatin, are dissociated from chromatin upon H3S10P and are associated with each other in mitotic cells. Due to the inherent limitations of mammalian cells for mutational analysis, neither this study nor previous ones (Fischle et al., 2005
; Hirota et al., 2005
) provided a direct causal relationship linking the chromatin association/dissociation properties of SRp20, ASF/SF2 and HP1 proteins with M-phase progression and chromosome segregation. However, M-phase progression and chromosome segregation defects have been noted in S. pombe
mutants defective in H3S10P (Mellone et al., 2003
; Wei et al., 1999
). Additionally in S. pombe
, the HP1 homolog, Swi6, is required for proper chromatin segregation (Pidoux and Allshire, 2004
Our studies in conjunction with the above results provide a strong correlation between SR and HP1 proteins and their release from chromatin with proper chromosome segregation and M-phase progression. It is, therefore, reasonable to suggest that the coordinated removal of HP1 and ASF/SF2 during M-phase is probably necessary to allow access by factors required for mediating proper chromatin condensation and faithful chromosome segregation. In this context we note that although 14-3-3 proteins were shown to bind H3S10P, they did not significantly associate with hyperphosphorylated condensed chromosomes in mitotic cells. Since H3S10P is also implicated in transcriptional activation, it appears that 14-3-3 and H3S10P association may be restricted to transcription of inducible genes (Macdonald et al., 2005
We also found that removal of an M-phase block in cells depleted of ASF/SF2 significantly delayed both exit from G2/M and entry into G0/G1. There are several possibilities for why ASF/SF2 depleted cells, blocked in M-phase, would delay G0/G1 entry. The first is that ASF/SF2 depletion blocks the cells at G2, as suggested by Li et al. (Li et al., 2005
). These authors demonstrated that DT40-ASF cells depleted of ASF/SF2 induced a G2-block and an increase in apoptotic cell death by 72h post-tet treatment. However, it is important to note that our experimental time frame was significantly shorter (28.5h) than theirs and we did not see increased apoptotic cell death as indicated by the absence of a sub-G0/G1 peak in all time points analyzed within the experimental time frame (28.5h). This, by no means, eliminates the possibility that part of the G2/M increase we see in our cell cycle analysis is due to a G2 block and not an M-phase block. The second, more intriguing possibility for the delay of G0/G1 entry upon ASF/SF2 depletion and release from an M-phase block is because ASF/SF2 is not present to reassociate with chromatin in telophase, once H3S10P has diminished. ASF/SF2 reassociation with chromatin at the end of mitosis may be a trigger for M-phase completion and it may allow additional factors, such as HP1 proteins, to reassociate with chromatin as the cell enters G0/G1. We propose that inhibition of histone H3S10P in ZM-treated cells, ASF/SF2 and SRp20, as well as HP1 proteins, are prevented from dissociating from mitotic chromatin and thus exacerbating the effect we see with ASF/SF2 depletion alone.
In summary, this work has established a novel association of two SR proteins, SRp20 and ASF/SF2, with interphase chromosomes; demonstrates their release from hyperphosphorylated mitotic chromosomes and reassociation with post-mitotic chromatin; and provides insight into our evolving understanding of the function of H3S10P. The ability of the SR proteins to associate with and dissociate from chromosomes in a histone H3 modification-selective manner may account for G2/M cell cycle arrest and defects in G0/G1 accumulation. Significantly, the SR proteins have similar association/dissociation characteristics as HP1 proteins. ASF/SF2 and HP1 proteins associate in mitotic cells and knockdown of ASF/SF2 led to retention of HP1 proteins on mitotic chromatin, suggesting a mechanistic link between the release of ASF/SF2 and HP1 proteins from mitotic chromatin. Future studies will investigate the underlying mechanisms of SR protein association/dissociation from chromatin and the potential role of SR proteins in directly regulating chromatin function and cell cycle progression.