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Human cytomegalovirus IE1 protein alters the higher-order chromatin structure by targeting the acidic patch of the nucleosome.

Fang Q, Chen P, Wang M, Fang J, Yang N, Li G, Xu RM - Elife (2016)

Bottom Line: Human cytomegalovirus (hCMV) immediate early 1 (IE1) protein associates with condensed chromatin of the host cell during mitosis.We have determined the structure of the chromatin-tethering domain (CTD) of IE1 bound to the nucleosome core particle, and discovered that IE1-CTD specifically interacts with the H2A-H2B acidic patch and impairs the compaction of higher-order chromatin structure.Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Human cytomegalovirus (hCMV) immediate early 1 (IE1) protein associates with condensed chromatin of the host cell during mitosis. We have determined the structure of the chromatin-tethering domain (CTD) of IE1 bound to the nucleosome core particle, and discovered that IE1-CTD specifically interacts with the H2A-H2B acidic patch and impairs the compaction of higher-order chromatin structure. Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections.

No MeSH data available.


Related in: MedlinePlus

Assessment of the quality of reconstituted nucleosomal array.(A) Nucleosomal arrays corresponding to ~1 μg DNA were cleaved with indicated amount of micrococcal nuclease (MNase). (B) EM analysis of the reconstituted nucleosomal array (Bar: 100 nm).DOI:http://dx.doi.org/10.7554/eLife.11911.009
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fig4s1: Assessment of the quality of reconstituted nucleosomal array.(A) Nucleosomal arrays corresponding to ~1 μg DNA were cleaved with indicated amount of micrococcal nuclease (MNase). (B) EM analysis of the reconstituted nucleosomal array (Bar: 100 nm).DOI:http://dx.doi.org/10.7554/eLife.11911.009

Mentions: The acidic patch of the nucleosome has been implicated in mediating higher-order chromatin folding via interaction with the N-terminal tail of histone H4 (Luger et al., 1997a; Schalch et al., 2005). Our previous cryo-EM structure of 30-nm chromatin fiber reveals that N-terminal tails of histone H4 are involved in inter-nucleosomal contacts between the tetranucleosomal structural units through the acidic patches of adjacent nucleosomes (Song et al., 2014). Since IE1-CTD is bound at the acidic patch, it is conceivable that IE1 binding may interfere with proper folding of the 30-nm chromatin fiber. To determine the extent by which the folding of chromatin fiber is affected by IE1 binding, we incubated IE1-CTD with the in vitro reconstituted 30-nm chromatin fiber assembled with an array of 12 tandem nucleosomes carrying repeats of 177 bp 601 DNA in the presence of linker histone H1, and analyzed the sample by analytical ultracentrifugation in sedimentation velocity (AUC) (Song et al., 2014). The nucleosomal arrays used for reconstituting 30-nm chromatin fiber were highly saturated and homogeneous, as examined by micrococcal nuclease (MNase) digestion and electron microscopy (Figure 4—figure supplement 1A and B). For comparison, the same batch of nucleosomal array (without H1) and 30-nm chromatin fiber (with H1) were used in AUC analysis. AUC experiments showed that, in the absence of IE1-CTD, the nucleosomal array sedimented with a median sedimentation coefficient Save (sedimentation coefficient at 50% boundary fraction) of 36 ± 1 S, and the 30-nm chromatin fiber sedimented at 51.5 ± 0.6 S (Figure 3A). In the presence of IE1-CTD, the 10-nm nucleosomal array was unaffected while Save of the 30-nm chromatin fiber shifted from 51.5 to 48 S, indicating that the binding of IE1-CTD made the chromatin fiber more loosely folded (Figure 4A). It should be emphasized that the IE1-CTD-containing chromatin fiber represents an altered chromatin state different from both the extended 10-nm nucleosomal array and the folded 30-nm chromatin fiber. This chromatin-alteration property of IE1-CTD is shared by the full-length IE1 and fully depends on the presence of CTD (Figure 4B and C). Further AUC analyses with IE1-CTD mutants showed that they essentially retained the ability to decondense the 30-nm chromatin fiber, possibly due to their incomplete loss of NCP-binding abilities (Figure 4D, E and F). By contrast, chromatin fibers reconstituted with the E56R mutant of histone H2A, previously shown to be unable to interact with IE1-CTD, displayed no alteration of chromatin folding by IE1-CTD (Figure 4G). These observations indicate that the binding of IE1-CTD at the acidic patch of the nucleosome modulates the higher-order structure of chromatin. It should be pointed out that not all acidic patch-binding proteins affect chromatin folding, as LANA does not alter the folding of 30-nm chromatin fiber in our AUC analysis (Figure 4H).10.7554/eLife.11911.008Figure 4.Influence of IE1-CTD on higher-order chromatin structure.


Human cytomegalovirus IE1 protein alters the higher-order chromatin structure by targeting the acidic patch of the nucleosome.

Fang Q, Chen P, Wang M, Fang J, Yang N, Li G, Xu RM - Elife (2016)

Assessment of the quality of reconstituted nucleosomal array.(A) Nucleosomal arrays corresponding to ~1 μg DNA were cleaved with indicated amount of micrococcal nuclease (MNase). (B) EM analysis of the reconstituted nucleosomal array (Bar: 100 nm).DOI:http://dx.doi.org/10.7554/eLife.11911.009
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Related In: Results  -  Collection

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fig4s1: Assessment of the quality of reconstituted nucleosomal array.(A) Nucleosomal arrays corresponding to ~1 μg DNA were cleaved with indicated amount of micrococcal nuclease (MNase). (B) EM analysis of the reconstituted nucleosomal array (Bar: 100 nm).DOI:http://dx.doi.org/10.7554/eLife.11911.009
Mentions: The acidic patch of the nucleosome has been implicated in mediating higher-order chromatin folding via interaction with the N-terminal tail of histone H4 (Luger et al., 1997a; Schalch et al., 2005). Our previous cryo-EM structure of 30-nm chromatin fiber reveals that N-terminal tails of histone H4 are involved in inter-nucleosomal contacts between the tetranucleosomal structural units through the acidic patches of adjacent nucleosomes (Song et al., 2014). Since IE1-CTD is bound at the acidic patch, it is conceivable that IE1 binding may interfere with proper folding of the 30-nm chromatin fiber. To determine the extent by which the folding of chromatin fiber is affected by IE1 binding, we incubated IE1-CTD with the in vitro reconstituted 30-nm chromatin fiber assembled with an array of 12 tandem nucleosomes carrying repeats of 177 bp 601 DNA in the presence of linker histone H1, and analyzed the sample by analytical ultracentrifugation in sedimentation velocity (AUC) (Song et al., 2014). The nucleosomal arrays used for reconstituting 30-nm chromatin fiber were highly saturated and homogeneous, as examined by micrococcal nuclease (MNase) digestion and electron microscopy (Figure 4—figure supplement 1A and B). For comparison, the same batch of nucleosomal array (without H1) and 30-nm chromatin fiber (with H1) were used in AUC analysis. AUC experiments showed that, in the absence of IE1-CTD, the nucleosomal array sedimented with a median sedimentation coefficient Save (sedimentation coefficient at 50% boundary fraction) of 36 ± 1 S, and the 30-nm chromatin fiber sedimented at 51.5 ± 0.6 S (Figure 3A). In the presence of IE1-CTD, the 10-nm nucleosomal array was unaffected while Save of the 30-nm chromatin fiber shifted from 51.5 to 48 S, indicating that the binding of IE1-CTD made the chromatin fiber more loosely folded (Figure 4A). It should be emphasized that the IE1-CTD-containing chromatin fiber represents an altered chromatin state different from both the extended 10-nm nucleosomal array and the folded 30-nm chromatin fiber. This chromatin-alteration property of IE1-CTD is shared by the full-length IE1 and fully depends on the presence of CTD (Figure 4B and C). Further AUC analyses with IE1-CTD mutants showed that they essentially retained the ability to decondense the 30-nm chromatin fiber, possibly due to their incomplete loss of NCP-binding abilities (Figure 4D, E and F). By contrast, chromatin fibers reconstituted with the E56R mutant of histone H2A, previously shown to be unable to interact with IE1-CTD, displayed no alteration of chromatin folding by IE1-CTD (Figure 4G). These observations indicate that the binding of IE1-CTD at the acidic patch of the nucleosome modulates the higher-order structure of chromatin. It should be pointed out that not all acidic patch-binding proteins affect chromatin folding, as LANA does not alter the folding of 30-nm chromatin fiber in our AUC analysis (Figure 4H).10.7554/eLife.11911.008Figure 4.Influence of IE1-CTD on higher-order chromatin structure.

Bottom Line: Human cytomegalovirus (hCMV) immediate early 1 (IE1) protein associates with condensed chromatin of the host cell during mitosis.We have determined the structure of the chromatin-tethering domain (CTD) of IE1 bound to the nucleosome core particle, and discovered that IE1-CTD specifically interacts with the H2A-H2B acidic patch and impairs the compaction of higher-order chromatin structure.Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Human cytomegalovirus (hCMV) immediate early 1 (IE1) protein associates with condensed chromatin of the host cell during mitosis. We have determined the structure of the chromatin-tethering domain (CTD) of IE1 bound to the nucleosome core particle, and discovered that IE1-CTD specifically interacts with the H2A-H2B acidic patch and impairs the compaction of higher-order chromatin structure. Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections.

No MeSH data available.


Related in: MedlinePlus