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Methylation of histone H4 at aspartate 24 by protein L-isoaspartate O-methyltransferase (PCMT1) links histone modifications with protein homeostasis.

Biterge B, Richter F, Mittler G, Schneider R - Sci Rep (2014)

Bottom Line: We generated H4D24me specific antibodies and showed that H4D24me is ubiquitously present in different mouse and human cells.Furthermore, we demonstrated that VprBP (HIV-1 viral protein R (Vpr)-binding protein), a chromo domain-containing protein, specifically recognizes H4D24me potentially implicating H4D24me in H4 degradation.Thus, this work links for the first time a histone modification with histone protein aging and histone homeostasis, suggesting novel functions for histone modifications beyond transcriptional regulation.

View Article: PubMed Central - PubMed

Affiliation: 1] Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, INSERM U 964, Université de Strasbourg, 67404 Illkirch, France [2] Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg im Breisgau, Germany [3] University of Freiburg, Fahnenbergplatz, 79085 Freiburg im Breisgau, Germany.

ABSTRACT
Histone modifications play crucial roles in modulating chromatin function and transcriptional activity. Due to their long half-life, histones can, in addition to post-translational modifications, also accumulate spontaneous chemical alterations, which can affect their functionality and require either protein repair or degradation. One of the major sources of such protein damage or ageing is the conversion of aspartate into isoaspartate residues that can then be methylated. Here, we characterize a novel histone modification, the methylation of histone H4 at aspartate 24 (H4D24me). We generated H4D24me specific antibodies and showed that H4D24me is ubiquitously present in different mouse and human cells. Our in vitro and in vivo data identified PCMT1 (Protein L-isoaspartate O-methyltransferase), an enzyme involved in protein repair, as a novel H4D24 specific histone methyltransferase. Furthermore, we demonstrated that VprBP (HIV-1 viral protein R (Vpr)-binding protein), a chromo domain-containing protein, specifically recognizes H4D24me potentially implicating H4D24me in H4 degradation. Thus, this work links for the first time a histone modification with histone protein aging and histone homeostasis, suggesting novel functions for histone modifications beyond transcriptional regulation.

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Stress-induced degradation of H4 methylated at D24.(a) H4D24me levels decrease after UV treatment. Irradiation of U2OS cells with 60 j/m2 followed by indicated recovery times. Non-irradiated cells (NI) serve as control. Ponceau staining, tubulin and H4 blot as loading controls. γH2A.X as control for DNA damage induction. (b) H4D24me accumulates after treatment of HeLa cells with a synthetic proteasome inhibitor MG132 (20 μM). H2BK120ub immunoblot serves as a control to monitor the proteasome inhibition. Histone H4 and GAPDH blots as loading controls. Note that PCMT1 levels are not changing. (c) Treatment with MG132 results in elevated levels of H4D24me in the cytoplasm, where proteosomal degradation takes place compared to the untreated cells. H4D24me immunoblot on cytoplasmic histones. H2A immunoblot is shown as loading control.
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f4: Stress-induced degradation of H4 methylated at D24.(a) H4D24me levels decrease after UV treatment. Irradiation of U2OS cells with 60 j/m2 followed by indicated recovery times. Non-irradiated cells (NI) serve as control. Ponceau staining, tubulin and H4 blot as loading controls. γH2A.X as control for DNA damage induction. (b) H4D24me accumulates after treatment of HeLa cells with a synthetic proteasome inhibitor MG132 (20 μM). H2BK120ub immunoblot serves as a control to monitor the proteasome inhibition. Histone H4 and GAPDH blots as loading controls. Note that PCMT1 levels are not changing. (c) Treatment with MG132 results in elevated levels of H4D24me in the cytoplasm, where proteosomal degradation takes place compared to the untreated cells. H4D24me immunoblot on cytoplasmic histones. H2A immunoblot is shown as loading control.

Mentions: In addition to its chromo domain, VprBP also contains an Armadillo-like domain, a Lis homology motif (LisH) and tandem WD40 repeats that are suggested to have important functions in the dimerization of VprPB and interaction with other proteins including DDB120 (Fig. 3.c). DDB1 is another WD40 repeat-containing protein, which is part of the UV-damaged DNA-binding complex (UV-DDB) that is crucial for the nucleotide excision repair (NER) of DNA damage lesions induced by UV irradiation, as well as environmental mutagens including oxidative stress24. Because of this, we addressed whether there is a potential link between H4D24me and DNA damage repair. For this purpose, we examined whether the levels of H4D24me are altered upon UV irradiation. We treated U2OS cells with UV irradiation (60 j/m2) followed by a recovery and monitored the H4D24me levels. We observed that global H4D24me levels were decreased upon UV irradiation compared to the non-irradiated (NI) cells (Fig. 4.a) and only slowly recovered. VprBP has also been shown to be a substrate recognition component of the DCX (DDB1-CUL4-X-box) E3 ubiquitin-ligase complex, which can specifically recognize mono-methylated lysines and can target them for proteosomal degradation25. This could suggest that the decrease we observed in H4D24me levels after UV irradiation is a result of the recognition of H4D24me by VprBP, which in turn could mediate H4 degradation. To investigate this possibility, we treated HeLa cells with a synthetic proteasome inhibitor (MG132) for up to 6 hours. As shown in Figure 4.b, this proteasome inhibition resulted in accumulation of H4D24me (Fig. 4.b) which suggests that histone H4 methylated at D24 could indeed be targeted for proteosomal degradation. In support of this, in the cells that were treated with the proteasome inhibitor, we detected elevated H4D24me levels in the cytoplasmic fraction, where the proteosomal degradation takes place, when compared to the untreated control cells (Fig. 4.c). Thus, our data suggest that H4D24me may potentially act in addition to protein repair also as a signal for histone degradation. Similar to our results, Kaur et al. reported the UV-induced degradation of replication factor Mcm10 to be mediated by VprBP-DDB1-CUL426.


Methylation of histone H4 at aspartate 24 by protein L-isoaspartate O-methyltransferase (PCMT1) links histone modifications with protein homeostasis.

Biterge B, Richter F, Mittler G, Schneider R - Sci Rep (2014)

Stress-induced degradation of H4 methylated at D24.(a) H4D24me levels decrease after UV treatment. Irradiation of U2OS cells with 60 j/m2 followed by indicated recovery times. Non-irradiated cells (NI) serve as control. Ponceau staining, tubulin and H4 blot as loading controls. γH2A.X as control for DNA damage induction. (b) H4D24me accumulates after treatment of HeLa cells with a synthetic proteasome inhibitor MG132 (20 μM). H2BK120ub immunoblot serves as a control to monitor the proteasome inhibition. Histone H4 and GAPDH blots as loading controls. Note that PCMT1 levels are not changing. (c) Treatment with MG132 results in elevated levels of H4D24me in the cytoplasm, where proteosomal degradation takes place compared to the untreated cells. H4D24me immunoblot on cytoplasmic histones. H2A immunoblot is shown as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4202215&req=5

f4: Stress-induced degradation of H4 methylated at D24.(a) H4D24me levels decrease after UV treatment. Irradiation of U2OS cells with 60 j/m2 followed by indicated recovery times. Non-irradiated cells (NI) serve as control. Ponceau staining, tubulin and H4 blot as loading controls. γH2A.X as control for DNA damage induction. (b) H4D24me accumulates after treatment of HeLa cells with a synthetic proteasome inhibitor MG132 (20 μM). H2BK120ub immunoblot serves as a control to monitor the proteasome inhibition. Histone H4 and GAPDH blots as loading controls. Note that PCMT1 levels are not changing. (c) Treatment with MG132 results in elevated levels of H4D24me in the cytoplasm, where proteosomal degradation takes place compared to the untreated cells. H4D24me immunoblot on cytoplasmic histones. H2A immunoblot is shown as loading control.
Mentions: In addition to its chromo domain, VprBP also contains an Armadillo-like domain, a Lis homology motif (LisH) and tandem WD40 repeats that are suggested to have important functions in the dimerization of VprPB and interaction with other proteins including DDB120 (Fig. 3.c). DDB1 is another WD40 repeat-containing protein, which is part of the UV-damaged DNA-binding complex (UV-DDB) that is crucial for the nucleotide excision repair (NER) of DNA damage lesions induced by UV irradiation, as well as environmental mutagens including oxidative stress24. Because of this, we addressed whether there is a potential link between H4D24me and DNA damage repair. For this purpose, we examined whether the levels of H4D24me are altered upon UV irradiation. We treated U2OS cells with UV irradiation (60 j/m2) followed by a recovery and monitored the H4D24me levels. We observed that global H4D24me levels were decreased upon UV irradiation compared to the non-irradiated (NI) cells (Fig. 4.a) and only slowly recovered. VprBP has also been shown to be a substrate recognition component of the DCX (DDB1-CUL4-X-box) E3 ubiquitin-ligase complex, which can specifically recognize mono-methylated lysines and can target them for proteosomal degradation25. This could suggest that the decrease we observed in H4D24me levels after UV irradiation is a result of the recognition of H4D24me by VprBP, which in turn could mediate H4 degradation. To investigate this possibility, we treated HeLa cells with a synthetic proteasome inhibitor (MG132) for up to 6 hours. As shown in Figure 4.b, this proteasome inhibition resulted in accumulation of H4D24me (Fig. 4.b) which suggests that histone H4 methylated at D24 could indeed be targeted for proteosomal degradation. In support of this, in the cells that were treated with the proteasome inhibitor, we detected elevated H4D24me levels in the cytoplasmic fraction, where the proteosomal degradation takes place, when compared to the untreated control cells (Fig. 4.c). Thus, our data suggest that H4D24me may potentially act in addition to protein repair also as a signal for histone degradation. Similar to our results, Kaur et al. reported the UV-induced degradation of replication factor Mcm10 to be mediated by VprBP-DDB1-CUL426.

Bottom Line: We generated H4D24me specific antibodies and showed that H4D24me is ubiquitously present in different mouse and human cells.Furthermore, we demonstrated that VprBP (HIV-1 viral protein R (Vpr)-binding protein), a chromo domain-containing protein, specifically recognizes H4D24me potentially implicating H4D24me in H4 degradation.Thus, this work links for the first time a histone modification with histone protein aging and histone homeostasis, suggesting novel functions for histone modifications beyond transcriptional regulation.

View Article: PubMed Central - PubMed

Affiliation: 1] Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, INSERM U 964, Université de Strasbourg, 67404 Illkirch, France [2] Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg im Breisgau, Germany [3] University of Freiburg, Fahnenbergplatz, 79085 Freiburg im Breisgau, Germany.

ABSTRACT
Histone modifications play crucial roles in modulating chromatin function and transcriptional activity. Due to their long half-life, histones can, in addition to post-translational modifications, also accumulate spontaneous chemical alterations, which can affect their functionality and require either protein repair or degradation. One of the major sources of such protein damage or ageing is the conversion of aspartate into isoaspartate residues that can then be methylated. Here, we characterize a novel histone modification, the methylation of histone H4 at aspartate 24 (H4D24me). We generated H4D24me specific antibodies and showed that H4D24me is ubiquitously present in different mouse and human cells. Our in vitro and in vivo data identified PCMT1 (Protein L-isoaspartate O-methyltransferase), an enzyme involved in protein repair, as a novel H4D24 specific histone methyltransferase. Furthermore, we demonstrated that VprBP (HIV-1 viral protein R (Vpr)-binding protein), a chromo domain-containing protein, specifically recognizes H4D24me potentially implicating H4D24me in H4 degradation. Thus, this work links for the first time a histone modification with histone protein aging and histone homeostasis, suggesting novel functions for histone modifications beyond transcriptional regulation.

Show MeSH
Related in: MedlinePlus