Limits...
AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage.

Lee JH, Kang BH, Jang H, Kim TW, Choi J, Kwak S, Han J, Cho EJ, Youn HD - Nucleic Acids Res. (2015)

Bottom Line: H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal.AKT1 was more effective than AKT2 in phosphorylating H3-T45.Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3' end of DNA damage-activated genes to facilitate transcriptional termination.

View Article: PubMed Central - PubMed

Affiliation: National Creative Research Center for Epigenome Reprogramming Network, Department of Biomedical Sciences, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea.

Show MeSH

Related in: MedlinePlus

H3-T45 phosphorylation signal is most abundant near the TTS. MCF10A cells were treated with 0.4 μg/ml ADR for 18 h and analyzed by ChIP-seq. (A) Functional annotation of H3-T45 phosphorylation peak. (B) Pie chart showing the proportion of transcript coordinates for H3-T45 phosphorylation peaks (1041 regions). Proportion of transcript coordinates for H3-T45 phosphorylation peaks were compared with RefSeq transcripts. (C) Average profiles for phosphorylated Pol II-S2, S5, H3K36me3 and phosphorylated H3-T45 were plotted around ADR-induced H3-T45 phosphorylation-enriched genes (610 genes). (D) Phosphorylated RNA Pol II-S2 and phosphorylated H3-T45 ChIP peak distribution. (E and F) ChIP binding profiles of indicated genes. Scale data ranges are indicated on the right side of the individual track. Red boxes indicate the highest peak of phosphorylated H3-T45 signal. (G) Real-time qPCR analysis of CDKN1A mRNA in DMSO/ADR-treated MCF10A cells. (H) ChIP assay covering the CDKN1A locus above with the indicated antibodies. (I) ChIP-qPCR of the indicated gene locus with anti-phosphorylated H3-T45 and anti-phosphorylated RNA Pol II-S2. (J) ChIP-qPCR of promoter and TTS of CDKN1A, using anti-pan AKT. (K) ChIP-qPCR using anti-phosphorylated AKT-S473. qPCR was performed with complementary primers to the TTS of the indicated genes. ChIP-pPCR values were normalized with 1% input DNA. Real-time qPCR and ChIP assay data shown are the average values of at least three independent experiments. Standard deviations are indicated as error bars. * P < 0.05, **P < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482061&req=5

Figure 3: H3-T45 phosphorylation signal is most abundant near the TTS. MCF10A cells were treated with 0.4 μg/ml ADR for 18 h and analyzed by ChIP-seq. (A) Functional annotation of H3-T45 phosphorylation peak. (B) Pie chart showing the proportion of transcript coordinates for H3-T45 phosphorylation peaks (1041 regions). Proportion of transcript coordinates for H3-T45 phosphorylation peaks were compared with RefSeq transcripts. (C) Average profiles for phosphorylated Pol II-S2, S5, H3K36me3 and phosphorylated H3-T45 were plotted around ADR-induced H3-T45 phosphorylation-enriched genes (610 genes). (D) Phosphorylated RNA Pol II-S2 and phosphorylated H3-T45 ChIP peak distribution. (E and F) ChIP binding profiles of indicated genes. Scale data ranges are indicated on the right side of the individual track. Red boxes indicate the highest peak of phosphorylated H3-T45 signal. (G) Real-time qPCR analysis of CDKN1A mRNA in DMSO/ADR-treated MCF10A cells. (H) ChIP assay covering the CDKN1A locus above with the indicated antibodies. (I) ChIP-qPCR of the indicated gene locus with anti-phosphorylated H3-T45 and anti-phosphorylated RNA Pol II-S2. (J) ChIP-qPCR of promoter and TTS of CDKN1A, using anti-pan AKT. (K) ChIP-qPCR using anti-phosphorylated AKT-S473. qPCR was performed with complementary primers to the TTS of the indicated genes. ChIP-pPCR values were normalized with 1% input DNA. Real-time qPCR and ChIP assay data shown are the average values of at least three independent experiments. Standard deviations are indicated as error bars. * P < 0.05, **P < 0.001.

Mentions: H3-T45 lies in the N-terminus of the first helix of H3 and constitutes the nucleosome entry/exit point. This residue is assumed to have significant function, because it makes contact with genomic DNA (28–30). MCF10A cells were treated with ADR, and genome-wide ChIP-seq was performed with phosphorylated H3-T45 antibody, yielding a set of genes that contained phosphorylated H3-T45 (Supplementary Table S2). In our functional annotation analysis, most phosphorylated H3-T45-positive regions lay in cellular stress-responsive genes (Figure 3A and Supplementary Figure S7); the signal appeared primarily in the 3′ UTR of the gene-coding region (Figure 3B, C and Supplementary Figure S8). The specific pattern of H3-T45 phosphorylation, which centered around the transcription termination site (TTS), resembled that of phosphorylated RNA polymerase II CTD-Ser2 (Pol II-S2)/transcription termination factors (11,31). In comparing ChIP-seq profiles (Supplementary Table S2), we noted that over 67% of H3-T45 phosphorylation overlapped with RNA Pol II-S2 phosphorylation (Figure 3D and E). Housekeeping genes, such as GAPDH and HPRT1, were phosphorylated H3-T45-negative (Figure 3F), with lower RNA Pol II-S2 phosphorylation levels than H3-T45-positive genes (Figure 3E and F; RNA Pol II-S2 scale data ranges are 21 and 19 on CDKN1A and MDM2 versus 7.3 and 2.9 on GAPDH and HPRT1, respectively).


AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage.

Lee JH, Kang BH, Jang H, Kim TW, Choi J, Kwak S, Han J, Cho EJ, Youn HD - Nucleic Acids Res. (2015)

H3-T45 phosphorylation signal is most abundant near the TTS. MCF10A cells were treated with 0.4 μg/ml ADR for 18 h and analyzed by ChIP-seq. (A) Functional annotation of H3-T45 phosphorylation peak. (B) Pie chart showing the proportion of transcript coordinates for H3-T45 phosphorylation peaks (1041 regions). Proportion of transcript coordinates for H3-T45 phosphorylation peaks were compared with RefSeq transcripts. (C) Average profiles for phosphorylated Pol II-S2, S5, H3K36me3 and phosphorylated H3-T45 were plotted around ADR-induced H3-T45 phosphorylation-enriched genes (610 genes). (D) Phosphorylated RNA Pol II-S2 and phosphorylated H3-T45 ChIP peak distribution. (E and F) ChIP binding profiles of indicated genes. Scale data ranges are indicated on the right side of the individual track. Red boxes indicate the highest peak of phosphorylated H3-T45 signal. (G) Real-time qPCR analysis of CDKN1A mRNA in DMSO/ADR-treated MCF10A cells. (H) ChIP assay covering the CDKN1A locus above with the indicated antibodies. (I) ChIP-qPCR of the indicated gene locus with anti-phosphorylated H3-T45 and anti-phosphorylated RNA Pol II-S2. (J) ChIP-qPCR of promoter and TTS of CDKN1A, using anti-pan AKT. (K) ChIP-qPCR using anti-phosphorylated AKT-S473. qPCR was performed with complementary primers to the TTS of the indicated genes. ChIP-pPCR values were normalized with 1% input DNA. Real-time qPCR and ChIP assay data shown are the average values of at least three independent experiments. Standard deviations are indicated as error bars. * P < 0.05, **P < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482061&req=5

Figure 3: H3-T45 phosphorylation signal is most abundant near the TTS. MCF10A cells were treated with 0.4 μg/ml ADR for 18 h and analyzed by ChIP-seq. (A) Functional annotation of H3-T45 phosphorylation peak. (B) Pie chart showing the proportion of transcript coordinates for H3-T45 phosphorylation peaks (1041 regions). Proportion of transcript coordinates for H3-T45 phosphorylation peaks were compared with RefSeq transcripts. (C) Average profiles for phosphorylated Pol II-S2, S5, H3K36me3 and phosphorylated H3-T45 were plotted around ADR-induced H3-T45 phosphorylation-enriched genes (610 genes). (D) Phosphorylated RNA Pol II-S2 and phosphorylated H3-T45 ChIP peak distribution. (E and F) ChIP binding profiles of indicated genes. Scale data ranges are indicated on the right side of the individual track. Red boxes indicate the highest peak of phosphorylated H3-T45 signal. (G) Real-time qPCR analysis of CDKN1A mRNA in DMSO/ADR-treated MCF10A cells. (H) ChIP assay covering the CDKN1A locus above with the indicated antibodies. (I) ChIP-qPCR of the indicated gene locus with anti-phosphorylated H3-T45 and anti-phosphorylated RNA Pol II-S2. (J) ChIP-qPCR of promoter and TTS of CDKN1A, using anti-pan AKT. (K) ChIP-qPCR using anti-phosphorylated AKT-S473. qPCR was performed with complementary primers to the TTS of the indicated genes. ChIP-pPCR values were normalized with 1% input DNA. Real-time qPCR and ChIP assay data shown are the average values of at least three independent experiments. Standard deviations are indicated as error bars. * P < 0.05, **P < 0.001.
Mentions: H3-T45 lies in the N-terminus of the first helix of H3 and constitutes the nucleosome entry/exit point. This residue is assumed to have significant function, because it makes contact with genomic DNA (28–30). MCF10A cells were treated with ADR, and genome-wide ChIP-seq was performed with phosphorylated H3-T45 antibody, yielding a set of genes that contained phosphorylated H3-T45 (Supplementary Table S2). In our functional annotation analysis, most phosphorylated H3-T45-positive regions lay in cellular stress-responsive genes (Figure 3A and Supplementary Figure S7); the signal appeared primarily in the 3′ UTR of the gene-coding region (Figure 3B, C and Supplementary Figure S8). The specific pattern of H3-T45 phosphorylation, which centered around the transcription termination site (TTS), resembled that of phosphorylated RNA polymerase II CTD-Ser2 (Pol II-S2)/transcription termination factors (11,31). In comparing ChIP-seq profiles (Supplementary Table S2), we noted that over 67% of H3-T45 phosphorylation overlapped with RNA Pol II-S2 phosphorylation (Figure 3D and E). Housekeeping genes, such as GAPDH and HPRT1, were phosphorylated H3-T45-negative (Figure 3F), with lower RNA Pol II-S2 phosphorylation levels than H3-T45-positive genes (Figure 3E and F; RNA Pol II-S2 scale data ranges are 21 and 19 on CDKN1A and MDM2 versus 7.3 and 2.9 on GAPDH and HPRT1, respectively).

Bottom Line: H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal.AKT1 was more effective than AKT2 in phosphorylating H3-T45.Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3' end of DNA damage-activated genes to facilitate transcriptional termination.

View Article: PubMed Central - PubMed

Affiliation: National Creative Research Center for Epigenome Reprogramming Network, Department of Biomedical Sciences, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea.

Show MeSH
Related in: MedlinePlus