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Suppression and recovery of BRCA1-mediated transcription by HP1γ via modulation of promoter occupancy.

Choi JD, Park MA, Lee JS - Nucleic Acids Res. (2012)

Bottom Line: Herein, we show that HP1γ interacts with breast cancer type 1 susceptibility protein (BRCA1) and regulates BRCA1-mediated transcription via modulation of promoter occupancy and histone modification.Time-lapse studies on promoter association and histone methylation after DNA damage revealed that HP1γ accumulates at the promoter before DNA damage, but BRCA1 is recruited at the promoter after the damage while promoter-resident HP1γ is disassembled.HP1γ/SUV39H1 restoration at the promoter results in BRCA1 disassembly and histone methylation, after which transcription repression resumes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Science and Technology College of Natural Sciences Ajou University, Suwon, Korea.

ABSTRACT
Heterochromatin protein 1γ (HP1γ) is a chromatin protein involved in gene silencing. Herein, we show that HP1γ interacts with breast cancer type 1 susceptibility protein (BRCA1) and regulates BRCA1-mediated transcription via modulation of promoter occupancy and histone modification. We used several HP1γ mutants and small interfering RNAs for histone methyltransferases to show that BRCA1-HP1γ interaction, but not methylated histone binding, is important in HP1γ repression of BRCA1-mediated transcription. Time-lapse studies on promoter association and histone methylation after DNA damage revealed that HP1γ accumulates at the promoter before DNA damage, but BRCA1 is recruited at the promoter after the damage while promoter-resident HP1γ is disassembled. Importantly, HP1γ assembly recovers after release from the damage in a BRCA1-HP1γ interaction-dependent manner and targets SUV39H1. HP1γ/SUV39H1 restoration at the promoter results in BRCA1 disassembly and histone methylation, after which transcription repression resumes. We propose that through interaction with BRCA1, HP1γ is guided to the BRCA1 target promoter during recovery and functions in the activation-repression switch and recovery from BRCA1-mediated transcription in response to DNA damage.

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Related in: MedlinePlus

Recruitment of HP1γ at the Gadd45α promoter via its interaction with BRCA1, post-release from DNA damage. (A) Immunoblots for BRCA1 were performed with anti-BRCA1 antibody using extracts from cells harvested at the indicated time points (before [−] and 18 h after etoposide treatment (Eto 18 h), and post-release from etoposide treatment [+6, +12 and +24 h]) to ensure depletion of endogenous BRCA1 by siBRCA1. The constant amount of α-tubulin protein is shown as a quantitative control for BRCA1 proteins. (B) ChIP analyses of the Gadd45α promoter were performed using anti-HP1γ antibody after transfection of 293T cells with siRNA against BRCA1 as described in (A). BRCA1-depleted cells were harvested at the indicated time points: before (−), after (Eto 18 h) and at release from etoposide treatment (+6, +12 and +24 h). The recruitment level before etoposide treatment in mock knockdown cells (siCon) was arbitrarily set to 1. Etoposide treatment for 18 h is indicated as the shadow box. (C) ChIP analysis of the Gadd45α promoter was performed using anti-FLAG antibody throughout the cycle of DNA damage (etoposide treatment for 18 h, release from etoposide [Eto 18 h] and post-release [+6, +12, and +24]) after transfection of 293T cells with siRNAs against BRCA1 (siBRCA1) and endogenous HP1γ (siHP1γ) and FLAG-tagged wild-type or mutant HP1γ resistant to siRNA (Res HP1γWT, Res HP1γV22A, or Res HP1γW164A) as described in Figure 6D. (D) and (E) ChIP analyses of the Gadd45α promoter were performed as described in (A) using anti-H3K9me3 (D) and H3K9ac antibodies (E) after transfection of 293T cells with siRNA against BRCA1. The recruitment level before etoposide treatment in mock knockdown cells (siCon) was arbitrarily set to 1. Etoposide treatment for 18 h is indicated as the shadow box. (F) Proposed model for HP1γ targeting the Gadd45α promoter in an H3K9me-independent but BRCA1 interaction-dependent manner during recovery after release from DNA damage. (a) In the quiescent unstressed state, HP1γ and histone-modifying enzymes including histone deacetylases and HMTs are located on the Gadd45α promoter to repress transcription. (b) When DNA is damaged, BRCA1 is recruited into the Gadd45α promoter and activates the transcription. (c) and (d) After release from DNA damage, HP1γ is recruited at the BRCA1 target promoter via its direct or indirect (such as involving additional protein(s) indicated as X) interaction with BRCA1 accompanied by BRCA1 disassembly and recruitment of histone-modifying enzymes. Finally, the promoter occupancy and chromatin context are restored to quiescence.
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gks947-F7: Recruitment of HP1γ at the Gadd45α promoter via its interaction with BRCA1, post-release from DNA damage. (A) Immunoblots for BRCA1 were performed with anti-BRCA1 antibody using extracts from cells harvested at the indicated time points (before [−] and 18 h after etoposide treatment (Eto 18 h), and post-release from etoposide treatment [+6, +12 and +24 h]) to ensure depletion of endogenous BRCA1 by siBRCA1. The constant amount of α-tubulin protein is shown as a quantitative control for BRCA1 proteins. (B) ChIP analyses of the Gadd45α promoter were performed using anti-HP1γ antibody after transfection of 293T cells with siRNA against BRCA1 as described in (A). BRCA1-depleted cells were harvested at the indicated time points: before (−), after (Eto 18 h) and at release from etoposide treatment (+6, +12 and +24 h). The recruitment level before etoposide treatment in mock knockdown cells (siCon) was arbitrarily set to 1. Etoposide treatment for 18 h is indicated as the shadow box. (C) ChIP analysis of the Gadd45α promoter was performed using anti-FLAG antibody throughout the cycle of DNA damage (etoposide treatment for 18 h, release from etoposide [Eto 18 h] and post-release [+6, +12, and +24]) after transfection of 293T cells with siRNAs against BRCA1 (siBRCA1) and endogenous HP1γ (siHP1γ) and FLAG-tagged wild-type or mutant HP1γ resistant to siRNA (Res HP1γWT, Res HP1γV22A, or Res HP1γW164A) as described in Figure 6D. (D) and (E) ChIP analyses of the Gadd45α promoter were performed as described in (A) using anti-H3K9me3 (D) and H3K9ac antibodies (E) after transfection of 293T cells with siRNA against BRCA1. The recruitment level before etoposide treatment in mock knockdown cells (siCon) was arbitrarily set to 1. Etoposide treatment for 18 h is indicated as the shadow box. (F) Proposed model for HP1γ targeting the Gadd45α promoter in an H3K9me-independent but BRCA1 interaction-dependent manner during recovery after release from DNA damage. (a) In the quiescent unstressed state, HP1γ and histone-modifying enzymes including histone deacetylases and HMTs are located on the Gadd45α promoter to repress transcription. (b) When DNA is damaged, BRCA1 is recruited into the Gadd45α promoter and activates the transcription. (c) and (d) After release from DNA damage, HP1γ is recruited at the BRCA1 target promoter via its direct or indirect (such as involving additional protein(s) indicated as X) interaction with BRCA1 accompanied by BRCA1 disassembly and recruitment of histone-modifying enzymes. Finally, the promoter occupancy and chromatin context are restored to quiescence.

Mentions: Transcription of the Gadd45α gene is usually repressed, and its transcription is activated/derepressed by BRCA1 via recruitment of BRCA1 to the promoter when DNA damage occurs (16,28). To study the role of BRCA1 in the HP1γ disassembly/assembly at the promoter, we monitored the level of HP1γ and H3K9me throughout the repression-activation-recovery cycle (during DNA damage, at release from DNA damage and post-release) in BRCA1-depleted cells (Figure 7). Before DNA damage, the level of HP1γ accumulated at the promoter in the BRCA1-depleted cells was slightly greater than that in the presence of BRCA1 (control siRNA; Figure 7B), suggesting that BRCA1 is not required for the HP1γ assembly at the BRCA1 target promoter in the absence of DNA damage and that HP1γ can be efficiently recruited and accumulated at the promoter in BRCA1-deficient cells. HP1γ fall-off from the promoter barely occurred in the etoposide-treated BRCA1 knockdown cells (Figure 7B), possibly because available BRCA1 is insufficient for HP1γ fall-off after DNA damage through competitive occupancy of the promoter DNA. The release from etoposide treatment had little effect on HP1γ recruitment in BRCA1-depleted cells, unlike its rapid recruitment in the presence of BRCA1 (Figure 7B). These results suggest that BRCA1 is required for HP1γ to be disassembled and reassembled at the promoter after DNA damage and post-release for recovery, respectively, because HP1γ rarely falls off and thus its restoration at the promoter is unnecessary post-release.Figure 7.


Suppression and recovery of BRCA1-mediated transcription by HP1γ via modulation of promoter occupancy.

Choi JD, Park MA, Lee JS - Nucleic Acids Res. (2012)

Recruitment of HP1γ at the Gadd45α promoter via its interaction with BRCA1, post-release from DNA damage. (A) Immunoblots for BRCA1 were performed with anti-BRCA1 antibody using extracts from cells harvested at the indicated time points (before [−] and 18 h after etoposide treatment (Eto 18 h), and post-release from etoposide treatment [+6, +12 and +24 h]) to ensure depletion of endogenous BRCA1 by siBRCA1. The constant amount of α-tubulin protein is shown as a quantitative control for BRCA1 proteins. (B) ChIP analyses of the Gadd45α promoter were performed using anti-HP1γ antibody after transfection of 293T cells with siRNA against BRCA1 as described in (A). BRCA1-depleted cells were harvested at the indicated time points: before (−), after (Eto 18 h) and at release from etoposide treatment (+6, +12 and +24 h). The recruitment level before etoposide treatment in mock knockdown cells (siCon) was arbitrarily set to 1. Etoposide treatment for 18 h is indicated as the shadow box. (C) ChIP analysis of the Gadd45α promoter was performed using anti-FLAG antibody throughout the cycle of DNA damage (etoposide treatment for 18 h, release from etoposide [Eto 18 h] and post-release [+6, +12, and +24]) after transfection of 293T cells with siRNAs against BRCA1 (siBRCA1) and endogenous HP1γ (siHP1γ) and FLAG-tagged wild-type or mutant HP1γ resistant to siRNA (Res HP1γWT, Res HP1γV22A, or Res HP1γW164A) as described in Figure 6D. (D) and (E) ChIP analyses of the Gadd45α promoter were performed as described in (A) using anti-H3K9me3 (D) and H3K9ac antibodies (E) after transfection of 293T cells with siRNA against BRCA1. The recruitment level before etoposide treatment in mock knockdown cells (siCon) was arbitrarily set to 1. Etoposide treatment for 18 h is indicated as the shadow box. (F) Proposed model for HP1γ targeting the Gadd45α promoter in an H3K9me-independent but BRCA1 interaction-dependent manner during recovery after release from DNA damage. (a) In the quiescent unstressed state, HP1γ and histone-modifying enzymes including histone deacetylases and HMTs are located on the Gadd45α promoter to repress transcription. (b) When DNA is damaged, BRCA1 is recruited into the Gadd45α promoter and activates the transcription. (c) and (d) After release from DNA damage, HP1γ is recruited at the BRCA1 target promoter via its direct or indirect (such as involving additional protein(s) indicated as X) interaction with BRCA1 accompanied by BRCA1 disassembly and recruitment of histone-modifying enzymes. Finally, the promoter occupancy and chromatin context are restored to quiescence.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526311&req=5

gks947-F7: Recruitment of HP1γ at the Gadd45α promoter via its interaction with BRCA1, post-release from DNA damage. (A) Immunoblots for BRCA1 were performed with anti-BRCA1 antibody using extracts from cells harvested at the indicated time points (before [−] and 18 h after etoposide treatment (Eto 18 h), and post-release from etoposide treatment [+6, +12 and +24 h]) to ensure depletion of endogenous BRCA1 by siBRCA1. The constant amount of α-tubulin protein is shown as a quantitative control for BRCA1 proteins. (B) ChIP analyses of the Gadd45α promoter were performed using anti-HP1γ antibody after transfection of 293T cells with siRNA against BRCA1 as described in (A). BRCA1-depleted cells were harvested at the indicated time points: before (−), after (Eto 18 h) and at release from etoposide treatment (+6, +12 and +24 h). The recruitment level before etoposide treatment in mock knockdown cells (siCon) was arbitrarily set to 1. Etoposide treatment for 18 h is indicated as the shadow box. (C) ChIP analysis of the Gadd45α promoter was performed using anti-FLAG antibody throughout the cycle of DNA damage (etoposide treatment for 18 h, release from etoposide [Eto 18 h] and post-release [+6, +12, and +24]) after transfection of 293T cells with siRNAs against BRCA1 (siBRCA1) and endogenous HP1γ (siHP1γ) and FLAG-tagged wild-type or mutant HP1γ resistant to siRNA (Res HP1γWT, Res HP1γV22A, or Res HP1γW164A) as described in Figure 6D. (D) and (E) ChIP analyses of the Gadd45α promoter were performed as described in (A) using anti-H3K9me3 (D) and H3K9ac antibodies (E) after transfection of 293T cells with siRNA against BRCA1. The recruitment level before etoposide treatment in mock knockdown cells (siCon) was arbitrarily set to 1. Etoposide treatment for 18 h is indicated as the shadow box. (F) Proposed model for HP1γ targeting the Gadd45α promoter in an H3K9me-independent but BRCA1 interaction-dependent manner during recovery after release from DNA damage. (a) In the quiescent unstressed state, HP1γ and histone-modifying enzymes including histone deacetylases and HMTs are located on the Gadd45α promoter to repress transcription. (b) When DNA is damaged, BRCA1 is recruited into the Gadd45α promoter and activates the transcription. (c) and (d) After release from DNA damage, HP1γ is recruited at the BRCA1 target promoter via its direct or indirect (such as involving additional protein(s) indicated as X) interaction with BRCA1 accompanied by BRCA1 disassembly and recruitment of histone-modifying enzymes. Finally, the promoter occupancy and chromatin context are restored to quiescence.
Mentions: Transcription of the Gadd45α gene is usually repressed, and its transcription is activated/derepressed by BRCA1 via recruitment of BRCA1 to the promoter when DNA damage occurs (16,28). To study the role of BRCA1 in the HP1γ disassembly/assembly at the promoter, we monitored the level of HP1γ and H3K9me throughout the repression-activation-recovery cycle (during DNA damage, at release from DNA damage and post-release) in BRCA1-depleted cells (Figure 7). Before DNA damage, the level of HP1γ accumulated at the promoter in the BRCA1-depleted cells was slightly greater than that in the presence of BRCA1 (control siRNA; Figure 7B), suggesting that BRCA1 is not required for the HP1γ assembly at the BRCA1 target promoter in the absence of DNA damage and that HP1γ can be efficiently recruited and accumulated at the promoter in BRCA1-deficient cells. HP1γ fall-off from the promoter barely occurred in the etoposide-treated BRCA1 knockdown cells (Figure 7B), possibly because available BRCA1 is insufficient for HP1γ fall-off after DNA damage through competitive occupancy of the promoter DNA. The release from etoposide treatment had little effect on HP1γ recruitment in BRCA1-depleted cells, unlike its rapid recruitment in the presence of BRCA1 (Figure 7B). These results suggest that BRCA1 is required for HP1γ to be disassembled and reassembled at the promoter after DNA damage and post-release for recovery, respectively, because HP1γ rarely falls off and thus its restoration at the promoter is unnecessary post-release.Figure 7.

Bottom Line: Herein, we show that HP1γ interacts with breast cancer type 1 susceptibility protein (BRCA1) and regulates BRCA1-mediated transcription via modulation of promoter occupancy and histone modification.Time-lapse studies on promoter association and histone methylation after DNA damage revealed that HP1γ accumulates at the promoter before DNA damage, but BRCA1 is recruited at the promoter after the damage while promoter-resident HP1γ is disassembled.HP1γ/SUV39H1 restoration at the promoter results in BRCA1 disassembly and histone methylation, after which transcription repression resumes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Science and Technology College of Natural Sciences Ajou University, Suwon, Korea.

ABSTRACT
Heterochromatin protein 1γ (HP1γ) is a chromatin protein involved in gene silencing. Herein, we show that HP1γ interacts with breast cancer type 1 susceptibility protein (BRCA1) and regulates BRCA1-mediated transcription via modulation of promoter occupancy and histone modification. We used several HP1γ mutants and small interfering RNAs for histone methyltransferases to show that BRCA1-HP1γ interaction, but not methylated histone binding, is important in HP1γ repression of BRCA1-mediated transcription. Time-lapse studies on promoter association and histone methylation after DNA damage revealed that HP1γ accumulates at the promoter before DNA damage, but BRCA1 is recruited at the promoter after the damage while promoter-resident HP1γ is disassembled. Importantly, HP1γ assembly recovers after release from the damage in a BRCA1-HP1γ interaction-dependent manner and targets SUV39H1. HP1γ/SUV39H1 restoration at the promoter results in BRCA1 disassembly and histone methylation, after which transcription repression resumes. We propose that through interaction with BRCA1, HP1γ is guided to the BRCA1 target promoter during recovery and functions in the activation-repression switch and recovery from BRCA1-mediated transcription in response to DNA damage.

Show MeSH
Related in: MedlinePlus