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The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin.

Giri S, Aggarwal V, Pontis J, Shen Z, Chakraborty A, Khan A, Mizzen C, Prasanth KV, Ait-Si-Ali S, Ha T, Prasanth SG - Elife (2015)

Bottom Line: The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells.Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites.We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Champaign, United States.

ABSTRACT
Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. Its role in heterochromatin organization remained elusive. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affect replication timing, preferentially at the late-replicating regions. We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure.

No MeSH data available.


ORCA is a scaffold for G9a-Suv39H1 complexes.(A) a–b HA-IP in control and ORCA-depleted U2OS cells co-expressing with HA-G9a and Flag-Suv39H1. (B) a TIRF images of GFP SiMPull in control and ORCA-depleted U2OS cells co-transfected with YFP-Suv39H1 and mCherry-G9a. The same lysates incubated with biotinylated HA Ab served as the control. b Average number of YFP fluorescent molecules per imaging area (5000 μm2). c The % of mCherry-G9a pulled down by YFP Suv39H1 in control and ORCA KD. (C) a TIRF images of GFP SiMPull in U2OS cells transiently transfected with YFP-Suv39H1, mCherry-G9a, and T7-ORCA full-length or truncation mutant 1–270 or 128–647. The same lysates incubated with biotinylated HA Ab served as the control. b Average number of YFP fluorescent molecules per imaging area (5000 μm2). c The % of mCherry-G9a pulled down by YFP-Suv39H1. The % of mCherry-G9a pulled down by YFP-Suv39H1 in WT-ORCA is 25 ± 1%; 1–270 ORCA is 14 ± 3%; and 128–647 ORCA is 29 ± 6%. Scale bars, 20 μm. Error bars represent s.d., n = 3. **p < 0.01, ***p < 0.001.DOI:http://dx.doi.org/10.7554/eLife.06496.013
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fig6: ORCA is a scaffold for G9a-Suv39H1 complexes.(A) a–b HA-IP in control and ORCA-depleted U2OS cells co-expressing with HA-G9a and Flag-Suv39H1. (B) a TIRF images of GFP SiMPull in control and ORCA-depleted U2OS cells co-transfected with YFP-Suv39H1 and mCherry-G9a. The same lysates incubated with biotinylated HA Ab served as the control. b Average number of YFP fluorescent molecules per imaging area (5000 μm2). c The % of mCherry-G9a pulled down by YFP Suv39H1 in control and ORCA KD. (C) a TIRF images of GFP SiMPull in U2OS cells transiently transfected with YFP-Suv39H1, mCherry-G9a, and T7-ORCA full-length or truncation mutant 1–270 or 128–647. The same lysates incubated with biotinylated HA Ab served as the control. b Average number of YFP fluorescent molecules per imaging area (5000 μm2). c The % of mCherry-G9a pulled down by YFP-Suv39H1. The % of mCherry-G9a pulled down by YFP-Suv39H1 in WT-ORCA is 25 ± 1%; 1–270 ORCA is 14 ± 3%; and 128–647 ORCA is 29 ± 6%. Scale bars, 20 μm. Error bars represent s.d., n = 3. **p < 0.01, ***p < 0.001.DOI:http://dx.doi.org/10.7554/eLife.06496.013

Mentions: Next, we addressed if ORCA facilitates the assembly of the multimeric H3K9 KMT megacomplex. To address this, the association between the components of the KMT megacomplex, namely G9a and Suv39H1, was evaluated in cells that were treated with control or ORCA siRNAs. Flag-G9a and HA-Suv39H1 were expressed, and HA IP was carried out in control and ORCA-depleted (ORCA KD) cells. ORCA KD showed close to 50% reduction of Suv39H1 that co-immunoprecipitated with G9a (Figure 6Aa,Ab). This observation suggested that the stability of the KMT complex requires ORCA. We used SiMPull to obtain an accurate quantitative estimate of the reduction (Figure 6Ba–Bc). YFP-Suv39H1 and mCherry-G9a were expressed in cells depleted of ORCA. GFP pull-down was carried out, and the number of mCherry-G9a molecules associated with Suv39H1 was calculated (Figure 6Bb,Bc). ORCA KD led to ∼50% reduction in the complexes containing YFP-Suv39H1 and mCherry-G9a (note, 24 ± 3% mCherry-G9a pulled down by YFP-Suv39H1 in control vs 15 ± 1% in ORCA knock-down cells; Figure 6Bc). These results support the argument that ORCA acts as a scaffold protein that is necessary for stabilizing a subset of the complexes containing multiple H3K9 KMTs.10.7554/eLife.06496.013Figure 6.ORCA is a scaffold for G9a-Suv39H1 complexes.


The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin.

Giri S, Aggarwal V, Pontis J, Shen Z, Chakraborty A, Khan A, Mizzen C, Prasanth KV, Ait-Si-Ali S, Ha T, Prasanth SG - Elife (2015)

ORCA is a scaffold for G9a-Suv39H1 complexes.(A) a–b HA-IP in control and ORCA-depleted U2OS cells co-expressing with HA-G9a and Flag-Suv39H1. (B) a TIRF images of GFP SiMPull in control and ORCA-depleted U2OS cells co-transfected with YFP-Suv39H1 and mCherry-G9a. The same lysates incubated with biotinylated HA Ab served as the control. b Average number of YFP fluorescent molecules per imaging area (5000 μm2). c The % of mCherry-G9a pulled down by YFP Suv39H1 in control and ORCA KD. (C) a TIRF images of GFP SiMPull in U2OS cells transiently transfected with YFP-Suv39H1, mCherry-G9a, and T7-ORCA full-length or truncation mutant 1–270 or 128–647. The same lysates incubated with biotinylated HA Ab served as the control. b Average number of YFP fluorescent molecules per imaging area (5000 μm2). c The % of mCherry-G9a pulled down by YFP-Suv39H1. The % of mCherry-G9a pulled down by YFP-Suv39H1 in WT-ORCA is 25 ± 1%; 1–270 ORCA is 14 ± 3%; and 128–647 ORCA is 29 ± 6%. Scale bars, 20 μm. Error bars represent s.d., n = 3. **p < 0.01, ***p < 0.001.DOI:http://dx.doi.org/10.7554/eLife.06496.013
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fig6: ORCA is a scaffold for G9a-Suv39H1 complexes.(A) a–b HA-IP in control and ORCA-depleted U2OS cells co-expressing with HA-G9a and Flag-Suv39H1. (B) a TIRF images of GFP SiMPull in control and ORCA-depleted U2OS cells co-transfected with YFP-Suv39H1 and mCherry-G9a. The same lysates incubated with biotinylated HA Ab served as the control. b Average number of YFP fluorescent molecules per imaging area (5000 μm2). c The % of mCherry-G9a pulled down by YFP Suv39H1 in control and ORCA KD. (C) a TIRF images of GFP SiMPull in U2OS cells transiently transfected with YFP-Suv39H1, mCherry-G9a, and T7-ORCA full-length or truncation mutant 1–270 or 128–647. The same lysates incubated with biotinylated HA Ab served as the control. b Average number of YFP fluorescent molecules per imaging area (5000 μm2). c The % of mCherry-G9a pulled down by YFP-Suv39H1. The % of mCherry-G9a pulled down by YFP-Suv39H1 in WT-ORCA is 25 ± 1%; 1–270 ORCA is 14 ± 3%; and 128–647 ORCA is 29 ± 6%. Scale bars, 20 μm. Error bars represent s.d., n = 3. **p < 0.01, ***p < 0.001.DOI:http://dx.doi.org/10.7554/eLife.06496.013
Mentions: Next, we addressed if ORCA facilitates the assembly of the multimeric H3K9 KMT megacomplex. To address this, the association between the components of the KMT megacomplex, namely G9a and Suv39H1, was evaluated in cells that were treated with control or ORCA siRNAs. Flag-G9a and HA-Suv39H1 were expressed, and HA IP was carried out in control and ORCA-depleted (ORCA KD) cells. ORCA KD showed close to 50% reduction of Suv39H1 that co-immunoprecipitated with G9a (Figure 6Aa,Ab). This observation suggested that the stability of the KMT complex requires ORCA. We used SiMPull to obtain an accurate quantitative estimate of the reduction (Figure 6Ba–Bc). YFP-Suv39H1 and mCherry-G9a were expressed in cells depleted of ORCA. GFP pull-down was carried out, and the number of mCherry-G9a molecules associated with Suv39H1 was calculated (Figure 6Bb,Bc). ORCA KD led to ∼50% reduction in the complexes containing YFP-Suv39H1 and mCherry-G9a (note, 24 ± 3% mCherry-G9a pulled down by YFP-Suv39H1 in control vs 15 ± 1% in ORCA knock-down cells; Figure 6Bc). These results support the argument that ORCA acts as a scaffold protein that is necessary for stabilizing a subset of the complexes containing multiple H3K9 KMTs.10.7554/eLife.06496.013Figure 6.ORCA is a scaffold for G9a-Suv39H1 complexes.

Bottom Line: The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells.Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites.We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Champaign, United States.

ABSTRACT
Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. Its role in heterochromatin organization remained elusive. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affect replication timing, preferentially at the late-replicating regions. We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure.

No MeSH data available.