Limits...
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.


Depletion of ORCA alters the replication timing.(A) Patterns of BrdU incorporation in S phase. Examples of early (1), mid (2 and 3) and late (4 and 5) S patterns. Scale bar, 10µm. (B) Replication timing of genomic regions that show reduced H3K9me3 upon ORCA knockdown. Gray bars represent late replicating domains and black bars denote early replicating domains. (C) BrdU ChIP in S phase in control and ORCA knockdown cells. Note the changes in replication timing of CELSR3 (b) and FAM20A (c) upon loss of ORCA. C-FOS locus is used as a control region whose replication timing remains unaffected upon loss of ORCA (a). Fold enrichment in the graph represents the % input of BrdU ChIP over % input of rIgG ChIP.DOI:http://dx.doi.org/10.7554/eLife.06496.015
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4442312&req=5

fig7s1: Depletion of ORCA alters the replication timing.(A) Patterns of BrdU incorporation in S phase. Examples of early (1), mid (2 and 3) and late (4 and 5) S patterns. Scale bar, 10µm. (B) Replication timing of genomic regions that show reduced H3K9me3 upon ORCA knockdown. Gray bars represent late replicating domains and black bars denote early replicating domains. (C) BrdU ChIP in S phase in control and ORCA knockdown cells. Note the changes in replication timing of CELSR3 (b) and FAM20A (c) upon loss of ORCA. C-FOS locus is used as a control region whose replication timing remains unaffected upon loss of ORCA (a). Fold enrichment in the graph represents the % input of BrdU ChIP over % input of rIgG ChIP.DOI:http://dx.doi.org/10.7554/eLife.06496.015

Mentions: In general, chromatin at the nuclear periphery is significantly enriched with H3K9me2, whereas H3K9me3 is preferentially enriched around nucleolus (Yokochi et al., 2009). Typically, both of these regions replicate late during S-phase indicating that in general repressive histone marks-containing differentially condensed chromatin influences replication timing and chromatin positioning (Julienne et al., 2013). Therefore, we investigated whether the changes in H3K9me2 and H3K9me3 deposition in specific chromatin regions, upon ORCA depletion, also influences their replication timing. We depleted ORCA in U2OS cells and then synchronized the cells so as to analyze the spatio-temporal regulation of replication during S-phase (Figure 7A). Samples were collected at 4, 8, 12 hr post release from aphidicolin arrest with BrdU pulse-labeling prior to sample collection. This was followed by immunofluorescence to score for cells in early, mid, and late S-phase of cell cycle (Figure 7—figure supplement 1A). At 8 hr and 12 hr time points post-aphidicolin release, ORCA depletion caused dramatic reduction in cells showing late replication patterns (Figure 7B). BrdU (Bromodeoxyuridine)-PI flow cytometry results showed a significant reduction in BrdU incorporation in ORCA-depleted cells without significant changes in the early S-phase (Figure 7C). To determine if the changes in late replication pattern are a reflection of changes in the heterochromatin organization, we examined the replication timing of regions that showed a reduction in H3K9me2 and H3K9me3 upon ORCA KD. Initial analysis of the available repli-seq data set from various human cell lines in UCSC Genome Browser and ENCODE consortium revealed that the replication timing of large domains remains the same across cell lines. We, therefore, compared the H3K9me3 ChIP-seq data set to the HeLa repli-seq data set (Figure 7D). HeLa-S3 G1b and HeLa-S3 S1 are deep sequencing data sets for late G1 and early S replicating regions in HeLa-S3 cells (Hansen et al., 2010). The chromosomal regions that are replicating at these two stages are shown in black (early) and late (gray) along the length of the chromosome (Figure 7D and Figure 7—figure supplement 1B).10.7554/eLife.06496.014Figure 7.Loss of ORCA causes defects in heterochromatin organization.


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)

Depletion of ORCA alters the replication timing.(A) Patterns of BrdU incorporation in S phase. Examples of early (1), mid (2 and 3) and late (4 and 5) S patterns. Scale bar, 10µm. (B) Replication timing of genomic regions that show reduced H3K9me3 upon ORCA knockdown. Gray bars represent late replicating domains and black bars denote early replicating domains. (C) BrdU ChIP in S phase in control and ORCA knockdown cells. Note the changes in replication timing of CELSR3 (b) and FAM20A (c) upon loss of ORCA. C-FOS locus is used as a control region whose replication timing remains unaffected upon loss of ORCA (a). Fold enrichment in the graph represents the % input of BrdU ChIP over % input of rIgG ChIP.DOI:http://dx.doi.org/10.7554/eLife.06496.015
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4442312&req=5

fig7s1: Depletion of ORCA alters the replication timing.(A) Patterns of BrdU incorporation in S phase. Examples of early (1), mid (2 and 3) and late (4 and 5) S patterns. Scale bar, 10µm. (B) Replication timing of genomic regions that show reduced H3K9me3 upon ORCA knockdown. Gray bars represent late replicating domains and black bars denote early replicating domains. (C) BrdU ChIP in S phase in control and ORCA knockdown cells. Note the changes in replication timing of CELSR3 (b) and FAM20A (c) upon loss of ORCA. C-FOS locus is used as a control region whose replication timing remains unaffected upon loss of ORCA (a). Fold enrichment in the graph represents the % input of BrdU ChIP over % input of rIgG ChIP.DOI:http://dx.doi.org/10.7554/eLife.06496.015
Mentions: In general, chromatin at the nuclear periphery is significantly enriched with H3K9me2, whereas H3K9me3 is preferentially enriched around nucleolus (Yokochi et al., 2009). Typically, both of these regions replicate late during S-phase indicating that in general repressive histone marks-containing differentially condensed chromatin influences replication timing and chromatin positioning (Julienne et al., 2013). Therefore, we investigated whether the changes in H3K9me2 and H3K9me3 deposition in specific chromatin regions, upon ORCA depletion, also influences their replication timing. We depleted ORCA in U2OS cells and then synchronized the cells so as to analyze the spatio-temporal regulation of replication during S-phase (Figure 7A). Samples were collected at 4, 8, 12 hr post release from aphidicolin arrest with BrdU pulse-labeling prior to sample collection. This was followed by immunofluorescence to score for cells in early, mid, and late S-phase of cell cycle (Figure 7—figure supplement 1A). At 8 hr and 12 hr time points post-aphidicolin release, ORCA depletion caused dramatic reduction in cells showing late replication patterns (Figure 7B). BrdU (Bromodeoxyuridine)-PI flow cytometry results showed a significant reduction in BrdU incorporation in ORCA-depleted cells without significant changes in the early S-phase (Figure 7C). To determine if the changes in late replication pattern are a reflection of changes in the heterochromatin organization, we examined the replication timing of regions that showed a reduction in H3K9me2 and H3K9me3 upon ORCA KD. Initial analysis of the available repli-seq data set from various human cell lines in UCSC Genome Browser and ENCODE consortium revealed that the replication timing of large domains remains the same across cell lines. We, therefore, compared the H3K9me3 ChIP-seq data set to the HeLa repli-seq data set (Figure 7D). HeLa-S3 G1b and HeLa-S3 S1 are deep sequencing data sets for late G1 and early S replicating regions in HeLa-S3 cells (Hansen et al., 2010). The chromosomal regions that are replicating at these two stages are shown in black (early) and late (gray) along the length of the chromosome (Figure 7D and Figure 7—figure supplement 1B).10.7554/eLife.06496.014Figure 7.Loss of ORCA causes defects in heterochromatin organization.

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.