Limits...
CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells.

Athwal RK, Walkiewicz MP, Baek S, Fu S, Bui M, Camps J, Ried T, Sung MH, Dalal Y - Epigenetics Chromatin (2015)

Bottom Line: To investigate native CENP-A overexpression, we sought to uncover CENP-A-associated defects in human cells.A distinct class of CENP-A hotspots also accumulates at subtelomeric chromosomal locations, including at the 8q24/Myc region long-associated with genomic instability.These findings suggest that ectopic CENP-A nucleosomes could alter the state of the chromatin fiber, potentially impacting gene regulation and chromosome fragility.

View Article: PubMed Central - PubMed

Affiliation: Chromatin Structure and Epigenetics Mechanisms Unit, Center for Cancer Research, National Cancer Institute National Institutes of Health, 41 Center Drive, Bethesda, MD 20892 USA ; Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute National Institutes of Health, 41 Center Drive, Bethesda, MD 20892 USA.

ABSTRACT

Background: The histone H3 variant CENP-A is normally tightly regulated to ensure only one centromere exists per chromosome. Native CENP-A is often found overexpressed in human cancer cells and a range of human tumors. Consequently, CENP-A misregulation is thought to contribute to genome instability in human cancers. However, the consequences of such overexpression have not been directly elucidated in human cancer cells.

Results: To investigate native CENP-A overexpression, we sought to uncover CENP-A-associated defects in human cells. We confirm that CENP-A is innately overexpressed in several colorectal cancer cell lines. In such cells, we report that a subset of structurally distinct CENP-A-containing nucleosomes associate with canonical histone H3, and with the transcription-coupled chaperones ATRX and DAXX. Furthermore, such hybrid CENP-A nucleosomes localize to DNase I hypersensitive and transcription factor binding sites, including at promoters of genes across the human genome. A distinct class of CENP-A hotspots also accumulates at subtelomeric chromosomal locations, including at the 8q24/Myc region long-associated with genomic instability. We show this 8q24 accumulation of CENP-A can also be seen in early stage primary colorectal tumors.

Conclusions: Our data demonstrate that excess CENP-A accumulates at noncentromeric locations in the human cancer genome. These findings suggest that ectopic CENP-A nucleosomes could alter the state of the chromatin fiber, potentially impacting gene regulation and chromosome fragility.

No MeSH data available.


Related in: MedlinePlus

Genome-wide analysis of ectopic CENP-A demonstrates enrichment of CENP-A hotspots at DNase I hypersensitive sites. (A) Concordance analysis of chromatin immunoprecipitation (ChIP)-seq replicates of ectopic CENP-A IPs in normal colon, HeLa, and SW480 cells displays r2 > 0.9, indicative of high reproducibility. (B) Left: distribution analysis of hotspots in normal, HeLa and SW480 cells; Venn diagrams of hotspots from SW480, HeLa, or normal colon cells versus Mock immunoprecipitation (IP). Right: Genomic location of hotspots from left panel shown as a percentage of total. Legend below indicates genomic location. Fisher’s exact test was performed for comparison of HeLa and SW480 histograms: P = 0.0174 (C) Genome browser snapshots of ectopic CENP-A localization depict representative examples of CENP-A and DHS hotspots at genes overlapping with clusters of DNase I sensitivity, H3K27Acetylation and Transcription factor binding in the ENCODE data. Scale bars are in the upper left corner of each snapshot. (D) Distribution of HJURP hotspots in HeLa and SW480 cells. Venn diagram demonstrates 30 to 40% of HJURP hotspots overlap with ectopic CENP-A hotspots, but the number of HJURP hotspots in SW480 is very low.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4363203&req=5

Fig4: Genome-wide analysis of ectopic CENP-A demonstrates enrichment of CENP-A hotspots at DNase I hypersensitive sites. (A) Concordance analysis of chromatin immunoprecipitation (ChIP)-seq replicates of ectopic CENP-A IPs in normal colon, HeLa, and SW480 cells displays r2 > 0.9, indicative of high reproducibility. (B) Left: distribution analysis of hotspots in normal, HeLa and SW480 cells; Venn diagrams of hotspots from SW480, HeLa, or normal colon cells versus Mock immunoprecipitation (IP). Right: Genomic location of hotspots from left panel shown as a percentage of total. Legend below indicates genomic location. Fisher’s exact test was performed for comparison of HeLa and SW480 histograms: P = 0.0174 (C) Genome browser snapshots of ectopic CENP-A localization depict representative examples of CENP-A and DHS hotspots at genes overlapping with clusters of DNase I sensitivity, H3K27Acetylation and Transcription factor binding in the ENCODE data. Scale bars are in the upper left corner of each snapshot. (D) Distribution of HJURP hotspots in HeLa and SW480 cells. Venn diagram demonstrates 30 to 40% of HJURP hotspots overlap with ectopic CENP-A hotspots, but the number of HJURP hotspots in SW480 is very low.

Mentions: Sequencing of the mononucleosomal fraction obtained from chromatin input samples from each cell line confirmed equal and robust genomic representation in the extracts, which were comparable to other ENCODE data sets (Table 4). Reassuringly, mock IP ChIP-seq performed to rule out potential background signal identified a very small number of weak background-related hotspots (approximately 200). Furthermore, correlation analyses of replicates for normal colon, HeLa, and SW480 ectopic CENP-A ChIP-seq each demonstrated excellent concordance, with an r2 > 0.9 for each set of replicates (Figure 4A). From the pooled replicate concordant data, we next determined statistically significant, input-adjusted tags representing true ectopic CENP-A ‘hotspots’ in the genome, at a stringent false discovery rate (FDR) of 0.1%. This method yields a robust view of CENP-A occupancy after accounting for copy number variation often found in cancer genomes. Contrary to our expectation that CENP-A would be found exclusively at centromeres or heterochromatin, ectopic CENP-A hotspots localize to noncentromeric loci in normal colon, HeLa, and SW480 cells (Figure 4B, left panel). Indeed, the main difference was the number of hotspots found in each cell line, which generally corresponded to the overall level of CENP-A expression: whereas in normal colon cells, there are approximately 450 ectopic CENP-A hotspots, in HeLa cells there is a twofold increase to approximately 950, and in SW480 cells there is an almost sixfold increase over normal colon to approximately 2,850 hotspots (Figure 4B, left panel). These hotspots do not arise from background signal, as only a tiny fraction of the mock IP-hotspots correlated with any of the ectopic CENP-A hotspots above (Figure 4B, left panel).Table 4


CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells.

Athwal RK, Walkiewicz MP, Baek S, Fu S, Bui M, Camps J, Ried T, Sung MH, Dalal Y - Epigenetics Chromatin (2015)

Genome-wide analysis of ectopic CENP-A demonstrates enrichment of CENP-A hotspots at DNase I hypersensitive sites. (A) Concordance analysis of chromatin immunoprecipitation (ChIP)-seq replicates of ectopic CENP-A IPs in normal colon, HeLa, and SW480 cells displays r2 > 0.9, indicative of high reproducibility. (B) Left: distribution analysis of hotspots in normal, HeLa and SW480 cells; Venn diagrams of hotspots from SW480, HeLa, or normal colon cells versus Mock immunoprecipitation (IP). Right: Genomic location of hotspots from left panel shown as a percentage of total. Legend below indicates genomic location. Fisher’s exact test was performed for comparison of HeLa and SW480 histograms: P = 0.0174 (C) Genome browser snapshots of ectopic CENP-A localization depict representative examples of CENP-A and DHS hotspots at genes overlapping with clusters of DNase I sensitivity, H3K27Acetylation and Transcription factor binding in the ENCODE data. Scale bars are in the upper left corner of each snapshot. (D) Distribution of HJURP hotspots in HeLa and SW480 cells. Venn diagram demonstrates 30 to 40% of HJURP hotspots overlap with ectopic CENP-A hotspots, but the number of HJURP hotspots in SW480 is very low.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4363203&req=5

Fig4: Genome-wide analysis of ectopic CENP-A demonstrates enrichment of CENP-A hotspots at DNase I hypersensitive sites. (A) Concordance analysis of chromatin immunoprecipitation (ChIP)-seq replicates of ectopic CENP-A IPs in normal colon, HeLa, and SW480 cells displays r2 > 0.9, indicative of high reproducibility. (B) Left: distribution analysis of hotspots in normal, HeLa and SW480 cells; Venn diagrams of hotspots from SW480, HeLa, or normal colon cells versus Mock immunoprecipitation (IP). Right: Genomic location of hotspots from left panel shown as a percentage of total. Legend below indicates genomic location. Fisher’s exact test was performed for comparison of HeLa and SW480 histograms: P = 0.0174 (C) Genome browser snapshots of ectopic CENP-A localization depict representative examples of CENP-A and DHS hotspots at genes overlapping with clusters of DNase I sensitivity, H3K27Acetylation and Transcription factor binding in the ENCODE data. Scale bars are in the upper left corner of each snapshot. (D) Distribution of HJURP hotspots in HeLa and SW480 cells. Venn diagram demonstrates 30 to 40% of HJURP hotspots overlap with ectopic CENP-A hotspots, but the number of HJURP hotspots in SW480 is very low.
Mentions: Sequencing of the mononucleosomal fraction obtained from chromatin input samples from each cell line confirmed equal and robust genomic representation in the extracts, which were comparable to other ENCODE data sets (Table 4). Reassuringly, mock IP ChIP-seq performed to rule out potential background signal identified a very small number of weak background-related hotspots (approximately 200). Furthermore, correlation analyses of replicates for normal colon, HeLa, and SW480 ectopic CENP-A ChIP-seq each demonstrated excellent concordance, with an r2 > 0.9 for each set of replicates (Figure 4A). From the pooled replicate concordant data, we next determined statistically significant, input-adjusted tags representing true ectopic CENP-A ‘hotspots’ in the genome, at a stringent false discovery rate (FDR) of 0.1%. This method yields a robust view of CENP-A occupancy after accounting for copy number variation often found in cancer genomes. Contrary to our expectation that CENP-A would be found exclusively at centromeres or heterochromatin, ectopic CENP-A hotspots localize to noncentromeric loci in normal colon, HeLa, and SW480 cells (Figure 4B, left panel). Indeed, the main difference was the number of hotspots found in each cell line, which generally corresponded to the overall level of CENP-A expression: whereas in normal colon cells, there are approximately 450 ectopic CENP-A hotspots, in HeLa cells there is a twofold increase to approximately 950, and in SW480 cells there is an almost sixfold increase over normal colon to approximately 2,850 hotspots (Figure 4B, left panel). These hotspots do not arise from background signal, as only a tiny fraction of the mock IP-hotspots correlated with any of the ectopic CENP-A hotspots above (Figure 4B, left panel).Table 4

Bottom Line: To investigate native CENP-A overexpression, we sought to uncover CENP-A-associated defects in human cells.A distinct class of CENP-A hotspots also accumulates at subtelomeric chromosomal locations, including at the 8q24/Myc region long-associated with genomic instability.These findings suggest that ectopic CENP-A nucleosomes could alter the state of the chromatin fiber, potentially impacting gene regulation and chromosome fragility.

View Article: PubMed Central - PubMed

Affiliation: Chromatin Structure and Epigenetics Mechanisms Unit, Center for Cancer Research, National Cancer Institute National Institutes of Health, 41 Center Drive, Bethesda, MD 20892 USA ; Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute National Institutes of Health, 41 Center Drive, Bethesda, MD 20892 USA.

ABSTRACT

Background: The histone H3 variant CENP-A is normally tightly regulated to ensure only one centromere exists per chromosome. Native CENP-A is often found overexpressed in human cancer cells and a range of human tumors. Consequently, CENP-A misregulation is thought to contribute to genome instability in human cancers. However, the consequences of such overexpression have not been directly elucidated in human cancer cells.

Results: To investigate native CENP-A overexpression, we sought to uncover CENP-A-associated defects in human cells. We confirm that CENP-A is innately overexpressed in several colorectal cancer cell lines. In such cells, we report that a subset of structurally distinct CENP-A-containing nucleosomes associate with canonical histone H3, and with the transcription-coupled chaperones ATRX and DAXX. Furthermore, such hybrid CENP-A nucleosomes localize to DNase I hypersensitive and transcription factor binding sites, including at promoters of genes across the human genome. A distinct class of CENP-A hotspots also accumulates at subtelomeric chromosomal locations, including at the 8q24/Myc region long-associated with genomic instability. We show this 8q24 accumulation of CENP-A can also be seen in early stage primary colorectal tumors.

Conclusions: Our data demonstrate that excess CENP-A accumulates at noncentromeric locations in the human cancer genome. These findings suggest that ectopic CENP-A nucleosomes could alter the state of the chromatin fiber, potentially impacting gene regulation and chromosome fragility.

No MeSH data available.


Related in: MedlinePlus