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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

Ectopic CENP-A clusters at a large domain at the subtelomeric 8q24/Myclocus in colorectal cancer cells and tumors. (A) CENP-A hotspots cluster (indicated by gray boxes, additional examples in Figure 7) at subtelomeric regions of chromosome 8 in SW480 cells, but not in normal colon cells. (B) Chromatin immunoprecipitation (ChIP)-seq profiles (tag density peaks) and input-adjusted hotspot analysis (vertical bars below each profile) demonstrate CENP-A distribution relative to input chromatin upon the 30 MB domain spanning the cytogenetic band 8q24 in SW480, HeLa, and normal colon cells (note that input tag density peaks reflect increased copy number of this region in SW480 cells, but hotspot analysis takes into account copy number variation). Base numbers above indicate the genomic location, and horizontal lines below the genome browser profile indicate position of fluorescent in situ hybridization (FISH) probes used for FISH/co-IF in (C). (C) Upper left panel: qtPCR graph demonstrating CENP-A and CENP-C enrichment at the 8q24 locus in SW480 cells (positions of PCR primers are indicated in (A), negative control (NC) primers were selected from chromosomes 1 and 11 from regions with no CENP-A peaks based on ChIP-seq data). Upper right panel contains cytological analysis of metaphase chromosome spreads stained with FISH probe for 8q24 (in green), indicating amplification of this region in SW480 cells. One of these translocated 8q24 loci (green) associates with CENP-A (red) (middle left panel) and the inner kinetochore protein CENP-C (red) (middle right panel). Combining FISH and IF in three primary colon tumors demonstrates that CENP-A (red) co-localizes with one of the amplified regions of 8q24/Myc (green) in the tumor (right bottom panel) but not in normal tissue (left bottom panel). White insets and arrowheads point to co-localization spots detected by Image J automated co-localization algorithm. Quantification is provided in Table 7. (D) Additional controls for the FISH/IF experiments in (C). First panel depicts FISH of 8q24 to metaphase spreads of normal human lymphocytes, demonstrating specific hybridization to 2 subtelomeric locations. Second panel depicts Co-IF/FISH of native centromere 8 and CENP-A on metaphase chromosome spreads in SW480 cells demonstrating that native centromere 8 still contains CENP-A and is active. Remaining panels demonstrate that CENP-A co-localizes to one of the amplified and translocated regions of 8q24/Myc in SW480 cells (right panels), but not in HeLa (left panels) cells. Quantification of this data is presented in Table 7. Merge of DAPI (blue or gray), 8q24 or Myc (green) and CENP-A (red) is indicated for each cell line at the top of each image. Automated co-localization analysis was performed using Image J; white is indicative of co-localization shown as insets.
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Fig8: Ectopic CENP-A clusters at a large domain at the subtelomeric 8q24/Myclocus in colorectal cancer cells and tumors. (A) CENP-A hotspots cluster (indicated by gray boxes, additional examples in Figure 7) at subtelomeric regions of chromosome 8 in SW480 cells, but not in normal colon cells. (B) Chromatin immunoprecipitation (ChIP)-seq profiles (tag density peaks) and input-adjusted hotspot analysis (vertical bars below each profile) demonstrate CENP-A distribution relative to input chromatin upon the 30 MB domain spanning the cytogenetic band 8q24 in SW480, HeLa, and normal colon cells (note that input tag density peaks reflect increased copy number of this region in SW480 cells, but hotspot analysis takes into account copy number variation). Base numbers above indicate the genomic location, and horizontal lines below the genome browser profile indicate position of fluorescent in situ hybridization (FISH) probes used for FISH/co-IF in (C). (C) Upper left panel: qtPCR graph demonstrating CENP-A and CENP-C enrichment at the 8q24 locus in SW480 cells (positions of PCR primers are indicated in (A), negative control (NC) primers were selected from chromosomes 1 and 11 from regions with no CENP-A peaks based on ChIP-seq data). Upper right panel contains cytological analysis of metaphase chromosome spreads stained with FISH probe for 8q24 (in green), indicating amplification of this region in SW480 cells. One of these translocated 8q24 loci (green) associates with CENP-A (red) (middle left panel) and the inner kinetochore protein CENP-C (red) (middle right panel). Combining FISH and IF in three primary colon tumors demonstrates that CENP-A (red) co-localizes with one of the amplified regions of 8q24/Myc (green) in the tumor (right bottom panel) but not in normal tissue (left bottom panel). White insets and arrowheads point to co-localization spots detected by Image J automated co-localization algorithm. Quantification is provided in Table 7. (D) Additional controls for the FISH/IF experiments in (C). First panel depicts FISH of 8q24 to metaphase spreads of normal human lymphocytes, demonstrating specific hybridization to 2 subtelomeric locations. Second panel depicts Co-IF/FISH of native centromere 8 and CENP-A on metaphase chromosome spreads in SW480 cells demonstrating that native centromere 8 still contains CENP-A and is active. Remaining panels demonstrate that CENP-A co-localizes to one of the amplified and translocated regions of 8q24/Myc in SW480 cells (right panels), but not in HeLa (left panels) cells. Quantification of this data is presented in Table 7. Merge of DAPI (blue or gray), 8q24 or Myc (green) and CENP-A (red) is indicated for each cell line at the top of each image. Automated co-localization analysis was performed using Image J; white is indicative of co-localization shown as insets.

Mentions: In the genome-wide map of all CENP-A hotspots identified in this study, we noted a qualitative clustering of CENP-A hotspots in subtelomeric and pericentromeric regions (Figure 7 shows all chromosomes, Figure 8A focuses on one example, grey boxes denote clusters). Such regions have been previously associated with chromosomal breakpoints and translocations[58]. We chose one of these domains involving the cytoband 8q24 for further analysis, as it represents one of the most frequently rearranged regions in the cancer genome of many carcinomas and hematological malignancies[59, 60]. Furthermore, this region has long been associated with tumorigenesis[61, 62], and with chromosome instability[63]. From previously published cytogenetic SKY/CGH maps, it is known that the 8q24/Myc locus is amplified and translocates to multiple chromosome partners in SW480 cells but not in normal colon cells[64].Figure 7


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)

Ectopic CENP-A clusters at a large domain at the subtelomeric 8q24/Myclocus in colorectal cancer cells and tumors. (A) CENP-A hotspots cluster (indicated by gray boxes, additional examples in Figure 7) at subtelomeric regions of chromosome 8 in SW480 cells, but not in normal colon cells. (B) Chromatin immunoprecipitation (ChIP)-seq profiles (tag density peaks) and input-adjusted hotspot analysis (vertical bars below each profile) demonstrate CENP-A distribution relative to input chromatin upon the 30 MB domain spanning the cytogenetic band 8q24 in SW480, HeLa, and normal colon cells (note that input tag density peaks reflect increased copy number of this region in SW480 cells, but hotspot analysis takes into account copy number variation). Base numbers above indicate the genomic location, and horizontal lines below the genome browser profile indicate position of fluorescent in situ hybridization (FISH) probes used for FISH/co-IF in (C). (C) Upper left panel: qtPCR graph demonstrating CENP-A and CENP-C enrichment at the 8q24 locus in SW480 cells (positions of PCR primers are indicated in (A), negative control (NC) primers were selected from chromosomes 1 and 11 from regions with no CENP-A peaks based on ChIP-seq data). Upper right panel contains cytological analysis of metaphase chromosome spreads stained with FISH probe for 8q24 (in green), indicating amplification of this region in SW480 cells. One of these translocated 8q24 loci (green) associates with CENP-A (red) (middle left panel) and the inner kinetochore protein CENP-C (red) (middle right panel). Combining FISH and IF in three primary colon tumors demonstrates that CENP-A (red) co-localizes with one of the amplified regions of 8q24/Myc (green) in the tumor (right bottom panel) but not in normal tissue (left bottom panel). White insets and arrowheads point to co-localization spots detected by Image J automated co-localization algorithm. Quantification is provided in Table 7. (D) Additional controls for the FISH/IF experiments in (C). First panel depicts FISH of 8q24 to metaphase spreads of normal human lymphocytes, demonstrating specific hybridization to 2 subtelomeric locations. Second panel depicts Co-IF/FISH of native centromere 8 and CENP-A on metaphase chromosome spreads in SW480 cells demonstrating that native centromere 8 still contains CENP-A and is active. Remaining panels demonstrate that CENP-A co-localizes to one of the amplified and translocated regions of 8q24/Myc in SW480 cells (right panels), but not in HeLa (left panels) cells. Quantification of this data is presented in Table 7. Merge of DAPI (blue or gray), 8q24 or Myc (green) and CENP-A (red) is indicated for each cell line at the top of each image. Automated co-localization analysis was performed using Image J; white is indicative of co-localization shown as insets.
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Fig8: Ectopic CENP-A clusters at a large domain at the subtelomeric 8q24/Myclocus in colorectal cancer cells and tumors. (A) CENP-A hotspots cluster (indicated by gray boxes, additional examples in Figure 7) at subtelomeric regions of chromosome 8 in SW480 cells, but not in normal colon cells. (B) Chromatin immunoprecipitation (ChIP)-seq profiles (tag density peaks) and input-adjusted hotspot analysis (vertical bars below each profile) demonstrate CENP-A distribution relative to input chromatin upon the 30 MB domain spanning the cytogenetic band 8q24 in SW480, HeLa, and normal colon cells (note that input tag density peaks reflect increased copy number of this region in SW480 cells, but hotspot analysis takes into account copy number variation). Base numbers above indicate the genomic location, and horizontal lines below the genome browser profile indicate position of fluorescent in situ hybridization (FISH) probes used for FISH/co-IF in (C). (C) Upper left panel: qtPCR graph demonstrating CENP-A and CENP-C enrichment at the 8q24 locus in SW480 cells (positions of PCR primers are indicated in (A), negative control (NC) primers were selected from chromosomes 1 and 11 from regions with no CENP-A peaks based on ChIP-seq data). Upper right panel contains cytological analysis of metaphase chromosome spreads stained with FISH probe for 8q24 (in green), indicating amplification of this region in SW480 cells. One of these translocated 8q24 loci (green) associates with CENP-A (red) (middle left panel) and the inner kinetochore protein CENP-C (red) (middle right panel). Combining FISH and IF in three primary colon tumors demonstrates that CENP-A (red) co-localizes with one of the amplified regions of 8q24/Myc (green) in the tumor (right bottom panel) but not in normal tissue (left bottom panel). White insets and arrowheads point to co-localization spots detected by Image J automated co-localization algorithm. Quantification is provided in Table 7. (D) Additional controls for the FISH/IF experiments in (C). First panel depicts FISH of 8q24 to metaphase spreads of normal human lymphocytes, demonstrating specific hybridization to 2 subtelomeric locations. Second panel depicts Co-IF/FISH of native centromere 8 and CENP-A on metaphase chromosome spreads in SW480 cells demonstrating that native centromere 8 still contains CENP-A and is active. Remaining panels demonstrate that CENP-A co-localizes to one of the amplified and translocated regions of 8q24/Myc in SW480 cells (right panels), but not in HeLa (left panels) cells. Quantification of this data is presented in Table 7. Merge of DAPI (blue or gray), 8q24 or Myc (green) and CENP-A (red) is indicated for each cell line at the top of each image. Automated co-localization analysis was performed using Image J; white is indicative of co-localization shown as insets.
Mentions: In the genome-wide map of all CENP-A hotspots identified in this study, we noted a qualitative clustering of CENP-A hotspots in subtelomeric and pericentromeric regions (Figure 7 shows all chromosomes, Figure 8A focuses on one example, grey boxes denote clusters). Such regions have been previously associated with chromosomal breakpoints and translocations[58]. We chose one of these domains involving the cytoband 8q24 for further analysis, as it represents one of the most frequently rearranged regions in the cancer genome of many carcinomas and hematological malignancies[59, 60]. Furthermore, this region has long been associated with tumorigenesis[61, 62], and with chromosome instability[63]. From previously published cytogenetic SKY/CGH maps, it is known that the 8q24/Myc locus is amplified and translocates to multiple chromosome partners in SW480 cells but not in normal colon cells[64].Figure 7

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