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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

CENP-A is overexpressed in colon cancer SW480 cells, and associates with H3, ATRX, and DAXX. (A) Upper panel: Western blot (WB) analysis of total nuclear CENP-A, HJURP, ATRX, and DAXX, relative to core histone H4 across cell lines. Lower panel: quantification of CENP-A protein expression standardized to normal colon (replicate data from Table 1), error bars = SEM. (B) Upper panel: semi-quantitative PCR analysis of CENP-A mRNA expression in normal and SW480 cells in three replicates. Lower panel: quantification of the CENP-A mRNA expression normalized to β-actin from four experiments, error bars = SEM. (C) Scheme depicting strategy to separate centromeric from ectopic CENP-A nucleosomes and subsequent experiments performed with each fraction. (D) Micrococcal nuclease (MNase) ladders of input chromatin used subsequently in the primary CENP-B chromatin immunoprecipitation (ChIP) for normal, HeLa and SW480 cell lines demonstrates that mostly mono-, di-, and tri-nucleosomal arrays are present in the input chromatin. Slight variations in digestibility derive from intrinsic variability in the different cell lines’ biology. (E) WB slice panels show CENP-B, CENP-A, H2A.Z, and H3 protein analysis from CENP-B IP and sequential CENP-A IP from normal, HeLa, and SW480 cells. Mock immunoprecipitation (IP) indicates control immunoprecipitation with nonspecific IgG. Recombinant CENP-A (Rec. CpA) was used as a detection specificity control. Gray CENP-A arrow in third row indicates a band that was already present prior to H2A.Z probing. Data quantification is provided in Table 2. (F) WB for ATRX and DAXX in normal, HeLa, and SW480 CENP-B IPs versus ectopic CENP-A IPs (data summarized in Table 2).
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Fig1: CENP-A is overexpressed in colon cancer SW480 cells, and associates with H3, ATRX, and DAXX. (A) Upper panel: Western blot (WB) analysis of total nuclear CENP-A, HJURP, ATRX, and DAXX, relative to core histone H4 across cell lines. Lower panel: quantification of CENP-A protein expression standardized to normal colon (replicate data from Table 1), error bars = SEM. (B) Upper panel: semi-quantitative PCR analysis of CENP-A mRNA expression in normal and SW480 cells in three replicates. Lower panel: quantification of the CENP-A mRNA expression normalized to β-actin from four experiments, error bars = SEM. (C) Scheme depicting strategy to separate centromeric from ectopic CENP-A nucleosomes and subsequent experiments performed with each fraction. (D) Micrococcal nuclease (MNase) ladders of input chromatin used subsequently in the primary CENP-B chromatin immunoprecipitation (ChIP) for normal, HeLa and SW480 cell lines demonstrates that mostly mono-, di-, and tri-nucleosomal arrays are present in the input chromatin. Slight variations in digestibility derive from intrinsic variability in the different cell lines’ biology. (E) WB slice panels show CENP-B, CENP-A, H2A.Z, and H3 protein analysis from CENP-B IP and sequential CENP-A IP from normal, HeLa, and SW480 cells. Mock immunoprecipitation (IP) indicates control immunoprecipitation with nonspecific IgG. Recombinant CENP-A (Rec. CpA) was used as a detection specificity control. Gray CENP-A arrow in third row indicates a band that was already present prior to H2A.Z probing. Data quantification is provided in Table 2. (F) WB for ATRX and DAXX in normal, HeLa, and SW480 CENP-B IPs versus ectopic CENP-A IPs (data summarized in Table 2).

Mentions: Early reports of innate overexpression of CENP-A in colorectal tumors date back well over a decade[10]. Thus, we focused on well-characterized colorectal cancer cell lines derived from different stages of tumor progression, such as SW480, HT29, DLD-1, and HCT116, comparing them to normal colon cells. We also included HeLa cells, since they have long been used as a model for human centromere biology[28, 29]. We first examined total nuclear CENP-A protein across all the cell lines, using a sensitive fluorescence-based quantitative western blotting system (Figure 1A). Relative to normal colon cells, and standardized against internal amounts of the core histone H4, we observed CENP-A protein levels were slightly elevated in HeLa cells, lower in DLD-1, 1.35 fold overexpressed in HT29 and almost twofold overexpressed in the cell line SW480 (Figure 1A lower graph and Table 1 lists fold-values of all proteins tested in Figure 1A). To test whether the excess CENP-A protein in SW480 derived from excess mRNA, we next examined total CENP-A mRNA levels. Indeed, semi-quantitative PCR analysis indicated that CENP-A mRNA is almost fourfold overexpressed in SW480 cells compared to normal colon cells (Figure 1B, lower panel depicts graphical representation of 4 replicates). We next examined levels of the CENP-A chaperone Holliday junction recognition protein (HJURP), which is required for accurate loading of CENP-A to centromeres[30–32]. Surprisingly, HJURP levels do not follow those of CENP-A; the cell line possessing the most CENP-A (SW480) has normal amounts of HJURP (Table 1). This finding intrigued us because, under normal conditions, HJURP restricts CENP-A loading to centromeric nucleosomes. We wondered whether histone H3 variant chaperones were also misregulated. We assessed transcription-coupled histone chaperones ATRX and DAXX, and observed that both are overexpressed in most cancer cell lines relative to normal colon, ranging from three- to twentyfold excess protein (Figure 1A, Table 1). Thus, these data demonstrate that CENP-A gene expression is innately misregulated in some colorectal cancer cells. To examine the consequences of variable amounts of this key histone variant, we chose to focus the rest of the study on three cell lines, spanning normal (normal colon), moderate (HeLa), and high (SW480) levels of CENP-A protein.Figure 1


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)

CENP-A is overexpressed in colon cancer SW480 cells, and associates with H3, ATRX, and DAXX. (A) Upper panel: Western blot (WB) analysis of total nuclear CENP-A, HJURP, ATRX, and DAXX, relative to core histone H4 across cell lines. Lower panel: quantification of CENP-A protein expression standardized to normal colon (replicate data from Table 1), error bars = SEM. (B) Upper panel: semi-quantitative PCR analysis of CENP-A mRNA expression in normal and SW480 cells in three replicates. Lower panel: quantification of the CENP-A mRNA expression normalized to β-actin from four experiments, error bars = SEM. (C) Scheme depicting strategy to separate centromeric from ectopic CENP-A nucleosomes and subsequent experiments performed with each fraction. (D) Micrococcal nuclease (MNase) ladders of input chromatin used subsequently in the primary CENP-B chromatin immunoprecipitation (ChIP) for normal, HeLa and SW480 cell lines demonstrates that mostly mono-, di-, and tri-nucleosomal arrays are present in the input chromatin. Slight variations in digestibility derive from intrinsic variability in the different cell lines’ biology. (E) WB slice panels show CENP-B, CENP-A, H2A.Z, and H3 protein analysis from CENP-B IP and sequential CENP-A IP from normal, HeLa, and SW480 cells. Mock immunoprecipitation (IP) indicates control immunoprecipitation with nonspecific IgG. Recombinant CENP-A (Rec. CpA) was used as a detection specificity control. Gray CENP-A arrow in third row indicates a band that was already present prior to H2A.Z probing. Data quantification is provided in Table 2. (F) WB for ATRX and DAXX in normal, HeLa, and SW480 CENP-B IPs versus ectopic CENP-A IPs (data summarized in Table 2).
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Related In: Results  -  Collection

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Show All Figures
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Fig1: CENP-A is overexpressed in colon cancer SW480 cells, and associates with H3, ATRX, and DAXX. (A) Upper panel: Western blot (WB) analysis of total nuclear CENP-A, HJURP, ATRX, and DAXX, relative to core histone H4 across cell lines. Lower panel: quantification of CENP-A protein expression standardized to normal colon (replicate data from Table 1), error bars = SEM. (B) Upper panel: semi-quantitative PCR analysis of CENP-A mRNA expression in normal and SW480 cells in three replicates. Lower panel: quantification of the CENP-A mRNA expression normalized to β-actin from four experiments, error bars = SEM. (C) Scheme depicting strategy to separate centromeric from ectopic CENP-A nucleosomes and subsequent experiments performed with each fraction. (D) Micrococcal nuclease (MNase) ladders of input chromatin used subsequently in the primary CENP-B chromatin immunoprecipitation (ChIP) for normal, HeLa and SW480 cell lines demonstrates that mostly mono-, di-, and tri-nucleosomal arrays are present in the input chromatin. Slight variations in digestibility derive from intrinsic variability in the different cell lines’ biology. (E) WB slice panels show CENP-B, CENP-A, H2A.Z, and H3 protein analysis from CENP-B IP and sequential CENP-A IP from normal, HeLa, and SW480 cells. Mock immunoprecipitation (IP) indicates control immunoprecipitation with nonspecific IgG. Recombinant CENP-A (Rec. CpA) was used as a detection specificity control. Gray CENP-A arrow in third row indicates a band that was already present prior to H2A.Z probing. Data quantification is provided in Table 2. (F) WB for ATRX and DAXX in normal, HeLa, and SW480 CENP-B IPs versus ectopic CENP-A IPs (data summarized in Table 2).
Mentions: Early reports of innate overexpression of CENP-A in colorectal tumors date back well over a decade[10]. Thus, we focused on well-characterized colorectal cancer cell lines derived from different stages of tumor progression, such as SW480, HT29, DLD-1, and HCT116, comparing them to normal colon cells. We also included HeLa cells, since they have long been used as a model for human centromere biology[28, 29]. We first examined total nuclear CENP-A protein across all the cell lines, using a sensitive fluorescence-based quantitative western blotting system (Figure 1A). Relative to normal colon cells, and standardized against internal amounts of the core histone H4, we observed CENP-A protein levels were slightly elevated in HeLa cells, lower in DLD-1, 1.35 fold overexpressed in HT29 and almost twofold overexpressed in the cell line SW480 (Figure 1A lower graph and Table 1 lists fold-values of all proteins tested in Figure 1A). To test whether the excess CENP-A protein in SW480 derived from excess mRNA, we next examined total CENP-A mRNA levels. Indeed, semi-quantitative PCR analysis indicated that CENP-A mRNA is almost fourfold overexpressed in SW480 cells compared to normal colon cells (Figure 1B, lower panel depicts graphical representation of 4 replicates). We next examined levels of the CENP-A chaperone Holliday junction recognition protein (HJURP), which is required for accurate loading of CENP-A to centromeres[30–32]. Surprisingly, HJURP levels do not follow those of CENP-A; the cell line possessing the most CENP-A (SW480) has normal amounts of HJURP (Table 1). This finding intrigued us because, under normal conditions, HJURP restricts CENP-A loading to centromeric nucleosomes. We wondered whether histone H3 variant chaperones were also misregulated. We assessed transcription-coupled histone chaperones ATRX and DAXX, and observed that both are overexpressed in most cancer cell lines relative to normal colon, ranging from three- to twentyfold excess protein (Figure 1A, Table 1). Thus, these data demonstrate that CENP-A gene expression is innately misregulated in some colorectal cancer cells. To examine the consequences of variable amounts of this key histone variant, we chose to focus the rest of the study on three cell lines, spanning normal (normal colon), moderate (HeLa), and high (SW480) levels of CENP-A protein.Figure 1

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