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New clues to understand how CENP-A maintains centromere identity.

Sánchez P, Losada A - Cell Div (2011)

Bottom Line: Much effort has been devoted to understanding the mechanisms that drive the assembly of CENP-A containing nucleosomes exclusively onto centromeric DNA, as well as the peculiar structure of these nucleosomes.The spatial and temporal specificity of CENP-A deposition observed in human cells can be recapitulated in this in vitro system, making it suitable to dissect the precise role of the different factors that contribute to this pathway.Here, we discuss our results together with other recent advances in our understanding of the mechanisms that mediate centromere inheritance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Chromosome Dynamics Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain. alosada@cnio.es.

ABSTRACT
The centromere is a specialized chromosomal region that directs the formation of the kinetochore, a huge protein assembly that acts as the attachment site for spindle microtubules and carries out chromosome movement during cell division. Centromere loss or the presence of extra centromeres adversely affect chromosome segregation and may result in aneuploidy, a condition found in many human tumors and a major cause of miscarriages and birth defects. Consequently, understanding the basis of centromere determination and propagation is of great relevance to both fundamental and clinical research. In recent years, it has become clear that centromeres are defined by the presence of a histone H3 variant known as Centromere Protein A, CENP-A, or CenH3. Much effort has been devoted to understanding the mechanisms that drive the assembly of CENP-A containing nucleosomes exclusively onto centromeric DNA, as well as the peculiar structure of these nucleosomes. We have recently developed an immunofluorescence-based assay that measures CENP-A incorporation in the centromeres of chromosomes assembled in Xenopus egg extracts. The spatial and temporal specificity of CENP-A deposition observed in human cells can be recapitulated in this in vitro system, making it suitable to dissect the precise role of the different factors that contribute to this pathway. Here, we discuss our results together with other recent advances in our understanding of the mechanisms that mediate centromere inheritance.

No MeSH data available.


Related in: MedlinePlus

An assay to measure CENP-A incorporation in Xenopus egg extracts. Step 1: Extract preparation and depletion. Extracts are prepared from laid eggs arrested in metaphase II (mitotic extracts) and subjected to immunodepletion with specific antibodies against factor X. Step 2: Nuclei assembly. Sperm chromatin is added to mitotic extracts in two different tubes. In one tube (top), incubation proceeds for 80-120 min to get mitotic chromosomes. In another tube (bottom), calcium is added to drive entry into interphase 40 min after sperm addition. Incubation proceeds for 80 min to get interphase nuclei that have undergone replication. Step 3: Nuclei isolation. Equal volumes of the reaction mixtures in both tubes are combined, fixed and centrifuged over a coverslip placed at the bottom of a glycerol cushion. Step 4: Immunofluorescence. Coverslips are processed for immunofluorescence with an antibody against CENP-A and DNA is stained with DAPI. Step 5: Image acquisition and analysis. Images of a mass of mitotic chromosomes next to an interphase nucleus are acquired and CENP-A signals are quantitated using ImageJ software (http://rsb.info.nih.gov/ij/). The average Integrated Density (ID = average pixel intensity × area) is first calculated from the IDs of individual centromeres within each interphase nucleus (IDI) and neighboring mass of mitotic chromosomes (IDM) and then a ratio between the IDs of each imaged pair is obtained (IDr). Finally, the average ID ratio (IDr) of at least 15 pairs is calculated. The relative CENP-A loading efficiency of a depleted extract with respect to a mock depleted extract (considered 100%) is calculated and represented. If depletion of a factor X prevents loading of CENP-A and the defect is rescued by adding back the factor to the depleted extract, we conclude that factor X is involved in CENP-A incorporation.
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Figure 1: An assay to measure CENP-A incorporation in Xenopus egg extracts. Step 1: Extract preparation and depletion. Extracts are prepared from laid eggs arrested in metaphase II (mitotic extracts) and subjected to immunodepletion with specific antibodies against factor X. Step 2: Nuclei assembly. Sperm chromatin is added to mitotic extracts in two different tubes. In one tube (top), incubation proceeds for 80-120 min to get mitotic chromosomes. In another tube (bottom), calcium is added to drive entry into interphase 40 min after sperm addition. Incubation proceeds for 80 min to get interphase nuclei that have undergone replication. Step 3: Nuclei isolation. Equal volumes of the reaction mixtures in both tubes are combined, fixed and centrifuged over a coverslip placed at the bottom of a glycerol cushion. Step 4: Immunofluorescence. Coverslips are processed for immunofluorescence with an antibody against CENP-A and DNA is stained with DAPI. Step 5: Image acquisition and analysis. Images of a mass of mitotic chromosomes next to an interphase nucleus are acquired and CENP-A signals are quantitated using ImageJ software (http://rsb.info.nih.gov/ij/). The average Integrated Density (ID = average pixel intensity × area) is first calculated from the IDs of individual centromeres within each interphase nucleus (IDI) and neighboring mass of mitotic chromosomes (IDM) and then a ratio between the IDs of each imaged pair is obtained (IDr). Finally, the average ID ratio (IDr) of at least 15 pairs is calculated. The relative CENP-A loading efficiency of a depleted extract with respect to a mock depleted extract (considered 100%) is calculated and represented. If depletion of a factor X prevents loading of CENP-A and the defect is rescued by adding back the factor to the depleted extract, we conclude that factor X is involved in CENP-A incorporation.

Mentions: One central question of centromere biology is how is CENP-A deposited at centromeric chromatin. Members of a protein family named Scm3/HJURP (Holliday Junction Recognizing Protein), conserved from yeast to humans, have been proposed to act as CENP-A specific chaperones in yeast and human cells [7-15]. These proteins interact physically with CENP-A, can be found at centromeres and, most importantly, are required for CENP-A loading and maintenance. Many additional factors play a role in CENP-A incorporation, but how it actually happens remains unclear. To address this issue, we developed an assay to measure CENP-A incorporation using the Xenopus egg cell-free system [16]. In this immunofluorescence-based assay, nuclei assembled from sperm chromatin and taken at two different time points (e.g. mitosis and subsequent interphase) are combined and processed for immunofluorescence with a CENP-A specific antibody, imaged together, and the centromeric CENP-A signals measured to assess the average difference in intensity between the centromeres within each pair (see Figure 1). We first showed that, in the egg extracts, CENP-A incorporation occurs upon exit from mitosis but independently of DNA replication, same as in Drosophila embryos and human cells [17-20]. Specific immunodepletion of proteins involved in the deposition of other histone H3 variants (CAF-1 and HIRA [21]), did not affect significantly the incorporation of CENP-A. In contrast, we found that CENP-A deposition depends on Xenopus HJURP (xHJURP) and that human HJURP can replace xHJURP. In fact, xHJURP is stored in the oocyte cytoplasm in association with CENP-A, which supports the idea that they form a pre-assembly complex. Analysis of the crystal structure of the CENP-A binding domain of Scm3 in complex with CENP-A and H4 reveals that the interaction of Scm3/HJURP with the CENP-A/H4 dimer is not compatible with binding to DNA further supporting that this is indeed a pre-assembly complex [22].


New clues to understand how CENP-A maintains centromere identity.

Sánchez P, Losada A - Cell Div (2011)

An assay to measure CENP-A incorporation in Xenopus egg extracts. Step 1: Extract preparation and depletion. Extracts are prepared from laid eggs arrested in metaphase II (mitotic extracts) and subjected to immunodepletion with specific antibodies against factor X. Step 2: Nuclei assembly. Sperm chromatin is added to mitotic extracts in two different tubes. In one tube (top), incubation proceeds for 80-120 min to get mitotic chromosomes. In another tube (bottom), calcium is added to drive entry into interphase 40 min after sperm addition. Incubation proceeds for 80 min to get interphase nuclei that have undergone replication. Step 3: Nuclei isolation. Equal volumes of the reaction mixtures in both tubes are combined, fixed and centrifuged over a coverslip placed at the bottom of a glycerol cushion. Step 4: Immunofluorescence. Coverslips are processed for immunofluorescence with an antibody against CENP-A and DNA is stained with DAPI. Step 5: Image acquisition and analysis. Images of a mass of mitotic chromosomes next to an interphase nucleus are acquired and CENP-A signals are quantitated using ImageJ software (http://rsb.info.nih.gov/ij/). The average Integrated Density (ID = average pixel intensity × area) is first calculated from the IDs of individual centromeres within each interphase nucleus (IDI) and neighboring mass of mitotic chromosomes (IDM) and then a ratio between the IDs of each imaged pair is obtained (IDr). Finally, the average ID ratio (IDr) of at least 15 pairs is calculated. The relative CENP-A loading efficiency of a depleted extract with respect to a mock depleted extract (considered 100%) is calculated and represented. If depletion of a factor X prevents loading of CENP-A and the defect is rescued by adding back the factor to the depleted extract, we conclude that factor X is involved in CENP-A incorporation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104478&req=5

Figure 1: An assay to measure CENP-A incorporation in Xenopus egg extracts. Step 1: Extract preparation and depletion. Extracts are prepared from laid eggs arrested in metaphase II (mitotic extracts) and subjected to immunodepletion with specific antibodies against factor X. Step 2: Nuclei assembly. Sperm chromatin is added to mitotic extracts in two different tubes. In one tube (top), incubation proceeds for 80-120 min to get mitotic chromosomes. In another tube (bottom), calcium is added to drive entry into interphase 40 min after sperm addition. Incubation proceeds for 80 min to get interphase nuclei that have undergone replication. Step 3: Nuclei isolation. Equal volumes of the reaction mixtures in both tubes are combined, fixed and centrifuged over a coverslip placed at the bottom of a glycerol cushion. Step 4: Immunofluorescence. Coverslips are processed for immunofluorescence with an antibody against CENP-A and DNA is stained with DAPI. Step 5: Image acquisition and analysis. Images of a mass of mitotic chromosomes next to an interphase nucleus are acquired and CENP-A signals are quantitated using ImageJ software (http://rsb.info.nih.gov/ij/). The average Integrated Density (ID = average pixel intensity × area) is first calculated from the IDs of individual centromeres within each interphase nucleus (IDI) and neighboring mass of mitotic chromosomes (IDM) and then a ratio between the IDs of each imaged pair is obtained (IDr). Finally, the average ID ratio (IDr) of at least 15 pairs is calculated. The relative CENP-A loading efficiency of a depleted extract with respect to a mock depleted extract (considered 100%) is calculated and represented. If depletion of a factor X prevents loading of CENP-A and the defect is rescued by adding back the factor to the depleted extract, we conclude that factor X is involved in CENP-A incorporation.
Mentions: One central question of centromere biology is how is CENP-A deposited at centromeric chromatin. Members of a protein family named Scm3/HJURP (Holliday Junction Recognizing Protein), conserved from yeast to humans, have been proposed to act as CENP-A specific chaperones in yeast and human cells [7-15]. These proteins interact physically with CENP-A, can be found at centromeres and, most importantly, are required for CENP-A loading and maintenance. Many additional factors play a role in CENP-A incorporation, but how it actually happens remains unclear. To address this issue, we developed an assay to measure CENP-A incorporation using the Xenopus egg cell-free system [16]. In this immunofluorescence-based assay, nuclei assembled from sperm chromatin and taken at two different time points (e.g. mitosis and subsequent interphase) are combined and processed for immunofluorescence with a CENP-A specific antibody, imaged together, and the centromeric CENP-A signals measured to assess the average difference in intensity between the centromeres within each pair (see Figure 1). We first showed that, in the egg extracts, CENP-A incorporation occurs upon exit from mitosis but independently of DNA replication, same as in Drosophila embryos and human cells [17-20]. Specific immunodepletion of proteins involved in the deposition of other histone H3 variants (CAF-1 and HIRA [21]), did not affect significantly the incorporation of CENP-A. In contrast, we found that CENP-A deposition depends on Xenopus HJURP (xHJURP) and that human HJURP can replace xHJURP. In fact, xHJURP is stored in the oocyte cytoplasm in association with CENP-A, which supports the idea that they form a pre-assembly complex. Analysis of the crystal structure of the CENP-A binding domain of Scm3 in complex with CENP-A and H4 reveals that the interaction of Scm3/HJURP with the CENP-A/H4 dimer is not compatible with binding to DNA further supporting that this is indeed a pre-assembly complex [22].

Bottom Line: Much effort has been devoted to understanding the mechanisms that drive the assembly of CENP-A containing nucleosomes exclusively onto centromeric DNA, as well as the peculiar structure of these nucleosomes.The spatial and temporal specificity of CENP-A deposition observed in human cells can be recapitulated in this in vitro system, making it suitable to dissect the precise role of the different factors that contribute to this pathway.Here, we discuss our results together with other recent advances in our understanding of the mechanisms that mediate centromere inheritance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Chromosome Dynamics Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain. alosada@cnio.es.

ABSTRACT
The centromere is a specialized chromosomal region that directs the formation of the kinetochore, a huge protein assembly that acts as the attachment site for spindle microtubules and carries out chromosome movement during cell division. Centromere loss or the presence of extra centromeres adversely affect chromosome segregation and may result in aneuploidy, a condition found in many human tumors and a major cause of miscarriages and birth defects. Consequently, understanding the basis of centromere determination and propagation is of great relevance to both fundamental and clinical research. In recent years, it has become clear that centromeres are defined by the presence of a histone H3 variant known as Centromere Protein A, CENP-A, or CenH3. Much effort has been devoted to understanding the mechanisms that drive the assembly of CENP-A containing nucleosomes exclusively onto centromeric DNA, as well as the peculiar structure of these nucleosomes. We have recently developed an immunofluorescence-based assay that measures CENP-A incorporation in the centromeres of chromosomes assembled in Xenopus egg extracts. The spatial and temporal specificity of CENP-A deposition observed in human cells can be recapitulated in this in vitro system, making it suitable to dissect the precise role of the different factors that contribute to this pathway. Here, we discuss our results together with other recent advances in our understanding of the mechanisms that mediate centromere inheritance.

No MeSH data available.


Related in: MedlinePlus