A novel centromere monospecific serum to a human autoepitope on the histone H3‐like protein CENP‐A

Valdivia, Manuel M.; Figueroa, Javier; Iglesias, Concepción; Ortiz, Manuela

doi:10.1016/s0014-5793(97)01583-4

Cited by 32 publications

(30 citation statements)

References 30 publications

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“…The following antibodies were used: mouse anti-CENP-A (AN1); rabbit anti-CENP-A (Valdivia et al, 1998); rabbit anti-CENP-C (R554) and rabbit anti-H3K9ac (R607). Fluorophoreconjugated secondary antibodies were purchased from Jackson Labs.…”

Section: Generation Of Tetr Fusion Constructsmentioning

confidence: 99%

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

Bergmann

Jakubsche

Martins

et al. 2012

Journal of Cell Science

103

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SummaryHuman kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated 'centrochromatin', despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-kB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ,10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ,150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity -kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.

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Section: Generation Of Tetr Fusion Constructsmentioning

confidence: 99%

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

Bergmann

Jakubsche

Martins

et al. 2012

Journal of Cell Science

103

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“…Immunostaining and cytological analyses were performed as described in (Nakano et al, 2008) except that anti-CENP-A was as described in (Valdivia et al, 1998). A DeltaVision (Applied Precision, Issaquah, WA) system based on an Olympus IX-70 microscope stand (Melville, NY) coupled to a CH350 CCD camera (Photometrics, Tucson, AZ) and controlled through SoftWorx (Applied Precision) was used for image acquisition of transient transfection experiments.…”

Section: Immunostaining and Cytological Analysismentioning

confidence: 99%

Hierarchical Inactivation of a Synthetic Human Kinetochore by a Chromatin Modifier

Cardinale

Bergmann

Kelly

et al. 2009

MBoC

114

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We previously used a human artificial chromosome (HAC) with a synthetic kinetochore that could be targeted with chromatin modifiers fused to tetracycline repressor to show that targeting of the transcriptional repressor tTS within kinetochore chromatin disrupts kinetochore structure and function. Here we show that the transcriptional corepressor KAP1, a downstream effector of the tTS, can also inactivate the kinetochore. The disruption of kinetochore structure by KAP1 subdomains does not simply result from loss of centromeric CENP-A nucleosomes. Instead it reflects a hierarchical disruption of the outer kinetochore, with CENP-C levels falling before CENP-A levels and, in certain instances, CENP-H being lost more readily than CENP-C. These results suggest that this novel approach to kinetochore dissection may reveal new patterns of protein interactions within the kinetochore. INTRODUCTIONThe centromere/kinetochore is one of the most complex cellular substructures, with more than 80 protein components described to date (reviewed in Carroll and Straight, 2006;Cheeseman and Desai, 2008;Fukagawa, 2008;Vagnarelli et al., 2008). These components perform the complex job of attaching chromosomes to the mitotic spindle; ensuring that those attachments are correct; signaling to delay mitotic progression if they are not, and regulating the movements of the chromosomes toward the spindle poles in anaphase.The kinetochore is assembled at a unique locus on each natural chromosome. However, for organisms with regional centromeres (Pluta et al., 1995), this reflects only a preference¤, and not an absolute requirement for particular DNA sequences. Kinetochores can form on a wide range of singlecopy and repeated DNA sequences, leading to the conclusion that the ultimate determinants of kinetochore assembly are epigenetic. The long-term purpose of our studies is to determine the epigenetic "landscape" that promotes kinetochore assembly and its maintenance during cell divisions.Experiments including yeast genetics, RNA interference (RNAi) studies in mammalian cells, and gene knockout analysis in mouse and chicken DT40 cells have revealed that kinetochores assemble on a foundation of specialized chromatin containing the kinetochore-specific histone H3 variant CENP-A (Earnshaw and Rothfield, 1985). CENP-A is upstream of almost all other known components in the kinetochore assembly pathway. However, that pathway is multiplex, as recent studies in chicken and Drosophila show that inner kinetochore proteins CENP-H and -C are required for normal CENP-A loading or retention (Okada et al., 2006;Goshima et al., 2007;Erhardt et al., 2008).Our work was inspired by an approach first developed a number of years ago in which cloned fragments of human centromeric DNA were used to form human artificial chromosomes (HACs) in HT1080 fibrosarcoma cells (Harrington et al., 1997;Ikeno et al., 1998). Originally, HAC formation was only achieved with regular repeated arrays of ␣-satellite DNA containing CENP-B boxes Ohzeki et al., 2002;Okada et al., 2007)....

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“…Centromeres were detected with the human autoimmune anticentromere (ACA) serum EK at a 1:100 dilution. For detecting CENP-A we used the rabbit polyclonal serum SS2 raised against a synthetic peptide covering amino acid residues 3-17 of human CENP-A (Valdivia et al, 1998) at a 1:100 dilution. To detect CENP-E we used pAb1.6, a rabbit polyclonal serum that recognises the neck region (amino acids 256-817) of CENP-E (Lombillo et al, 1995;Parra et al, 2002), at a 1:400 dilution.…”

Section: Antibodiesmentioning

confidence: 99%

Involvement of the cohesin Rad21 and SCP3 in monopolar attachment of sister kinetochores during mouse meiosis I

Parra

Viera

Gómez

et al. 2004

Journal of Cell Science

152

179

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SCP3 is a meiosis-specific structural protein appearing at axial elements and lateral elements of the synaptonemal complex. We have analysed the behaviour of SCP3 and the cohesin subunit Rad21 in mouse spermatocytes by means of a squashing technique. Our results demonstrate that both proteins colocalize and are partially released from chromosome arms during late prophase I stages, although they persist at the interchromatid domain of metaphase I bivalents. Thus, Rad21 cannot be considered a `mitotic'-specific variant, but coexists with Rec8. During late prophase I SCP3 and Rad21 accumulate at centromeres, and together with the chromosomal passenger proteins INCENP and aurora-B kinase, show a complex `double cornet'-like distribution at the inner domain of metaphase I centromeres beneath the associated sister kinetochores. We have observed that Rad21 and SCP3 are displaced from centromeres during telophase I when sister kinetochores separate, and are not present at metaphase II centromeres. Thus, we hypothesise that Rad21, and the superimposed SCP3 and SCP2, are involved in the monopolar attachment of sister kinetochores during meiosis I, and are not responsible for the maintenance of sister-chromatid centromere cohesion during meiosis II as previously suggested.

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A novel centromere monospecific serum to a human autoepitope on the histone H3‐like protein CENP‐A

Cited by 32 publications

References 30 publications

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

Hierarchical Inactivation of a Synthetic Human Kinetochore by a Chromatin Modifier

Involvement of the cohesin Rad21 and SCP3 in monopolar attachment of sister kinetochores during mouse meiosis I

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