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Lampbrush chromosomes enable study of cohesin dynamics

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Abstract

The lampbrush chromosomes present in the nuclei of amphibian oocytes offer unique biological approaches for study of the mechanisms that regulate chromatin structure with high spatial resolution. We discuss fundamental aspects of the remarkable organization and plasticity exhibited by lampbrush chromosomes. We then utilize lampbrush chromosomes to characterize the chromosomal distribution and dynamics of cohesin, the four-protein complex (RAD21/MCD1/SCC1, SMC1, SMC3, SCC3/SA2) responsible for sister chromatid cohesion. We find that endogenous SMC3 and newly expressed hRAD21 co-localize on chromosomal axes, sites where sister chromatids are tightly paired. We present evidence suggesting that hRAD21 recruitment to lampbrush chromosomes is modulated by chromosomal SMC1 and SMC3. Notably, using a technique for de novo chromosome assembly, we demonstrate that both SMC3 and hRAD21 are recruited to single, unreplicated lampbrush chromatids. Finally, we used our novel method of analyzing the oocyte nucleus under oil combined with fluorescence recovery after photobleaching, to provide direct evidence that cohesin is highly dynamic at discrete, condensed chromosomal regions. Collectively, these data demonstrate that lampbrush chromosomes provide a unique and powerful tool for combining biochemical and cytological analyses for dissection of complex chromosomal processes.

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Abbreviations

EGFP:

enhanced green fluorescent protein

FRAP:

fluorescence recovery after photobleaching

LBC:

lampbrush chromosome

SMC:

structural maintenance of chromosomes

RNP:

ribonucleoprotein

RNAPII:

RNA polymerase II

YFP:

yellow fluorescent protein

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Acknowledgements

We thank Dr. T. Hirano (Cold Spring Harbor Laboratory) for his generous gift of the antibody directed against the amphibian SMC3. We are greatly indebted to Dr. J.G. Gall (Carnegie Institution of Washington) for providing the amazing images of LBC loops where the distribution patterns of histone H4 and RNAPII are compared. We would also like to thank him, as well as Dr. S.B. Patel, for very insightful discussions, and Dr. B. Beenders (UIUC) for the initial anti-cohesin antibody screen. We thank Dr. P.L. Jones (UIUC) for providing Xenopus sperm heads. Finally, we would like to thank our managing editor, Dr. Christian Haering, and the two anonymous reviewers for their very insightful and constructive critique of our manuscript. This work was a supported by a CAREER award from the National Science Foundation. Vincent Guacci is supported by the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Cell & Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Christopher Austin, Natalya Novikova & Michel Bellini

  2. Embryology, Carnegie Institution of Washington, Baltimore, MD, USA

    Vincent Guacci

  3. Department of Cell and Developmental Biology, School of Molecular and Cellular Biology, 601 South Goodwin Avenue, Room B107 CLSL, Urbana, IL, 61801, USA

    Michel Bellini

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  1. Christopher Austin
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  2. Natalya Novikova
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  3. Vincent Guacci
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  4. Michel Bellini
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Correspondence to Michel Bellini.

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Responsible Editor: Dr. Christian Haering.

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Austin, C., Novikova, N., Guacci, V. et al. Lampbrush chromosomes enable study of cohesin dynamics. Chromosome Res 17, 165–184 (2009). https://doi.org/10.1007/s10577-008-9015-9

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