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Perinuclear distribution of heterochromatin in developing C. elegans embryos

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Abstract

Specific nuclear domains are nonrandomly positioned within the nuclear space, and this preferential positioning has been shown to play an important role in genome activity and stability. Well-known examples include the organization of repetitive DNA in telomere clusters or in the chromocenter of Drosophila and mammalian cells, which may provide a means to control the availability of general repressors, such as the heterochromatin protein 1 (HP1). We have specifically characterized the intranuclear positioning of in vivo fluorescence of the Caenorhabditis elegans HP1 homologue HPL-2 as a marker for heterochromatin domains in developing embryos. For this purpose, the wavelet transform modulus maxima (WTMM) segmentation method was generalized and adapted to segment the small embryonic cell nuclei in three dimensions. The implementation of a radial distribution algorithm revealed a preferential perinuclear positioning of HPL-2 fluorescence in wild-type embryos compared with the diffuse and homogeneous nuclear fluorescence observed in the lin-13 mutants. For all other genotypes analyzed, the quantitative analysis highlighted various degrees of preferential HPL-2 positioning at the nuclear periphery, which directly correlates with the number of HPL-2 foci previously counted on 2D projections. Using a probabilistic 3D cell nuclear model, we found that any two nuclei having the same number of foci, but with a different 3D probabilistic positioning scheme, can have significantly different counts in the 2D maximum projection, thus showing the deceptive limitations of using techniques of 2D maximum projection foci counts. By this approach, a strong perinuclear positioning of HPL-2 foci was brought into light upon inactivation of conserved chromatin-associated proteins, including the HAT cofactor TRAPP.

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Abbreviations

2D:

Two-dimensional

3D:

Three-dimensional

DNA:

Deoxyribonucleic acid

GFP:

Green fluorescent protein

HAT:

Histone acetyl transferase

HDAC:

Histone deactetylase complex

HDA:

Histone deacetylase

HP1:

Heterochromatin protein 1

HPL-2:

Heterochromatin protein-like 2

kb:

Kilobase

Mi-2/NuRD:

NUcleosome remodeling and histone Deacetylase

Muv:

Multivulval

μm:

Micrometer

nm:

Nanometer

PI3K:

Phosphatidyl-inositol-3-kinase

Rb:

Retinoblastoma

RNA:

RiboNucleic acid

RNAi:

RNA interference

SIR:

Silencing information regulator

synMuv:

Synthetic multivulval

TIP60/NuA4:

histone acetyltransferase complex

TRRAP:

Transformation/transcription domain-associated protein

TSA:

Trichostatin A

WT:

Wavelet transform

WTMM:

Wavelet transform modulus maxima

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Acknowledgments

A.K. acknowledges the University of Maine new faculty startup funds as well as a grant from the Maine Cancer Foundation. C.V. is supported through a NSF-funded Research Experiment for Undergraduate at the University of Maine, NSF Fund CCF #0754951. The microscopy acquisition was realized on the Microscope Facility of IFR128 Biosciences Gerland-Lyon Sud, PLATIM. K.M. benefited from a fellowship from La Ligue Contre le Cancer.

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Author notes

  1. Vincent Coustham

    Present address: John Innes Centre, Colney, Norwich, NR4 7UH, UK

Authors and Affiliations

  1. Department of Mathematics and Statistics, University of Maine, Orono, ME, 04469, USA

    Jeremy Grant & Andre Khalil

  2. Graduate School of Biomedical Sciences, University of Maine, Orono, ME, 04469, USA

    Jeremy Grant & Andre Khalil

  3. Department of Electrical and Computer Engineering, University of Maine, Orono, ME, 04469, USA

    Craig Verrill

  4. Université de Lyon, F69007, Lyon, France

    Vincent Coustham, Alain Arneodo, Francesca Palladino & Karine Monier

  5. Laboratoire de Biologie Moléculaire de la Cellule, École Normale Supérieure de Lyon, Lyon, France

    Vincent Coustham & Francesca Palladino

  6. Laboratoire Joliot-Curie, École Normale Supérieure de Lyon, Lyon, France

    Alain Arneodo & Karine Monier

  7. Laboratoire de Physique, École Normale Supérieure de Lyon, Lyon, France

    Alain Arneodo

  8. Department of Physics and Astronomy, University of Maine, Orono, ME, 04469, USA

    Andre Khalil

  9. Institute for Molecular Biophysics, University of Maine, Orono, ME, 04469, USA

    Andre Khalil

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  1. Jeremy Grant
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Correspondence to Andre Khalil.

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Responsible Editor: Mary Delany

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Fig. S1

Three-dimensional sample animations showing how the distribution of simulated foci are positioned as a function of the radial probability parameter q. q has a value of 0 (peripheral positioning) (GIF 3763 kb)

Fig. S2

Three-dimensional sample animations showing how the distribution of simulated foci are positioned as a function of the radial probability parameter q. q has an intermediate value of 0.25 (GIF 4254 kb)

Fig. S3

Three-dimensional sample animations showing how the distribution of simulated foci are positioned as a function of the radial probability parameter q. q has an intermediate value of 0.50 (GIF 4795 kb)

Fig. S4

Three-dimensional sample animations showing how the distribution of simulated foci are positioned as a function of the radial probability parameter q. q has an intermediate value of 0.75 (GIF 3754 kb)

Fig. S5

Three-dimensional sample animations showing how the distribution of simulated foci are positioned as a function of the radial probability parameter q. q has a value of 1.0 (uniform random distribution) (GIF 3735 kb)

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Grant, J., Verrill, C., Coustham, V. et al. Perinuclear distribution of heterochromatin in developing C. elegans embryos. Chromosome Res 18, 873–885 (2010). https://doi.org/10.1007/s10577-010-9175-2

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