Characterization of chromatin domains by 3D fluorescence microscopy: An automated methodology for quantitative analysis and nuclei screening

Fluorescence microscopy has provided a route to qualitatively analyze features of nuclear structures and chromatin domains with increasing resolution. However, it is becoming increasingly important to develop tools for quantitative analysis. Here, we present an automated method to quantitatively determine the enrichment of several endogenous factors, immunostained in pericentric heterochromatin domains in mouse cells. We show that this method permits an unbiased characterization of changes in the enrichment of several factors with statistical significance from a large number of nuclei. Furthermore, the nuclei can be sorted according to the enrichment value of these factors. This method should prove useful to monitor events related to changes in the amount, rather than the presence or absence, of any factor. By adapting a few parameters, it could be extended to other nuclear structures and the benefit of using available software will permit its use in many biological labs.

[1]  Andrew J. Bannister,et al.  Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain , 2001, Nature.

[2]  Mao-Jiun J. Wang,et al.  Image thresholding by minimizing the measures of fuzzines , 1995, Pattern Recognit..

[3]  G. Karpen,et al.  Epigenetic regulation of heterochromatic DNA stability. , 2008, Current opinion in genetics & development.

[4]  F. Cordelières,et al.  A guided tour into subcellular colocalization analysis in light microscopy , 2006, Journal of microscopy.

[5]  G. Karpen,et al.  Epigenetic regulation of centromeric chromatin: old dogs, new tricks? , 2008, Nature Reviews Genetics.

[6]  B. Buendia,et al.  HP1γ associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes , 2000, Cytogenetic and Genome Research.

[7]  S. Kozubek,et al.  Differentiation-specific association of HP1α and HP1β with chromocentres is correlated with clustering of TIF1β at these sites , 2007, Histochemistry and Cell Biology.

[8]  P. Chambon,et al.  Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins. , 2001, Molecular cell.

[9]  D. Spector,et al.  Nuclear neighborhoods and gene expression. , 2009, Current opinion in genetics & development.

[10]  Max A. Viergever,et al.  Quantitative evaluation of convolution-based methods for medical image interpolation , 2001, Medical Image Anal..

[11]  C. Ponting,et al.  Regulation of chromatin structure by site-specific histone H3 methyltransferases , 2000, Nature.

[12]  A. Lamond,et al.  Toward a High-Resolution View of Nuclear Dynamics , 2007, Science.

[13]  G. Almouzni,et al.  Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells , 1998, The Journal of cell biology.

[14]  Tom Misteli,et al.  Biogenesis of nuclear bodies. , 2010, Cold Spring Harbor perspectives in biology.

[15]  Axel Imhof,et al.  PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state. , 2006, Molecular cell.

[16]  G. Almouzni,et al.  Mouse centric and pericentric satellite repeats form distinct functional heterochromatin , 2004, The Journal of cell biology.

[17]  James Allan,et al.  Formation of facultative heterochromatin in the absence of HP1 , 2003, The EMBO journal.

[18]  Nicholas Hamilton,et al.  Quantification and its Applications in Fluorescent Microscopy Imaging , 2009, Traffic.

[19]  S. Gasser,et al.  Silent chromatin at the middle and ends: lessons from yeasts , 2009, The EMBO journal.

[20]  Karl Mechtler,et al.  Loss of the Suv39h Histone Methyltransferases Impairs Mammalian Heterochromatin and Genome Stability , 2001, Cell.

[21]  R. Tsien,et al.  The Fluorescent Toolbox for Assessing Protein Location and Function , 2006, Science.

[22]  S. Kozubek,et al.  SUV39h-independent association of HP1β with fibrillarin-positive nucleolar regions , 2010, Chromosoma.

[23]  G. Almouzni,et al.  Histone chaperones, a supporting role in the limelight. , 2004, Biochimica et biophysica acta.

[24]  G. Almouzni,et al.  Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases , 2001, Nature Cell Biology.

[25]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[26]  Nathan Blow,et al.  Cell imaging: New ways to see a smaller world , 2008, Nature.

[27]  Karl Mechtler,et al.  Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins , 2001, Nature.

[28]  N. Otsu.  A threshold selection method from gray level histograms , 1979 .

[29]  C J Fuller,et al.  Image analysis benchmarking methods for high‐content screen design , 2010, Journal of microscopy.

[30]  Shyang Chang,et al.  A new criterion for automatic multilevel thresholding , 1995, IEEE Trans. Image Process..

[31]  A. Lamond,et al.  Nuclear speckles. , 2011, Cold Spring Harbor perspectives in biology.

[32]  G. Almouzni,et al.  HP1 and the dynamics of heterochromatin maintenance , 2004, Nature Reviews Molecular Cell Biology.

[33]  T. Cremer,et al.  Chromosome territories. , 2010, Cold Spring Harbor perspectives in biology.

[34]  S. Kozubek,et al.  Differentiation-specific association of HP1alpha and HP1beta with chromocentres is correlated with clustering of TIF1beta at these sites. , 2007, Histochemistry and cell biology.

[35]  En Li,et al.  Suv 39 h-Mediated Histone H 3 Lysine 9 Methylation Directs DNA Methylation to Major Satellite Repeats at Pericentric Heterochromatin , 2003 .

[36]  H. Burkhardt,et al.  Spatial quantitative analysis of fluorescently labeled nuclear structures: Problems, methods, pitfalls , 2008, Chromosome Research.

[37]  P. Fraser,et al.  Nuclear organization of the genome and the potential for gene regulation , 2007, Nature.