Reverse X—I inks PCR ofspccific DNA

Fig.5.5 ChIP analysis. Intact cells or tissue are exposed to a cross-linking chemical agent, e.g. formaldehyde, which covalently links (X) DNA and proteins within Angstroms of each other. A lysate is prepared and the chromatin/DNA is randomly sheared into fragments, generally smaller than 500 bp. The chromatin/DNA fragments can be immunoprecipitated with antibodies (Y) that recognize histones, specific post-translational modifications of histones, transcription factors, co-regulatory proteins or other chromatin-interacting proteins. Following immunoprecipitation, the cross-links are reversed by heating and DNA is extracted. The presence of a specific region of DNA within the immunoprecipitated materials is analyzed by PCR with gene-specific primers. Symbols are as follows: nucleosomes (brown, shaded barrels), DNA (black line), histone modifications: Ac (acetylated). Me (methylated), chromatin-associated proteins (green, red, orange and blue ovoid shapes) and colored arrows (PCR primers).

HAT activity are also positively affected by H3S10 phosphorylation, but inhibition of this modification by mutation of H3S10 has no direct effect on gene expression (Lo et al., 2000). However, H3S10 is needed to determine if the majority of effects ascribed to H3S10 phosphorylation are due to direct effects on transcription, its function in modulating acetylation or methylation of neighboring residues and/or its integral role in mitotic and meiotic condensation.

Chromatin and the Future

This chapter has addressed the basics of chromatin structure, how specific regulatory patterns are put into place, mechanisms of altering chromatin structure and the complex interplay of chromatin-interacting factors and regulators of transcription. We have emphasized that regulated gene expression is the outcome of an intricate choreography of transcription factors, chromatin structure alterations and modifications. The impact of chromatin as a key regulatory player is being felt across an amazing variety of endeavors in basic and clinical sciences. The study of chromatin structure and function has greatly expanded in recent years and continues to grow in excitement and implications for regulation of gene expression and numerous nuclear processes. New modifications of histone residues and their specific roles in regulation of gene expression and/or other cellular processes are being reported. The enzyme complexes that promote these modifications, the ones that erase them and their mechanisms of action continue to be identified. Perhaps the most exciting areas of chromatin research are these studies, as well as explorations of higher order chromatin structures and the basis for epigenetic/cellular memory. The study of chromatin is likely to continue as a growth industry for some time.


The authors are grateful for the contributions of R. Kori, early in development of this chapter, and to D. Wilkinson, for critical reading. R. Kelley and S. Stratton graciously provided unpublished data.

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