Methods 1999 Feb 01;172:173-87. doi: 10.1006/meth.1998.0728.

Functional analysis of chromatin assembled in synthetic nuclei.

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Numerous regulatory mechanisms contribute to the control of eukaryotic transcription. These controls are manifested through higher-order protein-DNA structure within the nucleus. In vitro assays have proven extremely useful in deciphering the potential regulatory roles of chromatin and nuclear structure in transcription. Embryonic egg extracts of Xenopus with their vast maternal stores and rapid cell-cycle oscillations can be exploited to recapitulate multiple layers of nuclear regulation. Incubation of cloned DNA templates in Xenopus egg extracts promotes a self-ordered assembly of physiologically spaced nucleosomes and synthetic nuclei structure formation. Interaction of membrane vesicles with chromatin leads to formation of a bilayer nuclear envelope encapsulating the DNA. These synthetic nuclei are functional organelles capable of active protein transport and a single round of semiconservative DNA synthesis. This system can be used to directly test the mechanisms by which trans-acting factors promote transcription on nucleosomal DNA, either during chromatin assembly or postassembly or in conjunction with remodeling machinery and/or DNA replication. The functional consequences of trans-acting factor interaction within synthetic nuclei are determined by a coupled in vitro transcription analysis. Immobilizing biotin end-labeled DNA templates on paramagnetic streptavidin beads greatly improves the flexibility of the system. The ease of chromatin-assembled template recovery allows the introduction of wash steps, buffer changes, and specific reaction optimization. These methods for reconstituting gene regulatory mechanisms in vitro are an attempt to strike a balance between biochemical accessibility and physiological relevance.

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