The presence of nucleosomes on a DNA template prevents initiation by RNA polymerase II in vitro

RNA was synthesized in vitro using HeLa cell nuclear extracts and circular DNA templates onto which varying numbers of nucleosomes had been reconstituted with Xenopus oocyte extracts. We found that fully reconstituted templates supported no specific initiation by RNA polymerase II; however, DNA exposed to the reconstitution extracts under conditions which did not allow nucleosome deposition was transcribed normally. A set of successively less reconstituted templates was also transcribed. No initiation occurred on reconstitutes with more than two-thirds of the physiological nucleosome density; reconstitutes with less than one-third of the physiological nucleosome density were transcribed as efficiently as naked DNA.     

Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate

Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha- amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205- 217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.