Functional nuclei and mitotic spindles are shown to assemble around DNA-coated beads incubated in Xenopus egg extracts. Bipolar spindles assemble in the absence of centrosomes and kinetochores, indicating that bipolarity is an intrinsic property of microtubules assembling around chromatin in a mitotic cytoplasm. Microtubules nucleated at dispersed sites with random polarity rearrange into two arrays of uniform polarity. Spindle-pole formation requires cytoplasmic dynein-dependent translocation of microtubules across one another. It is proposed that spindles form in the absence of centrosomes by motor-dependent sorting of microtubules according to their polarity.

Chromatin structure must be flexible to allow the binding of regulatory proteins and to accommodate different levels of gene activity. Chromatin assembled in a cell‐free system derived from Drosophila embryos contains an activity that hydrolyses ATP to render entire nucleosome arrays mobile. Nucleosome movements, most likely their sliding, occurred even in the presence of the linker histone H1. The dynamic state of chromatin in the presence of the activity and ATP globally increased the accessibility of nucleosomal DNA to incoming proteins. This unprecedented demonstration of energy‐dependent nucleosome mobility identifies a new principle which is likely to be fundamental to the mechanism of chromatin remodelling and the binding of regulatory proteins.

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Eukaryotic ribosomal gene promoters are preceded by a terminator element which is recognized by the transcription termination factor TTF-I. We have studied the function of this promoter-proximal terminator and show that binding of TTF-I is the key event which leads to ATP-dependent nucleosome remodeling and transcriptional activation of mouse rDNA pre-assembled into chromatin. We have analyzed TTF-I mutants for their ability to bind to free or nucleosomal DNA, and show that the DNA binding domain of TTF-I on its own is not sufficient for interaction with chromatin, indicating that specific protein features exist that endow a transcription factor with chromatin binding and remodeling properties. This first analysis of RNA polymerase I transcription in chromatin provides a clue for the function of the upstream terminator and establishes a dual role for TTF-I both as a termination factor and a chromatin-specific transcription activator.

Chromatin reconstituted in an extract from preblastoderm Drosophila embryos represses transcription by RNA polymerase II. We have assembled regularly spaced nucleosomes on DNA attached to paramagnetic beads enabling the efficient purification of chromatin templates for transcription studies. We have used diagnostic salt extractions to establish that transcriptional repression of immobilized chromatin was largely due to nucleosome cores. When purified H1 was incorporated into chromatin, resulting in increased repeat lengths to 200‐220 bp, the contribution of H1 to transcriptional repression was negligible. If more H1 was added no regularly spaced chromatin was obtained and only under these conditions was transcriptional inhibition by H1 apparent. We conclude that efficient repression of transcription by polymerase II in this system does not require the presence of histone H1.

HMG-D is an abundant chromosomal protein associated with condensed chromatin during the first nuclear cleavage cycles of the developing Drosophila embryo. We previously suggested that HMG-D might substitute for the linker histone H1 in the preblastoderm embryo and that this substitution might result in the characteristic less compacted chromatin. We have now studied the association of HMG-D with chromatin using a cellfree system for chromatin reconstitution derived from The vertebrate high mobility group proteins of the HMGB class (formerly termed HMG1/2) (1) are relatively abundant nuclear DNA-binding proteins that bend DNA substantially and appear to act primarily as architectural facilitators in the assembly of nucleoprotein complexes (recently reviewed in Refs. 2-4). There is direct evidence that these proteins facilitate the binding of transcription factors to their cognate sites both in vitro and in vivo, but any involvement in the assembly of histone-DNA complexes is less well documented. Circumstantial evidence indicates that certain functions of the vertebrate HMGB proteins (5-7) may parallel those of the linker histone H1 (8). Both have been shown to bind to linker DNA sequences (7, 9, 10), and both stabilize and bind to four-way junctions (11)(12)(13)(14). HMG-D is one of two Drosophila proteins closely related to the vertebrate HMGB proteins but in contrast to these proteins contains only a single HMG DNA-binding domain (6,15,16). The HMG domain is followed by a region that has basic sequences similar to the C-terminal domain of histone H1 and a short C-terminal acidic stretch also seen in HMGB proteins. The NMR structure of the HMG domain from HMG-D is very similar to the previously determined structure of the B domain of HMG1, which revealed the characteristic L-shaped fold formed by three ␣-helices (17-21).High mobility group proteins of the HMGB family bind DNA with little sequence specificity but recognize structural features in DNA (22-24), including cruciforms, kinks, DNA bulges, and bends (22,23,(25)(26)(27)(28). DNA footprint analysis and mutagenesis experiments suggest that HMG domain proteins bind in the minor groove (29). In addition, both crystallization and NMR studies show that HMG-D bending is achieved by the intercalation of hydrophobic residues at two different base steps (21, 30).The addition of histone H1 protein to an extended array of nucleosome core particles promotes its folding into a fiber with an approximate width of 30 nm (31-33). During early Drosophila development, histone H1 is not detectable (34 -36) until nuclear cycles 7/8 (37). Therefore, H1 is not involved in chromatin condensation in these earliest phases of Drosophila development characterized by rapid condensation-decondensation cycles. We have previously suggested that HMG-D could function as a linker protein in the absence of histone H1 in Drosophila (37). In support of this hypothesis, Wolffe and colleagues (10, 38) have postulated a similar role for the Xenopus HMG B1 protein. Thus, as the maternal pool of H...

To generate long arrays of nucleosomes within a topologically defined chromatin domain we have assembled minichromosomes on negatively supercoiled plasmid DNA with extracts from Drosophila preblastoderm embryos. These minichromosomes are dynamic substrates for energy-dependent nucleosome remodeling machines that facilitate the binding of various transcription factors but do not exhibit nucleosome positioning. In contrast, if such minichromosomes include the mouse mammary tumour virus (MMTV) promoter we find it wrapped around a nucleosome with similar translational and rotational position as in vivo . This structure precluded binding of NF1 to its cognate site at -75/-65 at salt concentrations between 60 and 120 mM, even in the presence of ATP, which rendered the NF1 site accessible to the restriction enzyme Hin fI. However, insertion of 30 bp just upstream of the NF1 site, which moves the site to the linker DNA, allowed ATP-dependent binding of NF1 to a fraction of the minichromosomes, even in the presence ofstoichiometric amounts of histone H1. The minichromosomes assembled in the Drosophila embryo extract reproduce important features of the native MMTV promoter chromatin and reveal differences in the ability of transcription factors and restriction enzymes to access their binding sites in positioned nucleosomes.

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