Abraham Worcel

Chromatin assembly in Xenopus oocytes: In vitro studies

We describe and characterize a complex reaction that catalyzes DNA supercoiling and chromatin assembly in vitro. A Xenopus oocyte extract supplemented with ATP and Mg++ converts DNA circles into minichromosomes that display a native, 200 bp periodicity. When supercoiled DNA is added to this extract it undergoes a time-dependent series of topological changes, which precisely mimic those found when the DNA is microinjected into oocytes. As judged by the conformation of the subsequently deproteinized DNA, the supercoiled DNA is first relaxed, in a reaction that takes 4 min, and then it is resupercoiled in a slower process that takes 4 hr. The relaxation is partially inhibited by EDTA, to an extent that suggests that that it is catalyzed by a type I DNA topoisomerase. The resupercoiling , on the other hand, requires ATP and Mg++, is completely inhibited by EDTA, and is inhibited by novobiocin in a manner that suggests it is catalyzed by a type II DNA topoisomerase. These findings, and the ones reported in the preceding paper ( Ryoji and Worcel , 1984), lead us to propose that chromatin assembly is an active, ATP-driven process.

The positive transcription factor of the 5S RNA gene induces a 5S DNA-specific gyration in xenopus oocyte extracts

We report that TFIIIA, the positive transcription factor of the 5S RNA gene, induces DNA gyration in Xenopus oocyte extracts. The reaction uses one molecule of TFIIIA per molecule of DNA and is highly specific for 5S DNA plasmids. DNA gyration also requires the oocyte supernatant, ATP, and Mg2+, and is inhibited by novobiocin, suggesting that it is catalyzed by a type II DNA topoisomerase. The chromatin assembled with TFIIIA is dynamic and rapidly relaxed by novobiocin; the chromatin assembled without TFIIIA is static and unaffected by novobiocin. The torsionally strained DNA is produced in a novel concerted reaction: all of the 5S DNA molecules gyrate at TFIIIA-5S DNA ratios equal to or above 1, and none of them gyrate at TFIIIA-5S DNA ratios below 1. We discuss the biological implications of this eukaryotic DNA gyration.

Assembly and properties of chromatin containing histone H1

The Xenopus oocyte supernatant (oocyte S-150) forms chromatin in a reaction that is affected by temperature and by the concentration of ATP and Mg. Under optimal conditions at 27 degrees C, relaxed DNA plasmids are efficiently assembled into supercoiled minichromosomes with the endogenous histones H3, H4, H2A and H2B. This assembly reaction is a gradual process that takes four to six hours for completion. Micrococcal nuclease digestions of the chromatin assembled under these conditions generate an extended series of DNA fragments that are, on average, multiples of 180 base-pairs. We have examined the effect of histone H1 in this system. Exogenous histone H1, when added at a molar ratio of H1 to nucleosome of 1:1 to 5:1, causes an increase in the micrococcal nuclease resistance of the chromatin without causing chromatin aggregation under these experimental conditions. Furthermore, the periodically arranged nucleosomes display longer internucleosome distances, and the average length of the nucleosome repeat is a function of the amount of histone H1 added, when this histone is present at the onset of the assembly process. In contrast, no major change in the length of the nucleosome repeat is observed when histone H1 is added at the end of the chromatin assembly process. Protein analyses of the purified minichromosomes show that histone H1 is incorporated in the chromatin that is assembled in the S-150 supplemented with histone H1. The amount of histone H1 bound to chromatin is a function of the total amount of histone H1 added. We define here the parameters that generate histone H1-containing chromatin with native nucleosome repeats from 160 to 220 base-pairs, and we discuss the implications of these studies.

Histone H1 represses transcription from minichromosomes assembled in vitro

We have previously shown that transcription from a Xenopus 5S rRNA gene assembled into chromatin in vitro can be repressed in the absence of histone H1 at high nucleosome densities (one nucleosome per 160 base pairs of DNA) (A. Shimamura, D. Tremethick, and A. Worcel, Mol. Cell. Biol. 8:4257-4269, 1988). We report here that transcriptional repression may also be achieved at lower nucleosome densities (one nucleosome per 215 base pairs of DNA) when histone H1 is present. Removal of histone H1 from the minichromosomes with Biorex under conditions in which no nucleosome disruption was observed led to transcriptional activation. Transcriptional repression could be restored by adding histone H1 back to the H1-depleted minichromosomes. The levels of histone H1 that repressed the H1-depleted minichromosomes failed to repress transcription from free DNA templates present in trans. The assembly of transcription complexes onto the H1-depleted minichromosomes protected the 5S RNA gene from inactivation by histone H1.

Structure of the two distinct types of minichromosomes that are assembled on DNA injected in Xenopus oocytes

DNA injected into germinal vesicles of Xenopus oocytes is assembled into two distinct types of minichromosomes. One type is soluble and behaves like conventional nucleosomal chromatin. The other type is insoluble, is sensitive to DNAase I and to micrococcal nuclease, lacks a canonical nucleosome repeat, and generates a half-nucleosome size limit digest with micrococcal nuclease. We suggest that these peculiar minichromosomes may be the ones that display the unconstrained, dynamic DNA supercoils in the living oocyte.

A simple procedure for parallel sequence analysis of both strands of 5′-labeled DNA

Ligation of a 5-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

Assembly of transcriptionally active chromatin in Xenopus oocytes requires specific DNA binding factors

Active minichromosomes assembled on injected 5S RNA gene clones are stable in Xenopus oocytes; endogenous 5S DNA specific factor(s) are required for their assembly. When somatic-type and oocyte-type 5S RNA gene clones are coinjected, the somatic genes are assembled into active minichromosomes, while most of the oocyte genes are assembled into inactive ones. The differential 5S RNA gene expression, which mimics that in somatic cells, appears to result from titration of 5S DNA specific factor(s) by the competing somatic 5S DNA, followed by histone mediated assembly of inactive chromatin on the oocyte 5S DNA. Stable minichromosomes are also assembled on a cloned histone H4 gene; again, intragenic DNA rearrangements affect the efficiency of assembly of active chromatin and differential gene expression occurs after coinjection of two or more H4 DNA constructs. We suggest that the H4 DNA molecules also compete for limiting quantities of specific DNA binding factor(s) required for the assembly of active H4 gene chromatin.

Mechanism of chromatin assembly in Xenopus oocytes

We have analyzed the chromatin assembly reaction catalyzed by the Xenopus oocyte extract (S-150). A 50 S complex is formed upon mixing the 17 S pUC DNA and the S-150. Mature histones are not detected in this complex, which contains relaxed DNA and protein, and generates subnucleosomal 7 S particles upon digestion with micrococcal nuclease. The relaxed nucleoprotein is gradually supercoiled into nucleosomal chromatin in the S-150, via a pathway that requires ATP and is blocked by novobiocin, and this process is accompanied by the appearance of mature histones H3 and H4. Isolated complexes also supercoil in vitro, which implies the complex is a kit that contains histone precursors, as well as topoisomerases and other enzymes required for assembly. We discuss the biological implications of these findings.

The role of DNA-mediated transfer of TFIIIA in the concerted gyration and differential activation of the Xenopus 5S RNA genes

The transcription factor of the 5S RNA gene, TFIIIA, induces gyration of oocyte- and somatic-type 5S DNA plasmids in Xenopus oocyte extracts, but oocyte 5S gyration requires a 5-fold higher TFIIIA concentration than somatic 5S gyration. Concomitant with the differential gyration at intermediate TFIIIA concentrations, the oocyte genes are repressed and the somatic genes become activated, a situation that mimics the one seen in Xenopus somatic cells. Data obtained with plasmids immobilized in agarose indicate that TFIIIA finds its site via a DNA-mediated transfer mechanism, and that all-or-none gyration is a consequence of TFIIIA transfer between 5S DNA sites. Based on these results, we present a model that explains the differential all-or-none activation of the 5S RNA genes.

Analysis of the chromatin assembled in germinal vesicles of Xenopus oocytes

We have injected circular DNA, labeled with 32P at a single restriction site, into germinal vesicles of Xenopus laevis oocytes in order to study the nucleosome arrangement on the assembled minichromosomes. Two types of genes were used in these studies, the somatic 5 S RNA gene unit of Xenopus borealis and the histone gene unit of Drosophila melanogaster. We find that injections of labeled DNA alone, at 1 ng DNA per oocyte, results in irregularly spaced nucleosomes and partially supercoiled DNA molecules. However, perfectly spaced nucleosomes are assembled and fully supercoiled DNA is recovered if 5 to 20 nanograms of cold vector DNA is coinjected with the labeled DNA. At the optimum chromatin assembly conditions, the nucleosomes are perfectly spaced with a 180 base-pair periodicity, but they are randomly positioned on the DNA. The assembly of a periodic chromatin structure is accompanied by a dramatic enhancement in the expression of the injected 5 S RNA gene.