Ulrich K. Laemmli

Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold

Data are presented for sequence-specific chromatin-loop organization in histone-depleted nuclei from Drosophila melanogaster Kc cells. We find one loop for each of the tandemly repeated histone gene clusters. The attachment site is localized in the A + T rich H1-H3 spacer on a 657 bp fragment. In the cluster of the hsp70 heat-shock genes, in both control and heat-shocked cells, we find two attachment sites in close proximity upstream of regulatory elements. The transcribed sequences are not associated with the nuclear scaffold in control or in heat-shocked cells. A family of attachment sites related by hybridization to those of the hsp70 genes was discovered.

Metaphase chromosome structure. Involvement of topoisomerase II.

SCI is a prominent, 170,000 Mr, non-histone protein of HeLa metaphase chromosomes. This protein binds DNA and was previously identified as one of the major structural components of the residual scaffold structure obtained by differential protein extraction from isolated chromosomes. The metaphase scaffold maintains chromosomal DNA in an organized, looped conformation. We have prepared a polyclonal antibody against the SC1 protein. Immunolocalization studies by both fluorescence and electron microscopy allowed identification of the scaffold structure in gently expanded chromosomes. The micrographs show an immunopositive reaction going through the kinetochore along a central, axial region that extends the length of each chromatid. Some micrographs of histone-depleted chromosomes provide evidence of the substructural organization of the scaffold; the scaffold appears to consist of an assembly of foci, which in places form a zig-zag or coiled arrangement. We present several lines of evidence that establish the identity of SC1 as topoisomerase II. Considering the enzymic nature of this protein, it is remarkable that it represents 1% to 2% of the total mitotic chromosomal protein. About 60% to 80% of topoisomerase II partitions into the scaffold structure as prepared from isolated chromosomes, and we find approximately three copies per average 70,000-base loop. This supports the proposed structural role of the scaffold in the organization of the mitotic chromosome. The dual enzymic and apparent structural function of topoisomerase II (SC1) and its location at or near the base of chromatin loops allows speculation as to its involvement in the long-range control of chromatin structure.

Chromatin boundaries in budding yeast: the nuclear pore connection.

Chromatin boundary activities (BAs) were identified in Saccharomyces cerevisiae by genetic screening. Such BAs bound to sites flanking a reporter gene establish a nonsilenced domain within the silent mating-type locus HML. Interestingly, various proteins involved in nuclear-cytoplasmic traffic, such as exportins Cse1p, Mex67p, and Los1p, exhibit a robust BA. Genetic studies, immunolocalization, live imaging, and chromatin immunoprecipitation experiments show that these transport proteins block spreading of heterochromatin by physical tethering of the HML locus to the Nup2p receptor of the nuclear pore complex. Genetic deletion of NUP2 abolishes the BA of all transport proteins, while direct targeting of Nup2p to the bracketing DNA elements restores activity. The data demonstrate that physical tethering of genomic loci to the NPC can dramatically alter their epigenetic activity.

Higher order metaphase chromosome structure: Evidence for metalloprotein interactions

One level of DNA organization in metaphase chromosomes is brought about by a scaffolding structure that is stabilized by metalloprotein interactions. Fast-sedimenting, histone-depleted structures (4000-7000 S), derived from metaphase chromosomes by extraction of the histones, are dissociated by metal chelators or by thiol reagents. The chromosomal (scaffolding) proteins responsible for constraining the DNA in this fast-sedimenting form are solubilized under the same conditions. Chromosomes isolated in a metal-depleted form, which generate slow-sedimenting, histone-depleted structures, can be specifically and reversibly stabilized by Cu2+, but not by Mn2+, Co2+, Zn2+ or Hg2+. Metal-depleted chromosomes can also be stabilized by Ca2+ (at 37 degrees C), but this effect is less specific than that of Cu2+. The scaffolding protein pattern that is reproducibly generated following treatment with Cu2+ is composed primarily of two high molecular weight proteins--Sc1 and Sc2 (170,000 and 135,000 daltons). The identification of this simple protein pattern has depended upon the development of new chromosome isolation methods that are highly effective in eliminating cytoskeletal contamination.

Metaphase chromosome structure: Bands arise from a differential folding path of the highly AT-rich scaffold

Using the highly AT-specific fluorochrome daunomycin, a longitudinal optical signal called AT queue, thought to arise from a line-up of the highly AT-rich scaffold-associated regions (SARs) by the scaffolding, was identified in native chromosomes. Fluorescence banding is proposed to result from a differential folding path of the AT queue during its progression from telomere to telomere. The AT queue is tightly coiled or folded in a Q band, the resulting transverse striations across the chromatid, which also represent Giemsa subbands, generating a bright AT-rich signal over the Q region. The R bands, in contrast, contain a more central (unfolded) AT queue, yielding an AT-dull signal over the R regions. The AT queue is identified by immunofluorescence against topoisomerase II (topo II) and HMG-I/Y as the scaffold of native chromosomes; the fluorescence signal from both proteins is akin to a detailed Q-type banding pattern. Native chromosomes appear assembled according to the loop-scaffold model.

SAR-dependent mobilization of histone H1 by HMG-I/Y in vitro: HMG-I/Y is enriched in H1-depleted chromatin.

An experimental assay was developed to search for proteins capable of antagonizing histone H1‐mediated general repression of transcription. T7 RNA polymerase templates containing an upstream scaffold‐associated region (SAR) were highly selectively repressed by H1 relative to non‐SAR control templates. This is due to the nucleation of H1 assembly into flanking DNA brought about by the numerous A‐tracts (AT‐rich sequences containing short homopolymeric runs of dA.dT base pairs) of the SAR. Partial, selective titration of these A‐tracts by the high mobility group (HMG) protein HMG‐I/Y led to the complete derepression of transcription from the SAR template by inducing the redistribution of H1 on to non‐SAR templates. SARs are associated with many highly transcribed regulated genes where they may serve to facilitate the HMG‐I/Y‐mediated displacement of histone H1 in chromatin. Indeed, HMG‐I/Y was found to be strongly enriched in the H1‐depleted subfraction which can be isolated from chromatin.

Preferential, cooperative binding of DNA topoisomerase II to scaffold-associated regions.

DNA elements termed scaffold‐associated regions (SARs) are AT‐rich stretches of several hundred base pairs which are known to bind specifically to nuclear or metaphase scaffolds and are proposed to specify the base of chromatin loops. SARs contain sequences homologous to the DNA topoisomerase II cleavage consensus and this enzyme is known to be the major structural component of the mitotic chromosome scaffold. We find that purified topoisomerase II preferentially binds and aggregates SAR‐containing DNA. This interaction is highly cooperative and, with increasing concentrations of topoisomerase II, the protein titrates quantitatively first SAR‐containing DNA and then non‐SAR DNA. About one topoisomerase II dimer is bound per 200 bp of DNA. SARs exhibit a Circe effect; they promote in cis topoisomerase II‐mediated double‐strand cleavage in SAR‐containing DNA fragments. The AT‐rich SARs contain several oligo(dA).oligo(dT) tracts which determine their protein‐binding specificity. Distamycin, which is known to interact highly selectively with runs of A.T base pairs, abolishes the specific interaction of SARs with topoisomerase II, and the homopolymer oligo(dA).oligo(dT) is, above a critical length of 240 bp, a highly specific artificial SAR. These results support the notion of an involvement of SARs and topoisomerase II in chromosome structure.

The Anchor-Away Technique: Rapid, Conditional Establishment of Yeast Mutant Phenotypes

The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addition of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addition, when examined functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biology to dissect the function of individual or gene pairs in synthetic, lethal situations.

The metaphase scaffold is helically folded: Sister chromatids have predominantly opposite helical handedness

We have studied the three-dimensional folding of the scaffolding in histone H1-depleted chromosomes by immunofluorescence with an antibody specific for topoisomerase II. Two different types of decondensed chromosomes are observed. The majority of the chromosomes are expanded, and the central fluorescence signal is surrounded by a large halo of chromatin. A much smaller number of chromosomes are more compact in length; they contain a smaller halo of chromatin and their scaffolds are not extended but folded into a genuine, quite regular helical coil. This conclusion is based on a three-dimensional structural analysis by optical sectioning. The number of helical coils is related to chromosome length. Surprisingly, sister chromatids have predominantly opposite helical handedness; that is, they are related by mirror symmetry.

Specific complex of human immunodeficiency virus type 1 rev and nucleolar B23 proteins: dissociation by the Rev response element.

The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.