Garry T. Morgan

Lampbrush chromosomes and associated bodies: new insights into principles of nuclear structure and function

The lampbrush chromosomes and assorted nuclear bodies of amphibian and avian oocytes provide uniquely advantageous and amenable experimental material for cell biologists to study the structure and function of the eukaryotic nucleus, and in particular to address the processes of nuclear gene expression. Recent findings discussed here include the molecular analysis of the actively elongating RNA polymerase complexes associated with lampbrush chromosome loops and of the association between loop nascent transcripts and RNA processing components. In addition, several types of chromosome structure that do not outwardly resemble simple extended loops and that may house novel nuclear functions have recently been studied in detail. Among these a type of chromosomal body that can also exist free in the oocyte nucleus, the Cajal body, has been shown to possess a range of characteristics that suggest it is involved in the assembly of macromolecular complexes required for gene expression. Homologous structures have also been described in somatic nuclei. Fundamental aspects of the looped organization exhibited by lampbrush as well as other chromosomes have also been addressed, most notably by the application of a technique for de-novo chromosome assembly.

A sequence motif conserved in diverse nuclear proteins identifies a protein interaction domain utilised for nuclear targeting by human TFIIS

The three structural domains of transcription elongation factor TFIIS are conserved from yeast to human. Although the N-terminal domain is not needed for transcriptional activity, a similar sequence has been identified previously in other transcription factors. We found this conserved sequence, the LW motif, in another three human proteins that are predominantly nuclear localized. We investigated two examples to determine whether the LW motif is actually a dedicated nuclear targeting signal. However, in one of the newly identified proteins, hIWS1 (human Iws1), a region containing classic nuclear localization signals (NLS) rather than the LW motif was necessary and sufficient for nuclear targeting in HeLa cells. In contrast, human TFIIS does not possess an NLS and only constructs containing the LW motif were efficiently targeted to nuclei. Moreover, mutations in the motif could cause cytoplasmic accumulation of TFIIS and enabled a structure/function assay for the domain based on the efficiency of nuclear targeting. Finally, GST pull-down assays showed that the LW motif is part of a protein-binding domain. We suggest that the targeting role the LW motif plays in TFIIS arises from its more general function as a protein interaction domain, enabling TFIIS to bind a carrier protein(s) that accomplishes nuclear import.

Localized co-transcriptional recruitment of the multifunctional RNA-binding protein CELF1 by lampbrush chromosome transcription units.

The highly-extended transcription units of lampbrush chromosomes (LBCs) offer unique opportunities to study the co-transcriptional events occurring on nascent transcripts. Using LBCs from amphibian oocytes, I investigated whether CELF1, an RNA binding protein involved in the regulation of alternative splicing, mRNA stability and translation, is localized to active transcription units. Antibodies raised against mammalian (CUG-BP1) and amphibian (EDEN-BP) CELF1 were used to immunostain LBC spreads prepared from several species, including Xenopus laevis and the axolotl Ambystoma mexicanum. Up to about 50 separate LBC loci were convincingly immunostained and it was clear that CELF1 was present in the nascent RNPs of lateral loops. Furthermore, myc-tagged CUG-BP1 expressed in microinjected axolotl oocytes was specifically targeted to nascent transcripts of loops that recruit endogenous CELF1. In many active transcription units CELF1 was distinctly localized, being first recruited by nascent transcripts only far downstream of the transcription start site and remaining associated until the end of transcription. Overall it appears possible that the multiple functions of CELF1 in regulating posttranscriptional gene expression could all be predetermined during transcription by virtue of a region-specific binding to the nascent transcripts of target genes.

Working with Oocyte Nuclei: Cytological Preparations of Active Chromatin and Nuclear Bodies from Amphibian Germinal Vesicles

The giant nucleus or germinal vesicle (GV) of amphibian oocytes presents a remarkable opportunity to examine nuclear structures in unprecedented levels of detail. By making use of spread preparations of GVs, it is possible to investigate the structure and function of transcription units in active chromatin and a variety of nuclear bodies, all within the limits of resolution of the light microscope. The basic method for producing GV spreads that is described here is based on simple manual dissection and, therefore, it permits the preparation of nuclear components that have suffered a minimum of experimental manipulation. The particular method described is based on the use of oocytes from a salamander, the axolotl, although the approach is robust and applicable with minor modification to two other model amphibian species, Xenopus laevis and X. tropicalis. One common approach to investigating the molecular organisation of oocyte nuclear structures by immunofluorescent staining of endogenous or exogenous polypeptides is also described.

Conservation of intergenic spacer length in ribosomal DNA of the tailed frog, Ascaphus truei

We have examined the organization of cloned rDNA [encoding ribosomal RNA (rRNA)] repeat units from the tailed frog, Ascaphus truei, and have compared rDNA spacer lengths in the genomes of eleven individuals from two widely-separated populations. This comparison has shown that the A. truei spacer is always very short (about 1.5 kb) and that it is remarkably constant in length. In none of the individuals tested were more than two spacer-length classes found and the maximum difference in spacer length found in comparisons both within single animals and across both populations was about 120 bp. We point out those structural features that may contribute to the unusual stability of this spacer and the consequent absence of the extensive length heterogeneities found amongst rDNA repeat units in most genomes.

Alterations in cloned Xenopus ribosomal spacers generated by high-frequency plasmid recombination

In an attempt to understand the mechanisms that have led to the complex intraspecific heterogeneity in spacer organisation found in Xenopus r.DNA, we have transformed Escherichia coli strains that exhibit high levels of plasmid recombination with Xenopus laevis r.DNA sequences inserted in pBR322. We have found that in these recBC sbcA strains, plasmid recombination results in the generation of many types of altered r.DNA spacer at a high frequency. The altered spacers result from two types of deletion/duplication events that occur in the promoter-derived sequences making up much of the spacer. The alterations caused by these events mimic in several ways the different types of organisation found among spacers in the genome. The sbcA-dependent plasmid recombination apparently provides a test system for reconstructing some of the recombinational events that operate during the evolution and maintenance of spacer DNA sequences in vivo.

Isolation and Analysis of Xenopus Germinal Vesicles

The giant nucleus or germinal vesicle (GV) of Xenopus oocytes provides an unusual opportunity to analyze nuclear structure and function in exquisite detail by light microscopy. Detailed here are two rapid procedures for using manually isolated GVs in combination with fluorescent reporter proteins to investigate the lampbrush chromosomes and nuclear bodies of oocytes. One procedure provides spreads of nuclear components in an unfixed and life-like, although not living, form. The other describes the isolation of intact, functional GVs directly into mineral oil offering possibilities for direct observation of nuclear dynamics.

Association of modified cytosines and the methylated DNA-binding protein MeCP2 with distinctive structural domains of lampbrush chromatin

The lampbrush chromosomes (LBCs) of the amphibian oocyte are the largest known chromosomes and offer the unique opportunity to visualize the distinctive closed and open chromatin structural domains with an unprecedented spatial resolution. In this study we investigated the association of DNA methylation and proteins interpreting methylation state, such as MeCP2, with LBCs. We expressed HA-tagged MeCP2 in Xenopus laevis oocytes, and we observed that while it predominantly targeted the transcriptionally inactive chromomeres, a minor fraction of HA-MeCP2 also associated with many of the transcriptionally active lateral loops of LBCs. We further demonstrated that the association of MeCP2 with LBCs is directly determined by its 5-methylcytosine-binding domain. We then defined the distribution of 5-methylcytosines (5mC) by immunostaining Xenopus and axolotl LBCs and confirmed the pattern suggested by the expression of HA-MeCP2 targeting of intense staining of the chromomeres and of many loop bases. In addition, we found that short interstitial regions of the active transcriptional units could be clearly stained for 5mC. Interestingly, these 5mC-positive regions corresponded precisely to segments of active transcription units from which RNA polymerase II and nascent transcripts were simultaneously absent. Together, these data support a model presenting MeCP2 as both a transcriptional repressor and activator.