Visualising transcription, gene positioning and reorganisation in the nucleus

Friday, 24 February 2006

Visualising transcription, gene positioning and reorganisation in the nucleus
Friday, 24 February 2006 09:30 - 16:30

Birkbeck College
Basement Lecture Theatre
43 Gordon Square
United Kingdom

Map and Directions

“In recent years there has been a huge advance in techniques that can visualize key events in the nuclei of single cells. These include primary transcription from individual alleles, and associated processes such as chromatin remodelling and relocalization of DNA loci, and localization of protein factors involved in gene activation and silencing.  The techniques include RNA-DNA -and - immuno-FISH (fluorescence in situ hybridization) respectively. This meeting will explore the practical application of these techniques, as well as the tremendous advances that they have enabled, particularly in the immunology field. They have identified new mechanisms such as intergenic transcription,  locus contraction and allele-specific heterochromatinisation in V(D)J recombination and allelic exclusion. They are particularly powerful for analysis of nuclear reorganisation and gene expression changes during immune cell development, and for study of monoallelic expression, for example for NK receptors, interleukins, antigen receptors.” - Co-Chair: Dr Anne Corcoran, Babraham Institute

9:15 - 9:45       Registration - Tea, Coffee and biscuits

9:45 - 10:00     Introduction by the Chair
Prof Peter Fraser - Babraham Institute, Cambridge, UK

10:00 - 10:30   Visualising gene transcription using RNA-FISH 
Dr. Daniel Bolland, Babraham Institute, UK

Since the majority of the transcribed mammalian genome is non-coding, including intergenic and antisense transcription, new approaches are required to understand these processes. RNA fluorescence in situ hybridisation has several advantages over traditional techniques for analysing transcription, including the ability to analyse primary transcripts in single cell nuclei, and to visualise each allele separately.

Recent FACS improvements have enabled RNA-FISH analysis of very small cell populations. Using RNA-FISH we have discovered intergenic antisense transcription across the mouse immunoglobulin heavy chain (Igh) locus that correlates strongly with V(D)J recombination. We propose this enhances accessibility of the chromatin for V(D)J recombination.

10.30 - 11:00   Modulation of Nucleosome Remodelling Activity by Histone Acetylation
Dr Joan Boyes - University of Leeds
Targeted chromatin remodelling is essential for many nuclear processes, including the regulation of V(D)J recombination. ATP-dependent nucleosome remodelling complexes are important players in this process whose activity must be tightly regulated. Evidence will be presented that histone acetylation modulates nucleosome remodelling complex activity to boost initiation of V(D)J recombination. Recombination requires nucleosome mobilisation from recombination signal sequences. Acetylation does not stimulate nucleosome mobilisation per se by CHRAC, ACF or their catalytic subunit, ISWI. Instead, we find the increased accessibility of acetylated chromatin regulates the ability of nucleosome remodelling complexes to access their nucleosome templates. We suggest this targeting plays an important role in regulating nucleosome remodelling complex activity.

11:00 - 11:30  
Morning Tea/coffee

11:30 - 12:00  Delineation of nucleolar trafficking signals using live cell and confocal microscopy

Dr Julian Hiscox, University of Leeds

The nucleolus is a dynamic sub-nuclear structure crucial to the successful functioning of a eukaryotic cell, and functions to promote ribosome sub-unit biogenesis, control cell growth and monitor cellular stress.  We have been using viruses as tools to probe the trafficking of nucleolar proteins and the effects of changes in nucleolar structure on function.  Our work has focused on the trafficking of the coronavirus nucleocapsid protein to the nucleolus and its interactions with nucleolar proteins and the tumor suppressor protein p53.

12:00 - 12:30   Cross talk between chromosome structure, nuclear architecure and gene expression

Dr Dean JacksonUniversity of Manchester

Gene expression is regulated at many levels, including: the distribution of genes in chromosomes, the way chromosomes are organised in the nucleus, epigenetic modifications of chromatin that correlate with gene activity and the dynamic properties of chromatin that facilitate the association of genes with active nuclear sites. We are exploring how nuclear architecture influences chromatin function. A key element is to understand the role of the nucleoskeleton. Recent experiments are revealing the structure and role of the nucleoskeleton and show how studies of the nucleoskeleton are beginning to demonstrate the importance of nuclear organisation in the regulation of nuclear function.

12:30 - 13:30   Lunch in the exhibition area


13:30 - 14:00   Nuclear re-organisation of Hox genes during ES cell differentiation and mouse   
Dr Celine Morey, Medical Research Council, Human Genetics Unit, Edinburgh UK

We use 4-colour DNA-FISH to study the nuclear behaviour of Hox genes in differentiating ES cells and mouse embryos. As for Hoxb genes, the colinear temporal expression of Hoxd genes is associated with their sequential extrusion outside of the chromosome territory and with decondensation of Hoxd chromatin, suggesting an evolutionary conserved chromosomal mechanism. Furthermore, the looping out from chromosome territories spreads away from Hoxd in both directions to encompass unrelated genes. The Global Control Region (GCR), located outside of Hoxd is involved in regulating later 5' Hoxd gene expression in novel vertebrate structures. It also appears to be a boundary to the spread of chromosome territory remodelling and has a constitutively decondensed chromatin structure.

14:00 - 14:30  Preferential associations between actively transcribing genes reveal transcriptional networks

Dr Lyubomira Chakalova - The Babraham Institute

We have studied the spatial organization of transcription by using RNA FISH, DNA FISH and immunostaining techniques. We have analyzed the nuclear organization of multiple genes from several mouse chromosomes in two cell types. Gene transcription is compartmentalized into discrete foci or ‘transcription factories’. We demonstrate that during transcription in vivo genes often co-localize to the same transcription factory. We show that individual genes co-associate with preferred partners in cis and in trans. Our data reveal physical transcription networks of active genes and show that transcription factories are major focal points for the non-random organisation of the genome.

14:30 - 15:00   Afternoon Tea/Coffee

15:00 - 15:30 
Genomics to Epigenomics: Ribosomal RNA Genes as a Paradigm Between Form and Function
Dr Numo Neves - Portugal

When analysing nuclear dynamics in terms of transcription, replication events and chromatin topology, an high level of functional structuring emerges. The dynamic behaviour of nuclear components, strongly linked to genome functioning, can be easily observed when analysing ribosomal RNA (rRNA) genes. Using Arabidopsis as a model-species for molecular and cytobiology, we will present our current model that integrates rDNA topology modulated by epigenetic mechanisms with the on/off states of these gene arrays. This model comprehends both DNA methylation and post-translational histone modifications tagged by chromatin remodelling enzymes and their relation to gene silencing events.

15:30 - 16:00  
A Euchromatic Epigenetic Mark In Rye B Chromosome Heterochromatin
Ms. Margarida Delgado, Sec. Genética, Portugal
We investigated the chromatin organization of the heterochromatic domain of the rye B chromosome (B) in several distinct cell types and in two different genomic environments, namely in rye itself and also when the B is introgressed into hexaploid wheat as an addition line. The rye B consistently presents a large Giemsa positive band at the terminal part of the long arm, which corresponds with a heavily DAPI stained block where the B-specific repetitive DNA sequences (D1100 and E3900) are located (Houben et al., 1996). In situ hybridization revealed that despite of these heterochromatic features this domain is decondensed at interphase in meristematic, differentiating and endopolyploid cells and this feature is particularly pronounced at pachytene. Immunolocalization of histone H3 trimethylated at lysine 4 (H3trimethylK4) further revealed that this region is highly enriched in this  epigenetic mark, characteristically associated with euchromatin and gene transcription, in both genomic backgrounds. The enrichment in H3trimethylK4 in this domain must be related to chromatin decondensation, since it lacks gene transcription. The results are discussed in terms of the dynamic and puzzeling nature of this domain and of B chromosome transmission.

16:00              Discussion and close

Registraton fees

  • Standard fee - £400
  • Academic fee - £199
  • Student fee- £140
  • IBMS members fee - £199

This meeting is CPD accredited


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    Sally Wheatland
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