Lab members

Christine J Farr
Group leader

Dr Michael Mimmack
Postdoc

Contact:
Dr Christine J. Farr
Address:
Dept of Genetics,
Downing Street,
Cambridge,
CB2 3EH,
England
Telephone:
+44 1223 333972
Fax: +44 1223 333992

To contact members of the group by email, please use the following formula:
first initial.surname
@gen.cam.ac.uk

Farr Lab
Chromosome Biology

Keywords

Vertebrate chromosome, centromere, kinetochore, topoisomerase 2, telomere, minichromosome

Overview

Research in the chromosome biology group involves the systematic manipulation of vertebrate cells, maintained in tissue culture, as a means of exploring the relationship between the structural organisation of chromosomes and their function. Current research focuses on:

• the functional organisation of vertebrate centromere domains
• the generation of mammalian mini-chromosomes
• the role of topoisomerase 2 at the centromere
• the role of Trf1 at the telomere

The Vertebrate Centromere and Topoisomerase 2

Dissection of human centromeres is difficult because of the lack of landmarks within highly repeated DNA. We have systematically manipulated a single human X centromere domain generating a large series of centric deletion derivatives (Spence et al. 2002). These have been examined at four levels: linear DNA structure; the distribution of constitutive centromere proteins, such as CENP-C and CENP-H; topoisomerase II cleavage activity and mitotic stability. We have determined that the human X major alpha-satellite locus, DXZ1, is asymmetrically organised with an active subdomain anchored ~150 kb in from the Xp-edge.

Fig. 1: Localisation of the kinetochore proteins CENP-C and CENP-H (green) on the Xp-side (white) of the human X centromeric α-satellite array DXZ1 (red) using immuno-FISH (Spence et al. 2002)

We have identified a major site of topoisomerase 2 cleavage within this domain that can shift if juxtaposed with a telomere, suggesting that this enzyme recognises an epigenetic determinant within the DXZ1 chromatin. We have identified a similar site of topo 2 cleavage activity buried within the centromeric DNA of human chromosome 11. This cleavage activity is concentrated within the centromere domain through an extended period of G2 and M, with levels declining in G1 and S (Spence et al. 2006).

Fig. 2: Summary of topoisomerase 2 cleavage site mapping within human centromeric  α‑satellite arrays (Floridia et al. 2000, Spence et al. 2002, Porter & Farr 2004, Spence et al. 2005)

As well as being associated with vertebrate centromeric DNA, others have mapped topo 2 cleavage sites close to candidate centromeric DNA sequences on the chromosomes of the parasite Plasmodium falciparum (Kelly, McRobert & Baker 2005). These observations suggest that topo 2 may have a fundamental part to play in centromere biology. Using a mutant human cell line conditionally‑lethal for the major chromatin-associated form of vertebrate topo 2, topo 2α (Carpenter & Porter, 2004), we have found that depletion of this isoform, while leading to a disorganised metaphase plate, does not have any obvious effect on general kinetochore assembly. Nevertheless, a linear minichromosome displayed a significantly increased rate of mis-segregation. A shortening of the distance across metaphase sister centromeres and the abnormal persistence of PICH‑coated connections between segregating chromatids suggests that this may be linked to structural alterations within the centromere domain.

Fig. 3: The effect of topoisomerase 2α depletion on anaphase PICH‑coated threads. Human HT1080 anaphase cells depleted  of topo 2α were co-strained for PICH (green) and CENP-B (red). Quantification of the number of PICH threads seen in anaphase cells expressing, or depleted of, topo 2α (Spence et al. 2007)

Minichromosomes and vector development

Minichromosomes, and mammalian artificial chromosomes, as well as allowing basic research into the requirements for mammalian chromosome function and stability during mitosis and meiosis, may also provide vectors for the introduction of large DNA into recipient cells.  Recently, considerable progress has been made in the generation of human and mouse-based minichromosomes, some of which offer the potential to be developed as vectors for gene delivery.  We have generated a series of linear minichromosomes ranging from ~10 Mb down to 450 kb. They are all derived from the centromeric region of the human X chromosome and were generated by homologous recombination and targeted seeding of de novo telomeres (Mills et al. 1999, Spence et al. 2005, 2006).

Trf1 and the vertebrate telomere

The chicken cell line DT40 is hyper‑recombinogenic and can be used to study the role of cell autonomous proteins through targeted genetic manipulation. Several chromosomally‑associated proteins have been characterised in chicken cells and appear to be functionally conserved between birds and mammals. We are generating and characterising DT40 cell lines mutant for the TTAGGG repeat binding factor, TRF1 (Konrad et al. 1999; Fillon et al. 2001; Faure et al. 2008; Cooley et al. submitted).

Current collaborations include

• Dr Andrew Porter, Imperial College London, UK
• Dr Ciaran Morrison, NUI-Galway, Ireland
• Dr Desmond J. Smith, UCLA, USA

Recent group publications

(lab members are underlined)

• Lim HN, Farr CJ (2004) Mammalian artificial chromosomes: an overview of their development and application. Methods Mol Biol (ed. Eridani, S) pp. 167-186 abstract
• Farr CJ (2004) Forward: Centromeres, kinetochores and the segregation of chromosomes. Chrom Res 12: 517-520 abstract
• Porter AC, Farr CJ (2004) Topoisomerase II: untangling its contribution at the centromere. Chrom Res 12: 569-583 abstract

2005

• Spence JM, Fournier REK, Oshimura M, Regnier V, Farr, CJ (2005) Topoisomerase II cleavage activity within the human DXZ1 and D11Z1 alpha-satellite arrays. Chrom Res 13: 637-648 abstract

2006

• Farr CJ, Spence JM  (January 2006 online version) Mammalian Artificial Chromosomes (MACs) in Encyclopedia of Life Sciences. Chichester: Wiley. http://www.els.net/[doi:10.1038/npg.els.0005671] abstract
• Baird DM, Farr CJ (2006) The organization and function of chromosomes. EMBO Reports 7: 372-6 abstract
• Spence JM, Mills W, Mann K, Huxley C, Farr CJ (2006) Increased missegregation and chromosome loss with decreasing chromosome size in vertebrate cells. Chromosoma 115: 60-74 abstract
• Vagnarelli P, Hudson DF, Ribeiro SA, Trinkle-Mulcahy L, Spence JM, Lai F, Farr CJ, Lamond AI,  Earnshaw WC (2006) Condensin and Repo-Man/PP1 co-operate in the regulation of chromosome architecture during mitosis. Nat Cell Biol 8: 1133-42 abstract

2007

• Spence JM, Phua HH, Mills W, Carpenter AJ, Porter ACG, Farr CJ  (2007) Depletion of topoisomerase IIalpha leads to shortening of the metaphase interkinetochore distance and abnormal persistence of PICH-coated anaphase threads. J Cell Sci 120: 3952-3964 abstract

2008

• Faure V, Wenner T, Cooley C, Bourke E, Farr CJ, Takeda S, Morrison CG (2008) Ku70 prevents genome instability resulting from heterozygosity of the telomerase RNA component in a vertebrate tumour line. DNA Repair 7: 713-724 abstract
• Park CC, Ahn S, Bloom JS, Lin A, Wang RT, Wu T, Sekar A, Khan AH, Farr CJ, Lusis AJ, Leahy RM, Lange K, Smith DJ (2008) Fine mapping of regulatory loci for mammalian gene expression using radiation hybrids. Nat Genet 40: 421-429 abstract

2009

• Ribeiro SA, Gatlin JC, Dong Y, Joglekar A, Cameron L, Hudson DF, Farr CJ, McEwen BF, Salmon ED, Earnshaw WC, Vagnarelli P (2009) Condensin Regulates the Stiffness of Vertebrate Centromeres. Mol Biol Cell Mar 4 [e-pub ahead of print]
• Cooley C, Baird KM, Faure V, Wenner T, Stewart JL, Modino S, Slijepcevic P, Farr CJ, Morrison CG (2009) Trf1 is Not Required for Proliferation or Functional Telomere Maintenance in Chicken DT40 Cells. Mol Biol Cell Mar 25 [e-pub ahead of print]

Funding

• Cancer Research UK

Complete Publication List can be found here

Page updated 9 April 2009