Making Better Artificial Chromosomes for Mammalian Cells
Short title MAKEMAC
Contract Number HPRN-CT-2000-00089
Start date 1/10/2000
Duration 36 Months
Report period 1-10-2000-30/9/2001
Co-ordinator:-
Howard J Cooke
MRC Human Genetics Unit
Western General Hospital
Crewe Road
Edinburgh EH4 2XU
Tel
Fax
Email
+44 131 467 8427
+44 131 343 2620
howard.cooke@hgu.mrc.ac.uk
A1 Scientific Highlights
The scientific highlights of the first year of support for this program are
detailed below. They put in place a number of resources of reagents and
expertise which will be used during the following stages of the workplan.
Broadly speaking they fall into three areas, basic work on the biology of
chromosome components such as telomeres and origins of replication and
the proteins which interact with them, generation of constructs for artificial
chromosome production and verification of delivery methods.
1)Basic Biology
a)Origins
Bi directional origins of replication have been described in a number of
regions of mammalian genomes – the focus of the work here is the lamin B2
at the 3’ end of the gene. We have shown that a 1.2kb sequence contains all
the sequence requirements for ectopic activity of the origin and a deletion
analysis of this DNA has been started. A 70 bp region with cell cycle
dependent characteristics represents the core of this sequence and deletion
and base composition changes have been made. We have shown that
specific proteins bind to this DNA sequence by using it for affinity purification
of a complex of more than 40 proteins of which only CdC6 and Orc2 bind
directly to the origin. Unknown proteins will be identified and sequenced by
HPLC/MS2
b)Telomeres
The dogma from yeast is that telomeric regions are transcriptionally silenced
but there is little evidence for this so far in mammalian cells. We have inserted
a GFP gene at subtelomeric loci and find a repression of expression
compared to euchromatic insertion. This repression is alleviated by the
histone deacetylase inhibitor tichostatin and by increased levels of the TRF1
protein which binds to telomeric repeats. This is of direct relevance to the
establishment of artificial chromosomes since silencing of marker genes could
result in a loss of efficiency of the process. Modulation of the levels of
telomere associated proteins may provide a way of circumventing this. We
have started the construction of a set of retroviruses expressing TRF1, TRF2,
TIN2 and Human SIR2 homologs, as well as mutant forms of these proteins.
We are making use of the pBabe-LoxP-Puro plasmid, a pBabe-derived
retroviral vector conferring resistance to puromycin and containing two loxP
sites that allow excision of the transgene upon expression of the Cre
recombinase enzyme from bacteriophage P1. We believe that the use of a
conditional expression system is essential to overcome deleterious
phenotypes resulting from the overexpression of the transgene.
An alternative to globally increasing the level of telomere associated proteins
in the cell is to use tethering to increase the concentrations locally. As a first
step toward the manipulation of tethered telomeric proteins in MACs, we
investigated the control of telomere length by the human telomeric proteins
TRF1 and TRF2. We established telomerase positive cell lines where one can
induce the tethering of these telomeric proteins to specific telomeres. We
demonstrate that their targeting leads to telomere shortening. This indicates
that these proteins act in cis to repress telomere elongation. Inhibition of
telomerase activity by a modified oligonucleotide did not further increase the
pace of telomere erosion caused by TRF1 targeting, suggesting that
telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting
and telomerase inhibition have additive effects.
2) Chromosome manipulations
a)De novo Chromosomes and constructs
Non of the existing “bottom up” artificial chromosome systems have been
shown to function in mouse. We have made a library from a chromosome
produced by telomere associated chromosome fragmentation which is
capable of function in mouse human and chicken cells. From this library we
have isolated a number of PAC clones which contain a variety of repeated
sequences including mouse minor satellite, human alphoid sequences and
parts of the human Y chromosome – the original starting material for the
chromosome truncation. Some of these clones contain telomeric sequences
in addition to centromeric ones and we have transfected these into a cell line
known to be competent for chromosome formation and have preliminary
evidence of MAC formation.
To develop a test gene for use in the MAC program we have first constructed
a system for retrofitting OriP and the EBNA1 gene into BACs or PACs and
are developing this approach to include other genes. The CFTR gene will be
the gene of choice and we have obtained the three BACs necessary to span
the gene and its control regions. They will be assembled using RecET
recombination based constructs.
b)Chromosome Modification
We have immortalised a lymphoblastoid line from an individual who carries a
chromosome 21 with a substantial deletion of the short arm. The p arm of this
chromosome is free of certain restriction sites and so fragments containing
the centromeric sequences and one telomere were co-transfected with a
telomer construct into a human line HT1080. Preliminary data suggest that a
new chromosome of 2-10Mb has been formed. To back up this approach to
cloning the 21 centromere we have constructed vectors forTAR cloning which
will allow us to create circular YACs containing the centromere of
chromosome 21.
3)DNA transfer into cells.
The ability to transfer large DNA molecules into cells with high efficiency is
critical for many of the component projects of our network. Usin a BAC
containing an eGFP reporter gene and a 130kb fragment of human DNA we
have been able to test a number of different cell lines and transfection
reagents. Efficiencies for transient transfection reached a maximum 9% of
cells. Numbers of cells transfected were proportional to the molar amounts of
DNA to which they were exposed. Using large amounts of high molecular
BAC DNAs poses problems of delivery to the cells because of precipitation
and agregation. We have been able to develop protocols which minimise
these problems but which do not affect the efficiency of transfection.
A2 Publications
Participants 6 and 7
1) Abdurashidova, G., Deganuto, M., Klima, R., Riva, S., Biamonti,G., Giacca,
M.,
Falaschi, A. Start sites of bi-directional DNA synthesis at the human lamin B2
origin. 2000. Science 287, 2023-2026.
2) de Stanchina, E., Gabellini, D., Norio, P., Giacca, M., Peverali,F.A., Riva,
S., Falaschi, A., Biamonti, G. Selection of homeotic proteins for binding to a
human DNA replication origin. 2000. J. Mol. Biol. 299, 667-680.
3)Davide Gabellini, Ivan N. Colaluca, Giuseppe Biamonti, Mauro Giacca,
Arturo Falaschi, Silvano Riva and Fiorenzo A. Peverali.
Cell Cycle-Dependent Degradation of HOXC10 Homeotic Protein by
Anaphase Promoting Complex/Cyclosome.
(submitted, 2001)
B1 – Research objectives
The research objectives are still relevant and achievable and progress
towards this has been made.
B2 – Research method
The research methods set out in the contract are basically unchanged but a
number of detailed refinements have been developed over the period since
the proposal was written.
B3 – Work Plan
The work plan tasks are in bold and our progress and deviation follows.
1. Determining what DNA efficiently makes centromeres in human cells
and in mouse cells. (Participant 1).
We have generated and analyzed a number of potential new centromeric
sequences in preparation for milestones b and c. We have obtained the telomeric
sequences from participant 3
2. Transfer of existing novo-chromosomes into mice and analysis.
(Participant 1).
Milestones
Despite substantial effort we have not been able to achieve this and are
discontinuing this effort
3. Better constructs for telomere formation by tethering telomere proteins
to non-telomere DNA. (Participant 3)
Milestones
We have made fusion proteins between telomere binding proteins such as TRF1
and TRF2 and DNA binding proteins such as the lactose repressor (24
months).
4. What effect do telomeres have on gene expression. (Participant 3)
Milestones
We have introduced constructs with a reporter GFP gene and with or without
different telomere
constructs into mammalian cells by transfection (12
months).
Assessment of gene expression with or without the telomere by FACs
analysis is underway
5. Construction and analysis of a library of replication origins. (Participant
6)
Milestones
The site of the lamin origin ahas been tightly defiend and mutagenesis is
underway to further functionally define this region. Affinity purification of
interacting proteins is proceeding and will lead to mass spectrometry analysis
in the next 12 months
6. Mapping replication origins in two regions of the human genome.
(Participant 6)
Milestones
Not yet started
7. A replication cassette that can be used in MAC constructs. (Participant
7)
Milestones
The site of the lamin origin ahas been tightly defiend and mutagenesis is
underway to further functionally define this region. Affinity purification of
interacting proteins is proceeding and will lead to mass spectrometry analysis
in the next 12 months
8. What effect do telomeres and centromeres have on replication.
(Participant 7)
Milestones
Not yet started
9. Assessing other centromere sequences in MACs (Participant 4)
Milestones
A candidate for a deleted version of the 21 chromosome is available and being
further characterised. Constructs for a recombination based approach to
clone the centromere from chromosome 21 have been constructed and are
being verified
10. Modification of deleted chromosome 21 (Participant 4)
Milestones
Not yet started
11. Construction of BACs with the intact human CFTR gene with and
without OriP and EBNA1. (Participant 2)
Milestones
We have made BACs carrying oriP and EBNA1 for Participant 5 to use as a test
system for delivery of big DNA.
The CFTR gene is partially reconstructed but needs further work to assemble the
complete control region.
12. Construction of MACs with loxP site and introduction of the CFTR gene
onto this. (Participant 2)
Milestones
This has been delayed due to having to assemble the CFTR gene from three
rather than two BACs
13. Delivery of large DNA to cells in vitro (Participant 5)
BACs carrying oriP and EBNA1 from Participant 2 for delivery to human cells by
a variety of different non-viral systems are now available
b) Biophysical analysis of the DNA particles with different packaging methods
(24 months).
We have started to assess the intactness of the BACs in the human cells by
rescue into E. coli or rare cutting restriction digestion of high molecular weight
DNA and pulsed field gel electrophoresis (24 months).
14. Delivery of large DNA to different cell types and in vivo (Participant 5)
Milestones
The delivery of BACs to cells in culture has been optimised
Organisation and management
B4.1
The network is managed by email and phone contact between the responsible
scientists, the co-ordinator and the young researchers. A web page for the
MAKEMAC project is hosted at www.hgu.mrc.ac.uk with a publicly accessible
area and a private area accessible only to members of the network. This
private area contains the detailed progress reports from each group. An email
group is established to enable broadcast emails to all network members.
Websites of the members are listed on this site.
B4.2-3
A first network meeting was held in February 2001 in Edinburgh over two days
to establish networking between the yourn researchers and the responsible
scientists. Bilateral meetings between participants 6 and 7 have ocurred
several times during this reporting period.
B5
5.1 Positions vacant were posted on the Edinburgh web site and advertised
twice in Nature. In addition the position for partner two was individually
advertised in Nature.
5.3 Young researchers are encouraged to make use of transferable skills
training locally and are trained by close personal interactions in the laboratory.
Training in the use of digital microscopy is provided. They present their work
at lab meetings and are required to give more formal presentations
approximately annually. Attendance at conferences is encouraged and
particularly where results merit a presentation. We expect exchanges
between labs to commence in the second and third years of the contract once
the scientific foundation is firmly laid.
B.6
Members of the network outside the Euro zone have suffered from a ~25%
reduction in the value of the funding since it was applied for. Internal salary
consistency means that there are no longer sufficient funds for many
activities. To preserve the core activities of young researcher training
resources have been focussed on the laboratory work.