CH391L/S2013 Taylor Pursell Feb 27 2013

Identification of Early Replicating Fragile Sites that Contribute to Genome Instability. Barlow JH, Faryabi RB, Callén E, Wong N, Malhowski A, Chen HT, Gutierrez-Cruz G, Sun HW, McKinnon P, Wright G, Casellas R, Robbiani DF, Staudt L, Fernandez-Capetillo O, Nussenzweig A. Cell. 2013 Jan 22. pii: S0092-8674(13)00008-1.

Background

Replication and Fragile Sites

The eukaryotic genome is divide into regions which are programmed to replicate at different times during S-phase of the cell cycle. Gene products important to the cell, or active genes, are replicated in the first half of s-phase, or are associated with early replication, while inactive genes are replicated during the second half of S-phase and are associated with late replication site firing [2].

Chromosomal fragile sites which are specific stretched of genetic material that are prone to gaps and breaks following replication leading to chromosome instability; these fragile sites are put into two classes: rare and common fragile sites. Rare fragile sites are those found in only a few individuals and are usually associated with genetic disorders (e.g. Fragile X syndrome) while common fragile sites (CFS) are found in all individuals [3]. Common fragile sites are can be associated with large genes, large AT rich regions, and stalled DNA replication [1,3].

B-Lymphocytes and DNA Damage

B-lymphocytes, or B-cells, originate in the bone marrow where their maturation occurs. Large B-cells circulated in the blood and become activated once an antigen binds to their surface receptor. Once activated, large B-cells undergo rapid proliferation and genomic rearrangements [1,4]. These genome changes come in two classes:

1. somatic hypermutation(SHM) in which point mutations are induced in the variable region of immunoglobulin (Ig) genes and
2. class switch recombination (CSR) where one constant region of Ig is deleted and replaced with another[1].

An enzyme celled activation-induced cytidine deaminase (AID) initiates these genome changes by deaminating cytosine residues on ssDNA that are exposed during Ig transcription. Although this is its primary function it is not uncommon for it to cause other DNA damage specifically to oncogenes like c-Myc [1].

Methods

ChIP and ChIP-Seq

Murian splenic B-cell samples were stimulated with LSP/IL4 to cause the cells to synchronously enter cell cycle. After 22 hours, cells should be in S phase (monitored by FACs). Cells were then treated with 10 mM hydroxyurea (HU) which arrests cells at G1/S phase and then cells were harvested. Cells were crosslinked in order to ensure any protein bound to DNA would remain bound. Cells were sonicated in order to break up cells and to fragment DNA. Somicated chromatin samples are then incubated with an antibody specific to the protein of interest (e.g. RPA, BRCA1, etc.) and magnetic breads specific to the constant region of the antibody used.

Once genetic material is harvested, it was prepared for micro sequencing using Illumina’s protocol for sample preparation and was analyzed using the Genome Analyzer IIx.

Fluorescent in-situ hybridization (FISH) analysis

Fluorescent probes that bind to complementary sequences on chromosomes are used to visualize abnormalities. Fluorescent microscopy can be used to find where the probe is bound. In this paper, blue is DAPI-stained DNA, green is the insert in the BAC (either ERFSs or CFSs) and red is telomeric DNA.

References

1. Barlow, J. H. et al. Identification of Early Replicating Fragile Sites that Contribute to Genome Instability. Cell 152:1-13 (2013)
2. Goren A, Cedar H. Replicating by the clock. Nature Reviews Molecular Cell Biology In Press 4: 25-32 (2003)
3. Durkin, S. G. & Glover, T. W. Chromosome Fraglie Sites. Annual Reviews Genetic 41: 169-192 (2007)
4. Parham, Peter. The Immune System. Garland Science, 2. (2005)