Technology & Methods Seminar: “Tiling Arrays - Probing Genome and Transcriptome Structure” On the use of Affymetrix Tiling Arrays for Comparative Genomic Hybridizations Norman Pavelka (Rong Li lab) Our next question was: What are the genomic changes that occurred in our e-strains and that were responsible for the observed phenotypic evolution? One way to fully address this question would be to perform a complete re-sequencing of the genome of the evolved strains, but (of course) this would be too expensive and too time-consuming. Besides of that, we did not expect that the timescale of our adaptive evolution experiment was sufficient to allow the accumulation of many single-nucleotide mutations. Rather, we reasoned that the observed rapid evolution of alternative cytokinesis mechanisms may have been caused by changes in copy numbers of DNA elements that in turn might have caused changes in gene expression. To analyze DNA copy number changes we used a technique called array-based Comparative Genomic Hybridization. Traditionally, CGH was performed by co-hybridizing differentially labeled DNA fragments from two samples of interest onto unlabelled metaphase chromosomes, used as probes. In this example, an amplification in this region would label the chromosome in green, a deletion in this region would color the chromosome in red, and the rest would light up as yellow. But traditional CGH has only a resolution of roughly 10Mb. Over the last 10 years, the technique has “evolved” to use microarrays instead of metaphase chromosomes as probes. Now, the resolution of aCGH depends only on how many probes you can spot on a chip, but in general has dramatically increased in respect to traditional CGH. March 29, 2007

Background: Role of MYO1 in cytokinesis Phenotype of yeast cells experiencing an acute loss of MYO1: Severe cytokinesis defect Impaired cell viability Phenotype of yeast cells experiencing a chronic loss of MYO1: Extremely heterogenous Occasionally: full recovery of cytokinesis proficiency and of growth ability

Biological question: What genome changes occurred in e-strains? Polyploidization? Aneuploidization? Interstitial deletions? Reciprocal translocations? Non-reciprocal translocations? Single-nucleotide mutations? Amplifications? Albertson & Pinkel, Hum Mol Genet (2003)

Method: array-based Comparative Genomic Hybridization (aCGH) Our next question was: What are the genomic changes that occurred in our e-strains and that were responsible for the observed phenotypic evolution? One way to fully address this question would be to perform a complete re-sequencing of the genome of the evolved strains, but (of course) this would be too expensive and too time-consuming. Besides of that, we did not expect that the timescale of our adaptive evolution experiment was sufficient to allow the accumulation of many single-nucleotide mutations. Rather, we reasoned that the observed rapid evolution of alternative cytokinesis mechanisms may have been caused by changes in copy numbers of DNA elements that in turn might have caused changes in gene expression. To analyze DNA copy number changes we used a technique called array-based Comparative Genomic Hybridization. Traditionally, CGH was performed by co-hybridizing differentially labeled DNA fragments from two samples of interest onto unlabelled metaphase chromosomes, used as probes. In this example, an amplification in this region would label the chromosome in green, a deletion in this region would color the chromosome in red, and the rest would light up as yellow. But traditional CGH has only a resolution of roughly 10Mb. Over the last 10 years, the technique has “evolved” to use microarrays instead of metaphase chromosomes as probes. Now, the resolution of aCGH depends only on how many probes you can spot on a chip, but in general has dramatically increased in respect to traditional CGH. U.C. Berkeley Division of Biostatistics Working Paper Series (2002), paper 106.

~6.5 million unique probes on the chip Technology: Affymetrix Yeast Tiling Arrays ~6.5 million unique probes on the chip ~12.5 million bp in the yeast genome And talking about how many spots you can put on a slide, Affymetrix holds an unmatched record. Today, they can synthesize up to 6.5 million unique oligonucleotide probes on a single chip. This allowed them to design a so-called GeneChip Tiling Array, able to interrogate the yeast genome at an amazing resolution: Every 5bp along the genome there is one Perfect-Match probe and one Mismatch probe designed to interrogate that position. This overlapping (or tiling) of the probes allows each nucleotide position to be interrogated by a 5- to 6-fold redundancy. These arrays have been used by other labs to analyze mRNA and identify transcription from previously unknown genes or for ChIP-on-chip studies to map transcription factor binding sites. We thought that they might be extremely useful for our project, if we could use them for aCGH. In this way, we could see if our e-strains carried gross chromosomal aberrations, such as aneuploidies, interstitial deletions or amplifications, non-reciprocal translocations, and possibly single-nucleotide mutations. Designed to interrogate the yeast genome with a 5bp resolution: Gresham et al., Science (2006)

Experimental protocol: Extraction of genomic DNA with Phenol / Chloroform / Isoamylalcohol “Controlled” fragmentation with DNase I (5 min at 37 °C) End-labeling with TdT and biotin-dUTP Hybridize on Affy chips Stain with streptavidin-PE Wash and scan chips Ladder 75 mU DNase I 150 mU DNase I 25 200 500 1000 2000 4000 6000 Fragment length (nt) Strain 7a-1 Strain 2b (wt)

2b (low DNase I, large fragments) 7a-1 (low DNase I, large fragments) 2b (high DNase I, small fragments) 7a-1 (high DNase I, small fragments)

Limitations: What genome changes can we see by aCGH? Polyploidization? Aneuploidization? Interstitial deletions? Reciprocal translocations? Non-reciprocal translocations? Single-nucleotide mutations? Amplifications? Albertson & Pinkel, Hum Mol Genet (2003)

Observation #1: Deletion of the MYO1 locus +1 MYO1 locus log2(ratio) -10 +1 log2(ratio) -10 A second piece of information provided by this array-CGH experiment, was that there was a progressive decay in signal intensity towards the end of each chromosome in all analyzed e-strains. We interpreted this finding as a shortening of the telomeres that is giving rise to a heterogeneous population of cells, each with its own specific telomere length. To test this hypothesis, we will perform Southern blots using probes designed to interrogate telomeric as well as subtelomeric regions. We can speculate that telomere instability might cause local gains or losses of genetic loci or local chromatin rearrangements that might in turn affect expression of genes residing at the ends of each chromosome. This could represent another mechanism by which variation can be introduced in an evolving population, upon which selection can then do its job. In addition, telomere instability is highly associated with genome instability and somatic evolution of cancer cells. Another important result we obtained from this CGH experiment, is that there was no sign of internal deletions or amplification, and neither of non-reciprocal translocations. The only other type of chromosomal aberration that might have occurred, but that CGH would have been unable to detect, is reciprocal translocation. But this type of aberration should be visible by pulse-field gel electrophoresis. Chromosome VIII

Observation #2: “Duplication” of the TRP1 locus +10 TRP1 log2(ratio) -1 +10 log2(ratio) -1 A second piece of information provided by this array-CGH experiment, was that there was a progressive decay in signal intensity towards the end of each chromosome in all analyzed e-strains. We interpreted this finding as a shortening of the telomeres that is giving rise to a heterogeneous population of cells, each with its own specific telomere length. To test this hypothesis, we will perform Southern blots using probes designed to interrogate telomeric as well as subtelomeric regions. We can speculate that telomere instability might cause local gains or losses of genetic loci or local chromatin rearrangements that might in turn affect expression of genes residing at the ends of each chromosome. This could represent another mechanism by which variation can be introduced in an evolving population, upon which selection can then do its job. In addition, telomere instability is highly associated with genome instability and somatic evolution of cancer cells. Another important result we obtained from this CGH experiment, is that there was no sign of internal deletions or amplification, and neither of non-reciprocal translocations. The only other type of chromosomal aberration that might have occurred, but that CGH would have been unable to detect, is reciprocal translocation. But this type of aberration should be visible by pulse-field gel electrophoresis. Chromosome IV Caveat #1: No information on where the signal comes from!

Caveat #2: Highly repetitive sequences! (aka “Saturation” effect) +1 log2(ratio) -1 +1 log2(ratio) A second piece of information provided by this array-CGH experiment, was that there was a progressive decay in signal intensity towards the end of each chromosome in all analyzed e-strains. We interpreted this finding as a shortening of the telomeres that is giving rise to a heterogeneous population of cells, each with its own specific telomere length. To test this hypothesis, we will perform Southern blots using probes designed to interrogate telomeric as well as subtelomeric regions. We can speculate that telomere instability might cause local gains or losses of genetic loci or local chromatin rearrangements that might in turn affect expression of genes residing at the ends of each chromosome. This could represent another mechanism by which variation can be introduced in an evolving population, upon which selection can then do its job. In addition, telomere instability is highly associated with genome instability and somatic evolution of cancer cells. Another important result we obtained from this CGH experiment, is that there was no sign of internal deletions or amplification, and neither of non-reciprocal translocations. The only other type of chromosomal aberration that might have occurred, but that CGH would have been unable to detect, is reciprocal translocation. But this type of aberration should be visible by pulse-field gel electrophoresis. Ty1 LTR Full-length Ty1 Full-length Ty1 -1 Chromosome II

Observation #3: Gradual loss of signal towards telomeres +1 log2(ratio) -1 +1 A second piece of information provided by this array-CGH experiment, was that there was a progressive decay in signal intensity towards the end of each chromosome in all analyzed e-strains. We interpreted this finding as a shortening of the telomeres that is giving rise to a heterogeneous population of cells, each with its own specific telomere length. To test this hypothesis, we will perform Southern blots using probes designed to interrogate telomeric as well as subtelomeric regions. We can speculate that telomere instability might cause local gains or losses of genetic loci or local chromatin rearrangements that might in turn affect expression of genes residing at the ends of each chromosome. This could represent another mechanism by which variation can be introduced in an evolving population, upon which selection can then do its job. In addition, telomere instability is highly associated with genome instability and somatic evolution of cancer cells. Another important result we obtained from this CGH experiment, is that there was no sign of internal deletions or amplification, and neither of non-reciprocal translocations. The only other type of chromosomal aberration that might have occurred, but that CGH would have been unable to detect, is reciprocal translocation. But this type of aberration should be visible by pulse-field gel electrophoresis. log2(ratio) -1 Full sequence of chromosome II

Observation #4: Aneuploidies And this is the first big picture we got after performing Tiling Array-CGH on the 10 e-strains that evolved the best strategies for cytokinesis and growth. What is plotted here is the log-ratio of the hybridization intensity of each analyzed e-strain in comparison to an ancestral wild-type control, as a function of chromosomal coordinate. These data clearly indicate that all 10 analyzed e-strains carry at least one aneuploid chromosome. Chromosomal gains and losses are apparently non-randomly distributed, with some chromosomes that seem to be preferentially duplicated or lost. See for example chromosomes 3 or 13, which are present in extra copies in the majority of the e-strains; or chromosomes 5 and 7 that are always present in normal number. This suggests that some chromosomes may contain genes important for the adaptation of yeast to the loss of MYO1, while other chromosomes may harbor genes that (if over-expressed) could be toxic for the cell. Interestingly, e-strains 7a-1, 7a-2 and 7a-3, which all derive from the same initial survivor spore share a conserved pattern of aneuploidy: all three e-strains carry additional copies of chromosomes 2, 3, 11, 13 and 15. This strongly indicates that the changes in chromosome copy number occurred in the very first passages, before the 3 strains were separated. It also indicates that after that initial aneuploidization, the karyotype was stably inherited by the future generations. This is consistent with the notion that cytokinesis defects can cause chromosome instability, and corroborates the hypothesis that strains that have adapted to the chronic loss of MYO1 are expected to have an increased fidelity in chromosome segregation as compared to strains that experience an acute loss of MYO1. Chr.

Caveat #3: “Dilution” effect And this is the first big picture we got after performing Tiling Array-CGH on the 10 e-strains that evolved the best strategies for cytokinesis and growth. What is plotted here is the log-ratio of the hybridization intensity of each analyzed e-strain in comparison to an ancestral wild-type control, as a function of chromosomal coordinate. These data clearly indicate that all 10 analyzed e-strains carry at least one aneuploid chromosome. Chromosomal gains and losses are apparently non-randomly distributed, with some chromosomes that seem to be preferentially duplicated or lost. See for example chromosomes 3 or 13, which are present in extra copies in the majority of the e-strains; or chromosomes 5 and 7 that are always present in normal number. This suggests that some chromosomes may contain genes important for the adaptation of yeast to the loss of MYO1, while other chromosomes may harbor genes that (if over-expressed) could be toxic for the cell. Interestingly, e-strains 7a-1, 7a-2 and 7a-3, which all derive from the same initial survivor spore share a conserved pattern of aneuploidy: all three e-strains carry additional copies of chromosomes 2, 3, 11, 13 and 15. This strongly indicates that the changes in chromosome copy number occurred in the very first passages, before the 3 strains were separated. It also indicates that after that initial aneuploidization, the karyotype was stably inherited by the future generations. This is consistent with the notion that cytokinesis defects can cause chromosome instability, and corroborates the hypothesis that strains that have adapted to the chronic loss of MYO1 are expected to have an increased fidelity in chromosome segregation as compared to strains that experience an acute loss of MYO1.

Possible observation #1: Non-reciprocal translocations? A second piece of information provided by this array-CGH experiment, was that there was a progressive decay in signal intensity towards the end of each chromosome in all analyzed e-strains. We interpreted this finding as a shortening of the telomeres that is giving rise to a heterogeneous population of cells, each with its own specific telomere length. To test this hypothesis, we will perform Southern blots using probes designed to interrogate telomeric as well as subtelomeric regions. We can speculate that telomere instability might cause local gains or losses of genetic loci or local chromatin rearrangements that might in turn affect expression of genes residing at the ends of each chromosome. This could represent another mechanism by which variation can be introduced in an evolving population, upon which selection can then do its job. In addition, telomere instability is highly associated with genome instability and somatic evolution of cancer cells. Another important result we obtained from this CGH experiment, is that there was no sign of internal deletions or amplification, and neither of non-reciprocal translocations. The only other type of chromosomal aberration that might have occurred, but that CGH would have been unable to detect, is reciprocal translocation. But this type of aberration should be visible by pulse-field gel electrophoresis. Dunham et al., PNAS (2002)

Possible observation #2: Single-nucleotide changes? Probes on the chip Genomic DNA 5 10 15 20 25 30 35 40 45 +10 -10 log2(ratio) 5 10 15 20 25 30 35 40 45 A second piece of information provided by this array-CGH experiment, was that there was a progressive decay in signal intensity towards the end of each chromosome in all analyzed e-strains. We interpreted this finding as a shortening of the telomeres that is giving rise to a heterogeneous population of cells, each with its own specific telomere length. To test this hypothesis, we will perform Southern blots using probes designed to interrogate telomeric as well as subtelomeric regions. We can speculate that telomere instability might cause local gains or losses of genetic loci or local chromatin rearrangements that might in turn affect expression of genes residing at the ends of each chromosome. This could represent another mechanism by which variation can be introduced in an evolving population, upon which selection can then do its job. In addition, telomere instability is highly associated with genome instability and somatic evolution of cancer cells. Another important result we obtained from this CGH experiment, is that there was no sign of internal deletions or amplification, and neither of non-reciprocal translocations. The only other type of chromosomal aberration that might have occurred, but that CGH would have been unable to detect, is reciprocal translocation. But this type of aberration should be visible by pulse-field gel electrophoresis. Gresham et al., Science (2006)

Summary: What can be seen by CGH on Tiling Arrays? Anything that causes a change in the copy number of a DNA segment, e.g. aneuploidies, deletions/amplifications, non-reciprocal translocations, etc. Mutations that affect the hybridization of multiple overlapping probes, i.e. single-nucleotide changes. What can not be seen by CGH? Anything that does not cause a change in the copy number of a DNA segment, e.g. polyploidization, reciprocal translocations etc. If probes are too long and non-overlapping, single-nucleotide mutations will not be detectable. What are the most common pitfalls? No information about where the signal actually comes from! No reliable information from probes hybridizing to highly-repetitive sequence (because of “saturation” effect)! If some chromosomes are gained or lost, this will affect the log-ratios also of all other chromosomes (because of “dilution” effect)! And this is the first big picture we got after performing Tiling Array-CGH on the 10 e-strains that evolved the best strategies for cytokinesis and growth. What is plotted here is the log-ratio of the hybridization intensity of each analyzed e-strain in comparison to an ancestral wild-type control, as a function of chromosomal coordinate. These data clearly indicate that all 10 analyzed e-strains carry at least one aneuploid chromosome. Chromosomal gains and losses are apparently non-randomly distributed, with some chromosomes that seem to be preferentially duplicated or lost. See for example chromosomes 3 or 13, which are present in extra copies in the majority of the e-strains; or chromosomes 5 and 7 that are always present in normal number. This suggests that some chromosomes may contain genes important for the adaptation of yeast to the loss of MYO1, while other chromosomes may harbor genes that (if over-expressed) could be toxic for the cell. Interestingly, e-strains 7a-1, 7a-2 and 7a-3, which all derive from the same initial survivor spore share a conserved pattern of aneuploidy: all three e-strains carry additional copies of chromosomes 2, 3, 11, 13 and 15. This strongly indicates that the changes in chromosome copy number occurred in the very first passages, before the 3 strains were separated. It also indicates that after that initial aneuploidization, the karyotype was stably inherited by the future generations. This is consistent with the notion that cytokinesis defects can cause chromosome instability, and corroborates the hypothesis that strains that have adapted to the chronic loss of MYO1 are expected to have an increased fidelity in chromosome segregation as compared to strains that experience an acute loss of MYO1.

Karin Zueckert-Gaudenz Allison Peak Chris Seidel Rong Li lab: Acknowledgements: Microarray group: Karin Zueckert-Gaudenz Allison Peak Chris Seidel Rong Li lab: Giulia Rancati Rong Li And this is the first big picture we got after performing Tiling Array-CGH on the 10 e-strains that evolved the best strategies for cytokinesis and growth. What is plotted here is the log-ratio of the hybridization intensity of each analyzed e-strain in comparison to an ancestral wild-type control, as a function of chromosomal coordinate. These data clearly indicate that all 10 analyzed e-strains carry at least one aneuploid chromosome. Chromosomal gains and losses are apparently non-randomly distributed, with some chromosomes that seem to be preferentially duplicated or lost. See for example chromosomes 3 or 13, which are present in extra copies in the majority of the e-strains; or chromosomes 5 and 7 that are always present in normal number. This suggests that some chromosomes may contain genes important for the adaptation of yeast to the loss of MYO1, while other chromosomes may harbor genes that (if over-expressed) could be toxic for the cell. Interestingly, e-strains 7a-1, 7a-2 and 7a-3, which all derive from the same initial survivor spore share a conserved pattern of aneuploidy: all three e-strains carry additional copies of chromosomes 2, 3, 11, 13 and 15. This strongly indicates that the changes in chromosome copy number occurred in the very first passages, before the 3 strains were separated. It also indicates that after that initial aneuploidization, the karyotype was stably inherited by the future generations. This is consistent with the notion that cytokinesis defects can cause chromosome instability, and corroborates the hypothesis that strains that have adapted to the chronic loss of MYO1 are expected to have an increased fidelity in chromosome segregation as compared to strains that experience an acute loss of MYO1.