Evolution of Gene Regulation in Mammalian Tissues · chicken). Numerous tissues are being...
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Evolution of Gene Regulation in Mammalian Tissues Nida, Amanda Socha, Or Zuk, Aviv Regev, Eric Lander Broad Institute, Cambridge, MA, USA Experimental RNA Reference RNA (pool of all tissues) 3’ 5’ AAAA 5’ 3’ AAAA AAAA AAAA TTTT TTTT Promoter Promoter Antisense Promoter Antisense Promoter 5’ 5’ 3’ 3’ MMLV‐RT (reverse transcriptase) and oligo dT‐ promoter primers are added for first and second strand cDNA synthesis 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ C C C C AAAA AAAA UUUU UUUU Cy3 Cy3 Cy5 Cy5 Antisense Promoter Antisense Promoter Cy3 Cy3 Cy5 Cy5 C C C C UUUU 3’ 3’ 5’ 5’ UUUU 2 nd Strand cDNA 1 st Strand cDNA 1 st Strand cDNA 2 nd Strand cDNA cRNA 2 nd strand cDNA cRNA 2 nd strand cDNA cRNA cRNA MMLV‐RT 3’ 3’ 5’ 5’ MMLV‐RT Experimental RNA Reference RNA Reference and samples are combined and fragmented Sample hybridized to array, scanned and features are extracted. T7 RNA polymerase T7 RNA polymerase RNA polymerase and NTPs (bases) are added, along with either Cy3 or Cy5 CTPs (flourescent cytosines), to amplify directly labled cRNA Labeled cRNA is purified using microfiltration. Conclusions [1] King, M.C. & Wilson, A. C. Evolution at two levels in humans and chimpanzees. Science 188, 107-116 (1975). [2] Rifkin, S.A., Houle, D., Kim, J. & White, K. P. A mutation accumulation assay reveals a broad capacity for rapid evolution of gene expression. Nature 438, 220-223 (2005). [3] Chan, E.T. et al. Conservation of core gene expression in vertebrate tissues. Journal of Biology 8:33 (2009). [4] Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc 4(1), 44-57 (2009). [5] Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 4(5), P3. (2003) [6] Reich M, Liefeld T, Gould J, Lerner J, Tamayo P, Mesirov JP (2006) GenePattern 2.0 Nature Genetics 38 no. 5 , 500-501 (2006). doi:10.1038/ng0506-500 . References Methods Results I would like to thank Amanda Socha and Or Zuk for mentoring me for the past six weeks. The assistance of Courtney French, Ido Amit, Aviv Regev and Eric Lander was also invaluable. I would also like to thank Megan Rokop, Kate MacSwain and Allison Martino for editing this poster, and for giving me the opportunity to participate in this internship. Acknowledgements For each tissue and gene set, we tested for change in regulation between human and mouse. Shown are the gene sets with significant change in expression. Variation in genotype does not play a solitary role in the diversity of mammals; instead, it has been hypothesized that differences in gene expression contribute significantly to the diversity of life [1]. Differentially expressed genes can explain the divergence in an organism’s phenotype. Meanwhile, conserved expression profiles among various species may indicate that the precise expression level of a certain gene or module is under purifying selection [2,3], thus indicating the evolutionary importance of these gene regulatory networks. At present, over 30 mammalian genomes are available, many of them recently sequenced. In order to gain further insight into variation of gene expression throughout evolution, we have chosen to use microarray technology. Microarrays are the method of choice in most large-scale whole-transcriptome studies because they allow analysis of an entire genome in a single experiment through use of high density oligo-nucleotide based arrays. Currently, a large scale comparative gene expression study is in progress to evaluate conserved and diverged gene regulatory systems within twelve vertebrates: ten eutherian mammals, one marsupial (the opossum) and one non-mammalian vertebrate (the chicken). Numerous tissues are being investigated in order to gain a fuller understanding of evolutionary patterns of gene expression. Introduction My goal is to compare gene expression between human and mouse in eight tissues: spleen, heart ,brain, kidney, liver, testes, ovary and thymus. Phylogenetic Tree of 44 Vertebrates Source: UCSC = part of my project = part of future project Spleen Heart Brain Kidney Liver Testes Ovary Thymus Vs. • Greater similarity of expression within a species (colored quadrants, upper right and lower left) • High correlation between thymus and spleen, especially in mouse (arrows) • Brain and cerebellum highly correlated to each other, but not to other tissues (blue perimeter) • Correlation between corresponding tissues among mouse and human (circled) Mouse Brain Mouse Ovary Mouse Thymus Mouse Spleen Mouse Heart Mouse Kidney Mouse Lung Mouse Cerebellum Human Testes Human Liver Human Ovary Human Thymus Human Spleen Human Heart Human Kidney Human Lung Human Cerebellum Mouse Brain Mouse Ovary Mouse Thymus Mouse Spleen Mouse Heart Mouse Kidney Mouse Lung Mouse Cerebellum Human Testes Human Liver Human Ovary Human Thymus Human Spleen Human Heart Human Kidney Human Lung Human Cerebellum Correlation Tissue Correlation Analysis shows inter-species and intra-species conservation patterns Gene Set Analysis reveals differences in expression between species • The gene and tissue correlation analyses demonstrate high conservation of expression between corresponding tissues in human and mouse. • We have identified sets of genes which exhibit high conservation of expression and have shown that they are enriched in developmental genes, ion transport and other important processes. • We have discovered gene sets which show a consistent difference in expression between human and mouse in certain tissues. These genes will be followed up on using experimental validation. • Further profiling in other mammalian species will be performed in order to track down the evolutionary changes in these genes’ expression patterns. Clustered Expression Heat Map shows conserved and diverged sets of genes • Many genes exhibit a high correlation in relative expression across all tissues, as is indicated in the general shift to the right compared to the randomized correlation. Analysis of highly conserved genes (corr. > 0.9) using DAVID [4,5] Process P value System development 1.6E‐9 Ion transport 1.1E‐12 Cell differentiation 1.1E‐7 Plasma membrane part 5.8E‐13 Symporter activity 7.5E‐6 Taxis 4.4E‐6 Cell adhesion 3.1E‐5 Motor protein 6.2E‐6 Mitochondrial envelope 7.63‐5 Cell junction 8.1E‐8 Gene correlation analysis shows conserved expression between human and mouse Up regulated in human Up regulated in mouse KEY to Clustered Expression Heat Map [6] **Diverged expression: Genes are up regulated in mouse cerebellum and down regulated in human cerebellum. **Conserved expression: Genes are down regulated in human and mouse kidneys. Corresponding tissues between species are clustered together, with the exception of spleen and thymus, and cerebellum and brain. ** ** ** **Conserved expression: Genes are down regulated in human and mouse ovaries. = conserved expression = diverged expression = down regulated = up regulated
Transcript of Evolution of Gene Regulation in Mammalian Tissues · chicken). Numerous tissues are being...
Evolution of Gene Regulation in Mammalian TissuesNida, Amanda Socha, Or Zuk, Aviv Regev, Eric Lander
Broad Institute, Cambridge, MA, USA
ExperimentalRNA
ReferenceRNA
(poolofalltissues)
3’5’ AAAA
5’ 3’AAAA AAAA
AAAA
TTTT
TTTT
Promoter
Promoter
AntisensePromoter
AntisensePromoter
5’
5’
3’
3’
MMLV‐RT(reversetranscriptase)andoligodT‐promoterprimersareaddedforfirstandsecond
strandcDNAsynthesis
5’
5’
3’
3’
3’
3’
5’
5’C C
C CAAAA
AAAAUUUU
UUUU
Cy3 Cy3
Cy5 Cy5
AntisensePromoter
AntisensePromoter
Cy3 Cy3
Cy5 Cy5
C C
CC UUUU
3’
3’
5’
5’
UUUU
2ndStrandcDNA
1stStrandcDNA
1stStrandcDNA
2ndStrandcDNA
cRNA
2ndstrandcDNA
cRNA
2ndstrandcDNA
cRNA
cRNA
MMLV‐RT
3’
3’
5’
5’
MMLV‐RT ExperimentalRNA
ReferenceRNA
Referenceandsamplesarecombined
andfragmented
Samplehybridizedtoarray,scannedand
featuresareextracted.
T7RNApolymerase
T7RNApolymerase
RNApolymeraseandNTPs(bases)areadded,alongwitheitherCy3orCy5CTPs(flourescentcytosines),toamplifydirectlylabledcRNA
LabeledcRNAispurifiedusingmicrofiltration.
Conclusions
[1] King, M.C. & Wilson, A. C. Evolution at two levels in humans and chimpanzees. Science 188, 107-116 (1975).[2] Rifkin, S.A., Houle, D., Kim, J. & White, K. P. A mutation accumulation assay reveals a broad capacity for rapidevolution of gene expression. Nature 438, 220-223 (2005).[3] Chan, E.T. et al. Conservation of core gene expression in vertebrate tissues. Journal of Biology 8:33 (2009).[4] Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVIDBioinformatics Resources. Nature Protoc 4(1), 44-57 (2009).[5] Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. DAVID: Database forAnnotation, Visualization, and Integrated Discovery. Genome Biol. 4(5), P3. (2003)[6] Reich M, Liefeld T, Gould J, Lerner J, Tamayo P, Mesirov JP (2006) GenePattern 2.0 Nature Genetics 38 no. 5 ,500-501 (2006). doi:10.1038/ng0506-500.
ReferencesMethods
Results
I would like to thank Amanda Socha and Or Zuk for mentoring me forthe past six weeks. The assistance of Courtney French, Ido Amit, AvivRegev and Eric Lander was also invaluable.I would also like to thank Megan Rokop, Kate MacSwain and AllisonMartino for editing this poster, and for giving me the opportunity toparticipate in this internship.
Acknowledgements
For each tissue and gene set, we tested for change in regulation betweenhuman and mouse. Shown are the gene sets with significant change inexpression.
Variation in genotype does not play asolitary role in the diversity ofmammals; instead, it has beenhypothesized that differences in geneexpression contribute significantly tothe diversity of life [1]. Differentiallyexpressed genes can explain thedivergence in an organism’sphenotype. Meanwhile, conservedexpression profiles among variousspecies may indicate that the preciseexpression level of a certain gene ormodule is under purifying selection[2,3], thus indicating the evolutionaryimportance of these gene regulatorynetworks.
At present, over 30 mammalian genomes are available, many of them recentlysequenced. In order to gain further insight into variation of gene expression throughoutevolution, we have chosen to use microarray technology. Microarrays are the method ofchoice in most large-scale whole-transcriptome studies because they allow analysis of anentire genome in a single experiment through use of high density oligo-nucleotide basedarrays.
Currently, a large scale comparative gene expression study is in progress toevaluate conserved and diverged gene regulatory systems within twelve vertebrates: teneutherian mammals, one marsupial (the opossum) and one non-mammalian vertebrate (thechicken). Numerous tissues are being investigated in order to gain a fuller understandingof evolutionary patterns of gene expression.
Introduction
My goal is to compare gene expression between human and mouse ineight tissues:
spleen, heart ,brain, kidney, liver, testes, ovary and thymus.
Phylogenetic Tree of 44 VertebratesSource: UCSC
= part of my project
= part of future project
Spleen Heart Brain Kidney Liver Testes Ovary Thymus
Vs.
• Greater similarity of expression within a species (colored quadrants, upper right and lower left)• High correlation between thymus and spleen, especially in mouse (arrows)• Brain and cerebellum highly correlated to each other, but not to other tissues (blue perimeter)• Correlation between corresponding tissues among mouse and human (circled)
Mouse B
rain
Mouse O
vary
Mouse Thym
us
Mouse Spleen
Mouse H
eart
Mouse K
idney
Mouse Lung
Mouse C
erebellum
Hum
an Testes
Hum
an Liver
Hum
an Ovary
Hum
an Thymus
Hum
an Spleen
Hum
an Heart
Hum
an Kidney
Hum
an Lung
Hum
an Cerebellum
Mouse BrainMouse Ovary
Mouse ThymusMouse Spleen
Mouse HeartMouse Kidney
Mouse LungMouse Cerebellum
Human TestesHuman Liver
Human OvaryHuman Thymus
Human SpleenHuman Heart
Human KidneyHuman Lung
Human Cerebellum
Correlation
Tissue Correlation Analysis shows inter-speciesand intra-species conservation patterns
Gene Set Analysis reveals differences inexpression between species
• The gene and tissue correlation analyses demonstrate highconservation of expression between corresponding tissues in human andmouse.• We have identified sets of genes which exhibit high conservation ofexpression and have shown that they are enriched in developmentalgenes, ion transport and other important processes.• We have discovered gene sets which show a consistent difference inexpression between human and mouse in certain tissues. These geneswill be followed up on using experimental validation.• Further profiling in other mammalian species will be performed inorder to track down the evolutionary changes in these genes’ expressionpatterns.
Clustered Expression Heat Map shows conservedand diverged sets of genes
• Many genes exhibit a high correlation in relative expression across all tissues, as isindicated in the general shift to the right compared to the randomized correlation.
Analysis of highlyconserved genes (corr. >0.9) using DAVID [4,5]
Process PvalueSystemdevelopment 1.6E‐9
Iontransport 1.1E‐12Celldifferentiation 1.1E‐7
Plasmamembranepart 5.8E‐13Symporteractivity 7.5E‐6
Taxis 4.4E‐6Celladhesion 3.1E‐5Motorprotein 6.2E‐6
Mitochondrialenvelope 7.63‐5Celljunction 8.1E‐8
Gene correlation analysis shows conservedexpression between human and mouse
Up regulatedin human
Up regulatedin mouse
KEY to Clustered Expression Heat Map [6]
**Diverged expression:Genes are up regulated inmouse cerebellum anddown regulated in humancerebellum.
**Conserved expression:Genes are down regulatedin human and mousekidneys.
Corresponding tissues between species are clusteredtogether, with the exception of spleen and thymus, andcerebellum and brain.
**
**
**
**Conserved expression:Genes are down regulatedin human and mouseovaries.
= conserved expression
= diverged expression
= down regulated
= up regulated
