- Email: [email protected]
Museum Epigenomics: Charting the Future by Unlocking the Past
TREE 2635 No. of Pages 5
Trends in Ecology & Evolution
Forum
Museum Epigenomics: Charting the Future by Unlocking the Past
needed to demonstrate the significance of epigenomic adaptation and effectuate its application in species management. One opportunity to fast-track the collection of temporal epigenomic data is Erin E. Hahn ,1,@ through the mobilisation of historical epigenomes (Figure 1). While museum Alicia Grealy ,1 specimens present numerous and subMarina Alexander ,1,@ and stantial challenges to molecular analyses Clare E. Holleley 1,*,@ (Figure 2) [4,5], they provide unparalleled records of biodiversity spanning the most Epigenomic state preserved in mu- recent and ongoing mass extinction. Here, we describe the potential of diverse seum specimens could be leveraged archival material to yield epigenomic to provide unique insights into gene information and highlight opportunities regulation trends associated with for future research that will allow muaccelerating environmental change seum epigenomics to flourish as an during the Anthropocene. We ad- emerging field.
dress the challenges facing museum epigenomics and propose a collabo- Once considered poor sources of DNA, rative framework for researchers and museum specimens are now recognised as a valuable and informative genomic curators to explore this new field. To mitigate the effects of the ongoing human-caused mass extinction (https:// ipbes.net/global-assessment), it is crucial to understand the adaptive capacity of species in response to rapid environmental change. Adaptation via evolution of the genome occurs on the timescale of many generations, by which time populations under stress may be driven to extinction. Alternatively, at short timescales, rapid adaptation can occur through dynamic interactions between the genome and the epigenome (see Glossary) [1–3]. Mapping epigenomic change across time and in response to environmental stressors may improve predictions of species resilience and long-term fitness, and reduce extinction risk through targeted management actions. For example, epigenetic data could facilitate earlier detection of stress responses, enable finer resolution analyses of population structure in populations with low genetic diversity (e.g., endangered and invasive species), and provide rapid feedback on the success of mitigation efforts. Long-term observation of natural populations is still
resource due to improved methodologies of extracting and sequencing degraded DNA [6]. In contrast, museum epigenomics has not yet been widely attempted or embraced. At the time of writing, we found just one epigenetic study examining museum specimens collected within the past 150 years [7]. This dearth of literature is indicative of the challenges associated with molecular research using museum specimens (Figure 2). Aging effects, preservation-related material degradation, and high contamination risk necessitate extensive protocol development and optimisation for the characterisation of epigenetic marks in archival tissues. Furthermore, the very feature that makes epigenomic marks so tantalisingly informative, their tissue-specific responsiveness to the environment, necessitates inclusion of extensive controls and can complicate experimental design when repurposing nonrandomly collected specimens. Nevertheless, we propose that examination of epigenomic marks in museum specimens is feasible, albeit heavily dependent on specimen type and preservation method.
Glossary Bisulfite sequencing: bisulfite treatment of DNA converts cytosines to uracils but leaves methylated cytosines (specifically, 5-methylcytosine) unchanged. Sequencing of bisulfite-treated DNA and comparison with untreated DNA reveals sites of DNA methylation. ChIP-Seq: chromatin immunoprecipitation followed by sequencing is used to analyse protein interactions with DNA. In the ChIP procedure, chromatin is crosslinked and antibodies against target proteins are used to enrich for bound DNA, which is then sequenced. Chromatin: the complex of DNA and proteins that packages DNA into chromosomes in eukaryotic cells. Crosslinking: tissue fixation is typically achieved through treatment with formalin (4% w/v formaldehyde in a buffered salt solution). Formaldehyde induces covalent chemical bonds within and between DNA and proteins. Cytosine deamination: removal of an amine group converts cytosine into uracil. This process occurs spontaneously in living cells but is corrected by the cellular machinery. Postmortem deamination accumulates spontaneously as DNA ages. DNA methylation: methylation is the addition of a methyl (CH3) group to the nucleotide base cytosine or, less commonly, adenine. Cytosine methylation most commonly occurs at the fifth position in the base’s six atom ring (5-methylcytosine). DNA methylation alters gene expression, however, the extent, placement and effect vary between taxa. Epigenome: chemical modifications to the genome that result in changes in gene expression that cannot be explained by changes in DNA sequence. While it is common in other fields to include a dependency upon heritability in a definition of epigenomics, we do not invoke this meaning because dynamic (plastic) changes that are not inherited may still be indicative of an adaptive response to environmental challenges. Histone modifications: histones are the predominant protein component of chromatin, acting as spools around which the DNA is wrapped. Chemical modifications are deposited on specific histones to regulate gene expression. Addition of methyl, acetyl, phosphate, and ubiquitin groups on histones can increase or decrease gene expression, depending on position and context. Mass spectrometry: a method of characterising proteins based on the mass-to-charge ratio of their molecular subunits. Mass spectrometry can be used to read the amino acid sequence of proteins, characterise protein interactions, and detect chemical modifications. Noncoding RNA: functional RNAs that are transcribed from DNA but not translated into protein. These RNAs act to regulate gene expression. Nucleosome: the primary packaging unit of DNA, comprised of a stretch of DNA coiled around a complex of eight histones. Positioning of nucleosomes along the DNA strand affects gene expression.
Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx
1
Trends in Ecology & Evolution
Cells employ a variety of epigenomic mechanisms to regulate gene expression, including DNA methylation, histone modifications, noncoding RNAs, and nucleosome positioning, the markers of which will be preserved to varying degrees in museum specimens (Figure 1). DNA methylation mapping is the most immediately transferable technique to museum epigenomics [7], as it is the most extensively studied epigenetic mark in nonmodel species and can be conducted using equipment standard to most molecular genetics laboratories. Gene expression can be measured directly through RNA and protein analyses, including whole transcriptome and proteome sequencing. Although more technically challenging, measurement of RNA, protein, and chromatin-level epigenomic marks has become routine practice in many molecular genetics laboratories and may also be transferable to museum specimens. The appropriate epigenomic marker to assay from museum specimens will depend on the information provided by the marker as well as the preservation of that marker in available specimens. To catalogue taxonomically diverse specimens, museums employ a variety of preservation methods, each presenting distinct technical challenges for molecular work, but potentially offering opportunities to recover epigenetic information (Figure 1). Existing protocols for epigenomic analysis can immediately be applied to flash frozen specimens (Figure 1) but such samples only span the past few decades, limiting temporal representation. In contrast, older, more abundant, and data-rich dry and fixed specimens present unique opportunities to characterise deeper historical trends in gene expression. Fixed soft tissue collections, in particular, have the potential to characterise tissue-specific phenotypes that may be more reflective of adaptation to environmental change than ossified tissue. Additionally, formalin- or ethanol-fixed 2
specimens may offer the only opportunity to retrieve historical epigenetic information from many species that are recognised as sentinels of ecosystem health (e.g., amphibians, marine life, plants, and insects). To access the epigenomic information contained in these specimens, it is necessary to rethink our aversion to formalin and ethanol preservation. Our optimistic outlook on the future of museum epigenomics stems from advances made in processing ancient [8] and formalin-fixed, paraffin-embedded (FFPE) tissue, both of which present significant challenges due to molecular degradation and modification. Degradation of museum specimens, ranging in age from tens to hundreds of years old, generally falls between that of ancient (thousands of years old) and fresh specimens. Thus, protocols developed for ancient or fresh specimens will need to be optimised for the unique molecular condition of museum specimens. Encouragingly, bisulfite sequencing has produced high resolution methylome mapping of ancient human remains aged 230 to 4500 years before present [9]. In some cases, patterns of age-related degradation can be informative of epigenomic state. In specimens greater than 4000 years old, cytosine deamination patterns enable mapping of DNA methylation [10–12] and genomic sequence coverage bias can be used to infer nucleosome positioning [11,12]. Characterisation of the patterns of degradation under specific preservation treatments may facilitate similar approaches in museum specimens. Also in ancient specimens, mass spectrometry has been used to characterise proteins from extinct species, for example, mastodon (Mammut americanum) [13] and RNA-Seq is possible from archaeological specimens, for example, ancient maize (Zea mays) kernels [14], supporting the feasibility of RNA and protein analyses in museum specimens. In addition to age-related molecular degradation, museum specimens
Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx
Proteome: the complement of proteins expressed by a cell, tissue, or organism. The proteome changes as gene expression is up- or downregulated and thus reflects proteins that are present in a given cell, tissue, or organism at a given time. Transcriptome: the complement of RNA transcripts expressed by a cell, tissue, or organism. The transcriptome can refer to both coding (messenger) and noncoding RNAs, however, it generally refers only to coding RNAs, those that will be translated into proteins. Voucher specimen: a representative sample retained as a reference for an identified taxon and deposited in an accessible and permanent collection facility.
preserved with formalin suffer from significant DNA degradation associated with crosslinking. Clinical medicine techniques for processing FFPE tissue for RNA-Seq [15], ChIP-Seq [16], and DNA methylation profiling [17] may be transferable to formalin-fixed ethanol-preserved collection items if optimised to contend with additional challenges. Whilst clinical FFPE samples are the closest modern analogue to formalin-fixed museum specimens, they are not exactly comparable, because FFPE tissue is typically very briefly fixed prior to paraffin embedding; whereas museum formalin samples are usually subjected to longer fixation and may suffer subsequent degradation during long-term ethanol storage. Additionally, museum preparation methods use a wider variety of fixatives (buffered and unbuffered) compared with standardised FFPE protocols. By unlocking the ‘vault’ containing historical epigenomic data, a plethora of environmental questions could be addressed. Temporal gene expression patterns and shifts from baseline epigenetic signatures could shed light on the biological impacts of global issues such as climate change, emerging pathogens, overexploitation, and pollution. Museum epigenomics could be used to understand, predict, and control the spread of invasive species, including crop pests. Through examination of voucher specimens, epigenotypes can be linked to
Trends in Ecology & Evolution
Trends in Ecology & Evolution
Figure 1. Museum Collections Preserve Signatures of Gene Expression and Elements of the Epigenome. (A) Traditional specimen preservation methods include drying and liquid fixation. Over the past few decades and with DNA analysis in mind, collections have adopted RNAlater and cryo-preservation of fresh tissue. Preservation method selection is driven by specimen type and the features to be preserved. (B) Museum specimens can provide direct measures of gene expression and complementary measures of epigenomic regulation. Theoretically, fresh museum specimens pose few additional processing challenges compared with fresh contemporary specimens. Fixed specimens may present significant barriers to epigenomic analyses due to intra- and intermolecular crosslinks induced by formalin. However, fixed specimens represent an extensive resource if formalin damage can be circumvented or exploited to infer epigenomic state. Analyses of dry specimens may be restricted to DNA methylation but opportunities may exist for nucleosome mapping and protein characterisation.
Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx
3
Trends in Ecology & Evolution
Trends in Ecology & Evolution
Figure 2. Collaborative Solutions to the Challenges Impeding Museum Epigenomics. Museum epigenomics faces a number of challenges inhibiting wide adoption. Here, we offer suggestions for both researchers and curators to facilitate collaboration in preparation for increasing interest in this promising research area. Research collections with embedded molecular and curatorial expertise are well-positioned to lead this effort. 4
Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx
Trends in Ecology & Evolution
phenotypic and genotypic data. Although promising, museum epigenomics faces technical challenges requiring concerted collaborative effort by researchers and curators to surmount (Figure 2). To extract informative epigenomic data from museum specimens, researchers must develop customised protocols and align biological questions with epigenetic marks, extraction technique, and specimens. Likewise, to facilitate advancement of museum epigenomics while increasing collection value and preserving resources, curators can identify suitable collection objects for use in optimisation and account for future epigenetic work in planning collection strategies and preservation workflows. Through collaboration, innovation, and resource sharing, we anticipate there will be an explosion of epigenomic studies using museum specimens in the near future. Acknowledgements The study was supported financially through CSIRO Environomics Future Science Platform funding awarded to C.H. This study was conceived by C.H.
All authors contributed to the writing and editing of the manuscript. Figures were prepared by A.G. and
6. 7.
E.H. We thank Andrea Wild for providing collections images for Figure 1 and Millie Menzies for assisting with artwork for Figure 2. We thank Olly Berry, Margaret Cawsey, and Leo Joseph for their valuable comments
8.
on the manuscript. 9. 1
National Research Collections Australia, Commonwealth Scientific Industrial Research Organisation, Canberra, ACT 2601, Australia 10.
*Correspondence: [email protected] (C.E. Holleley). @ Twitter: @erin_e_hahn (E.E. Hahn), @HolleleyClare (C.E. Holleley), and @Supergeek1979 (M. Alexander).
11.
https://doi.org/10.1016/j.tree.2019.12.005 12. Crown Copyright © 2019 Published by Elsevier Ltd. All rights reserved. 13. References 1. McNew, S.M. et al. (2017) Epigenetic variation between urban and rural populations of Darwin’s finches. BMC Evol. Biol. 17, 183 2. Sevane, N. et al. (2019) Genome-wide differential DNA methylation in tropically adapted Creole cattle and their Iberian ancestors. Anim. Genet. 50, 15–26 3. Banta, J.A. and Richards, C.L. (2018) Quantitative epigenetics and evolution. Heredity 121, 210–224 4. Wehi, P.M. et al. (2012) Artefacts, biology and bias in museum collection research. Mol. Ecol. 21, 3103–3109 5. Yeates, D.K. et al. (2016) Museums are biobanks: unlocking the genetic potential of the three billion specimens in the world’s biological collections. Curr. Opin. Insect Sci. 18, 83–88
14.
15.
16.
17.
Holmes, M.W. et al. (2016) Natural history collections as windows on evolutionary processes. Mol. Ecol. 25, 864–881 Rubi, T.L. et al. (2019) Museum epigenomics: characterizing cytosine methylation in historic museum specimens. Mol. Ecol. Resour. Published online November 04 2019. https://doi.org/10.1111/17550998.13115 Hanghøj, K. and Orlando, L. (2019) Ancient epigenomics. In Paleogenomics: Genome-Scale Analysis of Ancient DNA (Lindqvist, C. and Rajora, O.P., eds), pp. 75–111, Springer Smith, R.W.A. et al. (2015) Detection of cytosine methylation in ancient DNA from five Native American populations using bisulfite sequencing. PLoS One 10, e0125344 Gokhman, D. et al. (2014) Reconstructing the DNA methylation maps of the Neandertal and the Denisovan. Science 344, 523–527 Pedersen, J.S. et al. (2014) Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome. Genome Res. 24, 454–466 Hanghøj, K. et al. (2016) Fast, accurate and automatic ancient nucleosome and methylation maps with epiPALEOMIX. Mol. Biol. Evol. 33, 3284–3298 Asara, J.M. et al. (2007) Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry. Science 316, 280–285 Fordyce, S.L. et al. (2013) Deep sequencing of RNA from ancient maize kernels. PLoS One 8, e50961 Lee, J.H. et al. (2015) Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat. Protoc. 10, 442–458 Amatori, S. et al. (2018) Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology. Clin. Epigenetics 10, 143 Ludgate, J.L. et al. (2017) A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA. BMC Med. Genet. 10, 54
Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx
5
Trends in Ecology & Evolution
Forum
Museum Epigenomics: Charting the Future by Unlocking the Past
needed to demonstrate the significance of epigenomic adaptation and effectuate its application in species management. One opportunity to fast-track the collection of temporal epigenomic data is Erin E. Hahn ,1,@ through the mobilisation of historical epigenomes (Figure 1). While museum Alicia Grealy ,1 specimens present numerous and subMarina Alexander ,1,@ and stantial challenges to molecular analyses Clare E. Holleley 1,*,@ (Figure 2) [4,5], they provide unparalleled records of biodiversity spanning the most Epigenomic state preserved in mu- recent and ongoing mass extinction. Here, we describe the potential of diverse seum specimens could be leveraged archival material to yield epigenomic to provide unique insights into gene information and highlight opportunities regulation trends associated with for future research that will allow muaccelerating environmental change seum epigenomics to flourish as an during the Anthropocene. We ad- emerging field.
dress the challenges facing museum epigenomics and propose a collabo- Once considered poor sources of DNA, rative framework for researchers and museum specimens are now recognised as a valuable and informative genomic curators to explore this new field. To mitigate the effects of the ongoing human-caused mass extinction (https:// ipbes.net/global-assessment), it is crucial to understand the adaptive capacity of species in response to rapid environmental change. Adaptation via evolution of the genome occurs on the timescale of many generations, by which time populations under stress may be driven to extinction. Alternatively, at short timescales, rapid adaptation can occur through dynamic interactions between the genome and the epigenome (see Glossary) [1–3]. Mapping epigenomic change across time and in response to environmental stressors may improve predictions of species resilience and long-term fitness, and reduce extinction risk through targeted management actions. For example, epigenetic data could facilitate earlier detection of stress responses, enable finer resolution analyses of population structure in populations with low genetic diversity (e.g., endangered and invasive species), and provide rapid feedback on the success of mitigation efforts. Long-term observation of natural populations is still
resource due to improved methodologies of extracting and sequencing degraded DNA [6]. In contrast, museum epigenomics has not yet been widely attempted or embraced. At the time of writing, we found just one epigenetic study examining museum specimens collected within the past 150 years [7]. This dearth of literature is indicative of the challenges associated with molecular research using museum specimens (Figure 2). Aging effects, preservation-related material degradation, and high contamination risk necessitate extensive protocol development and optimisation for the characterisation of epigenetic marks in archival tissues. Furthermore, the very feature that makes epigenomic marks so tantalisingly informative, their tissue-specific responsiveness to the environment, necessitates inclusion of extensive controls and can complicate experimental design when repurposing nonrandomly collected specimens. Nevertheless, we propose that examination of epigenomic marks in museum specimens is feasible, albeit heavily dependent on specimen type and preservation method.
Glossary Bisulfite sequencing: bisulfite treatment of DNA converts cytosines to uracils but leaves methylated cytosines (specifically, 5-methylcytosine) unchanged. Sequencing of bisulfite-treated DNA and comparison with untreated DNA reveals sites of DNA methylation. ChIP-Seq: chromatin immunoprecipitation followed by sequencing is used to analyse protein interactions with DNA. In the ChIP procedure, chromatin is crosslinked and antibodies against target proteins are used to enrich for bound DNA, which is then sequenced. Chromatin: the complex of DNA and proteins that packages DNA into chromosomes in eukaryotic cells. Crosslinking: tissue fixation is typically achieved through treatment with formalin (4% w/v formaldehyde in a buffered salt solution). Formaldehyde induces covalent chemical bonds within and between DNA and proteins. Cytosine deamination: removal of an amine group converts cytosine into uracil. This process occurs spontaneously in living cells but is corrected by the cellular machinery. Postmortem deamination accumulates spontaneously as DNA ages. DNA methylation: methylation is the addition of a methyl (CH3) group to the nucleotide base cytosine or, less commonly, adenine. Cytosine methylation most commonly occurs at the fifth position in the base’s six atom ring (5-methylcytosine). DNA methylation alters gene expression, however, the extent, placement and effect vary between taxa. Epigenome: chemical modifications to the genome that result in changes in gene expression that cannot be explained by changes in DNA sequence. While it is common in other fields to include a dependency upon heritability in a definition of epigenomics, we do not invoke this meaning because dynamic (plastic) changes that are not inherited may still be indicative of an adaptive response to environmental challenges. Histone modifications: histones are the predominant protein component of chromatin, acting as spools around which the DNA is wrapped. Chemical modifications are deposited on specific histones to regulate gene expression. Addition of methyl, acetyl, phosphate, and ubiquitin groups on histones can increase or decrease gene expression, depending on position and context. Mass spectrometry: a method of characterising proteins based on the mass-to-charge ratio of their molecular subunits. Mass spectrometry can be used to read the amino acid sequence of proteins, characterise protein interactions, and detect chemical modifications. Noncoding RNA: functional RNAs that are transcribed from DNA but not translated into protein. These RNAs act to regulate gene expression. Nucleosome: the primary packaging unit of DNA, comprised of a stretch of DNA coiled around a complex of eight histones. Positioning of nucleosomes along the DNA strand affects gene expression.
Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx
1
Trends in Ecology & Evolution
Cells employ a variety of epigenomic mechanisms to regulate gene expression, including DNA methylation, histone modifications, noncoding RNAs, and nucleosome positioning, the markers of which will be preserved to varying degrees in museum specimens (Figure 1). DNA methylation mapping is the most immediately transferable technique to museum epigenomics [7], as it is the most extensively studied epigenetic mark in nonmodel species and can be conducted using equipment standard to most molecular genetics laboratories. Gene expression can be measured directly through RNA and protein analyses, including whole transcriptome and proteome sequencing. Although more technically challenging, measurement of RNA, protein, and chromatin-level epigenomic marks has become routine practice in many molecular genetics laboratories and may also be transferable to museum specimens. The appropriate epigenomic marker to assay from museum specimens will depend on the information provided by the marker as well as the preservation of that marker in available specimens. To catalogue taxonomically diverse specimens, museums employ a variety of preservation methods, each presenting distinct technical challenges for molecular work, but potentially offering opportunities to recover epigenetic information (Figure 1). Existing protocols for epigenomic analysis can immediately be applied to flash frozen specimens (Figure 1) but such samples only span the past few decades, limiting temporal representation. In contrast, older, more abundant, and data-rich dry and fixed specimens present unique opportunities to characterise deeper historical trends in gene expression. Fixed soft tissue collections, in particular, have the potential to characterise tissue-specific phenotypes that may be more reflective of adaptation to environmental change than ossified tissue. Additionally, formalin- or ethanol-fixed 2
specimens may offer the only opportunity to retrieve historical epigenetic information from many species that are recognised as sentinels of ecosystem health (e.g., amphibians, marine life, plants, and insects). To access the epigenomic information contained in these specimens, it is necessary to rethink our aversion to formalin and ethanol preservation. Our optimistic outlook on the future of museum epigenomics stems from advances made in processing ancient [8] and formalin-fixed, paraffin-embedded (FFPE) tissue, both of which present significant challenges due to molecular degradation and modification. Degradation of museum specimens, ranging in age from tens to hundreds of years old, generally falls between that of ancient (thousands of years old) and fresh specimens. Thus, protocols developed for ancient or fresh specimens will need to be optimised for the unique molecular condition of museum specimens. Encouragingly, bisulfite sequencing has produced high resolution methylome mapping of ancient human remains aged 230 to 4500 years before present [9]. In some cases, patterns of age-related degradation can be informative of epigenomic state. In specimens greater than 4000 years old, cytosine deamination patterns enable mapping of DNA methylation [10–12] and genomic sequence coverage bias can be used to infer nucleosome positioning [11,12]. Characterisation of the patterns of degradation under specific preservation treatments may facilitate similar approaches in museum specimens. Also in ancient specimens, mass spectrometry has been used to characterise proteins from extinct species, for example, mastodon (Mammut americanum) [13] and RNA-Seq is possible from archaeological specimens, for example, ancient maize (Zea mays) kernels [14], supporting the feasibility of RNA and protein analyses in museum specimens. In addition to age-related molecular degradation, museum specimens
Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx
Proteome: the complement of proteins expressed by a cell, tissue, or organism. The proteome changes as gene expression is up- or downregulated and thus reflects proteins that are present in a given cell, tissue, or organism at a given time. Transcriptome: the complement of RNA transcripts expressed by a cell, tissue, or organism. The transcriptome can refer to both coding (messenger) and noncoding RNAs, however, it generally refers only to coding RNAs, those that will be translated into proteins. Voucher specimen: a representative sample retained as a reference for an identified taxon and deposited in an accessible and permanent collection facility.
preserved with formalin suffer from significant DNA degradation associated with crosslinking. Clinical medicine techniques for processing FFPE tissue for RNA-Seq [15], ChIP-Seq [16], and DNA methylation profiling [17] may be transferable to formalin-fixed ethanol-preserved collection items if optimised to contend with additional challenges. Whilst clinical FFPE samples are the closest modern analogue to formalin-fixed museum specimens, they are not exactly comparable, because FFPE tissue is typically very briefly fixed prior to paraffin embedding; whereas museum formalin samples are usually subjected to longer fixation and may suffer subsequent degradation during long-term ethanol storage. Additionally, museum preparation methods use a wider variety of fixatives (buffered and unbuffered) compared with standardised FFPE protocols. By unlocking the ‘vault’ containing historical epigenomic data, a plethora of environmental questions could be addressed. Temporal gene expression patterns and shifts from baseline epigenetic signatures could shed light on the biological impacts of global issues such as climate change, emerging pathogens, overexploitation, and pollution. Museum epigenomics could be used to understand, predict, and control the spread of invasive species, including crop pests. Through examination of voucher specimens, epigenotypes can be linked to
Trends in Ecology & Evolution
Trends in Ecology & Evolution
Figure 1. Museum Collections Preserve Signatures of Gene Expression and Elements of the Epigenome. (A) Traditional specimen preservation methods include drying and liquid fixation. Over the past few decades and with DNA analysis in mind, collections have adopted RNAlater and cryo-preservation of fresh tissue. Preservation method selection is driven by specimen type and the features to be preserved. (B) Museum specimens can provide direct measures of gene expression and complementary measures of epigenomic regulation. Theoretically, fresh museum specimens pose few additional processing challenges compared with fresh contemporary specimens. Fixed specimens may present significant barriers to epigenomic analyses due to intra- and intermolecular crosslinks induced by formalin. However, fixed specimens represent an extensive resource if formalin damage can be circumvented or exploited to infer epigenomic state. Analyses of dry specimens may be restricted to DNA methylation but opportunities may exist for nucleosome mapping and protein characterisation.
Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx
3
Trends in Ecology & Evolution
Trends in Ecology & Evolution
Figure 2. Collaborative Solutions to the Challenges Impeding Museum Epigenomics. Museum epigenomics faces a number of challenges inhibiting wide adoption. Here, we offer suggestions for both researchers and curators to facilitate collaboration in preparation for increasing interest in this promising research area. Research collections with embedded molecular and curatorial expertise are well-positioned to lead this effort. 4
Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx
Trends in Ecology & Evolution
phenotypic and genotypic data. Although promising, museum epigenomics faces technical challenges requiring concerted collaborative effort by researchers and curators to surmount (Figure 2). To extract informative epigenomic data from museum specimens, researchers must develop customised protocols and align biological questions with epigenetic marks, extraction technique, and specimens. Likewise, to facilitate advancement of museum epigenomics while increasing collection value and preserving resources, curators can identify suitable collection objects for use in optimisation and account for future epigenetic work in planning collection strategies and preservation workflows. Through collaboration, innovation, and resource sharing, we anticipate there will be an explosion of epigenomic studies using museum specimens in the near future. Acknowledgements The study was supported financially through CSIRO Environomics Future Science Platform funding awarded to C.H. This study was conceived by C.H.
All authors contributed to the writing and editing of the manuscript. Figures were prepared by A.G. and
6. 7.
E.H. We thank Andrea Wild for providing collections images for Figure 1 and Millie Menzies for assisting with artwork for Figure 2. We thank Olly Berry, Margaret Cawsey, and Leo Joseph for their valuable comments
8.
on the manuscript. 9. 1
National Research Collections Australia, Commonwealth Scientific Industrial Research Organisation, Canberra, ACT 2601, Australia 10.
*Correspondence: [email protected] (C.E. Holleley). @ Twitter: @erin_e_hahn (E.E. Hahn), @HolleleyClare (C.E. Holleley), and @Supergeek1979 (M. Alexander).
11.
https://doi.org/10.1016/j.tree.2019.12.005 12. Crown Copyright © 2019 Published by Elsevier Ltd. All rights reserved. 13. References 1. McNew, S.M. et al. (2017) Epigenetic variation between urban and rural populations of Darwin’s finches. BMC Evol. Biol. 17, 183 2. Sevane, N. et al. (2019) Genome-wide differential DNA methylation in tropically adapted Creole cattle and their Iberian ancestors. Anim. Genet. 50, 15–26 3. Banta, J.A. and Richards, C.L. (2018) Quantitative epigenetics and evolution. Heredity 121, 210–224 4. Wehi, P.M. et al. (2012) Artefacts, biology and bias in museum collection research. Mol. Ecol. 21, 3103–3109 5. Yeates, D.K. et al. (2016) Museums are biobanks: unlocking the genetic potential of the three billion specimens in the world’s biological collections. Curr. Opin. Insect Sci. 18, 83–88
14.
15.
16.
17.
Holmes, M.W. et al. (2016) Natural history collections as windows on evolutionary processes. Mol. Ecol. 25, 864–881 Rubi, T.L. et al. (2019) Museum epigenomics: characterizing cytosine methylation in historic museum specimens. Mol. Ecol. Resour. Published online November 04 2019. https://doi.org/10.1111/17550998.13115 Hanghøj, K. and Orlando, L. (2019) Ancient epigenomics. In Paleogenomics: Genome-Scale Analysis of Ancient DNA (Lindqvist, C. and Rajora, O.P., eds), pp. 75–111, Springer Smith, R.W.A. et al. (2015) Detection of cytosine methylation in ancient DNA from five Native American populations using bisulfite sequencing. PLoS One 10, e0125344 Gokhman, D. et al. (2014) Reconstructing the DNA methylation maps of the Neandertal and the Denisovan. Science 344, 523–527 Pedersen, J.S. et al. (2014) Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome. Genome Res. 24, 454–466 Hanghøj, K. et al. (2016) Fast, accurate and automatic ancient nucleosome and methylation maps with epiPALEOMIX. Mol. Biol. Evol. 33, 3284–3298 Asara, J.M. et al. (2007) Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry. Science 316, 280–285 Fordyce, S.L. et al. (2013) Deep sequencing of RNA from ancient maize kernels. PLoS One 8, e50961 Lee, J.H. et al. (2015) Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat. Protoc. 10, 442–458 Amatori, S. et al. (2018) Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology. Clin. Epigenetics 10, 143 Ludgate, J.L. et al. (2017) A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA. BMC Med. Genet. 10, 54
Trends in Ecology & Evolution, Month 2019, Vol. xx, No. xx
5