Tamoxifen induction of Cre recombinase does not cause long-lasting or sexually divergent responses in the CNS epigenome or transcriptome: implications for the design of aging studies

Chucair-Elliott, Ana J.; Ocañas, Sarah R.; Stanford, David R.; Hadad, Niran; Wronowski, Benjamin; Otalora, Laura; Stout, Michael B.; Freeman, Willard M.

doi:10.1007/s11357-019-00090-2

Cited by 21 publications

(50 citation statements)

References 87 publications

(109 reference statements)

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“…This is consistent with the concept that mCH is concentrated in neurons 1 and provides more specific detail that this is true when astrocytes or microglial alone are examined. As previously reported for the brain 14, 25, 26 , no non-CG hydroxymethylation (hmCH) was detected in any of the samples analyzed (Figure 7D) . To uncover potential cell type-specific differences in mCG patterns, methylation across cell-type marker genes (from −4kb in respect to the TSS and +4b from the TES) was compared for astrocytes (Figure 7E) and microglia (Figure 7H) .…”

Section: Resultssupporting

confidence: 84%

“…While it is sometimes simplistically believed hydroxymethylation and non-CpG methylation are restricted to neurons these findings provide evidence that while CG hydroxymethylation and non-CG methylation levels may be lower in non-neuronal cell populations yet they are not absent. To date, there is no explanation for why hydroxymethylation is not evident in the non-CG context but as previously reported this modification is absent or at a level below detection 14, 25 .…”

Section: Discussionmentioning

confidence: 86%

“…Of note, the Aldh1l1-cre/ERT2; NuTRAP and Cx3cr1-cre/ERT2; NuTRAP models will be abbreviated when necessary, as Aldh1l1-NuTRAP and Cx3cr1-NuTRAP, respectively. Testing of Tamoxifen (Tam) administration, for cre induction, effects on the epigenome and transcriptome in the CNS found no long-lasting, significant effects on DNA modifications or gene expression 14 .…”

Section: Resultsmentioning

confidence: 99%

“…At ∼3 months of age, mice received a daily intraperitoneal (ip) injection of tamoxifen (Tam) solubilized in 100% sunflower seed oil by sonication (100 mg/kg body weight, 20 mg/ml stock solution, #T5648; Millipore Sigma, St. Louis, MO) for five consecutive days 12, 14 .…”

Section: Methodsmentioning

confidence: 99%

“…Protocols were carried out as previously described 14 . For each input, negative, and positive INTACT-isolated sample 400 ng of gDNA was brought up to 50 µl volume with 1X low-EDTA TE buffer and sheared with a Covaris E220 sonicator (Covaris, Inc., Woburn, MA) to an average 200 base pair size using the following settings: intensity of 5, duty cycle of 10%, 200 cycles per burst, 2 cycles of 60 seconds, at 7 °C.…”

Section: Methodsmentioning

confidence: 99%

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Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

Chucair-Elliott

Ocañas

Stanford

et al. 2019

Preprint

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23Epigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type 24 specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and 25 introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and 26Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling 27 and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific CNS cell types. 28Paired analysis of the transcriptome and DNA modifications in astrocytes and microglia 29 demonstrates differential usage of DNA methylation and hydroxymethylation in CG and non-CG 30 contexts that corresponds to cell type-specific gene expression. Application of this approach in 31 LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in 32 inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model 33 and the validation approaches presented can be applied to any CNS cell type for which a cell 34 type-specific cre is available. 36Significant advances are being made in understanding the epigenome and its relationship with 37 gene expression in the brain 1-3 . However, the lack of approaches for paired analysis of DNA and 38RNA profiles at the cell type-specific level within the same animal is a significant limitation for the 39 field, given that epigenetic processes differ across CNS cell types at the level of chromatin 40 organization and DNA modifications 1,4 . Obtaining enriched cell populations by flow sorting 41 requires cell surface markers but these markers can change with experimental conditions and cell 42 sorting causes molecular, morphological, and functional changes, such as cell activation, that 43 could confound studies 3,5,6 . Single cell approaches 7 may overcome some of the challenges of 44 cell sorting but the scale of such studies, partial genomic coverage, restriction to only certain types 45 of endpoints, and continued potential for brain dissociation artifacts are limitations. 46This has led to development of transgenic labeling approaches to isolate RNA or DNA from 47 specific cell types. Ribosome labeling and RNA isolation methods, such as Translating Ribosome 48Affinity Purification (TRAP 8 ), and ribosome tagging (RiboTag 9 ), are gaining acceptance across 49 neuroscience studies examining the transcriptome. Similar approaches have been developed to 50 transgenically tag and allow isolation of nuclei and thus DNA (Isolation of Nuclei TAgged in 51 Specific Cell Types, INTACT) 10 . However, using separate transgenic mouse strains for DNA and 52RNA endpoints is a complicated and resource intensive approach. 53Here we describe an approach where Nuclear Tagging and Translating Ribosome Affinity 54Purification (NuTRAP) 11 is combined with well-established cell-specific inducible cre-recombinase 55 expressing systems 12,13 to perform paired transcriptomic and epigenomic analyses of specific 56 CNS cell types in a temporally controllable manner from a single mouse. ...

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