Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)

Amin, Nirav M.; Greco, Todd M.; M., Kuchenbrod, Lauren; Rigney, Maggie M.; Chung, Mei I.; Wallingford, John B.; Cristea, Ileana M.; Conlon, Frank L.

doi:10.1242/dev.098327

Cited by 48 publications

(46 citation statements)

References 147 publications

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“…INTACT can enable profiling all of these levels of regulation from the same preparation of nuclei, as shown for mouse neuronal cells (Mo et al, 2015). Access to nuclei can also improve chromatin immunopurification and chromosome conformation studies (Rodriguez-Granados et al, 2016), enable RNAprotein interaction analyses (Foley et al, 2017), as well as refine proteomic analyses by allowing access to nuclei of discrete cell-or tissue-types (Amin et al, 2014). INTACT and other methods for isolation of nuclei vary in their advantages and disadvantages.…”

Section: Resultsmentioning

confidence: 99%

Nuclear Transcriptomes at High Resolution Using Retooled INTACT

et al. 2017

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Isolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here, we describe transfer of the isolation of nuclei from tagged specific cell types (INTACT) to the monocot rice (Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear-envelope-targeting domain of the nuclear tagging fusion (NTF) protein with an outer nuclear-envelope-anchored domain. This modified NTF was combined with codon-optimized Escherichia coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping, and profiling of the complete nuclear transcriptome, including a ribosomal RNA degradation procedure that minimizes pre-ribosomal RNA (pre-rRNA) transcripts. A high-resolution comparison of nuclear and steady-state poly(A) + transcript populations of seedling root tips confirmed the capture of pre-messenger RNA (pre-mRNA) and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell types of rice and other species.

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Section: Resultsmentioning

confidence: 99%

Nuclear Transcriptomes at High Resolution Using Retooled INTACT

et al. 2017

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“…With stable isotope labeling with amino acids in cell culture (SILAC), the entire cellular proteome can be evaluated, including global post-translational modifications 45 or for nuclear protein components from cardiomyocytes in Xenopus 46 . These proteomic approaches provide entry points to identify other important factors in cardiac biology.…”

Section: Cardiac Transcription Factors and Co-factorsmentioning

confidence: 99%

Investigating the Transcriptional Control of Cardiovascular Development

Kathiriya

Nora

Bruneau

2015

Circulation Research

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Transcriptional regulation of thousands of genes instructs complex morphogenetic and molecular events for heart development. Cardiac transcription factors (TFs) choreograph gene expression at each stage of differentiation by interacting with co-factors, including chromatin-modifying enzymes, and by binding to a constellation of regulatory DNA elements. Here, we present salient examples relevant to cardiovascular development and heart disease and review techniques that can sharpen our understanding of cardiovascular biology. We discuss the interplay between cardiac TFs, cis-regulatory elements and chromatin as dynamic regulatory networks, to orchestrate sequential deployment of the cardiac gene expression program.

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“…An alternate approach is INTACT (isolation of nuclei tagged in specific cell types; Deal and Henikoff, 2010), which uses affinity purification to isolate tagged nuclei. Captured nuclei can be used for gene expression, epigenomic, and proteomic profiling (Amin et al, 2014; Henry et al, 2012; Steiner et al, 2012). …”

Section: Introductionmentioning

confidence: 99%

Epigenomic Signatures of Neuronal Diversity in the Mammalian Brain

Mukamel

Davis

et al. 2015

Neuron

633

879

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SUMMARY Neuronal diversity is essential for mammalian brain function but poses a challenge to molecular profiling. To address the need for tools that facilitate cell-type-specific epigenomic studies, we developed the first affinity purification approach to isolate nuclei from genetically defined cell types in a mammal. We combine this technique with next-generation sequencing to show that three subtypes of neocortical neurons have highly distinctive epigenomic landscapes. Over 200,000 regions differ in chromatin accessibility and DNA methylation signatures characteristic of gene regulatory regions. By footprinting and motif analyses, these regions are predicted to bind distinct cohorts of neuron subtype-specific transcription factors. Neuronal epigenomes reflect both past and present gene expression, with DNA hyper-methylation at developmentally critical genes appearing as a novel epigenomic signature in mature neurons. Taken together, our findings link the functional and transcriptional complexity of neurons to their underlying epigenomic diversity.

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Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)

Cited by 48 publications

References 147 publications

Nuclear Transcriptomes at High Resolution Using Retooled INTACT

Nuclear Transcriptomes at High Resolution Using Retooled INTACT

Investigating the Transcriptional Control of Cardiovascular Development

Epigenomic Signatures of Neuronal Diversity in the Mammalian Brain

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