The Importance of Detailed Epigenomic Profiling of Different Cell Types within Organs

Figures & data

Figure 1. Mechanisms of epigenetic control.

The epigenome allows cell types with identical genomes (schematically depicted at the top) to exhibit differential gene expression patterns through a variety of mechanisms. They include, from left to right: regulatory RNAs of different types such as long noncoding RNAs, miRNAs and others; recruitment of transcription factors that can activate gene expression; enhancer-binding proteins that can activate gene expression by looping to promoters in cis or in trans (when different chromosomes interact); proteins like CTCF that mediate looping interactions in concert with other proteins such as cohesin and thereby support the three-dimensional organization of the genome; the binding of repressor proteins that can silence genes; DNA methylation that is usually absent in active regulatory regions such as promoters and enhancers (hydroxymethylation, thought to occur during DNA methylation removal, and other modifications can also occur); a variety of histone modifying enzymes that lead to protein interactions associated with different chromatin states; nucleosome remodeling complexes that can lead to the removal or deposition of nucleosomes in active and repressed genomic regions, respectively.

Figure 2. Diagram of the structure of the lung, resident cell types and the uses of cell type-specific epigenomes.

Only the right lung is shown. Airway and alveolar compartments and their cell types are indicated. The composition of human respiratory bronchioles has been poorly investigated but is thought to contain club cells and rare ciliated cells. At right, different applications for obtained epigenomes are indicated. At the bottom, an example is shown of epigenetic differences between lung cell types: histone 3 lysine 27 acetylation (H3K27Ac, a mark indicating an active enhancer) ChIP-seq data is compared between small airway epithelium, normal human lung fibroblasts and adult lung tissue. The presence of an enhancer is noted downstream of the FHAD gene in small airway epithelial cells but not in lung fibroblasts; the element is not clearly detectable in adult lung, which is a mix of many different cell types.

Table 1. Recommended epigenetic factors to be examined.

Epigenetic factor	Technique	Rationale	Feasible for low cell number approaches?
Noncoding RNA	Purification and whole transcriptome RNA sequencing (RNA-seq) adapted to different types of RNAs including miRNAs and long noncoding RNAs	Noncoding RNAs, including miRNAs and lncRNAs, are strongly implicated in gene regulation and are deregulated in disease processes. RNA-seq will simultaneously provide information on regulatory RNAs, gene expression, and splicing isoforms	Single cell RNA-seq is viable but may be very challenging for lncRNAs, many of which are lowly abundant. Single cell RNA-seq has been performed for lung cells [3] and other tissues
Open chromatin	Assay for transposase-accessible chromatin with high-throughput sequencing	This method requires fewer cells than other methods to map open chromatin, like measurement of DNase hypersensitive sites or formaldehyde-assisted identification of regulatory elements	Recently a single-cell method for ATAC-seq was published [4]
DNA methylation	Whole-genome bisulfite sequencing or nanopore-based sequencing	Genome-wide DNA methylation determination provides an unbiased assessment of the location of DNA methylation^†	Single cell bisulfite sequencing has been published [5], as well as solid state nanopore-based sequencing using the methyl-binding protein MBD1 [5]
H3K4me1	ChIP-seq	A histone mark associated with active and poised enhancer regions [6]	Low-cell-number approaches include iChIP-seq [7] or recovery via protection (RP)-ChIP-seq and favored amplification RP-ChIP-seq (FARP-ChIP-seq) [8]
H3K27Ac	ChIP-seq	A histone mark associated with active enhancers and promoters [6]	As above
H3K27me3	ChIP-seq	A histone mark indicative of Polycomb repression and gene inactivity [9]	As above
CTCF	ChIP-seq	CTCF is a chromatin-organizing protein involved in looping interactions between enhancers and promoters [10], among others	Due to the lower abundance of DNA-binding factor marks, low cell assays may not yet be within reach

^†Whole-genome bisulfite sequencing does not distinguish between methyl and hydroxyl-methyl C, an oxidized version of methylcytosine that is a likely intermediate in demethylation and that is common in the nervous system [11].

RNA: seq: RNA sequencing.

Wu AR , NeffNF , KaliskyTet al. Quantitative assessment of single-cell RNA-sequencing methods . Nat. Methods11 , 41 – 46 ( 2014 ).

 PubMed Web of Science ®Google Scholar

Buenrostro JD , WuB , ChangHY , GreenleafWJ . ATAC-seq: a method for assaying chromatin accessibility genome-wide . Curr. Protoc. Mol. Biol.109 , 21.29.1 – 9 ( 2015 ).

 PubMedGoogle Scholar

Smallwood SA , LeeHJ , AngermuellerCet al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity . Nat. Methods11 , 817 – 820 ( 2014 ).

 PubMed Web of Science ®Google Scholar

Roadmap Epigenomics Consortium , KundajeA , MeulemanWet al.Integrative analysis of 111 reference human epigenomes . Nature518 , 317 – 330 ( 2015 ).

 PubMed Web of Science ®Google Scholar

Lara-Astiaso D , WeinerA , Lorenzo-VivasEet al. Immunogenetics. Chromatin state dynamics during blood formation . Science345 , 943 – 949 ( 2014 ).

 PubMed Web of Science ®Google Scholar

Zheng X , YueS , ChenH , WeberB , JiaJ , ZhengY . Low-cell-number epigenome profiling aids the study of lens aging and hematopoiesis . Cell Rep.13 , 1505 – 1518 ( 2015 ).

 PubMed Web of Science ®Google Scholar

Bonasio R , TuS , ReinbergD . Molecular signals of epigenetic states . Science330 , 612 – 616 ( 2010 ).

 PubMed Web of Science ®Google Scholar

Dowen JM , FanZP , HniszDet al. Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes . Cell159 , 374 – 387 ( 2014 ).

 PubMed Web of Science ®Google Scholar

Globisch D , MünzelM , MüllerMet al. Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates . PLoS ONE5 , e15367 ( 2010 ).

 PubMed Web of Science ®Google Scholar