A focus on β Cells in Type 2 Diabetes
Pancreatic islet β cell dysfunction represents a core cause of most forms of diabetes. In type-1 diabetes (T1D), β cells become targeted for destruction by the immune system due to the production of auto-antigens. In type-2 diabetes (T2D), β cells lose their ability to function correctly, with an estimated 40-50% of β cell function lost by the time of T2D clinical diagnosis (Wysham and Shubrook). While obesity-induced cellular stress (Hudish et al.) and loss of epigenetic cellular identity (Cinti et al. and Son and Accili) play roles in the loss of β cell function, the mechanisms underlying sensitivity to/protection against obesity-related cellular stress remain largely unknown.
In progressive diseases such as T2D, populations of cells (in this case, β cells) progress from a functional to a dysfunctional state in a progressive manner, implying the existence of populations of fully functional, partially functional, and wholly dysfunctional cells at any given time, whose relative proportion shifts towards dysfunction over time. Therefore, we would expect heterogeneity within the cell population and the existence of continuous cellular paths from a functional to a dysfunctional state. The authors of our featured study (Wang et al.) identified β cell exposure to obesity as one (of many) settings where single-cell epigenetic profiling represents a critical requirement for a more profound understanding of disease mechanisms; here, the application of Paired-Tag identified a potential new target for the maintenance of β cell function in the presence of obesity-linked cell stress.
Applying Paired-tag to Explore Heterogeneity and Highlight Therapeutic Targets
The obesity-induced dysfunction of pancreatic islet β cells, which maintain glucose homeostasis by secreting insulin, contributes to T2D pathogenesis by inducing metabolic and inflammatory stress (Prentki et al. and Donath et al.). Both obesity and T2D represent significant challenges to public health systems; can a better understanding of these related conditions by analyzing single β cells facing obesity-induced stress lead to improved patient treatments?
Single-cell transcriptomic and epigenetic analyses have begun to explore the underlying mechanisms controlling the markedly heterogeneous results observed when analyzing the significant differences in gene expression, glucose-stimulated insulin secretion, and stress responses between normal and dysfunctional β cells. However, technical limitations have limited our understanding of how the epigenetic state and activity of distal regulatory elements known as gene enhancers (Schoenfelder and Fraser) contribute to heterogeneous gene expression and dysfunction in single β cells remains incompletely explored.
To overcome the known limitations of current analytical techniques and expand our knowledge regarding the heterogeneous nature of β cell dysfunction, researchers from the laboratory of Zong Wei (Mayo Clinic) took advantage of Paired-tag technology from Epigenome Technologies (Zhu et al.) to analyze transcriptomic and H3K4me1 or H3K27ac (enhancer-associated histone modifications) profiles from the same single β cells isolated from mice fed a normal diet (“lean”) or a high-fat diet (“obese”) to model metabolic/inflammatory stress.
Paired-Tag - Parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing - represents an exciting means to jointly profile histone modifications and gene expression at single-cell resolution and detect histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP–seq assays. Fascinatingly, the findings described in the Wei lab’s new Nature Communications study now confirm discoveries from previous single-cell islet studies; however, applying Paired-tag to its full potential revealed discrepancies in how H3K27ac and H3K4me1 regulate the gene expression dynamics associated with β cell dysfunction, which may significantly contribute to heterogeneity in β cell dysfunction (Wang et al.).
Paired-tag: Evaluating RNA Expression and H3K4me1 or H3K27ac in Pancreatic β cells
By comprehensively analyzing enhancer dynamics in β cells from islets isolated from lean and obese mice, Wang et al. simultaneously identified distinct gene expression signatures and enhancer epigenetic states correlating with elevated stress and subsequent β cell dysfunction in obese mice. The application of Paired-tag technology first revealed that stress-induced gene expression in obese mice accompanied concordant changes in H3K4me1 and H3K27ac at enhancer regions (describing so-called gene-regulatory networks). However, this advanced approach also described the existence of unique dynamics for H3K27ac and H3K4me1 alterations and the regulation of specific gene subsets by each modification. These results suggested that β cells exhibited substantial heterogeneity at the transcriptome and enhancer epigenetic status level driven by the obesity-induced stress contributing to β cell dysfunction and T2D pathogenesis.
Active gene enhancers generally possess high levels of H3K4me1 and H3K27ac (Creyghton et al. and Rada-Iglesias et al.); however, these two histone modifications typically display distinct localization patterns and dynamics determined by transcription factor binding and the balance between HAT/HDACs and HMT/KDMs. While bulk ChIP-seq studies (Kubo et al. and Kang et al.) have previously described discrepancies between H3K4me1 and H3K27ac occupancy, the findings of this Paired-tag-led study suggested that the observed discordance between these two histone modifications at gene enhancers contributes to the heterogeneity of stress-associated β cell dysfunction via heterogeneity in the levels of gene expression. Adding to the heterogeneity, the correlation between H3K27ac/H3K4me1 and transcription does not establish causality between enhancer activity and gene transcription, suggesting additional heterogeneity at the level of the genes affected.
Paired-tag: Defining the Importance of Primed Enhancers
Analysis of the Paired-tag dataset also supported the identification of H3K4me1Pos/H3K27acNeg “primed” enhancers (typically described in the context of developmental processes; Balsalobre and Drouin) in the β cells of lean mice (which were also occupied by the pioneering FoxA2 transcription factor). Fascinatingly, a similar analysis in obese mice highlighted the disappearance of primed enhancers, suppressing the expression of genes essential to metabolic functions and β cell identity and suggesting that the loss of these primed enhancers may present a distinct mechanism of cellular dysfunction. Furthermore, identifying FoxA2 as a vital modulator in the H3K4me1-RNA dataset (but not the H3K27ac-RNA dataset) suggested a key role for this transcription factor in regulating the heterogeneity of primed enhancers. Notably, the loss of the primed enhancer sites may also signify a reallocation of H3K4me1 to stress-induced enhancers, thereby indirectly contributing to stress-induced transcription.
Paired-tag Analysis Identifies a Potential Obesity-induced Type 2 Diabetes Treatment Approach
Finally, the authors employed intra-islet cell-cell communication analysis by applying NicheNet (Browaeys et al.) to Paired-tag data to identify the paracrine factors that regulate high-fat diet-induced transcriptome changes in β cells. Excitingly, the authors identified nerve growth factor (NGF) - previously uncharacterized in islets - as a protective paracrine signaling mechanism in β cells and revealed that NGF (with the help of the TrkA receptor) potently suppressed cell stress and ameliorated hyperglycemia in highly diabetic model mice, thereby suggesting an avenue for developing pharmacological approaches to mitigate damage from β cell stress.
The Power of Paired-tag: Bringing New Understanding to Obesity-induced Type 2 Diabetes
Applying Paired-Tag technology may enable quantum leaps forward in our understanding of disease development and significantly improve disease management. Here, the application of Paired-tag technology defined the mechanisms underpinning heterogeneity in β cell dysfunction when faced with obesity-associated stress, which supports a better understanding of T2D pathology and helped to reveal a potentially important therapeutic target.
So, what can Paired-tag technology offer to your research? See Nature Communications, October 2024, for more on the application of this innovative technology in this fascinating T2D study, and explore the Epigenome Technologies website to learn more about how our services, assays, and resources can boost your research.
By Stuart P. Atkinson
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