Beyond the Transcription Factor: The Under-Studied Role of Epigenomics in Lung Differentiation

Keywords::

• ATAC-seq
• chromatin dynamics
• chromatin structure
• DNA methylation
• epigenetic mechanisms
• lung development
• lung differentiation

The lung is one of the most complex organs in the human body. Composed of over a billion individual epithelial cells, this collaborative network facilitates gas exchange, regulates mucociliary clearance, defends against infection and maintains a host of other processes critical for our survival. Many decades of research have gone into defining the precise transcription factor cascades that first tracheal then branching morphogenesis employ to construct the epithelial component of our respiratory systems. Indeed, lineage tracing maps first developed in the mouse [1] have illuminated the origins of all epithelial cells in the adult lung, an important milestone in characterizing lung development and differentiation. Despite these achievements, studies enabled by recent technological breakthroughs in the isolation, culture and characterization of the lung epithelium are challenging our long-held definitions of the identities, forms and functions of lung epithelial cells [2–5]. Classical cell designations such as basal, goblet, club, ciliated and alveolar are being broken down by the application of revolutionary single-cell profiling technologies into specialized subtypes, intermediate cell states and highly plastic disease-driven stem populations [6,7]. When the meticulous approaches of so many of the world’s pre-eminent scientists have nonetheless overlooked these specialized, intermediate and transitory cell states that serve critical functions in determining cell fate, we must evaluate the latest scientific knowledge and technological innovations available and then reconsider the approach. To wholly understand lung composition and differentiation, the field must step beyond the transcription factor and prioritize the epigenomic landscape.

The dynamic topography of the epigenome, the structure upon which all signaling networks are built, is a key component of cell fate determination. Access to, compaction of and compartmentalization within DNA are the foundations of nuclear organization and subsequent gene regulation, something the embryonic development field has appreciated for more than three decades [8,9]. Researchers studying the biology of the normal lung epithelium have been slow to adopt this perspective, preferring instead to extensively characterize specific transcription factors regulating lung developmental processes as well as developmental changes in gene expression and protein distribution and function. While this approach has historically produced easily and reliably measurable results, data-dense and computationally complex epigenomic studies provide a much greater bank of nuanced information from which researchers may identify and characterize transcription factor relationships while remaining grounded in the context of development and differentiation.

Few studies have been conducted on the role epigenomic reorganization plays in lung epithelium differentiation, and the vast majority of those investigate the redistribution of epigenetic signatures between healthy and diseased tissue in such lung diseases as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and, of course, lung cancer. While there is an urgent need for disease therapies and while these studies have advanced our understanding of disease pathogenesis, prognostics and therapeutic options, they circumvent the core question of the epigenomic regulation of lung development and differentiation, the study of which has identified factors significant to both lung development and disease [10]. Studies that do characterize the epigenomic alterations occurring during normal lung differentiation can be roughly divided into three categories: those that investigate the regulators and targets of CpG DNA methylation [10–15]; those that investigate known chromatin remodeling complexes at specific loci important for lung function [12,16,17]; and those that investigate changes in histone or chromosomal architecture during differentiation, either in the context of a transcription factor of known interest in lung development [2] or by interrogating lung epithelial architecture [3,18–20].

The best-studied epigenomic feature of lung development is CpG DNA methylation – the addition of a methyl group to the cytosine within a CG dinucleotide. DNA methylation affects the expression of nearby target genes in a context-dependent manner. When it occurs within promoter regions in CG-dense areas known as islands, DNA methylation typically represses nearby gene expression by preventing core transcription factors and/or basal transcriptional machinery from binding to the DNA; when it occurs within gene bodies, DNA methylation increases transcriptional output, though the role of this phenomenon in lung differentiation remains poorly understood. During normal development, methylation patterning on the genome is both dynamic and highly regulated. Activity of the DNA methyltransferase family proteins regulates deposition and overall levels of DNA methylation and is opposed by loss of methylation which occurs through the activity of the TET DNA demethylase family. DNMT1 propagates DNA methylation patterning on the daughter strand during replication and is required for temporal and spatial regulation of branching morphogenesis of the lung in mice [11], while TET2, which is expressed at high levels in the final stages of mouse lung development [21], may act as its opposition, supporting a role for DNA methylation in the global regulation of gene expression during the formation of the lung parenchyma.

Beyond characterizing the functions and expression levels of proteins responsible for methylation patterning in the lung, several studies have also identified dynamically methylated loci of particular significance during lung development and differentiation. During alveolar septation, DNA methylation levels are altered around key factors known to be involved in lung differentiation, such as Wnt family members and SOX9 [13]. The statistical integration of DNA methylation, proteomic, gene and miRNA expression changes during alveolar maturation [10] has revealed key signaling hubs involving known transcription factors such as NKX2-1, GLI and CEBPA, as well as identifying previously uncharacterized roles for POU2F1, TMEM37 and the lncRNA MEG3, which plays a role in multiple lung diseases.

Despite these breakthroughs, the dynamics and targets of methylation patterning during lung development remain under-studied. Lung consortia rarely include DNA methylation profiling in their larger transcriptome-based investigations, though LUNGMAP has defied this trend with its inclusion of DNA methylation profiles for a few normal epithelial cell types and time points during mouse lung development [4]. Even this inclusion, however, represents minimal progress, as profiling of DNA methylation changes during development of the human lung is definitively under-reported as compared with mice. Furthermore, human lung methylation profiles exhibit wide variations that tend to be dependent on the population being studied [15]. These variations, which may contribute to observed gender and racial disparities in the occurrence of lung disease, highlight the wealth of information produced through, and the opportunities for therapeutically relevant discovery inherent in, epigenomic analysis of the developing lung.

Despite the paucity of DNA methylation research, studies profiling chromatin structure during lung epithelial differentiation have been rarer still, with most literature focused on alveolar epithelial differentiation. Two separate research groups studying mice [2] and humans [19,20] have profiled histone acetylation and methylation alterations in adjacent alveolar epithelial cell types: type 1 (AT1) cells and type 2 (AT2) cells. Both studies found that changes in the location, distribution and prevalence of enhancer elements were associated with differentiation of AT2 into AT1-like cells in vitro and with maintenance of cell identity in vivo. Additionally, mechanistic studies have revealed that proper lung development and differentiation require the temporally and spatially co-ordinated deposition of these histone marks by chromatin-remodeling complexes and factors that modulate chromatin-remodeling complex activity such as MCRIP1, JMJD3 and the EZH2-containing PRC [12,16,17].

The capacity for further discovery in the field of lung development cannot be overstated. Recent innovations in chromatin profiling technologies continue to provide opportunities for rapid advancement of what has been a chronically understudied field. A pair of publications employing a new chromatin accessibility profiling technology (single-cell assay for transposase-accessible chromatin using sequencing; scATAC-seq) recently shifted the long-standing paradigm of differentiation hierarchy in the alveolar epithelium by implicating AT1 cells, rather than the long-accepted AT2s or bronchioalveolar stem cells, as drivers of alveologenesis and regeneration following injury at birth [3,18]. While primary alveolar epithelial cells are notoriously difficult to isolate and have limited proliferative capacity in traditional 2D culture, recent advancements in the culture and immortalization of primary lung epithelium have the potential to revolutionize our understanding of the influence epigenetic changes exert during normal lung differentiation. The 3D organotypic culture of alveolar epithelial cells has expanded our capacity to perform profiling of these cell types [22,23], and developing advancements in the immortalization of alveolar epithelial cells [5] suggest great potential for increasing the quantity of purified alveolar cells one can profile and for providing a consistent starting material from which to generate epigenomic profiling.

Characterizing the development and diversity of the lung epithelium has been and remains an extraordinary undertaking, but emerging disciplines and novel technologies are providing a new frontier from which to tackle this decades-old puzzle. The epigenomic studies above, though limited, have begun to illuminate the mechanisms and factors governing normal lung development and differentiation; considering the shifts in our understanding facilitated by even limited epigenetic studies, there is significant potential for further advances. By following in the footsteps of the embryonic development field, by stepping beyond the transcription factor to consider the dynamic epigenomic landscape, we may take great strides in a relatively short time. It is our hope that the field embraces the central role epigenomic structure plays in the identity and function of the lung epithelium and widely adapts to the opportunities for progress that these technologies represent.

Financial & competing interests disclosure

The authors are supported by American Cancer Society (RSG-20-135-01) and US Department of Defense (W81XWH-21-1-0231). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

The authors are supported by American Cancer Society (RSG-20-135-01) and US Department of Defense (W81XWH-21-1-0231). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.