A Global Vista of the Epigenomic State of the Mouse Submandibular Gland

Abstract

The parotid, submandibular, and sublingual glands represent a trio of oral secretory glands whose primary function is to produce saliva, facilitate digestion of food, provide protection against microbes, and maintain oral health. While recent studies have begun to shed light on the global gene expression patterns and profiles of salivary glands, particularly those of mice, relatively little is known about the location and identity of transcriptional control elements. Here we have established the epigenomic landscape of the mouse submandibular salivary gland (SMG) by performing chromatin immunoprecipitation sequencing experiments for 4 key histone marks. Our analysis of the comprehensive SMG data sets and comparisons with those from other adult organs have identified critical enhancers and super-enhancers of the mouse SMG. By further integrating these findings with complementary RNA-sequencing based gene expression data, we have unearthed a number of molecular regulators such as members of the Fox family of transcription factors that are enriched and likely to be functionally relevant for SMG biology. Overall, our studies provide a powerful atlas of cis-regulatory elements that can be leveraged for better understanding the transcriptional control mechanisms of the mouse SMG, discovery of novel genetic switches, and modulating tissue-specific gene expression in a targeted fashion.

Keywords: ChIP-sequencing; epigenomics; gene expression; histone modification; regulatory regions; salivary glands.

Figure 1.

Characteristics of the epigenomic marks in the submandibular gland. (A) Clustered heatmap of Pearson correlation for the H3K27ac, H3K4me1, H3K4me3, and H3K27me3 chromatin immunoprecipitation (ChIP)–seq data based on the normalized read coverages across the genome-wide regions. (B) Metaplots of average ChIP-seq density of 4 histone marks at the transcription start sites (TSSs) of genes. (C) Barplots showing the percentage distribution patterns and locations of peaks of H3K27ac, H3K4me1, H3K4me3, and H3K27me3 relative to genes.

Figure 2.

The diverse nature of cis-regulatory elements in the submandibular salivary gland (SMG) and their potential transcriptional mediators. (A) Heatmap displaying 3 clusters of regulatory elements as defined by disparate combinatorial nature of the histone modification marks. (B) Bar plots displaying select top enriched GO Biological Processes associated with genes identified in each regulatory element cluster shown in panel A above. Genes were defined using GREAT (standard parameters) and subsequently used to find enriched gene sets using mSIGDB and marked for significance using the hypergeometric test and controlled for multiple hypothesis testing using false discovery rate (FDR) <0.05. (C) The top de novo motifs, derived from each regulatory element cluster shown in panel A above, as generated by MEME. Enriched motifs were determined by analysis of motif enrichment (AME) and using the Mouse UniProbe database.

Figure 3.

Foxc1 and Foxi2, members of the Fox family of transcriptions factors, are highly enriched in the submandibular salivary gland (SMG). (A) Heatmap showing the expression of Fox family members in mouse SMG and across a panel of mouse tissues and organs. Foxc1 and Foxi2 are selectively enriched in the salivary glands compared to other tissues. The samples were clustered by hierarchical clustering using Euclidean method as the distance measure with complete linkage. (B) Uniform manifold approximation and projection (UMAP) visualization of the different cell populations in the 12-wk female adult mouse SMG based on single-cell RNA-sequencing (scRNA-seq) analysis (Min et al. 2020). (C) Cell clusters were interrogated for the expression of Foxc1 and Foxi2. (D) Immunofluorescence staining of female adult mouse SMG. Foxc1 is predominantly expressed in the intercalated ductal cells as demonstrated by costaining with Sox9. Arrows highlight double positive cells. Scale bar: 37 µm.

Figure 4.

Super-enhancer landscape of the submandibular salivary gland (SMG). (A) Hockey stick plot displaying the ranked order of typical enhancers and super-enhancers as determined by the ranking order of super-enhancers (ROSE) algorithm. Names of selected genes are shown. (B) Table displaying the top enriched transcription factor (TF) motifs found within NFRs located in SMG super-enhancers. The analysis of motif enrichment (AME) program was used to perform the enrichment analysis of the Uniprobe motif database. (C) Boxplot displaying the distribution of expression of genes marked by either a super-enhancer or a typical enhancer. GREAT analysis was used to perform gene annotation of the 2 classes of regulatory elements. Trancscipts per million values, based on our previous RNA-sequencing studies performed in the SMG, were used to determine gene expression levels (Gluck et al. 2016). (D) Bar plots displaying selected top enriched pathways and biological processes from genes annotated to the SMG super-enhancers. The mSIGDB was used to examine gene set enrichment using the hypergeometric test, with a false discovery rate (FDR) cutoff of <0.05.

Figure 5.

Component signature genes of the submandibular salivary gland (SMG) are enriched for tissue-specific enhancers and super-enhancers. (A) Heatmap displaying the expression levels of all the genes that comprise the adult salivary gland gene signature across the select tissues used for epigenetic analysis and comparison. Far-left panel highlights genes that are associated by a SMG super-enhancer (red), typical enhancer (white), or a SMG-specific enhancer (black). (B) Heatmap showing the expression of transcription factors (TFs) that make up the SMG gene signature using the same analysis as described in panel A.