Transcriptional Profiling Reveals Gland-specific ...

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Physiological Genomics: RESEARCH ARTICLE │ General Interest

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Transcriptional Profiling Reveals Gland-specific Differential Expression in

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the Three Major Salivary Glands of the Adult Mouse

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Xin Gao,1,3¶ Maria S. Oei,1¶ Catherine E. Ovitt,4 Murat Sincan,2* and James E. Melvin1*

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Secretory Mechanisms and Dysfunctions Section, 2Office of the Clinical Director, National

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Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892

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20742

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Rochester, NY 14642

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Running Head: Salivary Gland-specific Differences in Gene Expression

Joint Institute for Food Safety and Applied Nutrition, University of Maryland, College Park, MD

Center for Oral Biology and Department of Biomedical Genetics, University of Rochester,

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¶

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*

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Address for correspondence: James E. Melvin, D.D.S., Ph.D.

These authors contributed equally to this paper Co-Senior Authors: M. Sincan and J.E. Melvin

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10 Center Drive

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Building 10/Room 1N-117B

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Bethesda, MD 20892

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Email: [email protected]

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Tel: (301) 402-1706

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Downloaded from www.physiology.org/journal/physiolgenomics by ${individualUser.givenNames} ${individualUser.surname} (216.165.126.118) on February 28, 2018. Copyright © 2018 American Physiological Society. All rights reserved.

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ABSTRACT

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Gao X, Oei MS, Ovitt CE, Sincan M, Melvin JE. Transcriptional Profiling Reveals

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Gland-specific Differential Expression in the Three Major Salivary Glands of the Adult Mouse.

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Physiol Genomics––RNA-seq was used to better understand the molecular nature of the biological

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differences among the three major exocrine salivary glands in mammals. Transcriptional profiling

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found that the adult murine parotid, submandibular and sublingual salivary glands express greater

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than 14,300 protein-coding genes, and nearly 2,000 of these genes were differentially expressed.

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Principle component analysis of the differentially expressed genes revealed three distinct clusters

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according to gland type. The three salivary gland transcriptomes were dominated by a relatively

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few number of highly expressed genes (6.3%) that accounted for more than 90% of transcriptional

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output. Of the 912 transcription factors expressed in the major salivary glands, greater than 90%

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of them were detected in all three glands, while expression for about 2% of them was enriched in

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an individual gland. Expression of these unique transcription factors correlated with sublingual

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and parotid specific subsets of both highly expressed and differentially expressed genes. Gene

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ontology analyses revealed that the highly expressed genes common to all glands were associated

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with global functions, while many of the genes expressed in a single gland play a major role in the

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function of that gland. In summary, transcriptional profiling of the three murine major salivary

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glands identified a limited number of highly expressed genes, differentially expressed genes and

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unique transcription factors that represent the transcriptional signatures underlying gland-specific

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biological properties.

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Keywords: RNA-seq; Exocrine Gland; Salivary Gland; Transcription Factors; Gene Expression

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INTRODUCTION

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THE PRIMARY function of salivary glands is to secrete saliva, a fluid mixture of water,

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electrolytes and proteins. The saliva secreted by mammals originates largely from three paired

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exocrine glands, the parotid, submandibular and sublingual salivary glands. Saliva lubricates and

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moistens the oral cavity to facilitate speech, swallowing and taste, initiates digestion, and inhibits

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dental decay and other opportunistic infections (1, 46). Although the three major salivary glands

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are similar in many respects, several functional characteristics are gland-specific. For example,

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saliva contains more than a thousand proteins, some of which are highly enriched or exclusively

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expressed by an individual salivary gland (10, 54). Moreover, although the essential ion and water

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transport proteins have been identified (16, 33), the differences in the fluid secretion mechanism

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among the different salivary glands have not been fully elucidated (23).

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Salivary glands are mainly composed of two cell types, i.e. acinar cells that are responsible

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for secreting most of the proteins, fluid and electrolytes found in saliva, and duct cells that

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primarily reabsorb the NaCl secreted by the acinar cells. Some of the gland-specific functional

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properties are associated with the distinctive cell types found in each salivary gland (2, 10, 33, 41,

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51). In most mammals, parotid acinar cells are exclusively serous in nature, while sublingual acinar

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cells are primarily mucous cells capped by a few serous cells. The acinar cells of the rodent

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submandibular gland are seromucous and their duct cells display sexual dimorphism (41).

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The genetic and molecular basis for the functional differences among the three major

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salivary glands remains unclear, but can likely be associated with the regulation of salivary gland-

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specific gene expression by different groups of transcription factors. Transcription factors are the

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central regulators of gene expression. Approximately 1,600 transcription factors comprise greater

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than 7% of the protein-coding genes in the mouse genome, which makes this family the single

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largest gene family (20). Mammalian genes are typically flanked by distinct binding sites for

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several transcription factors that work in unison to regulate expression of each gene. While

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globally expressed transcription factors often regulate housekeeping functions (28), others may

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control specific biological tasks, like organ development (25, 29, 47). Aberrant expression of

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transcription factors also frequently correlates with tumorigenesis (6, 31). However, expression

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data are lacking for the specific transcription factors that maintain the adult gland-specific

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phenotype for each of the three major salivary glands. 3


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The current study evaluated the transcriptional profiles of the three murine salivary glands

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using RNA-seq to better understand the molecular and genetic nature of their well-recognized

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biological differences. We found that the expression patterns of gland-specific protein-coding

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genes agree with the adult male mouse submandibular gland transcriptome (12) and the salivary

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gland secretion literature, validating our RNA-seq data. Transcriptome variation across the three

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murine salivary glands correlated with the differential co-expression of a limited number of

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transcription factors and salivary gland-specific protein-coding genes.

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MATERIALS AND METHODS

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General Methods. Chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA)

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unless otherwise stated. Experiments were performed using 12-weeks old male and female

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C57BL/6J mice (Jackson Laboratory, ME) that were housed in pathogen-free, micro-isolator cages

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with free access to laboratory chow and water with a 12-hour light/dark cycle. Animal procedures

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were approved by the Animal Care and Use Committee of the National Institute of Dental and

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Craniofacial Research, National Institutes of Health (ASP 16-802).

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RNA preparation and next-generation sequencing. Six adult mice (3 male and 3 female)

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were euthanized by exposure to CO2, followed by cervical dislocation, and the parotid,

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submandibular and sublingual glands immediately removed and placed in RNAlater Stabilization

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Solution (Thermo Fisher Scientific, Waltham, MA, catalog #AM7020) at 4°C. The submandibular

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and sublingual glands are encapsulated, and thus, were easily separated from the surrounding

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tissue, while the parotid gland is more diffuse and was removed with the aid of a dissecting

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microscope to exclude surrounding connective tissue. A single parotid and submandibular gland

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were used from each animal. The sublingual glands are considerably smaller than the other two

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major salivary glands, thus the left and right glands were pooled from each animal. The 18 gland

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samples were shipped overnight to Otogenetics Corporation (Atlanta, GA). Total RNA was

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extracted using the RNeasy Micro Kit (Qiagen, Valencia, CA, catalog #74004). The integrity and

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purity of total RNA were assessed using Agilent Bioanalyzer or TapeStation and OD260/280.

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cDNA was generated from high quality total RNA using the SMARTer PCR cDNA Synthesis Kit

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with modified oligo(dT) primers (Clontech Laboratories, Inc., Mountain View, CA, catalog

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#634926), and adapters were removed by digestion with RsaI. The resulting 1-2 μg of cDNA were 4


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fragmented using Bioruptor (Diagenode Inc., Denville, NJ) and profiled using Agilent Bioanalyzer

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or TapeStation. Illumina libraries were made from qualified fragmented cDNA using the

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SPRIworks HT Reagent Kit (Beckman Coulter, Inc., Indianapolis, IN, catalog #B06938). The

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quality, quantity, and size distribution of the Illumina libraries were determined using Agilent

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Bioanalyzer or Tapestation. The libraries were then submitted for Illumina HiSeq2500 sequencing

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according to the standard operation with v1 or v2 chemistry, and 100-106 nucleotide (nt) paired

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end reads were generated and checked for data quality using FastQC (Babraham Institute,

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Cambridge, UK). Each of the 18 sample libraries were sequenced at approximately 40 million

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reads.

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Analysis and Comparison of RNA-Seq Data.

The data generated by Otogenetics

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Corporation were subjected to analysis using the platform provided by DNAnexus (Mountain

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View, CA) and then: (1) mapped against the mouse mm10 reference genome with STAR 2.4.0j;

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(2) measured for expression levels of genes and transcripts with Cufflinks 2.2.1; and (3) compared

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across the three major salivary glands with Cufflinks/Cuffdiff 2.2.1 for significance (q500-fold more

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abundant in the SLG and SMG than in the PG, while Mucin 19/Muc19 was expressed at a 2,000-

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fold greater level in the SLG than in the PG and SMG.

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To further validate our RNA-seq data, we compared the adult male SMG transcriptome

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generated in the present study with the adult male SMG transcriptome previously reported (12) 6


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using the same age and mouse background (12-weeks old, C57BL/6). Gluck et al. identified 246

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protein-coding genes enriched in the adult male SMG transcriptome, 220 of which were detected

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in our male SMG transcriptome (Supplemental Table 3). The 90% overlap in detected protein-

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coding genes in the two male SMG transcriptomes confirms the reproducibility of the RNA-seq

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method.

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Highly Expressed Genes in the Three Major Murine Salivary Glands. Among the

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expressed protein-coding genes, we selected the ones most highly expressed for further

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examination, as these genes generally hold keys to understanding the genetic and biochemical

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foundation of a given organ system. To identify the highly-expressed genes (HEGs) in each gland,

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the sorted FPKMs were individually graphed on a logarithmic scale and a log FPKM cutoff value

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of 4 was used, as revealed by the turning points on the graphs (blue lines; Supplemental Figure 2).

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We observed in each of the major salivary glands about 700 HEGs with log FPKM values greater

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than 4 (Figure 1). Allowing that the three major salivary glands have similar biological functions,

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it was therefore not surprising that 685 of the 901 total HEGs (about 76%) in the PG, SLG and

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SMG presented with high expression levels in at least two of the three salivary glands (Figure 1;

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list of HEGs given in Supplemental Table 4). In contrast, 7.8%, 11.0% and 5.2% of the HEGs (PG,

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SLG and SMG, respectively) displayed enriched expression in a single gland, indicating that the

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latter group of HEGs contributes to the unique transcriptomic signature of each gland. The 901

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different HEGs in the three salivary glands represent only about 6.3% of the total number of

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expressed genes (14,371), but these highly expressed genes dominated expression, contributing

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96.6%, 87.8%, and 93.4% of the transcriptional outputs in PG, SLG, and SMG, respectively (total

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HEG FPKMs divided by total FPKMs for each gland).

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Gene Ontology (GO) enrichment analyses (refinement P value