Discovery of a MUC3B gene reconstructs the membrane mucin gene cluster on human chromosome 7

Abstract

Human tissue surfaces are coated with mucins, a family of macromolecular sugar-laden proteins serving diverse functions from lubrication to the formation of selective biochemical barriers against harmful microorganisms and molecules. Membrane mucins are a distinct group of mucins that are attached to epithelial cell surfaces where they create a dense glycocalyx facing the extracellular environment. All mucin proteins carry long stretches of tandemly repeated sequences that undergo extensive O-linked glycosylation to form linear mucin domains. However, the repetitive nature of mucin domains makes them prone to recombination and renders their genetic sequences particularly difficult to read with standard sequencing technologies. As a result, human mucin genes suffer from significant sequence gaps that have hampered the investigation of gene function in health and disease. Here we leveraged a recent human genome assembly to characterize a previously unmapped MUC3B gene located at the q22 locus on chromosome 7, within a cluster of four structurally related membrane mucin genes that we name the MUC3 cluster. We found that MUC3B shares high sequence identity with the known MUC3A gene and that the two genes are governed by evolutionarily conserved regulatory elements. Furthermore, we show that MUC3A, MUC3B, MUC12, and MUC17 in the human MUC3 cluster are expressed in intestinal epithelial cells (IECs). Our results complete existing genetic gaps in the MUC3 cluster which is a conserved genetic unit in vertebrates. We anticipate our results to be the starting point for the detection of disease-associated polymorphisms in the human MUC3 cluster. Moreover, our study provides the basis for the exploration of intestinal mucin gene function in widely used experimental models such as human intestinal organoids and genetic mouse models.

Fig 1. Conservation of MUC3 cluster in Cercopithecoid and Hominoid superfamilies.

(A) The MUC3 cluster at locus q22 in human chromosome 7 in the GRCh38.p13 assembly is flanked by genes ACHE, TRIM56, and SERPINE1. (B) Members of the Cercopithecoid and Hominoid superfamilies, except for H. sapiens and G. gorilla, carry a MUC3 cluster consisting of MUC3A, MUC3B, MUC12, and MUC17 genes. (C) Presence of the MUC3B gene in MUC3 cluster in Catarrhini parvorder (filled black circles). Open circles indicated the presence of MUC3, MUC12, and MUC17 genes in the Platyrrhini parvorder. NA indicates a lack of sufficient sequence information for the detection of MUC3 cluster genes.

Fig 2. Evidence of a putative MUC3B gene in recent human genome assemblies.

(A) Exploration of PacBio sequencing of HX1 genome identified SMRT reads covering the intergenic region between MUC3A and putative MUC3B, an incomplete PTS sequence, and intergenic sequences between putative MUC3B and MUC12. (B) The T2T-CHM13 assembly contains a 60 kb gap between MUC3A and MUC12. (C) Sequence alignments of SEA, transmembrane (TM), and cytoplasmic tails (CT) of MUC3A and putative MUC3B show high sequence identity, nucleotide mismatches, and a conserved PDZ binding motif.

Fig 3. Comparison of genetic and structural features of MUC3A and MUC3B genes.

(A) Exon structure and length of exon 2 of MUC3A and MUC3B. (B) The evolutionary rate of N-terminal-, PTS- and C-terminal-encoding exons in MUC3A, MUC3B, MUC12, and MUC17 measured as gene content conservation (%) versus evolutionary distance (Mya). (C) Dot plot of pairwise sequence alignment of MUC3A and MUC3B identified imperfect (blue) and perfect (red) tandem repeat sequences in exon 2. (D) Sequence logo representation of 17 amino acids long consensus sequence in 166 and 549 perfect tandem repeats (TRs) in exon 2 of MUC3A and MUC3B, respectively.

Fig 4. Conserved regulatory elements upstream of MUC3A and MUC3B genes.

(A) Epigenetic analysis of the human small intestine and colon reveals a DNase I-sensitive cCRE and specific histone modifications surrounding the MUC3A transcription start site. (B) Single-cell analysis of human and mouse intestines shows gene expression of transcription factors in transporting IECs, with conserved binding sites upstream of MUC3A and MUC3B. (C) Binding sites for transcription factors STAT3 and HNF4A/G are completely conserved upstream of MUC3A and MUC3B in Cercopithecoid and Hominoid superfamilies.

Fig 5. Expression of MUC3B gene in the human intestine.

(A) Unique reads for MUC3A and MUC3B in RNA-sequencing data from human ileum, colon, and rectum mapped to T2T-CHM13 human genome assembly. (B) Gene expression of MUC3 cluster genes in human ileum, colon, and rectum. 2 samples per tissue segment. * p<0.05 as determined by two-way ANOVA, corrected for multiple comparisons using Tukey´s test. Data are presented as mean ± standard deviation (SD). (C) Specific primers amplify a 646 bp cDNA spanning exons 3–8 in MUC3A and MUC3B transcripts from the ileum of five individuals. MUC3A cDNA carries a PstI restriction site in exon 6 that distinguishes MUC3A from MUC3B transcripts. Agarose gel electrophoresis of PstI restriction digests of amplified cDNA from MUC3A and MUC3B transcripts results in 380 bp and 266 bp fragments from MUC3A cDNA. (D) Quantification of bands from agarose gel in C. n = 5 individuals. Data are presented as mean ± standard deviation (SD).