MIST1 and PTF1 Collaborate in Feed-Forward Regulatory Loops That Maintain the Pancreatic Acinar Phenotype in Adult Mice

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Mei Jiang,a Ana C. Azevedo-Pouly,a Tye G. Deering,a Chinh Q. Hoang,a Daniel DiRenzo,b David A. Hess,b Stephen F. Konieczny,b Galvin H. Swift,a Raymond J. MacDonalda Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USAa; Department of Biological Sciences, Bindley Bioscience Center and b Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA Downloaded from

Much remains unknown regarding the regulatory networks formed by transcription factors in mature, differentiated mammalian cells in vivo, despite many studies of individual DNA-binding transcription factors. We report a constellation of feed-forward loops formed by the pancreatic transcription factors MIST1 and PTF1 that govern the differentiated phenotype of the adult pancreatic acinar cell. PTF1 is an atypical basic helix-loop-helix transcription factor complex of pancreatic acinar cells and is critical to acinar cell fate speciﬁcation and differentiation. MIST1, also a basic helix-loop-helix transcription factor, enhances the formation and maintenance of the specialized phenotype of professional secretory cells. The MIST1 and PTF1 collaboration controls a wide range of specialized cellular processes, including secretory protein synthesis and processing, exocytosis, and homeostasis of the http://mcb.asm.org/ endoplasmic reticulum. PTF1 drives Mist1 transcription, and MIST1 and PTF1 bind and drive the transcription of over 100 downstream acinar genes. PTF1 binds two canonical bipartite sites within a 0.7-kb transcriptional enhancer upstream of Mist1 that are essential for the activity of the enhancer in vivo. MIST1 and PTF1 coregulate target genes synergistically or additively, depending on the target transcriptional enhancer. The frequent close binding proximity of PTF1 and MIST1 in pancreatic acinar cell chromatin implies extensive collaboration although the collaboration is not dependent on a stable physical interaction.

he mammalian pancreas is a mixed exocrine and endocrine MIST1 (encoded by Bhlha15) is a bHLH transcription factor on November 14, 2017 by PURDUE UNIV LIB TSS Tgland consisting of acini, ducts, and islets. Approximately 90% present selectively in serous secretory cells, including pancreatic of the mass of the adult pancreas is exocrine acinar tissue. Pancre- acinar cells (14, 15). In the pancreas, Ptf1a and Mist1 are expressed atic acinar cells are highly specialized for the synthesis and secre- selectively in acinar cells, and inactivation of either gene affects the tion of digestive enzymes that are flushed via ducts to the intestine differentiated acinar phenotype (16, 17). Mist1-null pancreatic for digestion of complex nutrients (1). Abundant rough endoplas- acinar cells have aberrant calcium signaling, reduced content of mic reticulum (ER) supports an extraordinary level of secretory digestive enzymes, and decreased expression of critical genes of protein synthesis (2). Maintenance of ER homeostasis without the the secretory pathway, such as Rab genes (18–20). These deficien- accumulation of misfolded and unfolded proteins is especially im- cies cause the disruption of apical-basal polarity, the failure of portant for acinar function and viability (3). Acinar cells are po- intercellular communication due to defective gap junctions, and a larized, with basal rough ER and an extensive supranuclear Golgi severe impairment of secretagogue-stimulated secretion. Further- apparatus for sorting and condensing newly synthesized secretory more, the absence of functional MIST1 causes progressive acinar proteins into secretory vesicles (zymogen granules) that fill the lesions and the acquisition of some ductal properties (15). Mice apical cellular domain nearest the luminal plasma membrane. Se- lacking MIST1 have a greatly reduced ability to recover from cretion is regulated to ensure an appropriate surge of digestive caerulein-induced acute pancreatitis (21) and increased sensitiv- enzyme release in response to feeding (1). ity to KRAS-driven adenocarcinoma (22). Several transcription factors, including PTF1 and MIST1, are Little is known of the regulatory networks formed by transcrip- known to play crucial roles in the specification, differentiation, tion factors in pancreatic acinar cells (23, 24). Large-scale studies and maturation of pancreatic acinar cells (4–6). PTF1 is a complex of transcription factors in unicellular organisms, including Esch- of three tightly associated DNA-binding subunits: the cell-type- restricted basic helix-loop-helix (bHLH) protein PTF1A, one of the common bHLH E proteins (e.g., E47) (7), and RBPJ or its Received 28 June 2016 Returned for modification 22 July 2016 paralog RBPJL (8, 9). All three subunits contribute to the recog- Accepted 10 September 2016 nition of an extended bipartite binding sequence consisting of an Accepted manuscript posted online 19 September 2016 E box bound by the PTF1A-E protein heterodimer and a TC box Citation Jiang M, Azevedo-Pouly AC, Deering TG, Hoang CQ, DiRenzo D, Hess DA, bound by the RBP subunit (8, 10, 11). Early pancreatic develop- Konieczny SF, Swift GH, MacDonald RJ. 2016. MIST1 and PTF1 collaborate in feed- ment requires the RBPJ form of the PTF1 complex (12), desig- forward regulatory loops that maintain the pancreatic acinar phenotype in adult mice. Mol Cell Biol 36:2945–2955. doi:10.1128/MCB.00370-16. nated PTF1-J. With the onset of acinar development, PTF1-J ac- Address correspondence to Raymond J. MacDonald, tivates Rbpjl, and RBPJL gradually replaces RBPJ in the complex [email protected]. (12). The RBPJL form (PTF1-L) is restricted to pancreatic acinar Supplemental material for this article may be found at http://dx.doi.org/10.1128 cells and controls the final stage of acinar differentiation. The ex- /MCB.00370-16. pression of genes encoding digestive enzymes and proteins in- For a companion article on this topic, see doi:10.1128/MCB.00366-16. volved in regulated exocytosis is reduced in Rbpjl-null pancreatic Copyright © 2016, American Society for Microbiology. All Rights Reserved. acinar cells (13).

December 2016 Volume 36 Number 23 Molecular and Cellular Biology mcb.asm.org 2945 Jiang et al. erichia coli and Saccharomyces cerevisiae, identified regulatory mo- Coimmunoprecipitation. The nuclear extract was derived from a tifs that recur in transcriptional networks, especially autoregula- whole pancreas (300 mg) of an ICR mouse by an NE-PER nuclear and tory and feed-forward loops (25, 26). For autoregulation, a cytoplasmic protein extraction kit (Thermo Fisher Scientific, Waltham, regulator binds and affects the transcriptional regulatory region of MA), except that the nuclear extraction reagent (NER) buffer was re- its own gene. Positive autoregulatory loops can create a stable, placed with a lysis buffer (20 mM HEPES, 1% Triton X-100, 150 mM complex biological system to maintain a cellular identity. Dual, NaCl, 5 mM EDTA, 5% glycerol, pH 7.4). The lysate was precleared and then immunoprecipitated with 3 ␮g of affinity-purified rabbit anti-mouse positive autoregulation of Ptf1a and Rbpjl by the PTF1-L complex PTF1A (8). The immunoprecipitate was resolved by SDS-PAGE and blot- in pancreatic acinar cells sustains the level of PTF1 (12, 27). A ted with goat anti-mouse PTF1A, rabbit anti-mouse RBPJL (8), or rabbit feed-forward loop consists of a pair of regulators in which one anti-human MIST1 (30). Goat anti-mouse PTF1A was a gift from Chris- controls the other and the two jointly control a third (target) gene. topher Wright (Vanderbilt University Medical Center, Nashville, TN). Downloaded from Feed-forward loops are highly represented in transcriptional net- ChIP-Seq. Chromatin immunoprecipitation with high-throughput works and control 10% of yeast genes (25) and more than 30% of sequencing (ChIP-Seq) was performed with chromatin isolated from E. coli genes (28). Feed-forward loops respond to sustained rather adult mouse pancreas, as previously described (13), using rabbit antibod- than transient stimuli, provide precise temporal control for devel- ies against either rat or human MIST1 (30, 31). Two independent ChIP- opmental decisions (29), and can be particularly effective in sup- Seq assays (one pancreas per ChIP-Seq experiment) were performed with port of the stability of a differentiated state. each MIST1 antibody. The sequence reads were aligned with Bowtie (32). The ChIP-Seq assays using anti-human MIST1 had 13.5 million and 54.9 Here, we report a multioutput, feed-forward regulatory system million aligned reads, and the ones using anti-rat MIST1 had 11.5 million consisting of PTF1, MIST1, and a set of shared target genes in and 18.6 million aligned reads. MIST1-binding peaks were called using http://mcb.asm.org/ pancreatic acinar cells. PTF1 binds and directly regulates Mist1 HOMER (33) with a false discovery rate (FDR) of Ͻ0.01 for the combined transcription, and together MIST1 and PTF1 bind and coregulate anti-rat MIST1 data sets and confirmed if overlapping peaks were present genes for a wide range of specialized pancreatic acinar cellular for the combined anti-human MIST1 data sets with an FDR of Ͻ0.05. The processes. We show that MIST1 and PTF1 coactivate additively or de novo discovery of MIST1 DNA-binding motifs was performed using synergistically, depending on the nature of the regulatory se- MEME (34). MIST1-bound regions were assigned to nearby genes using quences of the target gene. GREAT (35) with its default settings. Four ChIP-Seq assays were also performed with rabbit anti-mouse PTF1A (8) and with rabbit anti-mouse RBPJL (8). The raw data are avail- MATERIALS AND METHODS able through the NCBI Gene Expression Omnibus (GEO) database under on November 14, 2017 by PURDUE UNIV LIB TSS Generation of transient transgenic embryos and detection of transgene accession number GSE86262 (17). Identification of regions bound by ei- activities. Transient transgenic embryos bearing an ϳ0.7-kb genomic re- ther PTF1A or RBPJL followed the pipeline of MIST1 ChIP-Seq analysis gion 6 kb upstream of Mist1 (Mus musculus build 10 [mm10], chromo- described above. PTF1-bound regions were identified by the overlap of some 5 [chr5] positions 144183992 to 144184700) were generated, and PTF1A and RBPJL binding (peak width, 250 bp; overlap, Ն1 bp). The transgene activities were detected as previously described (27). Briefly, the probability of overlap between PTF1- and MIST1-bound regions was cal- enhancer of interest was placed 5= of the lacZ reporter gene and sent to the culated with the phyper function of the R project (www.r-project.org) and Transgenic Core Facility of the University of Texas Southwestern Medical based on 20.3 million base pairs of open chromatin, which is the median Center to generate transient transgenic embryos. Embryos were sacrificed value for DNase I-hypersensitive sites of a variety of mouse tissues (36). at embryonic day 12.5 (E12.5), E14.5, and E15.5 and stained for ␤-galac- RNA sequencing. Total RNA samples from two control (Mist1ϩ/Ϫ) tosidase activity. There are two PTF1-binding sites in the Mist1 enhancer. and three Mist1-KO (Mist1Ϫ/Ϫ) adult mouse pancreases were subjected To examine the ability of PTF1 to regulate the enhancer activity in vivo, to RNA sequencing (RNA-Seq) as previously described (13). The Mist1 the TC boxes were modified from TTCCCA to TTCAAT, and the E boxes knockout (Mist1-KO) mouse (Bhlha15TM1Skz) has been described previ- were modified from CANNTG to TCNNTA (mutations are underlined). ously (15). The sequence reads were aligned with TopHat (37). The con- Transient transgenic embryos carrying the enhancer with either the two trol libraries have 35 million and 24.4 million aligned reads, and the TC boxes or the two E boxes mutated were generated, collected at E15.5, Mist1-KO libraries have 33.1 million, 30.6 million, and 35.3 million and stained for ␤-galactosidase activity. The University of Texas South- aligned reads. HTSeq (38) was used to count the number of reads mapped western Institutional Animal Care and Use Committee approved all of the to each gene. Differential gene expression analysis was then performed animal experiments. with edgeR (39) (FDR of Ͻ0.05). Cufflinks (37) calculated the number of Immunohistochemistry. Whole-mount ␤-galactosidase-stained E15.5 reads per kilobase per million mapped reads (RPKM), a measure of the transient transgenic embryos were embedded in paraffin and sectioned. relative level of a transcript normalized to 1 million reads. RNA-Seq re-

Sections were deparafﬁnized and blocked with 0.3% H2O2 for 30 min and sults for three control and four Ptf1a conditional knockout (Ptf1a-cKO) 10% normal donkey serum buffer (Jackson ImmunoResearch, West mouse pancreases were deposited in the NCBI GEO database under ac- Grove, PA) for 1 h. Primary antibodies included goat anti-CPA1 (used at cession number GSE86261 (17). Differential gene expression analysis and 1:200; R&D Systems, Minneapolis, MN), rat anti-cytokeratin 19 (CK19) RPKM calculation followed the pipeline described above. (1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), guinea In silico functional analysis of genes controlled by both PTF1 and pig anti-insulin (1:1,000; Millipore, Billerica, MA), and guinea pig anti- MIST1. Among potential PTF1 and MIST1 target genes assigned by glucagon (1:1,000; Millipore). Primary antibodies, except anti-CK19, GREAT (26), our functional analysis focused on those with meaningful were applied overnight at 4°C, followed by the application of secondary expression in the normal pancreas (RPKM of Ն1). A value of 1 RPKM is antibodies (Jackson ImmunoResearch) for 30 min at room tempera- equivalent to ϳ1 mRNA molecule per cell, based on an RNA/DNA ratio of ture. Anti-CK19 was applied for1hatroom temperature. Immuno- 9 for normal pancreas and 6 pg of DNA per cell. localization was visualized by diaminobenzidine (DAB) staining Three important gene sets were collected, including pancreas-re- (Roche, Indianapolis, IN) and then eosin Y counterstaining (Sigma- stricted, acinus-related, and exocytosis genes. (i) The results of gene ex- Aldrich, St. Louis, MO). The percentage of immunostaining-positive cells pression proﬁling of 91 adult mouse cell types and tissues (NCBI accession in the ␤-galactosidase-positive (blue) cell population was counted for number GSE10246)(40) were analyzed using the affy package of R (41). each section. The mean and standard deviation of the percentage were Pancreas-restricted genes with the top two probe intensities in pancreas calculated for each marker. and with values at least 10-fold higher than those in the other tissues were

2946 mcb.asm.org Molecular and Cellular Biology December 2016 Volume 36 Number 23 MIST1 and PTF1 Maintain the Acinar Pancreas Phenotype chosen. (ii) A PubMed query for acinus-related genes was performed with (12), preferential occupancy by RBPJ at E15.5 (RBPJ/RBPJL, 11:1) an NCBI eUtils pipeline using a homemade PERL script. The keywords changed to a preference for RBPJL at E17.5 (1:6) and in adult mice were the following: (gene symbol OR gene synonyms) AND (acin*[TIAB] (1:3). The presence of RNA polymerase II and histone 3 dimethy- OR “gastric chief cell”[TIAB] OR “zymogenic cell”[TIAB] OR “plasma lated at lysine 4 (H3K4me2) at and surrounding these two sites in cell”[TIAB]). Gastric chief cells (also referred to as zymogenic cells) and embryonic and adult pancreatic chromatin indicates that this re- plasma cells are acinar cell-like. Our PubMed search added these cell types gion is likely a transcriptional enhancer (50, 51). to the keywords because genes functioning in these cells might also play a role in pancreatic acinar cells. (iii) Exocytosis-related genes were collected We note that this potential enhancer is located in the last intron from the Gene Ontology Database (http://geneontology.org), from pro- of Lmtk2. The expression of Lmtk2 during embryonic pancreatic teomic studies of mouse and rat pancreatic zymogen granules (42–44), development is constant, unlike the increasing expression of

and from reviews on exocytosis (45, 46). Experimental evidence for in- known PTF1 targets, including Mist1,asPtf1a expression in- Downloaded from volvement in exocytosis was collected for each gene with an NCBI eUtils creases (unpublished data). In addition, the continuous distribu- pipeline using a homemade PERL script. Genes without the support of tions of RNA polymerase II and H3K4me2 from the enhancer to experimental evidence or gene ontology were removed from the exocyto- the Mist1 promoter indicate that the enhancer and Mist1 pro- sis gene list. Hypergeometric tests were performed with the phyper func- moter are in the same regulatory block. Thus, this enhancer con- tion of R to examine the statistical significance of enrichment of the po- trols Mist1; however, we cannot rule out some effect on Lmtk2. tential MIST1 and PTF1 targets in the three gene sets. Qiagen’s Ingenuity The RBPJ and RBPJL forms of PTF1 recognize the same DNA- Pathway Analysis (IPA; Qiagen, Redwood City, CA) and WebGestalt analysis (47) were performed to discover pathways enriched in the MIST1 and binding sequence, which consists of an E box (CANNTG) and a TC box (TTCCCA) spaced one, two, or (infrequently) three heli- PTF1 target gene sets. http://mcb.asm.org/ Cell transfection assays of enhancer function. The enhancers of cal DNA turns apart (8, 10, 52). The PTF1-binding sequences Rab26 (ϩ739 to ϩ250), Cabp2 (Ϫ1172 to Ϫ502 relative to the transcrip- present near the center of each of the two PTF1-bound regions in tion start site [TSS]), Nfe2l2 (Ϫ740 to ϩ1198 and ϩ67896 to ϩ67648), the Mist1 enhancer are well conserved (including the spacing be- and Tead2 (ϩ825 to ϩ1893) were amplified from the C57BL/6 mouse tween the E and TC boxes) in mouse, rat, human, and four other genomic DNA and cloned into pGL3 (Promega, San Luis Obispo, CA). mammals. The proximal binding sequence is further conserved, Mutagenesis of the transcription factor binding sites was performed with except for a single nucleotide variation in the TC box, across eight overlap extension PCR. For PTF1-binding sites, the TC boxes were mod- additional mammalian species (see Fig. S1B in the supplemental ified from TCCCA to TCAAT, and the E boxes were modified from CA material). Conservation extends throughout the proposed en- NNTG to TCNNTA (mutations are underlined). MIST1-preferred E on November 14, 2017 by PURDUE UNIV LIB TSS boxes CA(G/T)(C/A)TG were modified to TC(G/T)(C/A)TA. The cDNA hancer and the gene arrangement of this region of mouse chr5 (see expression plasmids of mouse Ptf1a and Rbpjl have been described previ- Fig. S1C). ously (8). The full-length protein coding region with part of the 3= un- To test whether the conserved PTF1-bound sites are part of an translated region of Mist1 (bp 125 to 818) was amplified from mouse acinar transcriptional enhancer, the 0.7-kb region containing the genomic DNA, linked downstream of the 5= untranslated region of Xeno- two sites (Fig. 1A, gray shading) was linked to a lacZ reporter gene pus laevis ␤-globin mRNA, and inserted into the pcDNA3.1(ϩ) vector and injected into fertilized mouse eggs to examine activity in fetal between HindIII and XhoI restriction sites (Invitrogen). Transfection of mouse tissues (Fig. 2). Enhancer-driven ␤-galactosidase activity 293 human embryonic kidney cells (HEK293; American Type Culture was detected selectively in the pancreas for 12 of 29 (41%) E15.5 Collection CRL-1573) was performed using FuGene 6 (Promega) as pre- founder transgenic embryos (Fig. 2A) and in 5 of 14 (36%) E14.5 viously described (48). Each transfection was repeated three times. The transgenic embryos (see Fig. S2 in the supplemental material) but enhancer-driven firefly luciferase activity was normalized to the Renilla luciferase activity of cotransfected pRL-CMV (Promega). only very faintly in 1 of 10 E12.5 transgenic embryos (data not Accession numbers. Raw data have been deposited in the NCBI GEO shown). Thus, the enhancer was activated specifically and within database under accession numbers GSE86288 and GSE86289. the same developmental window as endogenous Mist1 expression between E12.5 and E13.5, shortly after the onset of acinar differ- RESULTS entiation (15, 53). Transgenic ␤-galactosidase activity was de- The PTF1 complex directs pancreas-specific transcription of tected in most acinar cells but not in ductal or endocrine cells (Fig. Mist1 via an upstream enhancer. PTF1 is crucial for pancreatic 3). The activity of the enhancer in the acinar cells of the pancreas organogenesis, including the differentiation of acinar cells (4, 13, and not elsewhere in the gut is the same as that of the endogenous 49). However, knowledge of the downstream genes controlled by Mist1 gene (15). Thus, the proper temporal and cell-type-re- PTF1 in mature acinar cells is largely limited to the genes of the stricted expression is further evidence that the enhancer drives secretory digestive enzymes (8, 13). Inactivation of Ptf1a in the Mist1 transcription in pancreatic acinar cells. adult pancreas (17) reduced Mist1 mRNA to 24% of the normal To test whether the activity of the Mist1 enhancer requires level after 6 days (0.24 Ϯ 0.13; P ϭ 3.4EϪ4). To determine PTF1 binding, we examined the effect of specific binding-site mu- whether Mist1 is a direct target of PTF1, we performed ChIP-Seq tations known to eliminate PTF1 binding of other pancreatic gene experiments with chromatin from adult pancreas using antibodies control regions (8, 27, 54). Minimal 3-bp mutations in both E against the PTF1 subunit PTF1A, RBPJ, or RBPJL. Two sites ap- boxes or both TC boxes largely eliminated the activity of the en- proximately 300 bp apart with strong binding for all three proteins hancer in vivo (Fig. 2B and C). No mice with transgenic mutant were identified 6 kb upstream of Mist1 (Fig. 1A), indicating a mix enhancers had extensive ␤-galactosidase staining of the pancreas; of PTF1-J and PTF1-L complexes at both sites in the acinar cell any residual activity was limited to much less than 1% of the pan- population. PTF1A, RBPJ, and RBPJL were also present at these creas. These results show that the enhancer requires binding of the sites in pancreatic chromatin during fetal development (see Fig. trimeric PTF1 complex; that is, binding of the PTF1A-E protein S1A in the supplemental material). Consistent with the previous heterodimer or an RBP alone is not sufficient. report that early pancreatic development requires PTF1-J and that MIST1 prefers binding to GC, TA, and GA/TC E boxes. RBPJL replaces RBPJ in the complex during late development MIST1 plays a critical role in regulating the secretory function of

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FIG 1 PTF1 binds to an enhancer present 6 kb upstream of Mist1 in the adult mouse pancreas. (A) Representative ChIP-Seq for binding of PTF1A, RBPJ, RBPJL, on November 14, 2017 by PURDUE UNIV LIB TSS RNA polymerase II (Pol II), and H3K4me2 at the Mist1 locus in chromatin from adult mouse pancreas. Distal (D) and proximal (P) PTF1-binding sites are present in the last intron of Lmtk2, 6 kb upstream of Mist1. The gray shading indicates the ϳ0.7-kb enhancer. (B) The nucleotide sequences of the two PTF1-binding sites are conserved in several mammals. Lowercase letters and dashes represent nucleotide differences and additions or deletions, respectively, compared to the mouse sequence. Mutations (lowercase and underlined) in the TC and E boxes (TCmut and Emut, respectively) of the PTF1 binding sites analyzed in this study are shown at the bottom.

the exocrine pancreas (18, 19); however, there is limited knowl- 523 MIST1-bound regions without a MIST1-preferred E box (E edge of MIST1 target genes (19). We performed four ChIP-Seq value, ϽEϪ140). However, this motif often occurs far away from experiments using two independently derived antibodies against the center of MIST1-bound regions and therefore is unlikely to be MIST1 to identify MIST1-bound sites in pancreatic chromatin. a novel MIST1-binding sequence. It may be the preferred binding The reproducibility among the four experiments across biological motif of an unknown factor that collaborates with MIST1. Fur- and experimental replicates and two independent MIST1 anti- thermore, the preferred RBP-binding TC boxes (TTYCY) were bodies (see Materials and Methods) was high (Pearson correlation also enriched in the MIST1-binding regions (E value, 2EϪ120) coefficients between 0.64 and 0.99) (see Table S1 in the supple- and consistent with nearby binding of PTF1. mental material). Both MIST1 antibodies detected 11,384 MIST1- MIST1 and PTF1 appear to coregulate gene expression inde- bound regions. Thirteen of 19 MIST1-bound regions previously pendent of a direct, stable physical interaction. The colocaliza- identified by ChIP-PCR (19) were also detected in our ChIP-Seq tion of MIST1 and PTF1 selectively in pancreatic acinar cells (8) analysis. and the similar effects of PTF1 and MIST1 deficiency on the acinar The predominant sequence motif present at MIST1-bound cell phenotype (13, 15) suggest that these two factors collaborate sites (E value, ϽEϪ3,000) (Fig. 4A) includes three possible E to establish and maintain cell-type-specific acinar cell functions. boxes, CAGCTG (termed GC E box), CATATG (TA E box), and To investigate the potential for direct collaboration, we compared CAGATG (GA/TC E box). Greater than 95% of MIST1-bound the genome-wide binding of MIST1 and PTF1A. The results of regions contain one of these three versions. MIST1 protein binds ChIP-Seq analyses for PTF1A and RBPJL identified a total of 6,750 GC and TA E boxes in electrophoretic mobility shift assays (31, cobound sites representing the presence of the trimeric PTF1-L 55). The GC E box is the prevailing MIST1 DNA binding motif in complex. Most of the PTF1-bound sites are between 50 kb and 500 vivo despite its lower affinity for MIST1 protein than the TA E box kb upstream or downstream of transcriptional start sites (TSSs) in vitro (55). The three E boxes are enriched at the center of (Fig. 4B). The distribution of MIST1-bound regions is similar. MIST1-bound regions (Fig. 4A), the preferred position of binding MIST1 and PTF1 had a high incidence of juxtaposed binding: 23% sequences for regions predicted from ChIP-Seq data. The seven of PTF1-binding centers had MIST1 bound within 200 bp (Fig. other E-box versions were distributed uniformly across the length 4C) (hypergeometric test P value, ϽEϪ300). of MIST1-bound sites, indicating that their occurrence is unspe- The enrichment of PTF1- and MIST1-bound regions with ac- cific. We note that the sequence CAGCAG(G/C) was present in tive chromatin marks (H3K4me2, acetylated histone 3 [H3ac], and

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FIG 2 In vivo activity of Mist1 enhancer at E15.5. The enhancer without alterations (A) or with mutations (B and C) in either the TC boxes (TCm) or E boxes (Em) was ligated upstream of a lacZ reporter and injected into fertilized mouse eggs. These eggs were then implanted in pseudopregnant female mice. Embryos on November 14, 2017 by PURDUE UNIV LIB TSS at E15.5 were stained for ␤-galactosidase (␤-Gal) activity. A representative staining photograph and the percentage of transgenic embryos showing pancreatic ␤-galactosidase activity are displayed for each transgene. A bar graph at the bottom summarizes the pancreas staining results for transgenic embryos. dp, dorsal pancreas; vp, ventral pancreas; d, duodenum; sp, spleen; st, stomach.

RNA polymerase II [50, 51, 56]) as well as the lack of the repressive mark H3K27me3 (histone 3 trimethylated at lysine 27) (51, 56)(Fig. 4E; see also Fig. S3 in the supplemental material) indicates that these two transcription factors mainly activate gene transcription. Totals of 9,082 and 5,365 genes are associated with MIST1- and PTF1-bound sites, respectively. Approximately half of PTF1-associated (51%) and MIST1-associated (47%) genes are expressed in the normal mouse pancreas (RNA-Seq RPKM of Ն1). To identify genes cobound by MIST1 and PTF1 with expression dependent on both factors, we compared the effects of the inactivation of Ptf1a and Mist1 on the pancreatic mRNA population. RNA-Seq analyses revealed that the mRNAs of 33% of the cobound genes decreased in adult pancreas after Ptf1a inactivation, and the mRNAs of 8.9% of the cobound genes decreased in Mist1-KO pancreas. A total of 157 genes were cobound by MIST1 and PTF1 and also codependent on the presence of both factors (see Table S2 in the supplemental material). Cobound and codependent target genes are designated coregulated. The binding of PTF1 and MIST1 was within 200 bp at more than half of the coregulated genes (Fig. 4D). Frequent close proximity of PTF1 and MIST1 binding sug- FIG 3 The enhancer activity is restricted to the acinar cells. To study cell gested a physical interaction between the transcription factors. A specificity of the Mist1 enhancer, immunohistochemistry was performed on sections from whole-mount ␤-galactosidase staining (blue) of two E15.5 tran- coimmunoprecipitation experiment with pancreatic nuclear ex- sient transgenic embryos bearing the wild-type enhancer. (A) CPA1 marks tracts readily detected the association between PTF1A and RBPJL acinar cells. Arrowheads, clear examples of forming acini. (B) CK19 (cytoker- but not of PTF1A with MIST1 (Fig. 4F). Thus, PTF1 and MIST1 atin 19) marks ductal cells. Arrowheads, ductal complexes; arrow and inset, coregulation does not require the formation of a stable complex ductal/acinar junction. (C) Glucagon plus insulin identifies differentiated endocrine cells. Arrowheads and inset, pre-islet endocrine clusters. Insets are containing the two factors. higher-magnification images. Scale bar, 50 ␮m. (D) Summary of the selective The collaboration of MIST1 and PTF1 is tied to the special- presence or absence of ␤-galactosidase in acinar cells (9,505/13,392, or 71%), ized characteristics of the acinar pancreas. To inquire about the ductal cells, and endocrine cells. GCG/INS, glucagon plus insulin.

December 2016 Volume 36 Number 23 Molecular and Cellular Biology mcb.asm.org 2949 Jiang et al. Downloaded from http://mcb.asm.org/ on November 14, 2017 by PURDUE UNIV LIB TSS FIG 4 Close localization between PTF1- and MIST1-bound regions. (A) MIST1 DNA-binding motifs. Distribution of the 10 possible E-box sequences relative to the centers of MIST1-bound regions. The occurrence was calculated for 10-bp windows. E boxes are named by their central two nucleotides. The inset shows the MIST1-binding motif identified with MEME (E value, 7EϪ3,310). (B) Distributions of PTF1- and MIST1-bound sites relative to transcription start sites (TSSs). (C) Distribution of PTF1-bound sites relative to the centers of MIST1-bound sites. (D) Distance between PTF1- and MIST1-bound sites associated with the MIST1- and PTF1-coregulated genes. (E) Heat map signatures (Ϯ2 kb) of PTF1A peak centers with MIST1 binding within 1 kb. Frequent colocalization of PTF1A and MIST1 with H3K4me2 and RNA polymerase II and the lack of H3K27me3 are consistent with preferential binding at transcriptional enhancers and promoters. (F) Coimmunoprecipitation of nuclear extract from adult mouse pancreas did not detect a stable physical association of MIST1 and PTF1. IP, immunoprecipitation; IB, immunoblotting. relevance of PTF1-MIST1 coregulation, we identified pancreatic scription factors bound in close proximity (38%; hypergeometric acinus-specific genes preferentially controlled by MIST1 and test P value, 2EϪ98). These results reveal the great extent of PTF1. In addition to RNA-Seq results (this report), data from MIST1 and PTF1 collaboration in regulating acinar cell processes. other sources were gleaned to provide a wide view of gene expres- We used Ingenuity Pathway Analysis and WebGestalt analysis sion. From an analysis of the microarray hybridization data for 91 of the 2,285 genes with occupancy of both MIST1 and PTF1 and mouse tissues and cell types (31), we identified 74 genes with ex- mRNA levels greater than 1 RPKM in normal pancreas to identify pression highly restricted to the pancreas (see Table S3 in the cellular pathways potentially coregulated by the two factors. This supplemental material). Of these, six are restricted to islet tissue analysis revealed that collaboration between PTF1 and MIST1 and therefore excluded from the PTF1 and MIST1 regulatory do- might control a wider range of pancreatic acinar functions, such as main. For the remaining 68, 70% (hypergeometric test P value, components of the protein synthetic machinery, regulation of 2EϪ20) had both PTF1 and MIST1 bound nearby (Fig. 5A), and translational efficiency, signal recognition particle (SRP)-depen- 40% were also codependent on the two factors. None of the endo- dent transfer of secretory proteins into the ER, ER stress, intracel- crine genes were coregulated. Among the pancreas-restricted lular transport, redox homeostasis, protein degradation, and cell/ genes are those of the secretory digestive enzymes and their se- tissue organization (Fig. 5C). Particularly enriched were genes for creted accessory proteins. More than half of these (17 of 30) are processes that compose the synthesis, processing and packaging of both cobound and codependent (see Fig. S4 in the supplemental the secretory enzymes (see Fig. S4 in the supplemental material), material), indicating the importance of the collaboration between the oxidative stress response (see Fig. S5), and exocytosis (see Ta- the two transcription factors in support of the quintessential func- ble S4). tion of pancreatic acinar cells. MIST1 and PTF1 coactivate gene transcription additively or Casting a wider net, PubMed database searches identified 4,895 synergistically. To explore the collaborative activities of MIST1 genes active in secretory cells. Of these, 2,053 were expressed in the and PTF1, we examined their actions at the control regions of four normal pancreas (RPKM of Ն1), and 69% of these genes were representative target genes by cell transfection analyses. The en- associated with either PTF1 or MIST1 binding in pancreatic chro- hancer-like control regions of Rab26, Tead2, Cabp2, and Nfe2l2 matin (Fig. 5B). Moreover, a significant fraction had both tran- have characteristic in vivo binding of PTF1A, RBPJL, and RNA

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FIG 5 Collaboration between MIST1 and PTF1 in the maintenance of the pancreatic acinar phenotype. (A and B) Pancreas-restricted genes (A) and acinus- on November 14, 2017 by PURDUE UNIV LIB TSS related genes (B) were enriched for associated MIST1 and PTF1 binding. PϩM, PTF1 and MIST1 bound. (C) Cellular pathways overrepresented in the set of MIST1- and PTF1-cobound genes, identified by IPA or KEGG pathway (via WebGestalt) analyses (Benjamini-Hochberg-adjusted Fisher’s exact test P value, Ͻ0.01). Gray bars, KEGG pathways (73); black bars, IPA pathways (Qiagen, Redwood City, CA). ERK, extracellular signal-regulated kinase; MAPK, mitogen- activated protein kinase; PI3K, phosphatidylinositol 3-kinase; AMPK, AMP-activated protein kinase; FAK, focal adhesion kinase; TGF-␤, transforming growth factor ␤. polymerase II, as well as the presence of H3K4me2 (Fig. 6A to D). sum of the two individual activation values reflects MIST1-PTF1A Each of these control regions could be activated by PTF1A or synergy at this enhancer. For the Cabp2 enhancer, PTF1A did not MIST1 expression in transfected HEK293 cells (Fig. 6E). HEK293 increase reporter activity, yet the addition of PTF1A enhanced the cells are devoid of PTF1A, MIST1, and RBPJL but have RBPJ and activation by MIST1; this increase depended on the presence of common E proteins, which can form active PTF1 complexes with intact PTF1-binding sites (see Fig. S6C in the supplemental mate- added PTF1A (5). rial). In cotransfection of RBPJL and PTF1A expression plasmids, PTF1A and MIST1 activated the Rab26 control region in an PTF1-L was no more effective than PTF1-J on the Cabp2 en- additive manner (Fig. 6E). Although the promoter-only control hancer, and both were equally effective in combination with was induced 2-fold by the combination of both factors, the induc- MIST1 (see Fig. S6C). Consistent with the augmented expression tion with the enhancer present was greater than 9-fold (t test P of Nfe2l2 in acinar cells and the presence of MIST1 and PTF1 value, Ͻ0.01) and independent of the effects on the promoter (see binding at the Nfe2l2 locus, both factors activated the enhancer Fig. S6B in the supplemental material). Mutation of the PTF1 or modestly, and together their effect was synergistic (Fig. 6E). MIST1 binding sites in the Rab26 control region abolished the induction by the corresponding factor as well as the additive effect DISCUSSION (Fig. 6E). Varying the level of cotransfected expression vectors The long-term stability of a cellular phenotype is maintained over a 9-fold range altered the magnitude of the induction pro- through the transcriptional control of cell-type-specific gene ex- portionally without changing the characteristic response patterns pression programs. In this report, we describe an extensive feed- of the mutants (see Fig. S6A), demonstrating that the levels of forward loop system that helps maintain cellular physiology and PTF1A and MIST1 tested were below saturation and within an differentiation in a mature, terminally differentiated cell type in appropriate response range. These results showed that MIST1 and vivo. Feed-forward loop regulation is an effective and widespread PTF1 coregulation of the Rab26 enhancer is dependent on their strategy of transcriptional control (57). A feed-forward loop in a binding to defined sites. gene regulatory network comprises two transcriptional regula- PTF1A and MIST1 together induced the activity of Tead2, tors, one of which binds and activates the gene of the second, and Cabp2, and Nfe2l2 control regions to an extent greater than the a target gene bound and regulated by both transcriptional regula- sum of their separate effects (Fig. 6E). PTF1A and MIST1 activated tors (Fig. 7). Positive coherent feed-forward loops in which both the Tead2 enhancer 3.8- and 2.1-fold, respectively; together, the transcriptional regulators are activators are the most prevalent induction was 8.9-fold. Total induction greater than the simple (58). The utility of feed-forward loops has been described for pro-

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FIG 6 MIST1 and PTF1 coregulate gene expression either additively or synergistically. (A to D) Representative ChIP-Seq for PTF1A, RBPJL, MIST1, and RNA polymerase II binding and H3K4me2 in adult pancreatic chromatin at the Rab26, Tead2, Cabp2, and Nfe2l2 loci. Potential binding sites of MIST1 and PTF1 are indicated with vertical lines and arrows, respectively. A schematic of MIST1- and PTF1-binding sites is shown in Fig. S6D in the supplemental material. The gray shading indicates genomic regions cloned and used in transfections. (E) Luciferase reporter constructs were transfected into HEK293 cells together with the expression vector for PTF1A and/or MIST1. The normalized luciferase activities were compared with those of the corresponding controls without addition of PTF1A and MIST1 to measure effects on the regulatory regions. WT, unmodiﬁed Rab26 enhancer; mMIST1, mutation of the three MIST1 sites; mPTF1, mutation of the E and TC boxes of the PTF1 site; mP-mM, combined mutations of MIST1 and PTF1 sites (see Materials and Methods). Error bars indicate standard errors of the means. *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. Asterisks within the bars, added factors versus the cognate control without the factors (open bars); asterisks above the bars, combined factors versus individual factors.

karyotic gene transcription networks (26), in which they rapidly occurs in mature, differentiated mammalian cells. PTF1 and provide maximal levels of induced gene expression in response to MIST1 form reiterative feed-forward loops with a large battery of carbon and energy sources and other environmental stimuli and target genes that function prominently in the maintenance of the resist responses to biologic noise (59). Feed-forward loops are also acinar cell phenotype and homeostasis. prominent in developmental programs of metazoans (60), where Whereas PTF1A is the master transcriptional regulator of pan- they provide regulatory resistance to transient inductive signals creatic acinar cell identity and differentiation (62, 63), MIST1 has and thereby reduce spurious cellular fate changes, help stabilize a more restricted role in the completion and optimization of aci- fate decisions once made, and enforce differentiation programs nar differentiation. MIST1 provides this specialization function (61). In this study, we showed that feed-forward regulation also for serous exocrine cell types of multiple organs (64). Within this

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equivalent responsibilities to PTF1 form similar reiterated feed- forward regulatory systems with MIST1 to provide the homolo- gous secretory mechanisms for these acinus-like cells as well. It seems likely that this general regulatory strategy extends to other mature cell types using additional scaling factors analogous to MIST1 for the coregulation of other differentiation states. The mechanisms that maintain the phenotype of adult cells are of potential biomedical relevance since the disruption of cellular phenotype and identity is a common basis of organ failure and

cancer. Indeed, the induction of chronic or acute pancreatitis Downloaded from FIG 7 PTF1A, RBPJL, and MIST1 collaborate via a two-stage, multioutput causes the loss of PTF1A and MIST1 and concomitant decrease of feed-forward loop system. (A) A simple positive feed-forward loop with two acinar differentiation (68–70). Moreover, PTF1A and MIST1 dis- transcription factors. (B) Feed-forward loop considering the PTF1-L complex appear in mouse and human pancreatic preneoplastic lesions as a single factor. (C) Underlying regulatory relationships illustrate two feed- forward stages (PTF1A collaborating with RBJL and MIST1) as well as auto- (PanINs) of cancer patients, and inactivation of Ptf1a or Mist1 regulatory components (PTF1 regulates both Ptf1a and Rbpjl). greatly enhances activated KRAS-driven transformation of pancreatic acinar cells in mouse models of pancreatic adenocarcinoma (22, 71, 72). An understanding of the mechanisms for the context, PTF1 and MIST1 control two broad classes of genes: maintenance of the acinar phenotype may inform interventions to http://mcb.asm.org/ those with expression highly restricted to acinar cells of the pan- prevent further degradation of acinar cells during pancreatitis or creas and others more widely expressed and highly induced in to resist the neoplastic transformation of pancreatic adenocarci- other professional secretory cells. The highly restricted genes in- noma. clude the hallmark secretory enzymes and cofactors. The more broadly expressed genes encode proteins involved in secretory ACKNOWLEDGMENTS protein production, processing, protein folding, and cellular We thank Robert Hammer and the University of Texas Southwestern stress responses. The PTF1-MIST1 partnership extends to both Transgenic Core for transgenic embryos, Jessica Williams and the Molec- gene classes. The PTF1-MIST1 loop is integrated in a more ex- ular Pathology Core for histological services, and Quan-Zhen Li and tended regulatory motif with PTF1A and RBPJL, providing addi- Ward Wakeland of the Genomics and Microarray Core for RNA and ChIP on November 14, 2017 by PURDUE UNIV LIB TSS tional properties that are expected to stabilize the acinar gene ex- sequencing. We are grateful to Christopher Wright for goat anti-mouse pression pattern (Fig. 7C). PTF1A and Jason Mills for rabbit anti-human MIST1 antibodies. We also The collaboration of MIST1 and the PTF1 complex satisfies the thank fellow lab members Mike Hale and Jumin Xue for their generous criteria for a reiterative system of feed-forward control (Fig. 7)as advice and expert assistance. This study was supported by National Institutes of Health grants R01 follows. (i) PTF1 controls Mist1 directly. PTF1 binds and activates DK55489 (S.F.K.), R01 CA124586 (S.F.K.), and R01 DK61220 (R.J.M.). Mist1 transcription through two sites in the Mist1 pancreatic enhancer; those sites are required for the activity of the enhancer in FUNDING INFORMATION pancreatic acinar cells in vivo. (ii) MIST1 and PTF1 coregulate This work, including the efforts of Stephen F. Konieczny, was funded by target genes. We identified genes with PTF1 and MIST1 bound HHS | NIH | National Cancer Institute (NCI) (R01 CA124586). This less than 200 bp apart within regions with chromatin properties of work, including the efforts of Raymond J. MacDonald, was funded by transcriptional enhancers and tested four of the regions in cell HHS | NIH | National Institute of Diabetes and Digestive and Kidney transfection assays. Tests in vitro showed that all possessed en- Diseases (NIDDK) (R01 DK61220). This work, including the efforts of hancer activity, that they were activated by PTF1A and MIST1 in Stephen F. Konieczny, was funded by HHS | NIH | National Institute of either an additive or synergistic manner, and that the activation Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK55489). depended on the retention of PTF1 and MIST1 binding site se- REFERENCES quences. (iii) The PTF1-MIST1 loop is reiterated. MIST1 and PTF1 cobind more than 2,000 target genes. Ptf1a inactivation af- 1. Gorelick FS, Jamieson JD. 2012. Structure-function relationships in the pancreatic acinar cell, p 1341–1360. In Johnson LR, Ghishan FK, Kaunitz fects (increases or decreases) nearly half of these genes, and the JD, Merchant JL, Said HM, Wood JD (ed), Physiology of the gastrointes- absence of MIST1 affects one-seventh; 157 genes are affected in tinal tract, 5th ed, vol 2. Academic Press, London, United Kingdom. common. 2. Case M. 1978. 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