NFIC regulates ribosomal biology and ER stress in pancreatic acinar cells and suppresses PDAC initiation

TL;DR: In this paper, NFIC binding sites are found at very short distances from NR5A2-bound genomic regions and both proteins co-occur in the same complex and NFIC dampens the ER stress program through its binding to ER stress gene promoters and is required for complete resolution of Tunicamycin-mediated ER stress.

Abstract: Tissue-specific differentiation is driven by specialized transcriptional networks. Pancreatic acinar cells crucially rely on the PTF1 complex, and on additional transcription factors, to deploy their transcriptional program. Here, we identify NFIC as a novel regulator of acinar differentiation using a variety of methodological strategies. NFIC binding sites are found at very short distances from NR5A2-bound genomic regions and both proteins co-occur in the same complex. Nfic knockout mice show reduced expression of acinar genes and, in ChIP-seq experiments, NFIC binds the promoters of acinar genes. In addition, NFIC binds to the promoter of, and regulates, genes involved in RNA and protein metabolism; in Nfic knockout mice, p-RS6K1 and p-IEF4E are down-regulated indicating reduced activity of the mTOR pathway. In 266-6 acinar cells, NFIC dampens the ER stress program through its binding to ER stress gene promoters and is required for complete resolution of Tunicamycin-mediated ER stress. Normal human pancreata from subjects with low NFIC mRNA levels display reduced epxression of genes down-regulated in Nfic knockout mice. Consistently, NFIC displays reduced expression upon induced acute pancreatitis and is required for proper recovery after damage. Finally, expression of NFIC is lower in samples of mouse and human pancreatic ductal adenocarcinoma and Nfic knockout mice develop an increased number of mutant Kras-driven pre-neoplastic lesions.

Summary

INTRODUCTION

• Pancreatic acinar cells are highly specialized protein synthesis factories that have a well-developed rough endoplasmic reticulum (ER), a prominent Golgi complex, and abundant secretory granules 1 .
• Monoallelic or homozygous inactivation of several acinar transcriptional regulators in the germline, the embryonic pancreas, or the adult pancreas can result in compromised acinar function that favors loss of cellular identity and poises acinar cells for transformation upon activation of mutant KRas 10, 11, 12 .
• Additional roles have been proposed through the regulation of Trp53 15, 16, 17 .
• (which was not certified by peer review) is the author/funder.
• Using a combination of omics analyses and studies in knockout mice and cultured cells, the authors now uncover novel roles of NFIC as a regulator of acinar function whose major impact is at the level of the ER stress response in murine and human pancreas.

Identification of novel transcription factors involved in the regulation of pancreatic

• To discover novel transcription factors that might cooperate with known acinar regulators (e.g. PTF1A, GATA6, NR5A2, and MIST1), the authors reanalyzed publicly available ChIP-sequencing data and used HOMER to search for motifs enriched in the sequencing reads.
• Motifs corresponding to RBPJ/RBPJL and HNF1, known regulators of acinar differentiation, were also enriched, thus validating the strategy applied.
• Of all NFI family members, NFIC is expressed at highest levels in both mouse and human pancreas ; therefore, the authors focused on NFIC for further study.
• Analysis of the published ChIP-Seq data revealed several PTF1A and NR5A2 peaks in the proximal Nfic promoter and the binding was confirmed by ChIP-qPCR , strongly suggesting that Nfic is a PTF1A and NR5A2 target.
• The copyright holder for this preprint this version posted August 9, 2021.

DISCUSSION

• Increasing evidence supports an "analog" differentiation model whereby additional TFs are required for "completion" of this process.
• The conservation of spacing between NR5A2 and NFIC motifs among NR5A2 target genes supports transcriptional cooperation.
• Comparison of NR5A2 ChIP-Seq data from embryonic and adult pancreas indicates that the role of NFIC is mainly in the latter, supporting its requirement for completion of acinar maturation and highlighting a functional role distinct from that of NR5A2.
• NFI proteins were first proposed to be involved in the regulation of ubiquitous genes but they can also regulate tissue-specific genes 13 , including CEL in the mammary gland and DSPP in odontoblasts 14, 19 .
• These findings suggest that the activation of pro-inflammatory phenotypes in mice in which acinar cells fail to acquire normal maturation can result from both direct (NR5A2 and GATA6) 4, 7, 10, 12 and indirect (PTF1A and NFIC) mechanisms, in agreement with the ChIP-Seq data available.

NFIC regulates expression of ribosomal genes and mitigates ER stress in the

• Accordingly, a coordinated down-regulation of gene sets related to the digestive process and to protein metabolism and oxidative phosphorylation occurs in adult Nfic -/pancreata.
• The mTOR pathway is a central actor in protein biosynthesis and autophagy and, therefore, a candidate mediator of this phenotype (reviewed in [33]).
• The authors found reduced levels of P-RS6K1, its substrate P-RS6, and P-EIF4E, together with an up-regulation of P-ERK, in acinar cells of Nfic -/mice.
• The finding that NFIC -but not NR5A2 -binds the promoter of ER stress genes suggests a distinct role of the former in this process.
• A re-analysis of published data shows that Nfic is down-regulated in Nr5a2 -/hepatocytes in basal conditions (fold change of -0.54) 35 , suggesting that the deficient response to TM in Nr5a2 -/hepatocytes might partially occur through Nfic down-regulation.

NFIC is dynamically regulated during pancreatitis and cancer.

• A failure to achieve complete acinar maturation is associated with more severe damage and delayed recovery during caerulein-mediated pancreatitis, as shown upon inactivation of Gata6, Mist1, and Ptf1a in the pancreas 4, 44 .
• The role of NFIC in acinar cell differentiation and mitigation of ER stress suggested a contribution during tumorigenesis.
• The copyright holder for this preprint this version posted August 9, 2021.
• TFs previously described, the role of NFIC is restricted to the adult pancreas and distinctly affects RNA and protein metabolism and the UPR-mediated ER stress.

MATERIAL AND METHODS

• All crosses were maintained in a predominant C57BL/6 background.
• In brief, animals were weighed before the procedure and caerulein was administered intraperitoneally.
• For the glucose tolerance test, male mice were fasted for 16 h and basal glycaemia was measured in tail blood.
• For most experiments, >5 mice per group were used.

Histology, immunofluorescence (IF) and immunohistochemical (IHC) analyses.

• Histological processing was performed using standard procedures.
• After washing with PBS, sections were mounted with Prolong Gold Antifade Reagent (Life Technology).
• Sections were incubated with 2% BSA-PBS for 1h at room temperature, and then with the primary antibody overnight at 4 ºC.
• Briefly, areas of interest (AOI) were selected for quantification and then exported as individual TIFF images.
• For quantification of KI67 + positive cells, at least 10 random images from each pancreas were selected and only positive acinar cells were quantified.

Quantitative RT-PCR (RT-qPCR).

• Total RNA was treated with DNase I for 30 min at 37 °C and cDNAs were prepared according to the manufacturer's specifications, using the TaqMan reverse transcription reagents (Applied (which was not certified by peer review) is the author/funder.
• For immunoprecipitation of proteins from fresh total pancreas lysates, a piece of mouse pancreas was isolated and minced in 50 mM Tris-HCl pH 8, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40 containing 3× phosphatase inhibitor cocktail (Sigma-Aldrich) and 3× EDTA-free complete protease inhibitor cocktail .
• Afterwards, beads were washed three times with coupling buffer and once with 1 M Tris pH 9.
• Protein lysates (10-15 mg, tissues) were then incubated overnight at 4 °C with antibody-coated dynabeads (Thermo Fisher Scientific).

Chromatin immunoprecipitation (ChIP).

• Pancreas tissue was minced, washed with cold PBS supplemented with 3× protease and phosphatase cocktail inhibitors, and then fixed with 1% formaldehyde for 20 min at room temperature.
• The supernatant was collected after centrifugation and chromatin was sonicated with a Covaris instrument for 40 min (20% duty cycle; 10% intensity; 200 cycle), yielding DNA fragments with a bulk size of 300-500 bp.
• Data were analysed using the nextpresso pipeline http://bioinfo.cnio.es/nextpresso/).

Gene Set Enrichment Analysis (GSEA).

• A ranking metric [-log10(p value)/sign(log2FoldChange)] was used to generate a ranked gene list from the DEseq output.
• The resulting libraries were sequenced on Illumina HiSeq 2500, v4 Chemistry.
• The copyright holder for this preprint this version posted August 9, 2021.
• Among the two replicates, the one with highest number of identified target genes was taken: replicate 1 of NFIC ChIP-Seq in GM12878, NFIC ChIP-Seq in HepG2 and NFIC ChIP-Seq in SK-N-SH and replicate 2 of NFIC ChIP-Seq in ECC1 cell line.

Figures

Figure 7. NFIC restrains formation of preneoplastic lesions in the pancreas. (A) 826 IHC analysis of NFIC expression shows down-regulation in PanINs (right) and tumor 827 cells compared to adjacent normal acinar cells (left). (B) NFIC mRNA analysis of tumor 828 samples and normal adjacent tissue assessed by microarrays (Janky et al., 2016) 829 showing reduced expression in tumor samples. (C,D) IHC analysis of NFIC expression 830 in human PDAC specimens showing reduced expression in tumoral cells (arrow) 831 compared to normal adjacent tissue or stromal cells (arrowheads). (E,F) Histological 832 analysis of the pancreas of KRasG12V or KRasG12V;Nfic-/- 14-20 week old mice showing 833 increased number of PanINs and area occupied by pre-neoplastic lesions 834 (n>5/genotype). 835

Full text

NFIC regulates ribosomal biology and ER stress in pancreatic acinar cells
1
and suppresses PDAC initiation
2
3
4
Isidoro Cobo,
1,2
Sumit Paliwal,
1
Júlia Melià-Alomà,
1,3
Ariadna Torres,
1,3
5
Jaime Martínez-Villarreal,
1
Fernando García,
4
Irene Millán,
1,2
Natalia del Pozo,
1,2
Joo-
6
Cheol Park,
5
Ray J. MacDonald,
6
Javier Muñoz,
4
and Francisco X. Real
1-3
7
8
9
Short title: NFIC in pancreatic homeostasis and cancer
10
11
1
Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre-CNIO,
12
Madrid, Spain
13
2
CIBERONC, Madrid, Spain
14
3
Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra,
15
Barcelona, Spain
16
4
Proteomics Unit, Spanish National Cancer Research Centre-CNIO, Madrid, Spain.
17
ProteoRed - ISCIII
18
5
Department of Oral Histology-Developmental Biology, School of Dentistry, Seoul
19
National University, Seoul, Korea
20
6
Department of Molecular Biology, University of Texas Southwestern Medical Center,
21
Dallas, TX, USA
22
23
24
25
Correspondence: Francisco X. Real
26
Centro Nacional de Investigaciones Oncológicas-CNIO
27
Melchor Fernández Almagro, 3
28
28029-Madrid, Spain
29
E-mail: preal@cnio.es
30
31
32
Conflicts of interest: none to declare
33
34
35
Funding: This work was supported, in part, by grants SAF2011-29530, SAF2015-70553-
36
R, and RTI2018-101071-B-I00 from Ministerio de Ciencia, Innovación y Universidades
37
(Madrid, Spain) (co-funded by the ERDF-EU) and RTICC from Instituto de Salud Carlos
38
III (RD12/0036/0034) to FXR. IC was recipient of a Beca de Formación del Personal
39
Investigador from Ministerio de Economía y Competitividad (Madrid, Spain). The
40
research leading to these results has received funding from People Programme (Marie
41
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint

2
Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-
42
2013) (REA grant agreement n° 608765”). SP was supported by a Juan de la Cierva
43
Programme fellowship from Ministerio de Ciencia, Innovación y Universidades. IM was
44
supported by a Fellowship from Fundació Bancaria La Caixa (ID 100010434) (grant
45
number LCF/BQ/ES18/11670009). CNIO is supported by Ministerio de Ciencia,
46
Innovación y Universidades as a Centro de Excelencia Severo Ochoa SEV-2015-0510.
47
48
Statement of author contributions
49
IC: study concept and design; acquisition of data; analysis and interpretation of data;
50
statistical analysis; drafting of the manuscript;
51
SP: acquisition of data; analysis and interpretation of data; drafting of the manuscript
52
JMA: acquisition of data; analysis and interpretation of data; drafting of the manuscript
53
AT: acquisition of data; analysis and interpretation of data;
54
JMV: analysis and interpretation of data;
55
FG: acquisition of data; analysis and interpretation of data;
56
IM: analysis and interpretation of data;
57
NdP: technical support and acquisition of data;
58
JCP: material support;
59
RJM: critical revision of the data and important intellectual content;
60
JM: acquisition of data; analysis and interpretation of data;
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FXR: study concept and design; analysis and interpretation of data; drafting of the
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manuscript; overall study supervision; obtained funding.
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All authors provided input about manuscript content.
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Accession numbers: RNA sequencing data have been deposited in GEO with
65
accession number GSE126907 and NFIC ChIP sequencing data have been deposited
66
in GEO with accession number GSE181098
67
68
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint

3
ABSTRACT
69
70
Tissue-specific differentiation is driven by specialized transcriptional networks.
71
Pancreatic acinar cells crucially rely on the PTF1 complex, and on additional
72
transcription factors, to deploy their transcriptional program. Here, we identify NFIC as a
73
novel regulator of acinar differentiation using a variety of methodological strategies. NFIC
74
binding sites are found at very short distances from NR5A2-bound genomic regions and
75
both proteins co-occur in the same complex. Nfic knockout mice show reduced
76
expression of acinar genes and, in ChIP-seq experiments, NFIC binds the promoters of
77
acinar genes. In addition, NFIC binds to the promoter of, and regulates, genes involved
78
in RNA and protein metabolism; in Nfic knockout mice, p-RS6K1 and p-IEF4E are down-
79
regulated indicating reduced activity of the mTOR pathway. In 266-6 acinar cells, NFIC
80
dampens the ER stress program through its binding to ER stress gene promoters and is
81
required for complete resolution of Tunicamycin-mediated ER stress. Normal human
82
pancreata from subjects with low NFIC mRNA levels display reduced epxression of
83
genes down-regulated in Nfic knockout mice. Consistently, NFIC displays reduced
84
expression upon induced acute pancreatitis and is required for proper recovery after
85
damage. Finally, expression of NFIC is lower in samples of mouse and human pancreatic
86
ductal adenocarcinoma and Nfic knockout mice develop an increased number of mutant
87
Kras-driven pre-neoplastic lesions.
88
89
Word count: 211
90
91
Keywords: NFIC, pancreas, acinar differentiation, ribosome, endoplasmic reticulum
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stress, unfolded protein response, transcriptional networks, pancreatitis, pancreatic
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cancer
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95
Abbreviations: ChIP, chromatin immunoprecipitation; DEG, differentially expressed
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genes; EMT, epithelial-mesenchymal transition; ER, endoplasmic reticulum; GSEA,
97
Gene set enrichment analysis; IF, immunofluorescence; IHC; immunohistochemistry;
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PDAC, pancreatic ductal adenocarcinoma; TF, transcription factor; TM, tunicamycin;
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UPR, unfolded protein response.
100
101
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint

4
INTRODUCTION
102
103
Pancreatic acinar cells are highly specialized protein synthesis factories that
104
have a well-developed rough endoplasmic reticulum (ER), a prominent Golgi complex,
105
and abundant secretory granules
1
. Acinar differentiation is contingent on the activity of a
106
master regulator, the adult PTF1 complex, composed of the pancreas-specific
107
transcription factors (TFs) PTF1A and RPBJL and the ubiquitous protein E47
2,3
. PTF1
108
binds the proximal promoter of genes coding for digestive enzymes, secretory proteins
109
and other TFs, and activates their expression. The PTF1 complex is the main driver of
110
acinar differentiation but additional TF with tissue-restricted expression patterns are
111
implicated in the fine-tuning of this process, including GATA6
4
, MIST1
5
, and
112
NR5A2/LRH-1
6,7
. Acinar cells play a crucial role in acute and chronic pancreatitis, two
113
common and disabling conditions. Recent work using genetic mouse models has shown
114
that, upon expression of mutant KRas, acinar cells can be the precursors of Pancreatic
115
Intraepithelial Neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC)
8,9
.
116
Our laboratory and others have shown that the acinar differentiation program acts
117
as a tumor suppressor in the pancreas. Monoallelic or homozygous inactivation of
118
several acinar transcriptional regulators in the germline, the embryonic pancreas, or the
119
adult pancreas can result in compromised acinar function that favors loss of cellular
120
identity and poises acinar cells for transformation upon activation of mutant KRas
10,11,12
.
121
The tumor suppressive function of these TF is not obvious because the exocrine
122
pancreas has a large functional reserve, i.e. massive alterations in cellular function need
123
to occur in order to be reflected in histological or clinical changes.
124
Here, we use bioinformatics tools to identify NFIC as a novel acinar regulator.
125
NFIC is a member of the nuclear factor I family of TFs that regulate both ubiquitous and
126
tissue-restricted genes
13
. In the mammary gland, NFIC activates the expression of milk
127
genes involved in lactation
14
. Furthermore, it acts as a breast cancer tumor suppressor,
128
as it directly represses the expression of Ccnd1 and Foxf1, a potent inducer of epithelial-
129
mesenchymal transition (EMT), invasiveness, and tumorigenicity. Additional roles have
130
been proposed through the regulation of Trp53
15,16,17
. The physiological role of NFIC has
131
been best studied in dentinogenesis, since Nfic
-/-
mice develop short molar roots and
132
display aberrant odontoblast differentiation and dentin formation
18
. NFIC regulates
133
odontoblast-related genes, including Dssp
19
, Wnt
20
, and hedgehog signaling
21
.
134
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint

5
Using a combination of omics analyses and studies in knockout mice and cultured
135
cells, we now uncover novel roles of NFIC as a regulator of acinar function whose major
136
impact is at the level of the ER stress response in murine and human pancreas. Unlike
137
most other TFs previously identified as required for full acinar function, NFIC belongs to
138
a novel family of acinar regulators with tissue-wide expression. NFIC dysregulation
139
sensitizes the pancreas to damage and neoplastic transformation.
140
141
142
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint