Nfib Regulates Transcriptional Networks That Control the Development of Prostatic Hyperplasia

Magdalena M Grabowska; Stephen M Kelly; Amy L Reese; Justin M Cates; Tom C Case; Jianghong Zhang; David J DeGraff; Douglas W Strand; Nicole L Miller; Peter E Clark; Simon W Hayward; Richard M Gronostajski; Philip D Anderson; Robert J Matusik

doi:10.1210/en.2015-1312

. 2015 Dec 17;157(3):1094–1109. doi: 10.1210/en.2015-1312

Nfib Regulates Transcriptional Networks That Control the Development of Prostatic Hyperplasia

Magdalena M Grabowska ^1,^✉, Stephen M Kelly ¹, Amy L Reese ¹, Justin M Cates ¹, Tom C Case ¹, Jianghong Zhang ¹, David J DeGraff ¹, Douglas W Strand ¹, Nicole L Miller ¹, Peter E Clark ¹, Simon W Hayward ¹, Richard M Gronostajski ¹, Philip D Anderson ¹, Robert J Matusik ¹

PMCID: PMC4769366 PMID: 26677878

Abstract

A functional complex consisting of androgen receptor (AR) and forkhead box A1 (FOXA1) proteins supports prostatic development, differentiation, and disease. In addition, the interaction of FOXA1 with cofactors such as nuclear factor I (NFI) family members modulates AR target gene expression. However, the global role of specific NFI family members has yet to be described in the prostate. In these studies, chromatin immunoprecipitation followed by DNA sequencing in androgen-dependent LNCaP prostate cancer cells demonstrated that 64.3% of NFIB binding sites are associated with AR and FOXA1 binding sites. Interrogation of published data revealed that genes associated with NFIB binding sites are predominantly induced after dihydrotestosterone treatment of LNCaP cells, whereas NFIB knockdown studies demonstrated that loss of NFIB drives increased AR expression and superinduction of a subset of AR target genes. Notably, genes bound by NFIB only are associated with cell division and cell cycle. To define the role of NFIB in vivo, mouse Nfib knockout prostatic tissue was rescued via renal capsule engraftment. Loss of Nfib expression resulted in prostatic hyperplasia, which did not resolve in response to castration, and an expansion of an intermediate cell population in a small subset of grafts. In human benign prostatic hyperplasia, luminal NFIB loss correlated with more severe disease. Finally, some areas of intermediate cell expansion were also associated with NFIB loss. Taken together, these results show a fundamental role for NFIB as a coregulator of AR action in the prostate and in controlling prostatic hyperplasia.

The prostate gland is a walnut-sized organ located at the base of the bladder in men, and its proper development depends on androgen receptor (AR) signaling in response to testicular androgens (reviewed in Ref. 1). Studies of prostatic development in mouse have determined that prostate gland development and homeostasis are also dependent on forkhead box A1 (FOXA1) (2, 3) expression, probably due to the physical interaction between FOXA1 and AR (4). FOXA1 is a “pioneer factor” that opens chromatin structures (5) and regulates AR-mediated gene expression after exposure to androgens (4). Subsequent studies have demonstrated that additional FOXA1 cofactors, such as upstream stimulatory factor 2 and nuclear family I (NFI) family members can modulate prostate-specific gene expression (6 –8).

The NFI family is composed of 4 members, NFIA, NFIB, NFIC, and NFIX, which can bind DNA as homodimers or heterodimers (9). NFI family members are variably expressed (10) and have nonredundant functions during murine development (11 –17), suggesting that in some organs, specific NFI family members are required for proper development. Although NFI family members drive differentiation of stem cells during development, they also appear to play a critical role in maintaining stem cell quiescence in some adult tissues (18). Our previous studies have demonstrated that NFI transcription factors can modulate four AR target genes (TMPRSS2, PSA, NKX3.1, and FKBP5) in LNCaP prostate cancer cells (8). However, the genome-wide association of NFI family members and NFIB specifically in androgen-dependent prostate cancer cells is unknown.

In the mouse, mesenchymal Nfib expression is required to promote epithelial lung cell differentiation (19), and Nfib knockout mice die shortly after birth due to lung hypoplasia (11). Along with immature lungs, Nfib knockout mice also exhibit a host of neural differentiation defects, such as agenesis of the corpus callosum and loss of glial populations (14). Importantly, some Nfib heterozygous animals exhibit similar phenotypes, suggesting Nfib haploinsufficiency (14). Nfib has been implicated in adipocyte differentiation (20), neural stem cell differentiation (21, 22), and cortical development (21), in part mediated by its repression of enhancer of zeste homologue 2 (23). Although Nfib supports differentiation in the brain and lung development, it appears to maintain the “stem-ness” of melanocyte stem cells via a complex mechanism whereby Nfib in hair follicle stem cells represses endothelin 2 expression (24). Thus, the role of Nfib appears to be context dependent, with Nfib maintaining stem-ness in adult tissues but supporting differentiation during organogenesis.

The prostate gland is composed of an epithelial compartment, which includes basal, luminal, and rare neuroendocrine cell types, as well as a stromal compartment that separates adjacent glands. During prostatic development, most epithelial cells coexpress basal (cytokeratin [KRT] 14, KRT5, and p63) and luminal markers (KRT8 and KRT18), but as development concludes, these markers become largely exclusive (25). Based on the transient nature and coexpression of basal/luminal markers, these cells are referred to as intermediate cells. Intermediate cells have been proposed by numerous investigators to be in the process of differentiating into luminal cells (26, 27). Cell labeling studies in mice revealed that postnatally labeled Krt14-positive basal cells give rise to 66.5% of luminal cells, whereas basal cells labeled 2 weeks after birth give rise to 22.6% of luminal cells (28), suggesting that basal to luminal cell differentiation is largely quiescent in homeostatic adult tissues. This observation is consistent with morphologic observations of prostate gland development in mice, where most branching morphogenesis is complete 2 weeks after birth (29).

Basal to luminal cell differentiation is also observable in adult rodent prostate. Prostatic intermediate cells were first referred to as “intermediate” because their cellular morphology was an intermediate between basal and luminal cells, as observed through electron microscopy during murine prostate gland regeneration after castration (30). Intermediate cells described on the basis of cytokeratin expression were identified several years later in the rat prostate during prostate gland regeneration after castration (27). Basal to luminal cell differentiation via intermediate cells also occurs at a low rate after injury such as castration and regeneration via administration of hormone or in a setting of chronic inflammation (31, 32). Therefore, the presence of intermediate cells can indicate a critical defect in the differentiation process or increased epithelial cell turnover in response to injury.

Because of the repression of several AR target genes by NFIB (8) and of AR itself by an unspecified NFI (33), we hypothesized that NFIB will frequently co-occupy genomic locations with AR and FOXA1 and suppress prostatic hyperplasia. In this study, we have explored the genome-wide role of NFIB in androgen-dependent cancer cells through chromatin immunoprecipitation (ChIP) followed by DNA sequencing (ChIP-Seq) and validated results through quantitative real-time PCR (qRT-PCR). To determine the consequences of Nfib loss in the prostate in vivo, we have examined Nfib wild-type, heterozygous, and knockout prostates, which, because of the perinatal lethality of germline Nfib knockout mice (11), were rescued and grafted into sexually mature syngeneic hosts. We have also examined human benign prostatic hyperplasia (BPH) samples to determine whether loss of NFIB was associated with more severe BPH and/or lower urinary tract symptoms (LUTS) as well as intermediate cell expansion.

Materials and Methods

ChIP-Seq

LNCaP cells were plated in 150-mm dishes and grown to subconfluence for 3 days. Cells were then washed with Hanks' balanced salt solution (Gibco) and incubated in 10% charcoal-stripped serum (Atlanta Biologicals) medium overnight. After overnight incubation, 4 × 10⁷ LNCaP cells were treated with 10 nM dihydrotestosterone (DHT) for 2 hours, crossed-linked with 37% formaldehyde for 9 minutes, neutralized with glycine for 5 minutes, and then processed using the SimpleChIP Enzymatic Chromatin IP Kit (magnetic beads; Cell Signaling Technology) in duplicate largely according to the manufacturer's instructions. Optimizations for the kit protocol include 25 minutes for the micrococcal nuclease digestion using 10 μL of micrococcal nuclease and 4 20-second pulses using the VirTis Virsonic 100 Ultrasonic Cell Disruptor set to 6. ChIP antibodies included N-terminal AR (2 μg; Abcam ab74272), FOXA1 (2 μg; Abcam ab23738), NFIB (5 μg; Sigma HPA003956), and normal rabbit IgG (5 μg; Cell Signaling Technology 2729, included in the kit). DNA isolated from the ChIP was validated by qRT-PCR for enrichment of target promoters and submitted to HudsonAlpha for quality control, library generation, and next-generation sequencing; 50-bp single-end sequencing was performed on a HiSeq 2000 (Illumina) instrument.

Sequencing quality was evaluated by FastQC (34), aligned to the human genome hg19 using Bowtie 2 (35, 36), peaks were called using HOMER (37), and a Venn diagram was created using VennDiagram (38). Peaks that were common to both replicates were used. AR, FOXA1, and/or NFIB peaks were considered overlapping if transcription factor binding occurred ±100 bp, as modeled on the probasin promoter, PB ARR (−244 to −96 bp), which requires 148 bp to confer prostate-specific gene expression (39). Binding sites were sorted based on the genomic location bound and quantitated in Excel (Microsoft) and graphed in GraphPad Prism. Raw ChIP-Seq data were deposited at the National Center for Biotechnology Information (NCBI) as BioProject PRJNA276666.

To determine whether transcription factor binding associated with changes in gene expression, previously published RNA sequencing data (40) were analyzed. This study analyzed LNCaP cells treated with 10 nM DHT for 4 hours (40), which is 2 hours longer than our ChIP-Seq analysis, thereby theoretically reflecting changes in gene expression due to transcription factor binding. All genes associated with transcription factor binding sites were included, regardless of the genomic location where they were bound. The NCBI Gene Expression Omnibus (GEO) edgeR output was imported into R for annotation and annotated using AnnotationDbi (41) and org.Hs.eg.db (42) human genome annotation database. Genes were considered to be differentially expressed if they had a greater than 2-fold change and a P value of <.01. For analysis of differentially regulated genes bound by AR, FOXA1, and NFIB, genes associated with transcription factor–bound genomic loci were associated with genes differentially expressed by RNA-sequencing (RNA-Seq) by sorting in Excel and graphing in GraphPad. Genes associated with NFIB-only binding underwent functional annotation utilizing the DAVID Bioinformatics Resource from the National Institute of Allergy and Infectious Diseases (NIAID) (43, 44). Functional annotations were considered significant if the P value was <.05, and enriched pathways with more than 10 genes are listed.

For motif analysis, motifs from HOMER (37) were read into R, using features in the XML package and screened against 100-bp sequences around peaks identified by ChIP-Seq. To determine whether binding to their cognate motifs was functionally important, gene expression analysis was performed on previously published LNCaP microarray data (16 hours of 1 nM DHT) (45). Three microarrays were imported into R, using the affy package in Bioconductor (46). Arrays were analyzed for quality (see Supplemental Methods) and then normalized using the MAS5 algorithm to identify which genes were expressed (P > .06) or not (P < .04). Microarray data were annotated using the annotation package in R (47) and the microarray-specific (hgu133plus2) annotation database (48). For bioinformatics details, see Supplemental Methods.

NFIB knockdown studies

Transient transfections of LNCaP cells with previously validated nontargeting small interfering RNA or small interfering NFIB (8) were performed in Lipofectamine 2000 (Invitrogen). Cell lines were transfected for 3 days in serum-containing or charcoal-stripped media, depending on the experimental requirements. For DHT induction experiments, on day 3, ethanol (vehicle) or DHT was added to charcoal-stripped medium to a final concentration of 10 nM and incubated for 4 hours. Cells were then collected, RNA was extracted by the RNeasy kit (QIAGEN), and RNA was converted to cDNA using the GoScript Reverse Transcription System (Promega). qRT-PCR was performed using a MyiQ Single Color Real-Time PCR Detection System (Bio-Rad). Results were analyzed using the cycle threshold (ΔΔC_T) method, first normalized to glyceraldehyde-3-phosphate dehydrogenase and then small interfering values for the gene of interest. Statistical analysis was performed using the Mann-Whitney U test in GraphPad Prism. Primers are listed in Supplemental Table 1.

Mouse breeding and renal grafting

Prostatic tissue rescue was used as described previously (2). In brief, Nfib heterozygous (Nfib^−/+) mice were bred by Nfib^−/+ mice, and at embryonic day 18, dams were sacrificed, and urogenital sinus containing prostatic rudiments from male embryos was isolated and grafted under the renal capsule in syngenic sexually mature male host mice for 6 or 12 weeks. Six Nfib^−/− grafts were compared with 13 Nfib^−/+ and 12 Nfib^+/+ grafts. To determine the consequence of Nfib knockout on the androgen withdrawal response, syngeneic hosts bearing 6-week-old grafts underwent orchiectomy (surgical castration) and were maintained another 2 weeks before killing. Seven Nfib^−/− grafts were compared with 9 Nfib^−/+ and 6 Nfib^+/+ grafts. All animal procedures were performed in accordance with approved Institutional Animal Care and Use Committee protocols. (For an excellent technical guide to mouse urogenital development and dissection, see Ref. 49.)

Immunohistochemistry and immunofluorescence

Hematoxylin and eosin staining, immunohistochemistry, and immunofluorescence were performed as described previously (3). Antibodies used included AR N-20 (Santa Cruz, sc-816), FOXA1 C-20 (Santa Cruz, sc-6553), KRT8/18 (Fitzgerald, 20R-CP004), KRT5 EP1601Y (Abcam, ab52635), NFIB (Sigma, HPA003956), and p63 4A4 (Santa-Cruz, sc-8431). Concentration details can be found in the antibody table (Table 1). Masson's trichrome staining was performed using the Sigma-Aldrich Accustain Trichrome Stains (Masson) Kit, according to the manufacturer's instructions. Hematoxylin and eosin and immunohistochemistry images were white-balanced, whereas immunofluorescence images were black-balanced and layered in Photoshop (Adobe).

Table 1.

Antibody Table

Peptide/Protein Target	Antigen Sequence (If Known)	Name of Antibody	Manufacturer, Catalog No., and/or Name of Individual Providing the Antibody	Species Raised in; Monoclonal or Polyclonal	Dilution Used
AR	Human N terminus of AR	N-20	Santa-Cruz, sc-816	Rabbit polyclonal	1:1000 (IHC)
FOXA1	Human C terminus of FOXA1	C-20	Santa-Cruz, sc-6553	Goat polyclonal	1:1000 (IHC)
NFIB	VKSGVFNVSELVRVSRTPITQGTGVNFPIGEIPSQPYYHDMNSGVNLQRSLSSPPSSKRPKTISID ENMEPSPTGDFYPSPSSPAAGSRTWHERDQDMSSPTTMKKPEKPLFSSASPQDSSPRLSTFPQHHHPGIPGVAHSVISTRTP	NFIB	Sigma, HPA003956	Rabbit polyclonal	1:1000 (IHC); 1:100 (IF)
p63	Human 1–205	4A4	Santa-Cruz, sc-8431	Mouse monoclonal	1:100 (IF)
KRT5	Synthetic human peptide	EP1601Y	Abcam, ab52635	Rabbit monoclonal	1:1000 (IHC); 1:100 (IF)
KRT8/18	Purified bovine cytokeratins 8,18	Cytokeratin 8 + 18	Fitzgerald, 20R-CP004	Guinea pig polyclonal	1:100 (IF)

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Abbreviations: IF, immunofluorescence; IHC, immunohistochemistry.

RNA-Seq

RNA was isolated from 2 Nfib^+/+ and 2 Nfib^−/− 6-week-old renal grafts using the RNeasy kit (QIAGEN). Next-generation sequencing was performed by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core, after RNA quantitation using the Qubit RNA assay and library preparation using the Illumina TruSeq mRNA-Seq Kit. Analysis was performed using DNAnexus (Amazon Web Services) running TopHat (50), which maps FASTQ reads to a reference genome, and Cuffdiff, which identifies statistically significant changes in expression, splicing, and promoter use. A false discovery rate threshold was set at 0.05 and the log₂FC threshold was set at 1.0 (determines a 2-fold change in gene expression). Differentially expressed genes underwent functional annotation using the DAVID Bioinformatics Resource from the NIAID (43, 44). Functional annotations were considered significant at a P value of <.05. Enriched pathways with more than 10 genes are listed.

Human tissue procurement

Deidentified human BPH samples were obtained from patients who underwent surgical intervention for progressive BPH (“surgical”; 12 samples) by holmium laser enucleation of the prostate or from the transition zone of patients who underwent prostatectomy for localized, low-grade peripheral zone prostate cancer (“incidental”; 12 samples). These deidentified samples and their institutional review board–approved acquisition have been described previously (51). Samples were stained for NFIB and scored by a pathologist (J.M.C.) for nuclear intensity (0, negative; 1, weak; 2, moderate; and 3, strong) and distribution (0, negative; 1, <33%; 2, 33%–66%; and 3, >66%) in the basal, luminal, and stromal compartments. The intensity and distribution scores were combined to generate a “sum NFIB staining score” and surgical vs incidental patient NFIB scores, and American Urological Association Symptom Scores (AUASS), a measure of BPH/LUTS severity, were compared with the Mann-Whitney U test (GraphPad Prism).

Results

NFIB is frequently associated with AR/FOXA1 binding sites

Previous studies have demonstrated that the NFI family members form a complex with AR and FOXA1, and in silico analysis revealed that 34.4% of AR/FOXA1 binding sites are associated with NFI consensus sequences (8). To determine the role of one specific NFI family member, NFIB, in mediating AR target gene expression, we treated an androgen-dependent prostate cancer cell line, LNCaP, with 10 nM DHT for 2 hours and then performed ChIP with antibodies against AR, FOXA1, NFIB, and rabbit IgG (control). After next-generation sequencing and peak mapping, the overlapping peaks were identified (Figure 1A).

Our in vitro analysis shows that NFIB occupies 21.73% of AR/FOXA1 binding sites (Figure 1B). From the perspective of NFIB binding, 64.31% of NFIB binding sites are associated with AR/FOXA1, 16.56% of binding sites occur independently of AR and/or FOXA1, 12.52% are associated with FOXA1, and 6.61% are associated with AR (Figure 1C). Conversely, from the perspective of total AR and FOXA1 binding sites, AR and FOXA1 are less frequently associated with NFIB binding sites (Supplemental Figure 1, A and B).

Next, we subdivided AR, FOXA1, and NFIB binding sites into 7 groups based on transcription factor occupancy (AR-only, AR/FOXA1, AR/FOXA1/NFIB, AR/NFIB, FOXA1/NFIB, FOXA1-only, and NFIB-only) and characterized the types of genomic elements bound (intragenic, intronic, exonic, promoter [defined as −1000 bp to +100 bp from the transcription start site], 3′-untranslated region, 5′-untranslated region, and transcription termination site [−100 bp to +1000 bp from the transcription termination site]). AR, FOXA1, and AR/FOXA1 had large numbers of binding sites compared with the numbers for other sets of transcription factors (Figure 1D). With the exception of NFIB-only binding sites, combinations of transcription factors had a high proportion of intragenic (43.78%–48.43%) and intronic (47.42%–51.11%) binding events, with promoter binding events being more limited and representing 0.82% to 1.87% of binding sites (Figure 1E and Supplemental Figure 1C). However, NFIB-only binding sites were 10 times more frequently associated with promoters, with NFIB-bound promoters representing 12.89% of all NFIB-only binding sites (Figure 1E and Supplemental Figure 1C).

To test whether the 7 transcription factor sets were associated with changes in gene expression after DHT treatment, we analyzed publically available RNA-Seq analysis of LNCaP cells after 4 hours of DHT treatment (40). This generated a list of up- or down-regulated genes that were compared with genes associated with the transcription factor binding sites we identified by ChIP-Seq. The number of differentially regulated genes fluctuated by each set of transcription factors, varying from 5 to 101 (Figure 1F). Most transcription factor binding sites were associated with increased gene expression in response to DHT (Figure 1G). However, for all transcription factor sets, the largest percentage of genomic elements bound was not associated with changes in gene expression after 4 hours of DHT treatment (Figure 1, H and I).

To identify additional transcription factors that may regulate AR, FOXA1, and NFIB target genes, motif analysis was performed, and a heat map of the motifs associated with transcription factor sets bound was generated, sorted by the percentage of motifs linked with NFIB-only ChIP-Seq sites as the standard (Figure 1J; see Supplemental Figure 1D for the complete list). Most of the motifs associated with NFIB-only binding sites were associated with other sets of transcription factors. Only a small subset of transcription factor motifs (PAX8, E2F4, SP1, PBX3, E2F7, and E2F) were unique to NFIB-only binding sites (Figure 1J and Supplemental Figure 1D) and only PBX3, E2F7, and E2F family members are expressed in LNCaP cells, based on gene expression analysis (Supplemental Figure 1F).

NFIB modulates AR and AR target gene expression

Our previous studies reported that knockdown of NFIB in LNCaP cells grown in the presence of serum and, therefore, DHT was sufficient to increase expression of NKX3–1 and FKBP5 (8); however, our ChIP-Seq data overlapped with mined microarray analysis determined that gene expression associated with AR/FOXA1/NFIB binding sites mostly increases in response to DHT. To resolve this inconsistency, we selected a set of genes associated with AR/FOXA1/NFIB binding sites and determined the consequence of transient NFIB knockdown on gene expression in response to DHT. Whereas TMPRSS2 and FKBP5 gene expression increased dramatically in response to DHT induction in the absence of NFIB (Figure 2A), NKX3-1, MAPK6, SYPL1, GREB1, and IL-1β expression was not affected (Figure 2A and Supplemental Figure 2A). Surprisingly, under these conditions, loss of NFIB resulted in further repression of PSA under charcoal-stripped conditions, as well as an inhibition of IL-6R expression in response to DHT (Figure 2A).

NFIB controls gene expression in LNCaP cells. A, Most AR/FOXA1/NFIB target genes are repressed by NFIB. Genes bound by AR/FOXA1/NFIB (*TMPRSS2*, *NKX3-1*, and *IL-6R*), AR/NFIB (*FKBP5*) in ChIP-Seq, or previously reported (*PSA*) were evaluated for changes in gene expression in response to DHT induction and transient *NFIB* knockdown. Changes in gene expression for small interfering nontargeting (siNT) vs small interfering NFIB (siNFIB) were compared for both ethanol (EtOH; vehicle) and DHT treatment. B, Most NFIB-bound genes are repressed. Genes bound by NFIB in promoter regions (*PCDHGC3*) or other regions (not bound by AR/FOXA1/NFIB: *FOXA1*, *KRT8*, *KIT*, and *TGFBR3;* also bound by AR/FOXA1/NFIB: *NR3C1*) were evaluated for changes in gene expression in response to transient *NFIB* knockdown. C, NFIB represses AR gene expression. AR expression was evaluated in response to transient *NFIB* knockdown. Additional genes queried that did not have statistically significant changes are shown in Supplemental Figure 2. *, P < .05; **, P < .01.

Analysis of genes bound by NFIB, but not by AR/FOXA1/NFIB, revealed that NFIB can promote or repress gene expression. ChIP-Seq revealed NFIB-only binding sites in the promoters of BUB1B, PCDHGC3, and CCNB1; however, only PCDHGC3 expression was affected by NFIB loss (Figure 2B and Supplemental Figure 2B). Analysis of nonpromoter NFIB binding sites demonstrated that NFIB also controls the expression of KIT, TGFBR3, and KRT8 (Figure 2B). Notably, although there were no ChIP-Seq–identified NFIB binding sites associated with the AR gene, knockdown of NFIB under serum-containing conditions resulted in a significant increase in AR expression (Figure 2C). Analysis of unique NFIB-only bound peaks through DAVID determined that NFIB-bound genes are frequently associated with cell division processes (Supplemental Figure 2C).

Nfib knockout drives prostatic hyperplasia

Based on the ability of NFIB to repress AR and AR target genes, we postulated that loss of NFIB in the prostate will drive hyperplasia. We therefore grafted embryonic day 18 urogenital sinus prostatic rudiments dissected from Nfib^−/−, Nfib^−/+, and Nfib^+/+ male mice under the renal capsule of syngeneic adult male mice for 6 or 12 weeks. After 6 weeks, some Nfib^−/− grafts had areas of hyperplasia (data not shown). However, 12-week-old grafts had a more severe phenotype. Of 6 Nfib^−/− grafts, 4 had focal areas of epithelial hyperplasia and stromal thickening (Figure 3A). Surprisingly, of 13 heterozygous mice, 8 exhibited epithelial hyperplasia and stromal thickening comparable to those for the homozygous knockout mice (Figure 3A). Increased stroma associated with hyperplastic epithelium was characterized by increased collagen deposition in both Nfib^−/− and Nfib^−/+ grafts (Supplemental Figure 3B).

Because of the phenotype in the heterozygous animals, we analyzed Nfib^−/−, Nfib^−/+, and Nfib^+/+ grafts by immunohistochemistry. Nfib-positive cells were observed in the basal, luminal, and stromal compartments in both the Nfib^−/+ and Nfib^+/+ grafts but, as expected, not detected in the knockout (Nfib^−/−) (Figure 2B). In the human prostate, NFIB mRNA expression has been reported previously to be basal cell–specific (52). Because the Nfib^−/− grafts were negative for Nfib staining (Figure 3B) and Nfib is similarly expressed in the adult mouse prostate (Supplemental Figure 3A), the specificity of the antibody was confirmed and demonstrated that Nfib is expressed more broadly in the mouse prostate than anticipated based on human expression profiling (52).

Areas of hyperplasia in the Nfib^−/− grafts were characterized by an expansion of Krt5-positive basal cells (Figure 3B). In the Nfib^−/+ grafts, similar areas of Krt5-positive basal cell hyperplasia were associated with loss of Nfib expression (Figure 3B). There was no difference in Foxa1 and Ar expression between the knockout, heterozygous, and wild-type Nfib grafts (Figure 3C). As in the normal prostate, Ar was expressed in all luminal cells, and some basal and stromal cells of Nfib^−/+ and Nfib^−/− grafts, whereas Foxa1 expression was limited to the luminal cells, as reported previously (3, 53). We did not observe any changes in Ki67 or cleaved caspase 3 in areas of hyperplasia or basal cell expansion (data not shown).

Nfib^−/− and Nfib^+/+ grafts were also characterized by RNA-Seq to determine which genes and pathways were perturbed; 138 genes were differentially expressed between the knockout and wild-type grafts (Supplemental Figure 4A). No statistically significant changes were observed in Ar expression in the Nfib^−/− grafts nor in canonical Ar target genes, such as Nkx3-1, Tmprss2, Fkbp5, or Pbsn. Based on analysis with the DAVID Bioinformatics Resource (43, 44), the biologic processes that are altered in response to loss of Nfib are clustered in ion transport, immune system response, regulation of transcription, and metabolic activities (Supplemental Figure 4B.i). Of these 138 genes, 18 were associated with genomic elements bound by NFIB in ChIP-Seq in LNCaP cells (Supplemental Figure 4A). Of these 18 genes, 8 were enriched in pathways associated with regulation of gene transcription (Supplemental Figure 4B.ii).

Nfib knockout supports the expansion of intermediate cells in the prostate

In the normal adult mouse prostate, expression of basal (Krt5) and luminal (Krt8 and 18 [Krt8/18]) markers is mutually exclusive (Figure 4A). However, 2 Nfib knockout and 1 Nfib heterozygous graft exhibited an expansion of an intermediate cell population, characterized by the coexpression of basal (Krt5) and luminal (Krt8/18) epithelial cell markers (Figure 4, B and C). These grafts were characterized by large glands with multiple epithelial cell layers that maintained Ar and Foxa1 expression (Supplemental Figure 4, C and D). In the Nfib^−/− grafts, this expansion encompassed either 1 entire gland (Figure 4B) or was localized to a small area of the graft. Similarly, in the Nfib^−/+ graft, the intermediate cell expansion was focal and associated with areas that had lost Nfib expression (Figure 4C and Supplemental Figure 4, C and D). These areas were also notable because they had an expansion of basal cells, based on the expression of the basal marker p63 (54). Interestingly, there appeared to be an inverse relationship between stromal and epithelial Nfib expression in the heterozygous grafts (Figure 4C, bottom panel).

Prostatic hyperplasia driven by Nfib knockout does not resolve in response to castration

To determine whether the hyperplasia observed in Nfib was driven by androgens, a second group of mice bearing Nfib grafts underwent surgical orchiectomy and were examined 2 weeks later. Nfib^+/+ grafts were characterized by involuted glands and a stromal expansion relative to those of epithelial cells (Figure 5A). Four of 9 Nfib^−/+ grafts and 5 of 6 Nfib^−/− grafts had large areas of hyperplasia (Figure 5A), frequently associated with adjacent very small glands (Figure 5A [Nfib^−/−] and Supplemental Figure 3C [Nfib^−/−]). The large hyperplastic areas were characterized by a lack of Nfib expression and Krt5 expansion (Figure 5B). No changes between Ar and Foxa1 were observed between Nfib^−/+, Nfib^+/+, and Nfib^−/− grafts (Figure 5C). Although individual intermediate cells were observable, no large areas of intermediate cell expansion were observed, and stroma in all 3 genotypes after castration was characterized by increased collagen deposition (Supplemental Figure 3C).

Figure 5. — Nfib knockout-driven hyperplasia does not resolve in response to castration. A, Prostatic hyperplasia caused by *Nfib* knockout is not responsive to androgen withdrawal. Mice bearing 6-week-old *Nfib* grafts underwent surgical castration. Wild-type grafts underwent involution, whereas heterozygous and knockout mice maintained areas of hyperplasia. H&E, hematoxylin and eosin. B, Areas of hyperplasia are characterized by Nfib loss and expansion of Krt5-positive basal cells. In heterozygous grafts, areas of Nfib loss are hyperplastic and associated with basal cell expansion. Adjacent areas that maintain Nfib expression are not hyperplastic and do not have a basal cell expansion. C, Foxa1 and Ar levels are unaffected by Nfib loss. Ar and Foxa1 levels appear consistent across castrated *Nfib* grafts.

NFIB expression is lost in the luminal epithelium of surgical BPH samples

Because rescued Nfib^−/+ and Nfib^−/− murine prostatic tissues develop hyperplasia, we postulated that loss of NFIB in the human prostate may correlate with progression of BPH in humans. We compared 2 patient populations referred to as incidental or surgical (previously reported in Ref. 51). Incidental patients had undergone radical prostatectomy for low-grade prostate cancer, and the transition zone was used in these studies (n = 12). Conversely, the surgical patients underwent the holmium laser enucleation of the prostate procedure to relieve symptoms of progressive BPH (n = 12). Nuclear NFIB was strongly expressed in the basal compartment and variably expressed in the luminal and stromal compartments of human prostate samples (Figure 6, A and B). In both patient groups, NFIB was strongly and consistently expressed in basal cells (Supplemental Figure 5B), but in surgical BPH, NFIB expression was globally decreased in the luminal compartment (Figure 6C). Some of these patients exhibited increased stromal NFIB staining, but the overall increase was not statistically significant (Supplemental Figure 5B).

Figure 6. — NFIB is lost in the luminal cells of human BPH patients with more severe disease. A, NFIB is expressed in incidental BPH. Incidental BPH patients express NFIB in the basal and luminal compartments, with limited staining in the stroma. B, NFIB is lost in luminal cells of surgical BPH patients. Surgical BPH patients lose NFIB expression in their luminal compartment. C, NFIB expression is decreased in the luminal cells of patients with severe BPH. Quantitation of NFIB loss in luminal cells during BPH progression, with patients broken down by incidental vs surgical BPH or mild/moderate vs severe AUASS, is shown. D, Intermediate cell expansion in incidental BPH. Some incidental BPH samples have areas of intermediate cell expansion (KRT5⁺, KRT8/18⁺, and p63⁺) associated with NFIB loss. E, Intermediate cell expansion in surgical BPH. Some surgical BPH samples have focal areas of intermediate cell expansion (KRT5⁺, KRT8/18⁺, and p63⁺), also associated with NFIB loss.

To determine whether NFIB loss was associated with more severe BPH symptoms and confirm that the observed NFIB loss was not associated with different surgical procedures, tissue processing times, or enrichment of the periurethral zone in the surgical samples, we divided patients into 2 groups of BPH/LUTS symptoms based on AUASS. According to the American Urological Association guidelines, patients with AUASS of <20 (n = 9) were considered to have mild/moderate symptoms, whereas patients with AUASS of ≥20 (n = 12) were considered to have severe symptoms (55). Three patients did not have an AUASS available and were excluded from the analysis. In our small cohort (n = 21), there was no statistically significant difference between incidental and surgical AUASS (Supplemental Figure 5C). BPH patients, either by grouping surgical and incidental patients together (n = 19) or by analysis of incidental patients alone (n = 12) who had severe BPH/LUTS, had decreased expression of luminal NFIB (Figure 6C). There were no statistically significant changes in basal or stromal nuclear NFIB staining between incidental and surgical or mild/moderate and severe AUASS (Supplemental Figure 5D).

Intermediate cell expansion and NFIB loss

Because we observed NFIB loss in the luminal compartment of human BPH samples, we postulated that loss of NFIB in BPH may also be associated with an expansion of intermediate cells. Both incidental and surgical samples contained focal areas of KRT5-positive basal cell expansion (data not shown). Five of 12 incidental BPH samples and 5 of 12 surgical BPH samples had focal areas of intermediate cell expansion. Of these patients, 1 incidental (Figure 6D) and 2 surgical (Figure 6E) BPH samples had focal areas of intermediate cell expansion with a loss of NFIB expression in the epithelial compartment (Figure 6, D and E). However, most of these intermediate cells observed in all patients maintained high levels of epithelial NFIB expression (Supplemental Figure 5D).

Discussion

To determine the global role of NFIB in the prostate, we have completed a systematic analysis of NFIB function in vitro and in vivo. ChIP-Seq analysis of AR, FOXA1, and NFIB binding sites of DHT-treated LNCaP cells revealed that 64.31% of NFIB binding sites are associated with AR/FOXA1 binding sites, suggesting that a large component of NFIB function is involved with the regulation of AR/FOXA1 target genes. Previous in silico studies showed that 34.4% of AR/FOXA1 binding sites are associated with NFI consensus sites in LNCaP cells (8). Our studies now show that 21.73% of AR/FOXA1 binding sites are bound by NFIB and a total of 31% of AR/FOXA1 binding sites are associated with NFI consensus sites as determined by motif analysis, suggesting that other NFI family members may regulate these AR/FOXA1 target genes.

Analysis of previously published RNA-Seq data comparing DHT treatment with vehicle (40) determined that after 4 hours, DHT treatment differentially regulated 336 transcripts. This is a small portion of the LNCaP transcriptome, but our analysis was designed to focus only on genes that changed rapidly by at least 2-fold in this short period of time. Of the 52 genes that are bound by AR/FOXA1/NFIB and are differentially expressed, 39 increased in response to DHT. Subsequent analysis of DHT induction of LNCaP cells transiently transfected with NFIB knockdown constructs demonstrated that NFIB plays a repressive role for the AR/FOXA1/NFIB complex in a small set of genes and suggests that NFIB serves as an attenuator of gene expression.

NFIB also binds genomic elements that are not associated with either AR or FOXA1 binding sites. These represent 16.6% of the total NFIB binding sites in DHT-treated LNCaP cells. NFIB-only binding sites are associated with promoters to a much higher extent than all other sets of transcription factors examined, suggesting that NFIB action often occurs proximal to the transcription start site. Previous studies have shown that AR-occupied regions that are associated with histone H3 acetylation have enhancer ability and are associated with consensus sequences that resemble FOXA1 and NFI binding sites (7). By genomic location, the data suggest that a subset of AR/FOXA1/NFIB binding sites reflect binding to putative enhancers, whereas NFIB-only binding sites reflect NFIB-mediated gene expression from the proximal promoter. Transient knockdown of NFIB expression in LNCaP cells results in variable expression of several NFIB-bound target genes, again suggesting that the role of NFIB in gene regulation is complex, and it may be regulated by NFIB's heterodimerization with other NFI family members or in association with additional transcription factors. Potential factors include RFX and SOX2, whose motifs were frequently associated within 100 bp of NFIB binding sites, and both have been reported previously to physically interact with NFIB (56, 57). Whereas SOX2 expression in LNCaP cells is limited, SOX2 is highly expressed in prostatic basal cells (58), as is NFIB, and, therefore, it is possible that NFIB and SOX2 can interact in basal cells to mediate gene expression. Significantly, because NFIB-only bound genes are associated with cell cycle and division processes, as determined by DAVID, and our knockdown studies determined that NFIB is largely repressive in these complexes, it is likely that NFIB serves as an inhibitor of cellular proliferation.

Consistent with this idea, Nfib^−/− and Nfib^−/+ prostatic grafts displayed areas of hyperplasia in both the stromal and epithelial compartments. In the heterozygous mice, areas of hyperplasia were associated with decreased Nfib expression and increased Krt5 expression, suggesting that the loss of Nfib drives the basal cell expansion. Both Nfib^−/− and Nfib^−/+ grafts expressed Ar and Foxa1, and RNA-Seq analysis of knockout and wild-type Nfib grafts did not reveal any statistically significant changes in expression of Ar or Ar target genes. This observation is inconsistent with our in vitro data, which suggest that NFIB regulates both AR and androgen-regulated genes through the AR/FOXA1/NFIB complex. This may simply be a reflection of comparing the gene expression by RNA-Seq of a mixture of cell types found in the grafts vs gene expression from a uniform, luminal cell line. However, it also raises the possibility that the hyperplasia we are observing in the Nfib^−/− and Nfib^−/+ grafts stems from additional factors beyond aberrant Ar activation. This possibility is supported by analysis of Nfib grafts hosted in castrated mice, which revealed that Nfib knockout–induced hyperplasia does not regress in response to castration.

Based on the analysis using DAVID, differentially regulated genes from Nfib^−/− grafts cluster into ion transport, immune system response, regulation of transcription, and metabolic activities. Alterations in these pathways are consistent with an increased proliferation rate and may explain the hyperplasia observed. However, the alterations in the immune system response, especially changes in the expression of chemokines such as CCL8, CXCL13, and CCL28 are particularly interesting because of the role of chronic inflammation in the pathogenesis and progression of BPH (51, 59).

In a small subset of Nfib^−/− and Nfib^−/+ grafts, there was an expansion of intermediate cells characterized by expression of both basal (Krt5) and luminal (Krt8/18) markers. In the heterozygous animal, there was a distinct interface between the intermediate cell expansion and normal prostatic epithelium, where the interface of the 2 cell types corresponded to a loss of Nfib expression. Although the loss of Nfib supports intermediate cell expansion in the mouse prostate, Nfib loss alone is not sufficient to induce it globally in all Nfib^−/− and Nfib^−/+ grafts. It is possible that analysis of the entire graft would reveal additional areas of intermediate cells, because examining individual tissue sections is not representative of the organ as a whole, and we might be missing intermediate cells in other regions. It is however more likely that this expansion is transient and resolves into areas of basal and luminal hyperplasia.

Although intermediate cells are normally associated with prostate development (25, 28), it is unlikely that we are observing a developmental phenotypic delay because the prostates of 6-week-old grafts did not have an intermediate cell expansion but some showed an epithelial cell hyperplasia (data not shown). If this was due to a delay in development, we would expect to see it by 6 weeks rather than having to wait until the 12-week-old grafts when severe hyperplasia develops. It is possible that, as in other adult tissue (24), Nfib is required to maintain the stem-ness of progenitor cells. Previous studies in the prostate have suggested that in postnatal development, there are bipotential basal progenitor cells that can give rise to luminal or basal cells (28). The epithelial hyperplasia observed may represent basal and luminal cell progenitors undergoing differentiation into basal or luminal cells, respectively, whereas the intermediate cell expansion may reflect the differentiation of a rare bipotential basal cell. Conversely, the expansion of intermediate cells may simply be a reflection of response to injury, as intermediate cells are observable in adult mice during prostate regeneration after castration and in response to chronic inflammation (31, 32).

Consistent with our observations in mice, human prostate sections also had stromal and epithelial cell expression of NFIB. BPH patients who underwent surgery or had higher AUASS, indicting more severe disease, exhibited a loss of NFIB expression in the luminal compartment, which is consistent with the observation that loss of Nfib in mice drives hyperplasia. A subset of patients also had focal areas of intermediate cell expansion, some of which were associated with loss of NFIB expression. However, most areas of intermediate cell expansion in human BPH samples maintained NFIB expression, suggesting that NFIB loss alone is not sufficient to cause the intermediate cell expansion seen in human BPH. Moreover, consistent with the mouse studies, not all areas with loss of NFIB in human BPH showed intermediate cell expansion. Therefore, it is likely that although NFIB loss can support intermediate cell expansion, either there are additional alterations that must occur concomitantly with NFIB loss or NFIB loss must occur in the rare bipotential progenitor cell for intermediate cell expansion to occur.

Because NFIB loss is associated with more severe BPH and Nfib loss–supported hyperplasia fails to resolve in response to androgen withdrawal, the spontaneous silencing of the second allele of Nfib in heterozygous animals may have implications in human disease. The sharp demarcation between intermediate cells negative for Nfib located adjacent to normal epithelium positive for Nfib is dramatic, and this division seems to be clonal because there are stretches of continuous adjacent expansion of the cell types. Although loss of Nfib provides a proliferative advantage, how the second allele of Nfib is silenced remains unknown. Except for targeting by miR-21 (60), little is known about the regulation of NFIB expression or protein stability. miR-21 can be induced by NF-κB, AP-1, and AR, which have all been implicated in BPH pathogenesis (60 –62). Because AP-1 and miR-21 are both up-regulated in the surgical BPH patients (51), it is possible that NFIB loss in these areas is supported by transcriptional silencing by AP-1–induced miR-21. Alternatively, NFIB can also interact with ubiquitin C and small ubiquitin-like modifier 1 (63, 64), and these interactions may represent alternative mechanisms of controlling NFIB expression.

In summary, these studies have demonstrated that NFIB is globally associated with AR/FOXA1 binding sites in androgen-dependent prostate cancer cells, and genomic elements bound by NFIB are largely associated with increased expression in response to DHT treatment. However, NFIB appears to predominantly play a repressive role in and independent of the AR/FOXA1 complex. Loss of Nfib supports intermediate cell expansion and hyperplasia in the murine prostate, resulting in changes in expression of genes involved in the inflammatory response, ion transport, metabolism, and regulation of gene expression. In human BPH samples, loss of luminal NFIB expression is associated with more severe BPH, and NFIB loss is also associated with a subset of intermediate cells. These studies define a fundamental role for NFIB in regulating AR signaling and growth of the prostate. Future studies will explore whether alterations in NFIB are associated with failure of therapy in BPH and prostate cancer.

Acknowledgments

We greatly acknowledge Manik Paul for technical assistance with tissue processing, Dr Harold Love for BPH sample coordination, the Cooperative Human Tissue Network (CHTN) for providing human tissue samples, Ivo Violich (VANTAGE Core) for setting up the data analysis workflow in DNAnexus, and Dr Marie-Claire Orgebin-Crist for critical review of the article.

This work was supported by the National Institutes of Health (Grants T32-CA119925 to M.M.G., R01-DK067049 and P20-DK097782 to S.W.H., and R01-DK055748-15 to R.J.M.), the Department of Defense (Postdoctoral Training Award W81XWH-14-1-0312 to M.M.G.), the Joe C. Davis Foundation (R.J.M.), and the Vanderbilt-Ingram Cancer Center (Cancer Center Support Grant P30-CA068485). The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent official views of the National Cancer Institute, the National Institute of Diabetes and Digestive and Kidney Diseases, or the National Institutes of Health. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense or U.S. Army.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:

AR: androgen receptor
AUASS: American Urological Association Symptom Scores
BPH: benign prostatic hyperplasia
ChIP: chromatin immunoprecipitation
ChIP-Seq: chromatin immunoprecipitation followed by DNA sequencing
DHT: dihydrotestosterone
FOXA1: forkhead box A1
KRT/Krt: cytokeratin
LUTS: lower urinary tract symptoms
NIAID: National Institute of Allergy and Infectious Diseases
NCBI: National Center for Biotechnology Information
NFI: nuclear family I
qRT-PCR: quantitative real-time PCR
RNA-Seq: RNA-sequencing.

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[B1] 1. Marker PC, Donjacour AA, Dahiya R, Cunha GR. Hormonal, cellular, and molecular control of prostatic development. Dev Biol. 2003;253:165–174. [DOI] [PubMed] [Google Scholar]

[B2] 2. Gao N, Ishii K, Mirosevich J, et al. Forkhead box A1 regulates prostate ductal morphogenesis and promotes epithelial cell maturation. Development. 2005;132:3431–3443. [DOI] [PubMed] [Google Scholar]

[B3] 3. DeGraff DJ, Grabowska MM, Case TC, et al. FOXA1 deletion in luminal epithelium causes prostatic hyperplasia and alteration of differentiated phenotype. Lab Investig. 2014;94:726–739. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Gao N, Zhang J, Rao MA, et al. The role of hepatocyte nuclear factor-3 α (forkhead box A1) and androgen receptor in transcriptional regulation of prostatic genes. Mol Endocrinol. 2003;17:1484–1507. [DOI] [PubMed] [Google Scholar]

[B5] 5. Cirillo LA, Lin FR, Cuesta I, Friedman D, Jarnik M, Zaret KS. Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell. 2002;9:279–289. [DOI] [PubMed] [Google Scholar]

[B6] 6. Sun Q, Yu X, Degraff DJ, Matusik RJ. Upstream stimulatory factor 2, a novel FoxA1-interacting protein, is involved in prostate-specific gene expression. Mol Endocrinol. 2009;23:2038–2047. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7. Jia L, Berman BP, Jariwala U, et al. Genomic androgen receptor-occupied regions with different functions, defined by histone acetylation, coregulators and transcriptional capacity. PLoS One. 2008;3:e3645. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8. Grabowska MM, Elliott AD, DeGraff DJ, et al. NFI transcription factors interact with FOXA1 to regulate prostate-specific gene expression. Mol Endocrinol. 2014;28:949–964. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9. Kruse U, Sippel AE. Transcription factor nuclear factor I proteins form stable homo- and heterodimers. FEBS Lett. 1994;348:46–50. [DOI] [PubMed] [Google Scholar]

[B10] 10. Chaudhry AZ, Lyons GE, Gronostajski RM. Expression patterns of the four nuclear factor I genes during mouse embryogenesis indicate a potential role in development. Dev Dyn. 1997;208:313–325. [DOI] [PubMed] [Google Scholar]

[B11] 11. Gründer A, Ebel TT, Mallo M, et al. Nuclear factor I-B (Nfib) deficient mice have severe lung hypoplasia. Mech Dev. 2002;112:69–77. [DOI] [PubMed] [Google Scholar]

[B12] 12. Murtagh J, Martin F, Gronostajski RM. The nuclear factor I (NFI) gene family in mammary gland development and function. J Mammary Gland Biol Neoplasia. 2003;8:241–254. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Nfib Regulates Transcriptional Networks That Control the Development of Prostatic Hyperplasia

Magdalena M Grabowska

Stephen M Kelly

Amy L Reese

Justin M Cates

Tom C Case

Jianghong Zhang

David J DeGraff

Douglas W Strand

Nicole L Miller

Peter E Clark

Simon W Hayward

Richard M Gronostajski

Philip D Anderson

Robert J Matusik

Abstract

Materials and Methods

ChIP-Seq

NFIB knockdown studies

Mouse breeding and renal grafting

Immunohistochemistry and immunofluorescence

Table 1.

RNA-Seq

Human tissue procurement

Results

NFIB is frequently associated with AR/FOXA1 binding sites

Figure 1.

NFIB modulates AR and AR target gene expression

Figure 2.

Nfib knockout drives prostatic hyperplasia

Figure 3.

Nfib knockout supports the expansion of intermediate cells in the prostate

Figure 4.

Prostatic hyperplasia driven by Nfib knockout does not resolve in response to castration

Figure 5.

NFIB expression is lost in the luminal epithelium of surgical BPH samples

Figure 6.

Intermediate cell expansion and NFIB loss

Discussion

Acknowledgments

Footnotes

References

ACTIONS

PERMALINK

RESOURCES

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