extfieldsofstudy: Biology Medicine

text:
Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing drosophila, Drosophila suzukii

Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2), using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) homology-directed repair gene-editing. Ds-tra-2 ts2 mutants developed as normal fertile XX and XY adults at permissive temperatures below 20 °C, but at higher restrictive temperatures (26 to 29 °C) chromosomal XX females developed as sterile intersexuals with a predominant male phenotype, while XY males developed with normal morphology, but were sterile. The temperature-dependent function of the Ds-TRA-2ts2 protein was also evident by the up- and down-regulation of female-specific Ds-Yolk protein 1 (Ds-Yp1) gene expression by temperature shifts during adulthood. This study confirmed the temperature-dependent function of a gene-edited mutation and provides a new method for the more general creation of conditional mutations for functional genomic analysis in insects, and other organisms. Furthermore, it provides a temperature-dependent system for creating sterile male populations useful for enhancing the efficacy of biologically-based programs, such as the sterile insect technique (SIT), to control D. suzukii and other insect pest species of agricultural and medical importance.

. Strategy for generating the Ds-tra2 ts2 mutation and IE1hr5-DsRed marker insertion via CRISPR/Cas9 homology-directed repair. (a) Schematic (not to scale) showing the wild type Ds-tra-2 gene and downstream HA2 (homologous arm 2) sequence with expected recombination sites (X) for the Cas9/sgRNA complexes (top). Dotted lines from these sites extend to the donor construct recombination sites that include the HA1 Ds-tra-2 sequence mutagenized in vitro to create a Pro191Ser substitution (vertical red line) resulting in the mutated Ds-TRA2 ts2 temperature sensitive protein, in addition to the IE1hr5-DsRed marker gene and HA2 sequence. The recombined donor construct resulting in the genomic insertion of the Ds-tra2 ts2 mutation and marker gene is shown below. (b) Ventral view of a homozygous Ds-tra2 ts2 adult XY male (left) and XX female (right) reared at 16 °C under brightfield, and (c) DsRed epifluorescence illumination. See Methods for further details.
be the biogenesis of lysosome-related organelles complex 1 subunit 1 gene (GenBank: XM_017072992). To bias editing towards HDR, the ligase 4 gene, a component of the non-homologous end joining (NHEJ) repair pathway, was suppressed using dsRNA-mediated RNA interference (RNAi). A mixture of sgRNAs, Cas9 protein, Ds-ligase-4 dsRNA and donor construct was injected into pre-blastoderm embryos of a D. suzukii wild strain, yielding 81 G0 adult flies (36 males and 45 females). Of these, 55 flies were fertile after individual backcrosses to wild type, with four G0 lines yielding G1 progeny expressing whole body red fluorescence, resulting in a germ-line transmission rate of 7.3% (4/55) based on marker expression. From each G0 line, two to five red fluorescent G1 flies (Fig. 1c) were selected to establish independent G1 lines. After being inbred to homozygosity, six lines were analyzed by PCR and sequencing showing that all lines had an insertion of the IE1hr5-DsRed marker gene, however, only three lines (F26m1, F26f4 and M36f1, from two G0 lines) carried the correct Ds-tra-2 ts2 (CCT > TCT) mutation while the other three lines could not be confirmed, resulting in a minimum mutation rate of 3.6% (2/55).

Temperature sensitivity of Ds-TRA-2 protein function based on adult morphology and fertility.
To determine the effect of temperature on the mutated Ds-TRA-2 protein in terms of morphological sexual differentiation in chromosomal females and males, the homozygous mutant line, M36f1, was first reared continuously at the expected permissive temperature of 16 °C, in addition to 20 °C. This resulted in normal development and fertility in all XX females and XY males at both temperatures ( Fig. 1b; Table 1). However, when rearing was performed at the expected restrictive temperature of 29 °C, all mutants died during late pupation, as did wild type control flies. Rearing at 27 °C and 28 °C also resulted in inviability for the mutant line and a low survival rate for wild type (1-2%), with low levels of survival (5-10%) for both wild type and mutant flies first observed at 26 °C. At this temperature 80 surviving chromosomal female XX; Ds-tra-2 ts2 homozygotes exhibited an intersexual, though predominantly male (or pseudo-male), phenotype ( Fig. 2c and f), but male-specific wing spots were not apparent (Fig. 2c). However, in a separate experiment where XX; Ds-tra-2 ts2 individuals (n = 21), were reared at 22 °C and shifted to 29 °C as late pupae, wing spot pigmentation was observed at 3 days after eclosion (Fig. 2c inset; Supplementary Table S1). Compared to mature ovaries from WT females (Fig. 2i), XX; Ds-tra-2 ts2 pseudo-males had a single poorly differentiated gonadal lobe (Fig. 2j) and no internal male reproductive organs, similar to XX; Dm-tra-2 ts2 flies reared at 29 °C 1 . At 26 °C pseudo-males were also sterile with fully differentiated male-specific foreleg sex combs (Fig. 3c) and male-specific pigmentation on tergite 6 ( Fig. 3f), while tergite 5 lacked male-specific pigmentation and female-specific tergite 7 was observed. The differing sexual identity of the abdominal tergites, as well as other sex-specific tissues and structures, can most simply be attributed to a variable sensitivity to Ds-TRA-2 function that was diminished in pseudo-males reared at a sub-optimal restrictive temperature.
A detailed examination of the external genitalia revealed that pseudo-males exhibited terminalia similar to WT males (Fig. 4), including the anterior paramere (clasper), posterior paramere, aedeagus and anal plates (Fig. 4c). However, the basal apodeme of the aedeagus was not apparent (Fig. 4a) and the female-specific sternite 8 was observed, while the female-specific spermatheca was not observed. At 26 °C, XY; Ds-tra-2 ts2 chromosomal males had normal external morphology, but were sterile consistent with poorly differentiated abnormal testes that had a small number of immotile sperm (n = 15, Fig. 2h, Supplementary Video S1), compared to the long spiral testes containing motile sperm found in WT males (Fig. 2g, Supplementary Video S2).
To more precisely determine the temperature sensitivity of Ds-TRA-2 function in the mutant line, adult morphology and fertility were assessed in XY and XX; Ds-tra-2 ts2 adults that developed at rearing temperatures ranging from 16 °C to 26 °C (Table 1). As noted, at the permissive temperatures of 16-20 °C both XX females and XY males were morphologically normal (Fig. 1b) and fertile, but at 22 °C XX females first exhibited male sex combs and were sterile based on a failure to oviposit (n = 61), while WT females oviposited ≥78 fertile eggs/ female (n ≥ 39) when reared at 20-24 °C ( some exhibiting dysmorphic testes and sterility at 24-25 °C, that included all males above 25 °C. These results are consistent with Ds-TRA-2 function, or the quantity of functional protein, diminishing just above permissive temperature, with an increasing loss of Ds-TRA-2 activity with rising temperature. The sex-specificity of somatic tissue and germ-line function appeared to be more highly sensitive to loss of Ds-TRA-2 activity in XX females (i.e., onset of sex comb differentiation and sterility) than the influence on testis morphology and germ-line function (i.e., fertility) in XY males where the onset of defects were first detected at a higher temperature. Complete sterility in XY; Ds-tra-2 ts2 males could be achieved by rearing at 25.5 °C or above, but viability at these temperatures was severely limited. Thus, XY; Ds-tra-2 ts2 males were reared at 22 °C until pharate adulthood or adult eclosion resulting in viability of 80 to 90%, and shifted to 26 °C and/or 29 °C to determine if these late shifts to restrictive temperature would result in sterility (Supplementary Table S1). Sterility was achieved in 90 to 100% of the males, with greater sterility in those that were shifted earlier and to the higher temperature, although this resulted in lower viability.

Mating behavior of mutant intersexes and pseudo-males.
In D. melanogaster, temperature sensitive male courtship behavior and mating in XX individuals required a nearly null Dm-tra-2 genotype that used the stronger Dm-tra-2 ts1 allele over a Dm-tra-2 deficiency 11 . For XX; Ds-tra-2 ts2 pseudo-males, flies reared at 26 °C (n = 20), and shifted to 29 °C for 3d at eclosion from 26 °C (n = 15) or as late pupae (n = 23) did not attempt courtship or mating with wild type virgin females, while XY; Ds-tra-2 ts2 males reared at 26 °C (n = 22) did mate, but were sterile. At 22 °C, XX; Ds-tra-2 ts2 females developed female genital terminalia and did not attempt courtship with WT virgin females (n = 29), but were courted by WT and XY; Ds-tra-2 ts2 males who were rejected (n = 20).
Temperature sensitivity of Ds-TRA-2 function based on terminal sex-specific Yp1 expression during adulthood. Similar to studies on XX; Dm-tra-2 ts2 in D. melanogaster 5 , the influence of Ds-TRA-2 activity on molecular and biochemical aspects of terminal sexual differentiation could be explored by applying temperature shifts to mutant adults. For D. suzukii in particular, the ability of adults to survive at 29 °C, after rearing at 26 °C or below, also allowed a more definitive test of the optimal restrictive temperature on Ds-TRA-2 ts2 protein function. This was achieved by assessing the effect of temperature and temperature shifts on the sex specific expression of the D. suzukii yolk protein 1 (Ds-Yp1) gene in Ds-tra-2 ts2 mutants that, in D. melanogaster, is normally expressed only in female adult fat body and the ovarian follicular epithelium beginning at adult eclosion 16,17 .  To measure Ds-Yp1 transcript levels by qPCR, Ds-Yp1 was first identified by its similarity to D. melanogaster Yp1, having 94.3% amino acid sequence identity ( Supplementary Fig. S1), providing primer sites for analysis. Initial qPCR tests for the primers showed that transcript levels were similar in XX WT and F23f1 control females, which were >20-fold higher than transcript in XX; Ds-tra-2 ts2 pseudo-male adults reared at 26 °C ( Supplementary  Fig. S2). For experimental tests, Ds-Yp1 transcripts were measured in XX; Ds-tra-2 ts2 (M36f1) phenotypic females and WT females reared and then maintained at 16 °C until 3 d, 6 d, and 9 d after eclosion ( Fig. 5a; Supplementary  Table S2). Transcript levels were not significantly different between the mutant and WT females at these time points, though transcript increased between days 3 and 6, and then decreased in both WT and XX; Ds-tra-2 ts2 females by day 9, though more so in M36f1 females (P > 0.05, two-tailed, no significant difference at all time points; Supplementary Table S2). In contrast, when mutant and WT females reared at 16 °C were shifted to 29 °C at eclosion, after 3 days WT females expressed >27-fold higher levels of Ds-Yp1 transcript compared to XX; Ds-tra-2 ts2 females (Fig. 5b). Decreasing levels of transcript occurred in both groups at 6 d and 9 d, with negligible transcript detected in mutant females after 9 d at 29 °C. To determine whether the basal transcript level observed in mutant females at 29 °C for 3 days could be reversed to a more normal female phenotype by turning on Ds-TRA-2 function, sibling flies were shifted to 16 °C at 3 d and assayed at days 6 and 9 after eclosion (Fig. 5c). At 6 d the Ds-Yp1 transcript level in WT females decreased ~40% (presumably due to a slowed metabolism), but increased ~9-fold in XX; Ds-tra-2 ts2 females, with similar levels maintained in both groups at 9 d (P > 0.05, two-tailed). However, if females at 6 d at 16 °C were shifted to 29 °C until 9 d, transcript levels decreased by half in WT females, but were barely detectable in the XX; Ds-tra-2 ts2 females, decreasing by >40-fold from day 6 (P < 0.05, two-tailed; Fig. 5d).
These results showed that adult XX; Ds-tra-2 ts2 phenotypic females reared at 16 °C exhibit normal female expression of Ds-Yp1, but when sibling females were shifted from 16 °C to 29 °C, low diminished transcript levels resulted reaching non-detectable levels, normally expected in XY males, by day 9. Furthermore, Ds-Yp1 transcript levels increased and decreased according to shifts in temperature to permissive and restrictive conditions, respectively, indicating that the dynamic, and reversible, expression of Ds-Yp1 during adulthood is continuously Figure 5. Response of Ds-Yp1 transcript expression to temperature. Ds-Yp1 transcript expression (relative to internal Ds-His3 controls) measured by qPCR from wild type females (WT-f, blue bars), XX; Ds-tra2 ts2 females (M36f1, red bars), and wild type males (WT-m, green bars) in response to indicated temperature and temperature shifts during adulthood. All flies were reared at 16 °C until adult eclosion, and time points indicate days from eclosion and temperatures maintained for the previous three days. Graphs show transcript levels from adults continuously reared at 16 °C (graph a) and 29 °C (graph b); adults reared from 0-3 days at 29 °C (3d-29 °C; from graph b) and then shifted to 16 °C for an additional 3 days (6d-16 °C) and 6 days (9d-16 °C) (graph c); or shifted to 29 °C at 6 days (9d-29 °C) (graph d; sibling samples from 3d and 6d in graph c shown for comparison). Each bar represents the mean fold change from three biological replicates ± SD relative to sample M36f1 at 3d-29 °C. Asterisks (*) above M36f1 samples indicate t-test significance (two-tailed) compared to WT-f at the same time point: ***P < 0.001; *0.01 < P < 0.05. Unmarked M36f1 data are not significantly different from WT-f. Transcript levels and P values are also presented in Supplementary Table S2. regulated by Ds-TRA-2 function. Presumably this function is mediated by the more direct effect of Ds-TRA/ Ds-TRA-2 interactions on the female-specific expression of Ds-dsx (GenBank: XM_017077018.1) 4 , as demonstrated in D. melanogaster 20 .

Discussion
Here we report the use of CRISPR/Cas9 gene editing in D. suzukii to create a conditional temperature sensitive allele of the tra-2 sex determination gene, Ds-tra-2 ts2 , analogous to the chemically induced Dm-tra-2 ts2 missense point mutation in D. melanogaster 1 . This was achieved by comparing amino acid sequence identity for TRA-2 in both species (Supplementary Fig. S3), and identifying the common peptides that are substituted in Dm-tra-2 ts1 and Dm-tra-2 ts2 1,18 . Since Dm-tra-2 ts2 was shown to exhibit better fertility than Dm-tra-2 ts1 at permissive temperatures 1,18 , its mutation site was the initial choice for editing in D. suzukii for this study, though the tra-2 ts1 allele might also present advantages for application, or additional insights into function.
Similar to Dm-tra-2 ts2 , XX chromosomal females homozygous for Ds-tra-2 ts2 reared at permissive temperatures of 16-20 °C resulted in development of normal fertile females, while rearing at a restrictive temperature of 26 °C resulted in a sterile, though incomplete pseudo-male phenotype. The lack of full male phenotypic differentiation, unlike XX; Dm-tra-2 ts2 pseudo-males reared at 29 °C, was most likely the result of rearing D. suzukii at a lower sub-optimal restrictive temperature, since this species fails to complete pupal development when reared above 26 °C. Thus, complete loss of Ds-TRA-2 function at 26 °C was unlikely, though distinct male morphological differentiation was apparent based on complete foreleg sex comb development, posterior dorsal tergite 6 pigmentation, and wing spot pigmentation when shifted to 29 °C in early adulthood. The genital apparatus structures were intersexual, but predominantly male-like, and similar to XX; Dm-tra-2 ts2 pseudo-males, the gonads of XX; Ds-tra-2 ts2 pseudo-males were dysmorphic, possibly intersexual, and did not produce motile sperm 1,19 . Conversely, XY; Ds-tra-2 ts2 males reared at 26 °C exhibited normal male external morphology and mated, but were also sterile with dysmorphic testes consistent with the tra-2 null phenotype in D. melanogaster that is known to interfere with male germ-line development 12,13 . A notable difference between the tra-2 ts2 lines in the two species is that Dm-tra-2 ts2 XX females and XY males were highly sensitive to loss of Dm-TRA-2 activity, becoming sterile at 18 °C 18 , while Ds-tra-2 ts2 XX and XY individuals exhibited less sensitivity, becoming sterile at 22 °C and 26 °C, respectively.
While D. suzukii could not develop past late pupation at 29 °C, surviving adults at 26 °C or below could be reared at 29 °C, allowing the sex-specificity of downstream transcriptional products in adults to be assessed in response to Ds-TRA-2 ts2 function at the optimal restrictive temperature. This allowed a more accurate comparison of TRA-2 function in D. suzukii and D. melanogaster and its downstream regulation of sexual differentiation. In D. melanogaster, functional TRA-2 acts in concert with the transformer gene product to facilitate the female-mode of doublesex alternative intron-splicing, resulting in DSX-Female function throughout adulthood 20 . The continuous need for control by the sex-determination gene pathway throughout female development was, indeed, first inferred by experiments with Dm-tra-2 ts2 that showed that female-specific Yp transcription and synthesis in adults could be up-and down-regulated by manipulating temperature-dependent Dm-TRA-2 function 5 . For Ds-TRA-2 ts2 , we showed that Yp gene expression could be similarly regulated by temperature such that XX; Ds-tra-2 ts2 pseudo-males reared at 16 °C and shifted to 29 °C at adult eclosion exhibited basal Yp1 transcription levels, that could be transiently up-regulated to levels comparable to WT females after a shift to 16 °C, and subsequently down-regulated to basal levels by a shift to 29 °C. Together, these studies confirmed the expressivity and temperature-dependent conditional function of the gene-edited Ds-tra-2 ts2 mutation that also provided support for the continuous, and reversible, requirement for Ds-TRA-2 in both females and males for adult sexual differentiation and fertility throughout development.
Beyond providing a conditional system to better understand how sex-determination genes control sexual differentiation and behavior, the Drosophila tra-2 ts studies also present a highly efficient strategy for generating a sterile male population for SIT 6 . For this, a tra-2 ts mutant strain reared at permissive temperature would result in a normal breeding population of fertile males and females, whose chromosomal male and female progeny all develop as sterile phenotypic males at the restrictive temperature. Thus, ideally, a tra-2 ts mutant strain could provide a complete sterile male population in response to a shift in temperature, and provide the significant efficiency of all zygotes being used for release, requiring a considerably smaller breeding population.
Current control of D. suzukii does not include SIT 21 , and the temperature-sensitive sexing system described is less than optimal for this species due to post-larval inviability at 29 °C required for complete XX female sex reversal. However, XY; Ds-tra-2 ts2 males are sterile but sexually active at ambient temperatures above 25 °C, and could be reared at lower temperatures for improved viability, yet sterilized when shifted to higher restrictive temperatures as pharate adults. These sterile males could directly compete with wild males in the field, though the ability of their sibling XX; Ds-tra-2 ts2 pseudo-males to provide mating competition remains uncertain. Regardless of their ability to mate with females in the field, chromosomal females that develop as phenotypic males or intersexuals would still preclude the need to eliminate or sterilize these individuals before release, unlike normal females, since they would not require sterilization, nor compete with females in the field for mating with released sterile males, or cause oviposition damage to host plants. Thus, the programmatic and cost advantages of Ds-tra-2 ts2 -based sexing and sterility might still provide a level of efficiency that would support its incorporation into prospective SIT programs for D. suzukii. Nevertheless, the fertility and mating behavior of both XX and XY mutant adults would require further testing to ensure the efficacy of this system in response to temperature fluctuations under actual field conditions. This demonstration of temperature-dependent sex reversal established by gene-editing in D. suzukii also supports the notion that this could be accomplished in other insect species having conserved tra-2 cognate function and sequence identity. This is especially so for several tephritid fruit fly species, such as Ceratitis capitata (medfly), Anastrepha ludens (mexfly), and A. suspensa (caribfly), which have conserved sites for the TRA-2 Alanine and Proline peptides substituted in the tra-2 ts1 and tra-2 ts2 mutations, respectively 3,22 . Importantly, sex reversal of chromosomal females by RNAi-based knock-down of the tra and tra-2 cognates, resulting in XX pseudo-males that copulate with wild females, has already been demonstrated for medfly and caribfly 22,23 . Given that these tropical insects thrive at ambient temperatures of 29 °C or above, it is likely that full male differentiation could be achieved in XX; tra-2 ts individuals with the potential for successful mating with females in the field. However, the RNAi studies also showed that while tra-2 knock-down in XX caribflies resulted in sterile pseudo-males, XY males remained fertile, and in medfly both XX pseudo-males and XY males remained fertile. Thus, for tephritid species tra-2 ts males reared at restrictive temperatures might remain fertile, requiring an independent conditional system for male sterility, such as the tetracycline-dependent embryonic lethality system described for medfly and caribfly 24,25 .
In summary, proof of principle has been demonstrated in D. suzukii for the creation of a conditional temperature-sensitive allele by gene-editing that most likely can be extended to other temperature-sensitive missense mutations, and in a wide range of species known to have an orthologous gene 26 . That a very broad range of genes can be edited in this way in insects is supported by D. melanogaster mutational studies that showed that greater than 10% of X-linked lethal and semi-lethal mutations are temperature-sensitive 27 . For the tra-2 gene in particular, that is conserved among several orders of insects, synthetic creation of temperature-sensitive mutations has significant potential for detailed comparative functional analyses and, for pest species, enhancing the efficacy of programs to control their population size. While this and other possible applications remain prospective, the ability to create conditional alleles in a large number of genes known to be important to many aspects of reproduction, development and behavior should have a highly significant impact on our understanding of genome function and evolution.

Methods
All data generated or analysed during this study are included in this published article (and its Supplementary Information files). All sequence files are available from the GenBank database (accession numbers MF142759, MF142760, MF142761 and MF142762) after publishing of this work.
Fly rearing, fertility and mating tests. A wild type strain of D. suzukii originally collected in Wimauma, Florida, was reared under standard laboratory conditions as described 28 . Gene-edited transgenic homozygous flies were reared at 16 °C ± 0.2 °C (EchoTherm ™ IN40 incubator, Torrey Pines Scientific, Inc.), and tested at indicated temperatures ( ± 0.2 °C) for temperature sensitivity after 24 h and 48 h of egg collection at 16 °C. Fertility tests were performed by mating each mutant male to five wild type virgin females (n ≥ 15) and each mutant female to three WT males (n ≥ 20) followed by egg collections for 12-15 days. Progeny were reared to adulthood and screened for red fluorescence. Mating tests were performed by combining previously unmated 3 d adult flies for observation of courtship behavior including body orientation, wing vibration, licking and copulation for 6 h at mid-day (1000-1600), and for 4 h beginning at dawn (approx. 0700) of the following day.

Mutation generation via CRISPR/Cas9. The Ds-tra-2 gene was identified by a Blast search using
Dm-tra-2 mRNA (GenBank: NM_057416.3) as a query, and was identified in contig1010 from the D. suzukii genome (GenBank: XM_017071951) 4 . The putative Ds-tra-2 ts2 mutation site was localized to nt 571 in the CDS (GenBank: MF142759) based on alignment with the Dm-tra-2 ts2 amino acid sequence 1,18 , and confirmed with primers AH1001/AH1002 by RT-PCR and sequencing. Oligonucleotide primers were designed with Geneious ® 8.0.5 (Biomatters Ltd.) software and listed in Supplementary Table S3 with annealing conditions. sgRNA targets were identified by a manual search for NGG proto-spacer adjacent motif (PAM) sequences. Two targets were identified having high GC content within the six PAM-proximal nucleotides (PAMPNs) 29 and less similarity to other genomic sequences to improve efficiency and avoid off-target effects. sgRNAs were synthesized as previously described 30 using primers AH992, AH1053 and AH1054 (Supplementary Table S3). Their specificities were confirmed by embryonic injections and Surveyor tests ( Supplementary Fig. S2).
To create the donor construct, a 3.6 kb fragment from D. suzukii genomic DNA was cloned using Q5 ® High-Fidelity DNA Polymerase (New England Biolabs) with primers AH1020/AH1021, which included two homologous arms of more than 1 kb upstream and downstream from the two sgRNA target sites. The Ds-tra-2 ts2 point mutation was introduced by primers AH1027/AH1028. To avoid cleavage of the donor construct and mutagenesis after integration by CRISPR/Cas9, two single-nucleotide synonymous substitutions (G > C for sgRNA1 site; G > A for sgRNA2 site) were introduced into the two sgRNA target site PAM sequences, respectively, since an intact PAM sequence is necessary for successful double strand breakage 31 . Homologous arm 1 (HA1) was assembled using the GeneArt ® Seamless Cloning and Assembly Kit (Thermo Fisher Scientific) from three fragments, which were amplified with primer pairs AH1026/AH1027, AH1028/AH1029, and AH1030/ AH1031. HA2 was assembled from two fragments amplified with primer pairs AH1032/AH1033 and AH1034/ AH1035 with the Ds-tra-2 ts2 mutation and PAM substitutions confirmed by sequencing. Next, a fragment containing the IE1hr5-DsRed/SV40 marker gene was amplified from the piggyBac transformation vector plasmid, pB{IE1hr5-DsRed/SV40} with primers AH1044/AH1045, and the complete HA1 and HA2 fragments were amplified using primer pairs AH1026/AH1043 and AH1035/AH1046. The DsRed marker, HA1 and HA2 were then assembled with the GeneArt ® Kit and confirmed by sequencing.
The D. suzukii Ds-ligase4 gene (GenBank: XM_017079597) 4 was identified by Blastn alignment similarity to Dm-ligase4 from D. melanogaster (GenBank: NM_132679.3; protein alignment of the conceptual translation of these sequences shown in Supplementary Fig. S4). The online SnapDragon tool (http://www.flyrnai.org/ snapdragon) was used to design a 421 bp dsRNA fragment whose sequence was confirmed after PCR amplification with primers AH1003/AH1004 (GenBank: MF142760). The dsRNA was then synthesized as described previously 22,32 .


s2fieldsofstudy: Biology Medicine

text:
Role of a novel immune modulating DDR2-expressing population in silica-induced pulmonary fibrosis

Micro-injuries associated with chronic inhaled particle exposures are linked with activation of the immune response and are thought to contribute to progression of fibrotic disease. In the pulmonary environment, we have previously demonstrated a heterogeneous population of circulating fibroblast precursors (CFPs), which are defined by expression of the pan-leukocyte marker CD45 and the collagen receptor, discoidin domain receptor-2 (DDR2). This population is derived from the hematopoietic stem cell, expresses collagen, and has a fibroblastic morphology in vitro. Herein, we demonstrate a novel subset of CFPs expressing immune markers CD11b, CD11c, and major histocompatibility complex II (MHC II). The CFP population was skewed toward this immune marker expressing subset in animals with silica-induced pulmonary fibrosis. Data indicate that this CFP subset upregulates co-stimulatory molecules and MHC II expression in response to silica-induced fibrosis in vivo. Functionally, this population was shown to promote T cell skewing away from a Th1 response and toward a pro-inflammatory profile. These studies represent the first direct flow cytometric and functional evaluation of the novel immune marker expressing CFP subset in an exposure-induced model of pulmonary fibrosis. Elucidating the role of this CFP subset may enhance our understanding of the complex immune balance critical to mediating exposures at the pulmonary-host interface and may be a valuable target for the treatment of exposure-induced pulmonary fibrosis.

Introduction
Chronic exposures to inhaled particles, such as silica, are often linked with epithelial damage due to repeated physical micro-injuries to the lung epithelium. This continued injury stimulus results in initiation of pulmonary repair and activation of the inflammatory immune response [1,2]. In pulmonary fibrosis, this repair process becomes dysregulated and develops into a persistent fibrotic response [3,4]. While the immune cascade is known to be involved in fibrotic a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 disease, it is unclear how the inflammatory process and epithelial damage ultimately contribute to chronicity and progression of pulmonary fibrosis. In the pulmonary microenvironment, a careful balance between immune reactivity and steady-state must be maintained due to the lung epithelial interface between the external environment and the host. While the mechanisms behind this delicate balance are not yet fully understood, immune-modulating cells involved in maintaining this homeostatic state are known to have the unique ability to respond and adapt to microenvironmental changes in response to infection, exposures, and/or selfantigens. When antigens are detected and phagocytosed by immune populations including macrophages, myeloid-derived suppressor cells, or dendritic cells, expression of major histocompatibility complex II (MHC II), and immune co-stimulatory molecules CD80/CD86 are upregulated. This process results in activation of an immune response through binding and presentation of antigen to T cells.
It was recently reported that Discoidin Domain Receptor-2 (DDR2), an extracellular matrix sensing receptor (collagen receptor) was associated with this process resulting in promotion of CD86 expression [5]. While the mechanism(s) behind this association is unclear, a cell with the ability to both sense and respond to extracellular matrix may have the potential to play a significant role in the fibrotic response to exposures. Our laboratory has previously identified a circulating fibroblast precursor (CFP) population of cells defined by the co-expression of CD45 (a pan-leukocyte marker) and DDR2 [6][7][8]. These cells were demonstrated to have the ability to differentiate into mature fibroblasts and promote solid tumor progression [6,9]. In the pulmonary microenvironment, we have demonstrated a heterogeneous population of CFPs and DDR2 + cells that are derived from the hematopoietic stem cell, express collagen, and have a fibroblastic morphology [10]. While the ability of the CFP to give rise to fibroblasts has been established [6][7][8]10], the immune contribution of this population has not yet been explored nor have these cells been examined in the context of pulmonary fibrosis. Given that the CFP has been demonstrated to contain the fibrocyte population [7] and is derived from the myeloid lineage [6], we hypothesized that the CFP may also contribute to pulmonary immune function. Therefore, in the present study, we have employed a silica exposure-induced model of pulmonary fibrosis in order to phenotypically and functionally assess the immunologic role of CFPs in disease. Herein, we have identified a subset of CFPs (CD45 + DDR2 + cells) that express markers common to dendritic-like populations and other immune subsets such as monocytes and macrophages. These markers include CD11b, CD11c, MHC II, and the co-stimulatory molecules CD80 and CD86. The CFP population was skewed toward the CD11b + CD11c + subset and demonstrated increased co-stimulatory molecule expression in silica-induced pulmonary fibrosis. In addition, this population was found to promote T cell skewing away from a Th1 phenotype toward a pro-inflammatory response in fibrotic lung, suggesting that the CFP may be involved in the inflammatory/immune balance in the fibrotic pulmonary exposure response.

Ethics statement
Studies were conducted in strict accordance with the Veterans Affairs Institutional Animal Care and Use Committee (IACUC) under the approved ACORP #592. All efforts were made to minimize suffering. Animals were anesthetized with isoflurane prior to procedures, including euthanasia.

Silica-induced pulmonary fibrosis
Male B6.SJL-Ptprc a Pepc b /BoyJ (C57Bl/6J) mice, aged 10-14 weeks, 28-30 g body weight, were used for all studies. These mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and were bred in-house. Mice were anesthetized via isoflurane inhalation. A dose of 6 mg silica (Sigma-Aldrich, St. Louis, MO, USA) suspended in 60 μl of sterile 0.9% saline solution, or 60 μl of sterile 0.9% saline solution alone, was delivered via intra-tracheal instillation through a 24 gauge catheter. This dose represents~0.2 g/Kg and dosage and delivery are based on published models of silica instillation [11,12]. Silica suspension was vortexed immediately prior to instillation. Immediately following delivery of saline/silica the catheter was removed, animals were recovered in an upright position and were subsequently returned to their home cage. Eight weeks post-instillation, mice were anesthetized by isoflurane anesthesia and were euthanized via thoracotomy followed by exsanguination. Animals exhibiting a robust fibrotic response were selected for analysis.

Statistics
Comparison of CFP populations by flow cytometric analysis was based on Unpaired Student's T-test with Welch's Correction, where p 0.05 was considered significant. Data represent n!7 acquired in at least 2 experimental replicates. T cell cytokine data was compared by Unpaired Student's T-test, where p 0.05 was considered significant, with n!2 for T cell isolation and n!3 for sorted CFP populations, with at least 2 experimental replicates. Statistical analyses were performed using GraphPad Prism 5 Software (GraphPad Software, La Jolla, CA, USA).

Silica-induced model of pulmonary fibrosis
To address the immune role of CFPs in exposure-induced disease, a silica model of pulmonary fibrosis was employed. In this model, a silica suspension was instilled into the lungs of mice via intra-tracheal administration under isoflurane anesthesia. Eight weeks following silica-instillation, fibrotic nodules were visualized throughout the lungs and significant loss of normal lung architecture was observed versus saline only controls (Fig 1A-1L). Staining of lung sections with Picrosirius Red (PSR)/Fast Green demonstrated increased collagen deposition and presence of multiple fibrotic nodules in silica treated animals (arrow indicates representative nodule, Fig 1B) versus saline treated control animals ( Fig 1A). Polarized light images (Fig 1C and  1D) indicated formation of fibrotic nodules (arrow, Fig 1D) and deposition of thick and thin collagen fibrils in silica-instilled animals ( Fig 1D) versus saline controls ( Fig 1C). Masson's Trichrome stain (Fig 1E-1H) of silica exposed lungs also demonstrated increased collagen content, alveolar thickening, and multiple fibrotic nodules ( Fig 1F) versus saline controls (Fig 1E). Collagen fibrils were evident in fibrotic nodules (Fig 1H) of silica-instilled animals versus saline-instilled controls (Fig 1G). Herovici's stain (Fig 1I-1L) also demonstrated loss of normal lung architecture, apparent fibrotic nodules ( Fig 1J) and increased collagen deposition in the lungs of animals with silica-induced fibrosis ( Fig 1L) versus saline-instilled animals (Fig 1I and  1K). Contribution of a novel circulating fibroblast precursor subset in lungs In order to assess the contribution of the CFP population to fibrotic lung, lung digest from silica-or saline-instilled animals was analyzed by flow cytometry for the presence of CFPs (CD45 + DDR2 + cells) (Fig 2A, saline control, and Fig 2B, silica-induced pulmonary fibrosis, fourth panels). The CFP population was reduced in the lungs of mice with silica-induced pulmonary fibrosis versus saline controls (9.076% vs. 12.48%, respectively, ÃÃ p = 0.0065) (compare fourth panels Fig 2A and 2B). CD11b and CD11c expression are associated with immune populations; therefore, in order to assess the ability of the CFP population to contribute to the immune microenvironment of the lung, CFPs were examined for expression of these markers. Flow cytometric analysis of lung digest demonstrated that a subset of the CD45 + DDR2 + population exhibited co-expression of CD11b + and CD11c + (Fig 2A and 2B, last panels). The percentage of CD11b + CD11c + CFPs was significantly increased in the lungs of animals with silica-induced pulmonary fibrosis versus saline controls (42.41% vs. 34.11%, respectively, ÃÃ p = 0.0038) (compare last panels Fig 2A and 2B, quantified in Fig 2C). The phenotype of the CD11b + CD11c + subset of CFPs in saline exposed lungs was predominantly CD11b high (Fig 2D, left panel) and CD11c low (Fig 2E, left panel). In animals with silicainduced pulmonary fibrosis, there was a loss of the CD11b low population (population indicated by arrow, Fig 2D, compare left (saline) and right (silica) panels) while there was a decrease in the CD11c high population (population indicated by arrow, Fig 2E, compare left (saline) and right (silica) panels).
Phenotypic changes in immune co-stimulatory molecules on the CD11b + CD11c + subset of CFPs in silica-induced pulmonary fibrosis Immune populations such as dendritic cells and other antigen presenting populations exhibit increased expression of co-stimulatory molecules upon exposure. Given that the CD45 + DDR2 + CFP expresses markers associated with these immune populations, costimulatory molecule expression was assessed on CD45 + DDR2 + CFPs that expressed CD11b + CD11c + (immune CFP subset) from lung digest of silica-or saline-instilled animals. The co-expression of CD80/CD86 by this immune CFP subset was significantly increased in the fibrotic lungs of silica-exposed animals versus saline controls (27.38% vs. 13.68%, ÃÃ p = 0.0011) (Fig 3A, saline 3B, silica, quantified in 3C). Upregulation of MHC II is generally concomitant with upregulation of immune co-stimulatory molecules in response to exposures, therefore the expression of MHC II on this population was also examined. With silica-induced pulmonary fibrosis, there was increased expression of MHC II (75.71% silica vs. 62.21% saline, ÃÃÃ p = <0.0001) on the CD11b + CD11c + subset of CFPs (Fig 4A, quantified  in 4B).  Functional immune role of the CD11b + CD11c + subset of CFPs in silicainduced pulmonary fibrosis In order to determine the functional effects of this novel CD11b + CD11c + subset of CFPs on CD4 + and CD8 + T cell populations, CD11b + CD11c + CFPs were sorted from the lungs of saline or silica treated animals at eight weeks post-instillation. Sorted cells were co-cultured with naïve CD4 + or CD8 + T cells isolated from untreated animals in a ratio of 1:10. Following 48 hour co-culture, supernatant was collected and analyzed by flow cytometric multiplex bead array for expression of T cell-associated cytokines (Figs 5 and 6). T cell expression of interleukin-2 (IL-2), interferon-gamma (IFN-γ), TNF-alpha (TNF-α), and interleukin-17A (IL-17A) were examined. As a control, CD11b + CD11c + CFPs (1x10 4 cells) were plated alone, and demonstrated no detectable expression of the cytokines examined. There was a statistically significant decrease in the production of IL-2 by CD4 + T cells co-cultured with CD11b + CD11c + CFPs from silica-instilled (5.450 pg/ml) versus saline-instilled lungs (6.715 pg/ml, Ã p = 0.0318) (Fig 5A). Production of IL-2 by CD4 + T cells co-cultured with CD11b + CD11c + CFPs from silica instilled animals was also significantly less than produced by stimulated (PMA/ionomycin treated) CD4 + T cells (551.9 pg/ml, ÃÃÃ p<0.0001) (Fig 5A). In addition, IFN-γ expression by CD4 + T cells was decreased in co-cultures with CD11b + CD11c + CFPs from silica-treated animals (16.40 pg/ml) versus saline controls (19.50 pg/ml, Ã p = 0.0405, Fig 5B). This was also significantly less than observed with stimulated CD4 + T cell controls (16.40 pg/ml vs. 32.07 pg/ ml, respectively, ÃÃÃ p = 0.0004, Fig 5B).

Discussion
Current therapies for the treatment of pulmonary fibrosis merely act to slow disease progression without cure, or involve risky transplantation and life-long anti-rejection treatment. While an active area of ongoing research, data is conflicting with regard to targeting the immune system for the treatment of pulmonary fibrosis. While there is known involvement of the immune response in fibrosis, it is unclear whether immune suppression or immune activation is beneficial to treatment of the disease. In the clinical setting in the treatment of the idiopathic form of pulmonary fibrosis, immunotherapies achieved little to no therapeutic response thus far [4,13]. However, early attempts at immune based therapies were broadly targeted, administered late in disease, and dose and combination therapies complicated outcomes. Due to the known contribution of the inflammatory and immune response to exposures, an immune-based therapy that is specifically targeted may, in fact, prove to be effective in slowing or reversing pulmonary fibrosis. Our previous studies have led to the identification of an hematopoietic stem cell (HSC)-derived circulating fibroblast precursor (CFP) that is present in the lungs and contributes to the collagen producing pulmonary fibroblast population. The CFP population is unique in its co-expression of CD45 and the collagen receptor (DDR2)potentially enabling these cells to both sense and respond to a changing matricellular environment. In the present study we demonstrate a role for a novel immune subset of the CFP population in silica-induced pulmonary fibrosis. Together these studies indicate a unique dual role for CFPs, contributing to immune function in lung as shown herein, in addition to

DDR2-expressing cells in pulmonary fibrosis
contributing to a population of lung fibroblasts [10] suggesting that the CFP may be a valuable therapeutic target toward inhibition of fibrotic progression.
To examine the immune role of this novel population in pulmonary disease, we employed a silica-induced model of pulmonary fibrosis. Inhalation of silica particles results in a progressive, persistent, and robust, pro-fibrotic response in rodents [14]. This model is clinically relevant in both its non-resolving nature, and in the development of fibrotic nodules which resemble those observed in patients with exposure to occupational dusts and particulates [14]. Studies have shown that exposure to silica nanoparticles in vitro results in alterations in surface marker expression of CD54, CD80, CD86 and MHC II by immune cell populations [15]. Therefore, a silica-induced model of pulmonary fibrosis was employed in order to provide the first direct flow cytometric and functional evaluation of the immunological role of the novel CD11b + CD11c + CFP subset. Based on our findings, the CFP population is skewed toward the CD11b + CD11c + immune subset in the fibrotic disease state. This subset of CFPs exhibits a CD11b high phenotype which is suggestive of a monocytic/immature dendritic-like population, or a precursor closely related to a monocyte [16]. In addition, this population exhibits a CD11c low profile in disease, which may also indicate that an immature state of these cells is maintained in the lung even in advanced fibrosis (8 weeks). Further, an increase in the immune subset of CFPs expressing MHC II and co-stimulatory molecules CD80 and CD86 was observed, indicating that these CFPs exhibit an activated immune phenotype in exposureinduced fibrosis.
Several other groups have examined immune populations during development of pulmonary fibrosis in murine models. A study by Holian et. al. using a silica-induced model of pulmonary fibrosis examined the dynamics of antigen presenting populations in the lung during fibrotic progression [17]. This study found that there were early increases in the CD11c hi / MHC II hi population of dendritic cells and early declines in the CD11c hi /MHC II lo alveolar macrophage population. However, by 28 days post silica-instillation these populations had returned to levels consistent with controls and their studies did not reveal any changes in CD86 co-stimulatory molecule expression on either population following silica exposure. However, studies in a bleomycin model of pulmonary fibrosis by Soler's group demonstrated a sustained increase in CD11c + /MHC II + dendritic cells and increased expression of MHC II and CD86 throughout the fibrotic stage of bleomycin-induced disease [18]. Interestingly, the novel immune CFP subset identified herein was elevated at 8 weeks post silica-instillation and demonstrated significant increases in MHC II and co-stimulatory molecule expression, indicating either a unique immune signature of the CFP in response to silica-induced fibrosis or a cyclical immune reaction that may perpetuate the fibrotic response.
While the mechanism(s) driving the activated phenotype of the immune CFP subset are not yet known, studies by Lee et. al. have demonstrated that immature bone marrow-derived dendritic cells upregulate expression of CD86 in response to collagen exposure through DDR2 signaling [5]. Our studies have shown increased deposition of collagen with silica-induced pulmonary fibrosis, which is concurrent with the increased percentage of the CD11b + CD11c + subset of CFPs expressing CD80/CD86, suggesting that DDR2/collagen I signaling may play a role in activation of the immune CFP subset.
In addition to phenotypic changes in response to silica-induced pulmonary fibrosis, herein we demonstrate that the immune CFP subset functionally contributes to the immune microenvironment by promoting T cell skewing. Data presented demonstrate that pulmonary CFPs from silica-induced pulmonary fibrosis exhibit an inhibitory role toward the Th1 phenotype based on decreased production of IL-2 and IFN-γ by CD4 + and CD8 + T cells. Data also demonstrate promotion of a pro-inflammatory T cell phenotype based on production of TNF-α and IL-17A. This T cell cytokine profile has been associated with fibrotic progression (reviewed in [19]). Notably, the CD11b + CD11c + CFP subset promoted the production of IL-17A by both CD4 + and CD8 + T cells in both control and silica-instilled animals, suggesting that CFPs may skew toward an inflammatory Th17 phenotype based on production of this Th17-associated cytokine. IL-17 production by T cells has been implicated in the pathogenesis of pulmonary fibrosis [20,21], (reviewed in [4]). IL-17 was also shown to be elevated in the bronchoalveolar lavage of patients with idiopathic pulmonary fibrosis [17] and in a bleomycin animal model of pulmonary fibrosis [22], reviewed in [4]. Together, data demonstrate that the CFP subset skews the lung immune environment away from a Th1 phenotype and toward a pro-inflammatory environment in silica-induced pulmonary fibrosis which may contribute to fibrotic development and progression.
It has been demonstrated that bone marrow-derived populations are largely recruited in diseases exhibiting rapid turnover in the lung, especially in cases of chronic injury (reviewed in [23]). This suggests that the HSC-derived CFP population may play a significant role in the development and progression of pulmonary fibrosis. Importantly, the immune subset of this population has not previously been described, nor have the contribution(s) of this novel DDR2-expressing population to pulmonary fibrosis ever before been investigated. The discovery that a subset of CFPs is capable of immune modulating effects in disease is important in terms of our understanding of the plasticity of such populations depending on micro-environmental stimuli. Future examination of the mechanisms by which the CFP senses and responds to the matricellular and microenvironmental stimuli of the fibrotic lung may provide insight into the pathways driving the dual roles of the CFP and may uncover a pathobiological mechanism critical to the fibrotic response.


extfieldsofstudy: Chemistry Medicine

text:
Role of the luxS gene in bacteriocin biosynthesis by Lactobacillus plantarum KLDS1.0391: A proteomic analysis

Certain probiotic species of lactic acid bacteria, especially Lactobacillus plantarum, regulate bacteriocin synthesis through quorum sensing (QS) systems. In this study, we aimed to investigate the luxS-mediated molecular mechanisms of QS during bacteriocin synthesis by L. plantarum KLDS1.0391. In the absence of luxS, the ‘spot-on-the-lawn’ method showed that the bacteriocin production by L. plantarum KLDS1.0391 significantly decreased upon co-cultivation with L. helveticus KLDS1.9207 (P < 0.01) but did not change significantly when mono-cultivated. Furthermore, liquid chromatography-electrospray ionization tandem mass spectrometry analysis showed that, as a response to luxS deletion, L. plantarum KLDS1.0391 altered the expression level of proteins involved in carbohydrate metabolism, amino acid metabolism, fatty acid synthesis and metabolism, and the two-component regulatory system. In particular, the sensor histidine kinase AgrC (from the two-component system, LytTR family) was expressed differently between the luxS mutant and the wild-type strain during co-cultivation, whereas no significant differences in proteins related to bacteriocin biosynthesis were found upon mono-cultivation. In summary, we found that the production of bacteriocin was regulated by carbohydrate metabolism, amino acid metabolism, fatty acid synthesis and metabolism, and the two-component regulatory system. Furthermore, our results demonstrate the role of luxS-mediated molecular mechanisms in bacteriocin production.

1. Detection of the double chamber effect in co-cultivation of L. plantarum KLDS1.0391 and L.
helveticus KLDS1.9207 The membrane permeability was detected by the method described below. The results are shown in Table S1. Detection  "-" means that no strains are inoculated. "*" means that the number of cell in this chamber is not determined.
The bacterial growth was detected by the following method. L. plantarum KLDS1.0391 was inoculated (1%) into the double chamber and triangular flask. Sample was incubated in MRS broth at 37 °C for 24 h with gentle agitation (60 rpm). The culture was removed every 2 h, and the number of viable cell of L. plantarum KLDS1.0391 with time changes in two culture modes was detected. The results are shown in Fig. S3.
2. SDS-PAGE was carried out on vertical electrophoresis apparatus (SE260, GE-Healthcare) at 15 mA and per gel using a 12% (w/w) poly-acrylamide separating gel overlaid with a 5% stacking gel. Samples were boiled for 5 min in SDS-PAGE 5×(5:1, v/v) Sample Loading Buffer (10% SDS, 0.5% BPB, 50% Glycerol, 500 mM DTT, 250 mM Tris-HCl, pH 6.8), and centrifuged at 14000 g for 10 min. The supernatant was transferred to a new tube. Each lane on the gel received 20 μg of protein for electrophoresis at 5 mA for 60 min. After electrophoresis, the polypeptides were stained with Coomassie Brilliant Blue R250 and decolored with methanol/ acetic acid. Finally, the result of electrophoresis was analyzed by gel imaging system.
Filter-aided-sample-preparation procedure was described as followed. Briefly, each sample added DTT to a final concentration of 100 mM, boiled for 5min and cooled down to room temperature. Then, 200 μL UA buffer (8 M Urea, 150 mM Tris-HCl, pH 8.0) was added to remove the detergent, DTT and other low-molecular-weight components by repeated ultrafiltration (Microcon units, 30 kD) facilitated by centrifugation (14000 g, 15 min). Further, 100 μL 50 mM iodoacetamide (IAA, Bio-Rad) in UA buffer was added to prevent the reduced cysteine residues and stirred at 600 rmp for 1 min, and the samples were incubated for 30 min in total darkness and centrifuged at 14000 g for 10 min. The filter was washed twice with 100 μL UA buffer and 100 μL 25 mM NH4HCO3 buffer, respectively. Finally, the protein suspension was digested with 2 μg Trypsin (Promega) in 40 μL 25 mM NH4HCO3 buffer at 37 °C for 16-18 h, and the resulting peptides were collected as a filtrate. The peptides of each sample were first desalted with C18-SD Extraction Disk Cartridge and then estimated by UV light absorbance at 280 nm.
The detailed Q-Exactive MS requirements were described below. The result of MS were acquired higher-energy collisional dissociation (HCD) fragmentation by choosing the positive ion and precursor ions range from 300 m/z to 1800 m/z. The resolution for survey MS scans was set at a value of 70,000 with a 200 m/z, and the HCD spectra were acquired a resolution of 17,500 with m/z of 200. The m/z of peptides and peptide fragments were collected by full scan and selected the twenty data-dependent MS/MS scans. The Normalized Collision Energy of 30 eV was used to activate precursor irons, and the Underfill ratio, which indicates the Mix percentage of the target value likely to be attained to the Max fill time, was defined as 0.1%.
"-" means that no cells were detected, "+" means that cells were detected.


text:
An HNF4α–microRNA-194/192 signaling axis maintains hepatic cell function

Hepatocyte nuclear factor 4α (HNF4α) controls the expression of liver-specific protein-coding genes. However, some microRNAs are also modulated by HNF4α, and it is not known whether they are direct targets of HNF4α and whether they influence hepatic function. In this study, we found that HNF4α regulates microRNAs, indicated by marked down-regulation of miR-194 and miR-192 (miR-194/192) in liver-specific Hnf4a-null (Hnf4aΔH) mice. Transactivation of the shared miR-194/192 promoter was dependent on HNF4α expression, indicating that miR-194/192 is a target gene of HNF4α. Screening of potential mRNAs targeted by miR-194/192 revealed that expression of genes involved in glucose metabolism (glycogenin 1 (Gyg1)), cell adhesion and migration (activated leukocyte cell adhesion molecule (Alcam)), tumorigenesis and tumor progression (Rap2b and epiregulin (Ereg)), protein SUMOylation (Sumo2), epigenetic regulation (Setd5 and Cullin 4B (Cln4b)), and the epithelial-mesenchymal transition (moesin (Msn)) was up-regulated in Hnf4aΔH mice. Moreover, we also found that miR-194/192 binds the 3′-UTR of these mRNAs. siRNA knockdown of HNF4α suppressed miR-194/192 expression in human hepatocellular carcinoma (HCC) cells and resulted in up-regulation of their mRNA targets. Inhibition and overexpression experiments with miR-194/192 revealed that Gyg1, Setd5, Sumo2, Cln4b, and Rap2b are miR-194 targets, whereas Ereg, Alcam, and Msn are miR-192 targets. These findings reveal a novel HNF4α network controlled by miR-194/192 that may play a critical role in maintaining the hepatocyte-differentiated state by inhibiting expression of genes involved in dedifferentiation and tumorigenesis. These insights may contribute to the development of diagnostic markers for early HCC detection, and targeting of the miR-194/192 pathway could be useful for managing HCC.

Liver has diverse functions such as metabolism of carbohydrates, lipids, and proteins; detoxification of ammonia and xenobiotics; and synthesis of bile acids and blood coagulation factors. Various liver-enriched transcription factors such as hepatocyte nuclear factor 1 (HNF1), 2 HNF3, HNF4, and HNF6 are needed to maintain differentiated liver-specific functions (1). Of these, HNF4␣ is a member of the nuclear receptor superfamily and a key regulator involved in the transcription regulatory network in the liver (2,3), and re-expression of HNF4␣ in dedifferentiated hepatoma cells has the potential to revert cells back to the differentiated state (4). As Hnf4a-null mice are embryonic lethal during gastrulation (5), two types of liverspecific Hnf4a-null mice (Hnf4a ⌬H ) were established (6,7). Disruption of HNF4␣ in the fetal livers showed that hepatic HNF4␣ is essential for formation of a hepatic epithelium, liver morphogenesis, and liver homeostasis by positive regulation of a large number of genes during development into the mature liver (6). Disruption of HNF4␣ in the Hnf4a ⌬H mice led to diverse phenotypes such as fatty livers and elevation of serum bile acids and ammonia (7)(8)(9). Hnf4a ⌬H mice also exhibit increased spontaneous hepatocyte proliferation and increased susceptibility to diethylnitrosamine-induced liver cancer, indicating that HNF4␣ has tumor suppressor activity, although the mechanism has not been established (10,11). Because HNF4␣ regulates many liver-enriched genes via HNF4␣-binding sites in the promoter regions of the target genes and HNF4␣ is bound to about 12% of the gene promoters expressed in human hepatocytes (12), the HNF4␣-derived transcriptional network in the liver is expected to be considerably complicated.
The gene expression pattern in the livers of Hnf4a ⌬H mice is markedly changed largely due to loss of expression of direct HNF4␣ target genes that are down-regulated, giving rise to the altered phenotypes of these mice (13). For instance, reduced expression of ornithine transcarbamylase (Otc), encoded by a HNF4␣ target gene, caused elevated serum ammonia due to the inability to degrade ammonia (8). In contrast, many mRNAs are up-regulated in Hnf4a ⌬H mice. Although direct transcriptional repression by HNF4␣ itself could be considered as a mechanism by which these mRNAs are reduced in Hnf4a ⌬H mice, another possibility is the involvement of microRNAs (miRNAs) that could be under direct transcriptional control of HNF4␣. miRNAs are small non-coding RNAs involved in various physiological functions such as development, differentiation, inflammation, and tumorigenesis through their ability to down-regulate protein-coding gene expression by binding to target mRNAs and either promoting mRNA degradation or inhibiting mRNA translation (14,15). miRNAs have important roles in liver diseases such as viral hepatitis, steatohepatitis, liver fibrosis, polycystic liver disease, and hepatocellular carcinoma (HCC) (16,17).
Among the miRNAs, miR-122 is the most abundant in adult liver (18). Expression of miR-122 is reduced in HCC, and overexpression of miR-122 suppresses cell proliferation (19,20). Thus, miR-122 could function as a tumor suppressor gene against HCC. Expression of miR-122 positively correlates with HNF4␣ expression in HCC cell lines, and miR-122 is directly regulated by HNF4␣, indicating that the HNF4␣-directed miR-122 pathway could be essential for inhibition of hepatocyte proliferation and HCC development (21,22). Likewise, HNF4␣ directly regulates the expression of miR-134 and miR-134-suppressed oncogenic KRAS expression, revealing that miR-134 has a suppressive effect on HCC progression (23). Moreover, the following HNF4␣-miRNA inflammatory feedback circuits in human HCC have been proposed. HNF4␣ positively regulates miR-7 and miR-124 followed by suppression of IL6R, STAT3, and RelA-mediated NF-B pathways, eventually resulting in the up-regulation of HNF4␣ through down-regulation of miR-24, miR-629, or miR-21 (24,25). These results also indicate that lack of HNF4␣-controlled miRNAs could result in HCC development by activation of inflammatory signaling. These miRNAs were identified from studies using immortalized hepatocytes or HCC cell lines and thus could be biomarkers for HCC diagnosis and candidates for new target genes for HCC therapy. However, miRNAs regulated by HNF4␣ in normal hepatocytes but not in immortalized or transformed hepatocytes remain to be identified.

Down-regulation of hepatic miR-194 and miR-192 in Hnf4a ⌬H mice
HNF4␣ positively regulates the expression of many genes through HNF4␣-binding sites in promoter regions (1,3). A marked change of the mRNA expression pattern was observed in the livers of Hnf4a ⌬H mice (13), including many down-regulated mRNAs identified as encoded by direct HNF4␣ target genes (8,9,26). In addition, several up-regulated genes were also detected in Hnf4a ⌬H mice, but their mechanism of increase is not known. Although transcriptional repression of genes such as Snail, Slug, and HMGA2 by HNF4␣ was demonstrated (27), the mechanism of negative regulation of other genes by HNF4␣ has not been established. Another possibility for negatively regulated mRNAs is indirectly through miRNA. Indeed, HNF4␣ is known to regulate the expression of miR-7, miR-122, miR-124, and miR-134 (22)(23)(24)(25). Thus, expression profiling of hepatic miRNAs in Hnf4a-floxed (Hnf4a f/f ) and Hnf4a ⌬H mice was investigated by microarray analysis (supplemental Table  S1). Of 610 miRNAs, expression of 24 and 19 miRNAs was down-regulated or up-regulated more than 2-fold in Hnf4a ⌬H mice, respectively (Table 1). In general, because HNF4␣ positively regulates the target genes, down-regulated miRNAs in Hnf4a ⌬H mice could be direct target genes for HNF4␣. Although other miRNAs, including miR-34a and miR-301a, were increased in Hnf4a ⌬H mice, they were not investigated in the current study. They could be either directly repressed by HNF4␣ or the result of downstream events mediated by loss of expression of other direct HNF4␣ target genes.
The expression of miR-194 was decreased by 90% in Hnf4a ⌬H mice compared with Hnf4a f/f mice. miR-194 was also expressed at considerably higher levels in Hnf4a f/f mice when compared with other miRNAs. Similarly, the expression of miR-192 was also higher in Hnf4a f/f mice and markedly lower in Hnf4a ⌬H mice. Indeed, qRT-PCR quantification showed that hepatic expression of miR-194 and miR-192 was suppressed by about one-tenth in Hnf4a ⌬H mice (Fig. 1A). Expression of other liverenriched miRNAs such as miR-21, -101b, -130a, -193, -193b, -455, and -805 was also decreased in Hnf4a ⌬H mice (Fig. 1A). Others have reported that the expression of miR-194 and miR-192 is regulated under the same promoter, and tissue distribution of miR-194 and miR-192 is almost equivalent to that of HNF4␣ (28,29), suggesting that HNF4␣ could positively regulate the expression of miR-194 and miR-192. Therefore, further studies were performed on miR-194 and miR-192. No significant difference in hepatic expression of miR-7, miR-122, miR-124, and miR-134, which were reported as the target miRNAs for HNF4␣ (22)(23)(24)(25), was observed in Hnf4a ⌬H mice (supplemental Table S1).
In mice, mature miR-194 is located on two chromosomes with miR-194-1 and miR-194-2 clustered with miR-215 and miR-192, respectively. Expression of miR-215 was unchanged in Hnf4a ⌬H mice, and the expression level was very low as compared with miR-194 and miR-192 (Table 1). Thus, miR-194-2, but not miR-194-1, could be highly expressed in normal liver and preferentially suppressed in Hnf4a ⌬H mice. As expected, hepatic expression of pri-miR-194-1 was unchanged, whereas expression of pri-miR-194-2 was markedly suppressed in Hnf4a ⌬H mice (Fig. 1B). These data indicate that HNF4␣ could directly activate the miR-194/192 gene via a single promoter.

Regulation of hepatic miR-194/192 by HNF4␣ and HNF1␣
HNF1␣, another liver-enriched transcription factor, directly regulates the miR-194/192 gene through a binding site in the miR-194/192 promoter region (28). Accordingly, qRT-PCR analysis using the livers of Hnf4a ⌬H mice and Hnf1a-null mice was performed to determine which factor, HNF1␣ or HNF4␣, predominates in controlling expression of the miR-194/192 gene. Hepatic expression of both miRNAs in Hnf1a-null mice was reduced to 50%, and the remnant levels were much higher when compared with Hnf4a ⌬H mice (Fig. 1C). Because HNF4␣ positively regulates Hnf1a in the liver (2), decreased expression of miR-194/192 in Hnf4a ⌬H mice could be due to decreased expression of both Hnf1a and Hnf1a. Therefore, the expression of Hnf4a and Hnf1a mRNAs in Hnf1a-null mice was also investigated. The expression of Hnf4a was reduced to 3%, and the expression of Hnf1a mRNA was reduced to 45% in Hnf4a ⌬H mice (Fig. 1D). Conversely, expression of Hnf1a mRNA was not detected, and expression of Hnf4a mRNA was reduced to 42% in Hnf1a-null mice (Fig. 1E), revealing that the remaining expression of Hnf1a in Hnf4a ⌬H mice and Hnf4a in Hnf1a-null mice is similar. Hence, HNF4␣ could be a more essential factor for controlling expression of miR-194/192 as compared with HNF1␣ in the liver.
p53 also has the potential to transcriptionally control the miR-194/192 gene (29,30). However, p53 was up-regulated in Hnf4a ⌬H mice, whereas it was unchanged in Hnf1a-null mice (Fig. 2, A and C). Similarly, the expression of p21 (Cdkn1a) mRNA, encoded by a direct target gene for p53, was also upregulated in Hnf4a ⌬H mice and unchanged in Hnf1a-null mice (Fig. 2, B and D), indicating that up-regulation of p53 in Hnf4a ⌬H mice could not overcome repression of miR-194/192 expression caused by loss of HNF4␣.

The miR-194/192 gene promoter is transactivated by HNF4␣
To investigate the mechanism by which HNF4␣ regulates the miR-194/192 gene, the promoter region of the miR-194/192 gene was analyzed. As described above, HNF1␣ was already found to transactivate the miR-194/192 gene through an HNF1␣-binding site in colorectal adenocarcinoma-derived Caco-2 cells, and two regions located at Ϫ130 to Ϫ118 and Ϫ110 to Ϫ98 upstream of the HNF1␣-binding site were highly conserved among species (28), but the binding proteins were not identified. Because HNF4␣ binding to both regions was predicted by JASPAR, an open access database (Fig. 3A), promoter analysis was performed to determine whether HNF4␣ has the potential to transactivate the miR-194/192 promoter. In HCC-derived and endogenous HNF4␣-expressing HepG2 cells, the Ϫ70/ϩ21 fragment containing an HNF1␣-binding site was transactivated by 50-fold as compared with the promoterless vector pGL4.11 (Fig. 3B). The promoter activity of the Ϫ115/ϩ21 fragment containing the HNF1␣-binding site and a proximal HNF4␣-binding site was much higher, and that of the Ϫ162/ϩ21 fragment was highest in the presence of both HNF4␣-binding sites, indicating that the minimum promoter of the miR-194/192 gene is located between Ϫ162 and ϩ21. Introduction of mutations into the proximal (M1) or distal (M2) HNF4␣-binding site resulted in significant reduction of the activities, and mutations in both binding sites (M1 ϩ M2) did not transactivate the Ϫ162 and Ϫ2032/ϩ21 regions (Fig.  3C). By co-transfection with the HNF4␣ expression vector in HEK293T cells, the promoter activity was slightly induced in the Ϫ115/ϩ21 fragment containing the distal HNF4␣-binding site and strongly activated in the Ϫ162/ϩ21 fragment in the presence of both HNF4␣-binding sites (Fig. 3D). Furthermore, the induced activities in the Ϫ162 and Ϫ2032/ϩ21 promoters were markedly suppressed by introduction of mutations into one or both HNF4␣-binding sites (Fig. 3E). These results indi-cate that both HNF4␣-binding sites cooperatively transactivate the miR-194/192 gene.
To determine whether HNF4␣ can directly bind to both HNF4␣-binding sites in the miR-194/192 promoter, gel mobility shift analysis was performed (Fig. 4A). Nuclear extracts from HepG2 cells bound to the distal and proximal HNF4␣-binding sites (Fig. 4A, lane 2, lower arrows). This complex was diminished by the addition of excess amounts of the unlabeled competitor and OTC competitor that contains an HNF4␣-binding site (Fig. 4A, lanes 3 and 5) but not the competitor that contains mutations in the HNF4␣-binding site (Fig. 4A, lane 4). Moreover, the complex was supershifted by anti-HNF4␣ antibody (Fig. 4A, lane 6, upper arrow) but not the anti-C/EBP␣ antibody (lane 7). These results indicate that HNF4␣ binds to both HNF4␣-binding sites of the miR-194/192 promoter. Next, chromatin immunoprecipitation (ChIP) was used to determine whether HNF4␣ directly binds to the miR-194/192 promoter in vivo. We found that HNF4␣ was bound to the HNF4␣-binding sites in HepG2 cells (Fig. 4B). In addition, ChIP analysis using the livers of Hnf4a f/f and Hnf4a ⌬H mice indicated that hepatic HNF4␣ in Hnf4a f/f mice bound to the promoter region, but the binding was decreased in Hnf4a ⌬H mice (Fig. 4C). Similar results were obtained in the mouse Otc promoter that is dependent on an HNF4␣-binding site (8), suggesting that HNF4␣  physiologically binds to the promoter region of the miR-194/ 192 gene in humans and mice.

Identification of miR-194 and miR-192 target mRNAs
It was reported that miR-194 is involved in epithelial-mesenchymal transition (EMT) and HCC metastasis (31). In addition, miR-194 regulates mRNAs encoded by many genes including N-cadherin (Cdh2), Rac1, heparin-binding epidermal growth factor (Hbegf), tyrosine protein phosphatase non-receptor type 2 (Ptpn2), integrin ␣9 (Itga9), suppressor of cytokine signaling 2 (Socs2), DNA methyltransferase 3A (Dnmt3a), Fzd6, and talin 2 (Tln2) that play important roles in tumorigenesis, tumor progression, cell adhesion, cell migration, and EMT (31)(32)(33). miR-192 is also known to negatively regulate mRNAs encoding IGF-1, IGF-1R, and ZEB2 that are also involved in EMT and tumor progression and invasion (30,34). Therefore, qRT-PCR was performed to determine whether hepatic expression of these mRNAs was increased in Hnf4a ⌬H mice. Expression of Fzd6, Hbegf, Ptpn2, Dnma3a, Itga9, and Rac1 mRNAs was significantly increased, whereas expression of Socs2, Cdh2, Tln2, and Zeb2 mRNAs was unchanged in Hnf4a ⌬H mice (Fig. 5, A and C). Next, novel target genes for miR-194 and miR-192 were investigated using TargetScan, DIANA-microT, and miRDB databases, and the resulting high-scoring hits that overlapped such as Hnf1b, Gyg1, hook microtubule-tethering protein 3  6 and 7). The complex between HNF4␣ and the probe and the supershifted complex are indicated by the lower and upper arrows, respectively. B, chromatin immunoprecipitation using HepG2 cells was performed with anti-HNF4␣ antibody (lane 2) and normal goat IgG (lane 3) (left). Input DNA was used as a positive control (lane 1). The regions between Ϫ201 and Ϫ41 containing both HNF4␣-binding sites (ϩHNF4␣) and between ϩ16667 and ϩ16874 without HNF4␣-binding site (ϪHNF4␣) in the human miR-194/192 gene were amplified. The data from qPCR were normalized relative to the input and expressed as -fold enrichment (right). C, chromatin immunoprecipitation using the livers of Hnf4a ⌬H and Hnf4a f/f mice with anti-HNF4␣ antibody and normal goat IgG. The regions between Ϫ164 and Ϫ70 containing both HNF4␣-binding sites in the mouse miR-194/192 promoter (left), between Ϫ264 and Ϫ165 containing two HNF4␣-binding sites in the mouse Otc promoter (right), and between ϩ45820 and ϩ45893 without an HNF4␣-binding site in the mouse Hmgcs2 gene were amplified, respectively. The data from qPCR was normalized relative to the input and expressed as -fold enrichment over data from IgG control. Error bars represent S.D. Data are mean Ϯ S.D. of three independent experiments. *, p Ͻ 0.05. (Hook3), Setd5, selenophosphate synthetase 1 (Sephs1), Sumo2, Cln4b, and Rap2b were selected as the target candidates for miR-194 (supplemental Fig. S1, B-F). Regarding the candidates of miR-192 target gene mRNAs, Ereg, ADP-ribosylation factor guanine nucleotide-exchange factor 1 (Arfgef1), radical S-adenosyl methionine domain-containing 2 (Rsad2), Alcam, and Msn were selected (supplemental Fig. S2, G-I). As expected, the expression of all candidate mRNAs was significantly increased in Hnf4a ⌬H mice (Fig. 5, B and C). The expression of CLN4B protein, a candidate for miR-194 targeting, and ALCAM protein, a candidate for miR-192 targeting, was also increased more than 2-fold in Hnf4a ⌬H mice compared with Hnf4a f/f mice (Fig. 5D). Therefore, studies were done to determine whether miR-194 and miR-192 could directly interact with the 3Ј-UTR of these candidate mRNAs and inhibit the protein expression (Fig. 6). miR-194 and miR-192 markedly repressed the 3Ј-UTR activities in the presence of the complementary sequences of miR-194-and miR-192-binding sites (c-miR-194 and c-miR-192) (supplemental Fig. S2). The 3Ј-UTR activities of Fzd6, Gyg1, Setd5, Sumo2, Cln4B, and Rap2b using a luciferase reporter system were significantly inhibited by miR-194 (Fig. 6A, wild type (WT)). Because two miR-194-binding sites were predicted within the 3Ј-UTR of Fzd6 and Rap2b, mutations were introduced to disrupt the binding of miR-194 in both binding sites. Each mutant (M1 and M2) partially rescued the 3Ј-UTR activity, and both mutants (M1 ϩ M2) completely recovered the activities (Fig. 6A). The same results were also obtained in the mutated 3Ј-UTR regions of the Gyg1, Setd5, Sumo2, and Cln4B (Fig. 6B). Similarly, 3Ј-UTR activities of Ereg, Alcam, and Msn were suppressed in a miR-192-dependent manner, and the activities were recovered by introduction of mutations into the miR-192-binding sites (Fig. 6B). These results indicate that Fzd6, Gyg1, Setd5, Sumo2, Cln4B, and Rap2b are the target mRNAs for miR-194 and that Ereg, Alcam, and Msn mRNAs are targets for miR-192.

Discussion
In this study, HNF4␣ was found to directly activate the miR-194/192 gene promoter. Down-regulation of HNF4␣ target genes partially could explain the onset of phenotypes in Hnf4a ⌬H mice (7,8,26). From microarray analysis using livers of Hnf4a ⌬H mice (13), most mRNAs were markedly down-regulated, probably due to the loss of direct positive regulation of the respective genes by HNF4␣. However, up-regulation of many mRNAs was also observed in Hnf4a ⌬H mice. Thus, HNF4␣ might negatively regulate these genes via direct positive regulation of miRNAs that in turn could bind to destabilize mRNAs. HNF4␣ was shown to bind to the promoter region of miR-122, the most abundant miRNA in the liver, and transactivate the miR-122 in an HNF4␣-dependent manner (22). However, hepatic expression of miR-122 was unchanged in Hnf4a ⌬H mice. HNF4␣ knockdown also inhibited miR-122 expression in mouse liver (22), suggesting that transactivation of miR-122 by HNF4␣ would be transient in Hnf4a ⌬H mice as observed, probably due to secondary effects. Likewise, HNF4␣ positively regulates miR-7, miR-124, and miR-134 in human HCC and tumor-derived cell lines (23)(24)(25). However, unexpectedly, the hepatic expression of these miRNAs was also unchanged in Hnf4a ⌬H mice. Because the expression levels of miR-7, miR-124, and miR-134 were very low or undetectable in normal livers of Hnf4a f/f mice, HNF4␣ may not directly regulate these miRNAs as found in normal liver. Thus, HNF4␣ might regulate these miRNAs only during the later stages of HCC progression but not the early stages of HCC development because Hnf4a ⌬H mice do not exhibit early spontaneous HCC development.
Unlike miR-7, miR-122, and miR-134, miR-194 and miR-192 were not reported to be down-regulated in human HCC specimens used in these studies, indicating that down-regulation of miR-194 and miR-192 in the liver could be an early event in the onset of liver inflammation and pretumorigenesis. miR-194 is involved in the differentiation of intestinal epithelial cells, and HNF1␣ was also reported to transactivate miR-194/192 via an HNF1␣-binding site (28). HNF1␣ is a direct target for HNF4␣ (2), and hepatic expression of HNF1␣ was actually down-regulated in Hnf4a ⌬H mice as expected. Thus, both HNF4␣ and HNF1␣ might cooperatively control the miR-194/192 gene promoter. However, down-regulation of miR-194 and miR-192 in Hnf1a-null mice was not as pronounced when compared with Hnf4a ⌬H mice because sufficient HNF4␣ still remains in Hnf1a-null mice, indicating that HNF4␣ might be a predominant factor in regulation of the miR-194/192 gene as compared with HNF1␣.
p53 was also reported to control expression of the miR-194/ 192 gene via a p53-binding site (29,30). The p53-binding site is located upstream of the distal HNF4␣-binding site, and the promoter of Ϫ162/ϩ21 fragment including the p53-and HNF4␣-binding sites was activated in an HNF4␣-dependent manner, suggesting that p53 and HNF4␣ might synergistically transactivate the miR-194/192 gene. Expression of miR-194 and miR-192 was markedly down-regulated in a p53-dependent manner in B cell neoplasm multiple myeloma, which rarely expresses endogenous HNF4␣ (30). Conversely, the expression of miR-194 and miR-192 was slightly down-regulated in human HCC including mutated or deleted p53 without statistical significance as compared with wild-type p53 (34), indicating that HNF4␣ and HNF1␣ mainly transactivate the miR-194/192 gene despite p53 status.
EMT is an important process for cells to acquire tumor metastatic potential. Expression of miR-194 was also down-regulated in liver mesenchymal-like cancer cell lines as compared with liver epithelial cancer cell lines, and overexpression of miR-194 suppressed invasion and migration of hepatoma cells (31). Thus, miR-194 and miR-192 expression in liver might retain the differentiated status of hepatocytes and suppress proliferation, migration, and malignant transformation. Re-expression of HNF4␣ in dedifferentiated hepatoma cells resulted in reversion to differentiated phenotypes (4). Moreover, reduced expression of HNF4␣ correlated with human HCC progression (25), and forced expression of HNF4␣ reduced the proliferation and induced epithelial formation (25,35,36). These results strongly suggest that HNF4␣ is one of the most important factors to induce hepatocyte differentiation by direct transactivation of liver-specific genes and to inhibit dedifferentiation of hepatocytes by indirect negative regulation of oncogenes and EMT-related genes via direct up-regulation of miR-194 and miR-192. However, hepatic expression of EMTactivating genes including Cdh2 as an miR-194 target and Zeb2 as an miR-192 target mRNA was not increased in mice, showing that down-regulation of miR-194 and miR-192 in inflammatory and non-/pretransformed hepatocytes in Hnf4a ⌬H mice could not yet activate EMT-related genes because Hnf4a ⌬H mice die at about 8 weeks of age before showing tumorigenesis (7).
Others have reported that HNF1␣-regulated miR-194 repressed the 3Ј-UTR activity of FZD6 (32). In this study, two functional miR-194-binding sites were identified in the 3Ј-UTR of Fzd6 mRNA. FZD6 is a membrane receptor for Wnt ligand signaling and controls cell differentiation (37). Because overexpression of FZD6 was confirmed in human HCC (38) and hepatic expression of FZD6 was not significantly increased in HNF1␣-null mice (32), repression of FDZ6 expression through negative regulation by miR-194 via HNF4␣ could be important for suppression of tumorigenesis.
There are no reports that GYG1 in involved in HCC and metastasis. Glycogen synthesis starts from self-glucosylation of GYG1 followed by elongation of glucose chains by glycogen synthase (39). Hepatic expression of glycogen synthase 2, a limited enzyme for glycogen synthesis in the liver and a direct target for HNF4␣, was markedly decreased in Hnf4a ⌬H mice (40). Eventually, expression of GYG1 might be increased to compensate for reduced production of de novo glycogen synthesis in Hnf4a ⌬H mice.
CUL4B is the main component of Cullin4B-Ring ubiquitin ligase complex and promotes tumorigenesis (41) and SETD5 is a histone methyltransferase on the histone H4 tail at lysines 5, 8, and 12 in yeast (42). Loss of function mutations in the SETD5 gene were found in patients with intellectual disability (43); however, the physiological significance of H4 methylation by SETD5 remains unsolved. Histone H4 lysines 5, 8, and 12 are also acetylated by histone acetyltransferases (44), and methylation of these sites by SETD5 might change chromatin structure and influence transcription. HNF4␣-dependent suppression of Cul4B and Setd5 could partially control the epigenetic program and be important to retain the differentiated status in normal hepatocytes.
SUMO2 has an important role to regulate a variety of biological functions such as transcription, DNA repair, cell cycle, and subcellular transport by SUMOylation to the target proteins (45). Similar to an ES cell-based model of hepatocyte differentiation in which SUMO2-modified proteins were decreased during hepatocyte differentiation (46), increased activity of SUMOylation by SUMO2 could induce hepatocyte dedifferentiation in Hnf4a ⌬H mice.
RAP2B functions as a p53-mediated prosurvival factor. RAP2B was also found to be up-regulated in many types of tumors, but RAP2B had very weak transformation activity, indicating that RAP2B itself is not an oncogene but maintains the tumorigenic status (47). Because hepatic expression of p53 and Cdkn1a was increased in Hnf4a ⌬H mice, induced p53 might be responsible for additional induction of Rap2b mRNA.
In addition, Ereg, Alcam, and Msn mRNAs are targets for miR-192 in the liver. EREG is one of the ligands for epidermal growth factor receptor and is involved in inflammation and tumorigenesis (48). Knockdown of EREG suppressed cell growth in human hepatoma cell lines (49), and Ereg-null mice exhibited a significant reduction of tumor number and size in HCC model experiments (50), indicating that EREG may contribute to HCC progression. Unlike other target genes for miR-194 and miR-192 examined in this study, EREG is more highly expressed in hepatic stellate cells than in hepatocytes (50), suggesting that miR-192 synthesized in hepatocytes might be secreted into hepatic stellate cells followed by repression of EREG in normal liver. ALCAM, a cell adhesion molecule, is a marker of colorectal cancer stem cells (51). Although ALCAM expression was inhibited by miR-192 in a gastric cell line, the 3Ј-UTR of Alcam mRNA failed to respond to miR-192 (52). By contrast, the 3Ј-UTR activity of Alcam mRNA was dependent on a miR-192-binding site. ALCAM was overexpressed in human hepatoma cell lines, and silencing of ALCAM suppressed cell growth and promoted apoptosis (53), suggesting that ALCAM could accelerate tumor cell survival by an antiapoptotic role in HCC.
MSN functions as a cross-linker between the plasma membrane and actin filament. High expression of moesin was observed in breast cancers and implicated in EMT (54,55). However, expression of Msn mRNA was induced only 1.6-fold in Hnf4a ⌬H mice. Because other EMT-related mRNAs such as Cdh2 and Zeb2 were not induced in Hnf4a ⌬H mice, these EMT-related mRNAs might not be induced sufficiently in inflammatory and non-transformed hepatocytes of Hnf4a ⌬H mice.
In conclusion, HNF4␣ was found to repress the expression of a variety of mRNAs through positive regulation of miR-194 and miR-192 in non-transformed liver. Most of these mRNAs encode proteins involved in tumorigenesis, prosurvival, epigenetic change, and EMT. These observations revealed an HNF4␣ cascade that inhibits hepatocyte dedifferentiation. Further studies may contribute to the development of novel diagnostic markers for the early detection of HCC using the HNF4␣-miR-194/192 axis, and inhibition of the miR-194/192 targets by induced expression and/or activation of miR-194/ 192 could be useful for HCC therapy.

Animals
Hnf4a ⌬H mice carrying a mixed SvJ129, FVB, and C57BL/6 background were generated as described previously (7). All experiments were performed with 45-day-old male Hnf4a f/f and Hnf4a ⌬H mice. Hnf1a-null mice were also used (56). The mice were housed in a pathogen-free animal facility under a standard 12-h light/12-h dark cycle with ad libitum water and chow. All experiments with mice were carried out under Association for Assessment and Accreditation of Laboratory Animal Care guidelines with approval of the National Cancer Institute Animal Care and Use Committee and Gunma University Animal Care and Experimentation Committee.

miRNA microarrays
Total RNA from the livers of Hnf4a f/f and Hnf4a ⌬H mice was purified using the miRNeasy mini kit (Qiagen, Tokyo, Japan). Extracted RNA was labeled with Cy5 using the Label IT miRNA labeling kit (Takara Bio, Otsu, Japan). Labeled RNAs were hybridized onto 3D-Gene Mouse miRNA Oligo chips (v.11.1; Toray Industries, Tokyo, Japan). The annotation and oligonucleotide sequences of the probes conformed to the miRBase miRNA database (http://www.mirbase.org/). 3 After stringent washes, fluorescence signals were scanned with the ScanArray Express Scanner (PerkinElmer Life Sciences) and analyzed using GenePix Pro version 5.0 (Molecular Devices, Sunnyvale, CA). The raw data of each spot were normalized by subtraction of the mean intensity of the background signal determined by all blank spots' signal intensities of 95% confidence intervals. Measurements of both duplicate spots with signal intensities greater than two standard deviations (S.D.) of the background signal intensity were considered to be valid. A relative expression level of a given miRNA was calculated by comparing the signal intensities of the averaged valid spots with their mean value throughout the microarray experiments after normalization by their median values adjusted equivalently. miRNA microarray data are described in supplemental Table S1. The data are also available at the Gene Expression Omnibus under accession number GSE70516.

Reverse transcription and real-time PCR
Total RNA was extracted using TriPure Isolation Reagent (Roche Applied Science). Reverse transcription for mature miRNA was performed using a Mir-X miRNA First-Strand Synthesis kit (Clontech). Real-time PCR was performed on a LightCycler 480 system II (Roche Applied Science). cDNAs encoding mature miRNA and U6 RNA were amplified using a Mir-X miRNA qRT-PCR SYBR kit (Clontech). The levels of mature miRNA were expressed relative to U6 RNA as an internal control using the ⌬⌬Ct method. For detection of two transcripts for pri-miR-194 and mRNAs, cDNA from the livers of Hnf4a f/f and Hnf4a ⌬H mice was transcribed using ReverTraAce qPCR RT Master with gDNA Remover (Toyobo, Osaka, Japan), and real-time PCR was performed using FastStart SYBR Green Master (Roche Applied Science). The levels of expression were normalized relative to Gapdh mRNA as an internal control using the ⌬⌬Ct method. Sequences for the primers are shown in supplemental Table S2.

Cloning of the human miR-194/192 gene promoter region
The Ϫ2032, Ϫ1003, Ϫ162, Ϫ115, Ϫ70, and Ϫ51/ϩ21 fragments of the human miR-194/192 promoter containing KpnI and XhoI sites were amplified with genomic DNA from HepG2 cells by PCR and cloned into the luciferase reporter vector pGL4.11 (Promega, Madison, WI). Mutations were introduced into the HNF4␣-binding sites in the miR-194/192 reporter vector by PCR-based site-directed mutagenesis. Sequences for the primers are shown in supplemental Table S3.

Transient transfection and luciferase assay
HepG2 and HEK293T cells were cultured at 37°C in Dulbecco's modified Eagle's medium (Wako, Osaka, Japan) containing 10% fetal bovine serum (HyClone, Logan, UT) and 1,000 units of penicillin and 0.1 mg of streptomycin/ml (Sigma-Aldrich). For suspension transfection, miR-194 promoters cloned into pGL4.11 and pGL4.74 encoding Renilla luciferase regulated under herpes simplex virus thymidine kinase promoter (Promega) were transfected into HepG2 cells with polyethylenimine Max (Polyscience, Warrington, PA) as a transfection reagent. For co-transfection using HEK293T cells, HNF4␣ expression plasmid was used. After 48 h of transfection, the cells were washed with phosphate-buffered saline (PBS), and promoter activities were measured using the Dual-Luciferase Reporter Assay System (Promega).

Western blotting
Liver samples from Hnf4a f/f and Hnf4a ⌬H mice and HepG2 cells treated with siRNA against Hnf4a mRNA were homogenized in lysis buffer (7 M urea, 2 M thiourea, and 1% Triton X-100) and allowed to sit on ice for 30 min. The homogenate was centrifuged at 12,000 ϫ g for 30 min at 4°C, and the supernatants were used as whole-cell lysates. Total protein (40 g) with Laemmli sample buffer was incubated at 65°C for 15 min and fractionated by 10% SDS-polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue R-250 or blotted onto a PVDF membrane (GE Healthcare). The membrane was incubated for 1 h with mouse monoclonal antibodies against human HNF4␣ (Perceus Proteomics, Tokyo, Japan) and rabbit polyclonal antibodies against human CLN4B (GeneTex, Irvine, CA) and human ALCAM (Sigma-Aldrich). After washing, the membrane was incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling Technology, Tokyo, Japan), and the reaction product was visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce) on an ImageQuant LAS4000 (GE Healthcare). Expression of CLN4B and ALCAM proteins was quantified by densitometric analysis using ImageJ software, and the expression normalized by total protein in Hnf4a ⌬H was presented as expression differences relative to the Hnf4a f/f mice.

Gel mobility shift analysis
Nuclear extracts from HepG2 cells were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific), and gel shift analysis carried out using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific). The following double-stranded probes were used: wildtype and mutants of distal and proximal HNF4␣-binding sites for the human miR-194/192 promoter and the HNF4␣-binding site for the mouse Otc promoter as a positive control. Nuclear extracts and the 5Ј-biotin-labeled probe of the HNF4␣-binding sites for the miR-194/192 promoter (wild type) were added, and the reaction mixture was incubated on ice for 10 min. For competition experiments, a 50-fold excess of unlabeled probe was added to the reaction mixture, and the mixture was incubated on ice for 10 min prior to the addition of the 5Ј-biotin-labeled probe. For supershift analysis, anti-HNF4␣ or anti-C/EBP␣ antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the mixture, and the mixture was incubated on ice for 10 min after the addition of the 5Ј-biotin-labeled probe. DNA-protein complexes were fractionated by 7% PAGE and blotted onto a Biodyne B nylon membrane (Pall, Tokyo, Japan). After washing, complexes were visualized using the detection module in the kit on an ImageQuant LAS4000. Sequences for the primers are shown in supplemental Table S4.

Chromatin immunoprecipitation
HepG2 cells were fixed in 0.5% formaldehyde and quenched by 125 mM glycine at room temperature. After washing, cells were resuspended in 3 ml of lysis buffer 1 (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, 0.25% Triton X-100, and protease inhibitor (Roche Applied Science)) on ice for 10 min and then centrifuged at 1,400 ϫ g for 5 min. The cell pellet was resuspended in 3 ml of lysis buffer 2 (10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, and 5 mM EGTA) for 10 min at room temperature and then centrifuged at 1,400 ϫ g for 5 min. The cell pellet was resuspended in 1 ml of lysis buffer 3 (10 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 0.1% sodium deoxycholate). Liver samples were ground into pieces with a pestle and mortar under liquid nitrogen and fixed in PBS containing 20 mM sodium butyrate, 1% formaldehyde, and protease inhibitor mixture for 10 min at room temperature. After centrifugation, the pellet was resuspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% sodium dodecylsulfate, 20 mM sodium butyrate, and protease inhibitor). The cell lysate from HepG2 cells and liver samples was disrupted by sonication (UR-20P, Tomy, Tokyo, Japan) for 5 min on ice and then diluted 10-fold with RIPA ChIP buffer (10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 20 mM sodium butyrate, and protease inhibitor) followed by centrifugation at 20,000 ϫ g for 10 min. A small volume of the supernatant was stored at 4°C as the input sample. The remaining supernatant was precleared by adding of 15 l of a 50% slurry of protein G-Sepharose 4 Fast Flow (GE Healthcare) with sonicated salmon sperm DNA and rotated for 4 h at 4°C followed by centrifugation at 1,900 ϫ g for 5 min. The supernatant was divided into two aliquots and incubated with 4 g of anti-HNF4␣ antibody or control normal goat IgG (Santa Cruz Biotechnology) for 16 h at 4°C and incubated with 40 l of a 50% slurry of protein G-Sepharose 4 Fast Flow. The reaction mixture was centrifuged at 1,900 ϫ g for 5 min at 4°C, and the pellet was collected. The pellet was washed for 5 min at 4°C with 1 ml of RIPA-1 buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate), RIPA-1 buffer containing 300 mM NaCl, LiCl wash solution (10 mM Tris-HCl (pH 8.0), 0.25 M LiCl, 1 mM EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate), and Tris-EDTA. Then ChIP direct elution buffer (10 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM EDTA, and 0.5% SDS) was added to the pellet and incubated for 16 h at 65°C for decrosslinking. After treatment with RNase A for 30 min at 37°C and proteinase K for 2 h at 55°C, DNA was purified and amplified by PCR and real-time PCR using the ⌬⌬Ct method. Enrichment of HNF4␣ binding was normalized to the input samples and expressed as -fold enrichment as compared with the control normal IgG antibody. Sequences for the primers are shown in supplemental Table S4.

3-UTR assays
3Ј-UTRs of the mouse candidate genes for the miR-194 and miR-192 were amplified by PCR and cloned downstream of firefly luciferase gene in pGL3-control (Promega). As positive controls, complementary sequences for miR-194 and miR-192 (c-miR-194 and c-miR-192) were self-annealed and then cloned into the XbaI site of the pGL3-control. For 3Ј-UTR assay, pGL3-control/3Ј-UTR plasmids and pGL4.74 with 20 nM miR-194 or miR-192 mimic were co-transfected into HEK293T cells using HilyMax (Dojindo, Kumamoto, Japan) as a transfection reagent and assayed by the Dual-Glo Luciferase Assay System (Promega). Sequences for the primers of the 3Ј-UTRs are shown in supplemental Table S5.

Statistical analysis
All values are expressed as the mean Ϯ S.D. All data were analyzed by unpaired Student's t test for significant differences between the mean values of each group.