FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation

Primary cilia are sensory organelles essential for embryonic and postnatal development, and tissue homeostasis in adulthood. They are generated in a cell cycle-dependent manner and found on most cells of the body. Although cilia formation is intensively investigated virtually nothing is known about the transcriptional regulation of primary ciliation. We used here Odf2/Cenexin, encoding a protein of the mother centriole and the basal body that is mandatory for primary cilia formation, as the target gene for the identification of transcriptional activators. We identified a consensus binding site for Fox transcription factors (TFs) in its promoter region and focused here on the Fox family. We found transcriptional activation of Odf2 neither by FOXO TFs nor by the core TF for multiciliation, FOXJ1. However, we identified FOXA1 as a transcriptional activator of Odf2 by reporter gene assays and qRT-PCR, and showed by qWB that Foxa1 knockdown caused a decrease in ODF2 and CP110 proteins. We verified the binding sequence of FOXA1 in the Odf2 promoter by ChIP. Finally, we demonstrated that knockdown of FOXA1 affected primary cilia formation. We, thus, showed for the first time, that FOXA1 regulates primary ciliation by transcriptional activation of ciliary genes.


Results
ODF2 is essential for primary cilia formation. To validate the relevance of ODF2 for the formation of primary cilia, Odf2 knockdown via transfection with either a short-hairpin plasmid (sh3) or siRNA was performed 45 . The plasmid K07 or the scrambled non-target siRNA served as controls for sh3-or siRNA-mediated knockdown, respectively. For rescue, the human Cenexin plasmid (hCenexin 46 ), was co-transfected with either sh3 or Odf2 siRNA. Additionally, to identify transfected cells, the plasmid encoding histone H4 fused to Egfp (H4::GFP) was always co-transfected. Cells were fixed 24 h post-transfection and primary cilia immunologically decorated for the ciliary marker ARL13B (Fig. 1A). Primary cilia were manually counted by visual inspection and scanning through all focal planes taking into consideration only H4::GFP positive cells. We found a clear reduction in primary cilia when Odf2 was knocked down by either the sh3-plasmid or siRNA when compared to the controls (K07-plasmid or scrambled non-target siRNA, respectively) (Fig. 1B). We have counted ~ 17% primary cilia in K07-transfected cells compared to only ~ 5% in sh3-transfected cells (p = 0.000114 ***), and ~ 15% in the siRNA control cells versus 7% in Odf2 siRNA transfected cells (p = 0.022857 *). In contrast, the rescue experiment caused an increase in primary cilia to ~ 13% (sh3 + hCenexin, p = 0.000989 +++ to sh3), and ~ 21% (Odf2 siRNA + hCenexin, p = 0.005358 ++ to Odf2 siRNA). Our results, thus, confirmed ODF2 as being essential for primary cilia formation. FOXA1 is a transcriptional activator of the Odf2 promoter. We previously reported the characterization of the mouse Odf2 promoter and identified C/EBPα and the stress-activated JNK-pathway as transcriptional activators 47 . Here, we identified a putative binding site for the forkhead-box TFs at position − 1775 of the Odf2 promoter resembling the consensus binding sequence for FOXO (TT[G/A]TTTAC or GTAAA(T/C)AA; 48 ), or FOXA1 (T(G/A)TT(T/G)AC; 49 ). To verify whether TFs of the FOX-family are involved in transcriptional regulation of ciliary genes and formation of primary cilia, we first investigated the impact of forkhead-box TFs on the activation of the Odf2 promoter, which controls expression of the firefly luciferase reporter gene (2.2-pGL3) 47 . The reporter vector 2.2-pGL3 comprised 1.8 kb upstream of the transcriptional start site of the mouse Odf2 gene together with 358 bp of the transcribed region (− 1805/ + 358) positioned upstream of the firefly luciferase coding sequence. The reporter vector 2.2-pGL3 was co-transfected with the internal control vector phRL-SV40, which strongly expresses the Renilla luciferase under the SV40 promoter, and either without co-expression of TFs in the control probe or with, to investigate the effect of TFs on the activity of the Odf2 promoter. The relative activity of the reporter vector was first calculated by dividing the light signals of the firefly luciferase by the light signals of the Renilla luciferase used as an internal control. The relative activity was then related to the average of the relative activity of the control probe, i.e. the reporter vector 2.2-pGL3 without co-expression of any TF, which was set to 1, finally giving the fold change in expression. When compared to the activity of the control probe, we observed a significant increase in reporter gene activity in cycling cells by Foxa1 co-trans-Scientific Reports | (2022) 12:21468 | https://doi.org/10.1038/s41598-022-25966-w www.nature.com/scientificreports/ fection (p = 0.001565**), but not by co-transfection of Foxo1, Foxo3a, Foxj1, or co-transfection of both, Foxj1 and Rfx3 ( Fig. 2A). Additionally, our previous results reporting transcriptional activation by C/EBPα, and the JNK-pathway (MEKK1 + cJUN) were verified. The co-transfection of Foxa1 with either C/ebpα, Mekk1, cJun or combinations of these factors revealed that FOXA1 together with C/EBPα has no additive effect on the reporter activity compared to C/EBPα. FOXA1 together with MEKK1 significantly increased the reporter gene activity compared to FOXA1 (p = 0.026646*), but without further increase when compared to MEKK1. FOXA1 together with cJUN increased the reporter gene activity to a similar extent as MEKK1-activated cJUN, and significantly (p = 2.84466 × 10 -5 ****) when compared to FOXA1. The co-transfection of Mekk1, cJun, and Foxa1 caused a further significant upregulation of the reporter activity compared to cJUN (p = 0.004115**), cJUN + MEKK1 (p = 0.0002307***), and FOXA1 (p = 3.37212 × 10 -10 ****). These data indicated a positive interaction between FOXA1 and cJUN in the transcriptional activation of Odf2 that is reinforced by MEKK1-mediated activation of cJUN. According to Student's T-test, the co-expression of all four factors, MEKK1, cJUN, C/EBPα, and FOXA1 did not significantly increase the reporter gene activity compared to the co-expression of these factors but without FOXA1. The combination of FOXA1 with cJUN and C/EBPα did not cause an up-regulation of reporter gene activity as compared to FOXA1 alone. Since cilia formation is stimulated by serum starvation-induced cellular quiescence simultaneously with enhanced Odf2 promoter activity 47 we wondered whether FOX TFs are also activated by serum starvation. The Only transfected cells, whose nuclei stained green because of histone H4::GFP expression, were considered for ciliary counting. Cells were transfected with histone H4::gfp (b, green) and decorated for primary cilia with anti-ARL13B staining (a, red, arrows). Nuclei were stained with DAPI (c, blue), and the merged image is shown in d (cilia are marked by arrows). Scale bars in a-d of 10 µm. (B) The percentage of transfected cells that are ciliated is strongly decreased when Odf2 was knocked down by transfection of either the short hairpin plasmid sh3 or Odf2 siRNA compared to the controls, plasmid K07 or control siRNA, respectively. Co-transfection of the non-targeted hCenexin with either sh3 or Odf2 siRNA rescued primary cilia formation. Student's T-test (one-tailed, homoscedastic): p < 0.05 * or + , p < 0.01 ** or ++ , p < 0.001 *** or +++ . Symbols represent comparison to either the respective controls (K07 or control siRNA) * or to the respective knockdown (sh3 or Odf2 siRNA) + . Biological triplicates with a total of n counted cells: K07 n = 1,621, sh3 n = 1,591, sh3 + hCenexin n = 1,593, control siRNA n = 1,509, Odf2 siRNA n = 1,538, Odf2 siRNA + hCenexin n = 1,580. P-values: sh3 to K07: p = 0.000114***, sh3 + hCenexin to sh3 p = 0.000989 +++ , Odf2 siRNA to control siRNA p = 0.022857*, and Odf2 siRNA + hCenexin (rescue) to Odf2 siRNA p = 0.005358 ++ .  2B). In contrast, a significant increase was found for FOXA1 compared to the control (2.2-pGL3 + phRL; p = 0.000041 ****). Increased reporter gene activity was also observed by co-expression of FOXA1 and C/EBPα when compared to the control (2.2-pGL3 + phRL; p = 3.69035 × 10 -9 ****), or to C/EBPα (p = 1.54407 × 10 -5 ***) but not when compared to FOXA1. Significant activation of the reporter compared to the control (2.2-pGL3 + phRL) was observed by co-expression of FOXA1 and MEKK1 (p = 0.003581**) but not when compared to FOXA1 indicating that MEKK1 did not affect FOXA1. Co-expression of MEKK1, cJUN, and FOXA1 caused a strong upregulation of the Odf2 promoter compared to the control (2.2-pGL3 + phRL; p = 3.18068 × 10 -12 ****), to cJUN + MEKK1 (p = 0.0003696***), to MEKK1 + FOXA1 (p = 0.00084057***), or to FOXA1 (p = 0.0009507***) indicating an effect of MEKK1-activated cJUN and its putative positive interaction with FOXA1 on the activity of the Odf2 promoter that was also observed in cycling cells ( Fig. 2A).
To further verify FOXA1 as a transcriptional activator of the Odf2 promoter, the reporter vector 2.2-pGL3 was co-transfected with the Foxa1 expression plasmid, and either the negative control siRNA duplex, or one of the Foxa1 siRNA duplexes A, B, or C. The activities of the firefly luciferase reporter and the Renilla luciferase as the internal control were measured either 24 h post-transfection (cycling cells) or after 48 h cultivation in serum starvation medium (cell cycle-arrested cells). Calculation of the relative luminescence revealed an approximately 2.5-fold increase of the reporter gene activity when expression of FOXA1 was enforced compared to control cells in which only the reporter 2.2-pGL3 and the internal control phRL were co-transfected (Fig. 3). Co-transfection of the negative control siRNA did not significantly change the expression of the reporter vector. However, we observed reduced activity of the reporter vector when Foxa1 siRNA duplexes B or C were co-transfected while siRNA A was not effective. Though, significant reductions in reporter gene activity (p*) were obtained exclusively for the Foxa1 siRNA duplex C in both, cycling cells (p = 0.03953205*) as well as in quiescent cells (p = 0.0142835*). Since the FOXA1-mediated activation of the Odf2 promoter can be efficiently repressed by Foxa1 siRNA duplexes these data demonstrate the importance of FOXA1 for the transcriptional activation of Odf2, as well as the specificity of Foxa1 siRNA duplexes for the knockdown of FOXA1. FOXA1 binds to the Odf2 promoter. Reporter gene assays were performed to narrow down the binding site of FOXA1 in the Odf2 promoter. Fragments of the Odf2 promoter, cloned upstream of the firefly reporter gene in pGL3 as described in Pletz et al. 47 (Fig. 4B), were investigated for their responsiveness to the FOXA1 transcription factor. Here, FOXA1-induced transcriptional activation was related to the basal level of the respective reporter construct without Foxa1 co-transfection. Strong induction of transcription was observed for the promoter region − 1282 to − 1805 in clone 7.6 (~ 70 × when co-transfected with Foxa1 as compared to the control without FOXA1 overexpression), for the region − 1368 to − 1805 in clone 22.1 (~ 16x), and the region − 94 to − 1805 in clone 7.1 (~ 4x) ( Fig. 4 A), indicating that the FOXA1-binding site most likely is located in the region between − 1282 and − 1805 of the Odf2-promoter.

Expression of Fox transcription factors in NIH3T3 cells. Our data indicated FOXA1 as an activator
of Odf2 transcription in NIH3T3 fibroblasts. However, it was unknown whether Foxa1 or any other TF of the Fox-family is indeed expressed in NIH3T3 cells. We, therefore, verified the expression of Fox TFs by RT-PCR using cDNA prepared from NIH3T3 cells. Although no products were present after the first RT-PCR round, a nested PCR reaction using the first RT-PCR reaction as the template, revealed products of the expected lengths for all Fox TFs investigated (Fig. 5A). The correct amplifications were verified by sequencing PCR products. Thus, all four transcription factors investigated, Foxa1, Foxo1, Foxo3a, and Foxj1, were expressed in NIH3T3 cells albeit at a low level. Furthermore, the endogenous FOXA1 was detected immunologically inside the nuclei showing a speckled appearance that colocalized with nucleoli ( Fig. 5B, a and c).  we asked whether knockdown of Foxa1 could also be detected at the endogenous transcript level and whether a knockdown of FOXA1 would be accompanied by a reduction of Odf2 transcripts and also of ODF2 proteins. NIH3T3 cells were, thus, transfected with the Foxa1 siRNA, and transcripts of Foxa1 and Odf2 were quantified by qRT-PCR. We observed a reduction of Foxa1 transcripts in Foxa1 siRNA-transfected cells to ~ 0.5 × of that in control siRNA-transfected cells (Fig. 7A, p* 0.02727533). Furthermore, the reduction of Foxa1 transcripts was accompanied by a reduction of Odf2 transcripts to ~ 0.6 × compared to control siRNA-transfected cells ( Fig. 7A; p*** 0.00062325). These results, therefore, indicate that transcription of Odf2 is under control of FOXA1. Finally, we quantified ODF2 proteins in lysates obtained from cells transfected with either the control siRNA or one of the Foxa1 siRNA duplexes. We observed a significant reduction of ODF2 to approximately 0.6 × when   FOXA1 is necessary for primary cilia formation. Since the amount of ODF2 is crucial for primary cilia generation we asked whether FOXA1 is involved in cilia formation. NIH3T3 cells were transfected with either one of the three different siRNA duplexes (A, B, or C) or the scrambled negative control siRNA duplex. 24 h post-transfection the medium was exchanged for serum starvation medium to induce cilia formation, and cells were cultivated for another 24 h or 48 h. Primary cilia were then immunologically decorated for ARL13B and manually counted (Fig. 8A). siRNA-mediated knockdown of FOXA1 caused a reduction of primary cilia to ~ 0.8 × when cultivated in serum starvation medium for 24 h (p < 0.05 + or p < 0.01 ++ ). Cultivation in serum starvation medium for 48 h caused a reduction of primary cilia to ~ 0.8 × by Foxa1 siRNA A (p < 0.05*) and ~ 0.5 × in Foxa1 siRNA B or C transfected cells (both p < 0.01**) (Fig. 8B). The Foxa1 siRNA A has been turned out once more to be the least effective thus corroborating the data of the reporter gene assays (Fig. 3) and the Western blots (Fig. 6). However, it has to be kept in mind that the siRNA-mediated knockdown effects in reporter gene assays and Western blots were based on the reduction of the co-expressed Foxa1-plasmid which most likely accounts for the somehow differing results. Our results thus indicate that FOXA1 is mandatory for the formation of primary cilia.

Co-immune precipitation revealed no direct interaction between FOXA1 and cJUN. Our data
demonstrated that FOXA1 is a transcriptional activator of Odf2. Furthermore, reporter gene assays suggested a positive interaction between FOXA1 and cJUN. To verify, we transfected cells with expression plasmids either Foxa1::gfp or Foxa1::gfp and Mekk1, followed by capturing of FOXA1::GFP using immobilized anti-GFP antibodies. Although FOXA1::GFP (of ~ 77 kDa) was successfully captured, demonstrated by its presence in the eluate of the bead-bound fraction, neither cJUN nor MEKK1-phosphorylated cJUN of either 36-39 kDa or 42-45 kDa, respectively, were co-precipitated (Fig. 9A). Thus, neither the unphosphorylated (Fig. 9A, B) nor the phosphorylated cJUN (Fig. 9A) were found to directly interact with FOXA1, despite FOXA1::GFP and cJUN colocalised in the nuclei of NIH3T3 cells (Fig. 9C).

Discussion
Primary cilia are essential sensory organelles present on nearly all cells of the body. They are built in a cell cycledependent manner and are mainly found in quiescent cells. Generation of primary cilia depends on a crucial amount of ODF2/Cenexin, a basal body protein mandatory for cilia formation and viability [42][43][44] . Transcription of Odf2 is cell cycle-dependent with upregulation in serum-starved cells and thus correlated with primary cilia formation 47 . We have previously identified TFs controlling the transcription of Odf2 47,50 . However, although few TFs have been identified to be involved in primary ciliation, as RFX3, the TF network regulating the formation of primary cilia has still to be figured out 51 . Motile ciliogenesis is controlled by the master regulator FOXJ1   37,47 . However, Odf2 was down-regulated in RFX3-deficient ependymal cells of the mouse and possesses RFX3-binding sites identified by ChIP indicating that expression of Odf2 is regulated by RFX3 in multi-ciliogenesis 54 . Furthermore, opposed to multiciliogenesis primary ciliogenesis is FOXJ1-independent 55 . FOXJ1 belongs to the large family of evolutionarily conserved forkhead-box (Fox) TFs that have diverse functions in development and differentiation. FOX TFs are characterised by the conserved DNA-binding domain of ~ 110aa, denoted as fork head domain, first identified in FKH and the rat hepatocyte-enriched transcription factor HNF-3A 26,27 . Currently, more than 44 genes were annotated in both, mice and humans, and categorized into subclasses A to S. FOX proteins are essential TFs as the deletion of just one Fox gene very often leads to lethality, and mutations in Fox genes are associated with developmental disorders or diseases 31,56 . FOXJ1, alias HFH4, is the master regulator for motile cilia formation and is, therefore, essential for the execution of the specialized functions of epithelial cells harbouring motile cilia 32,33 .
The FOXO-proteins and FOXM1 function in cell cycle control. FOXM1 is a key regulator of both the G1/S phase and G2/M phase transition and is essential for proper mitotic progression 57 . Furthermore, FOXM1 is a component of the DREAM complex that inhibits transcription of target genes for cell proliferation in the quiescent state but promotes expression during the cell cycle 58 . The DREAM complex contacts DNA via the cell cycle genes homology region (CHR), which most commonly comprises the sequence TTT GAA 59 60,61 . Their function as cell cycle inhibitors suggested a causal relationship to the transcriptional regulation of primary cilia formation, which are mainly found in quiescent cells. As a candidate approach, we focused on the sub-distal appendage protein and marker of the mother centriole and the basal body, ODF2/Cenexin, because it is mandatory for cilia formation and transcriptionally upregulated in serum-starved, cell-cycle arrested cells [42][43][44]46,50 . A consensus binding sequence for the forkhead-box TFs FOXO and FOXA1 was identified in the promoter region of the mouse Odf2-gene 48,49 . However, neither FOXO1 or its constitutively active form FOXO1ADA, nor FOXO3A activated transcription of Odf2 in reporter gene assays. These data indicated that FOXO TFs are most likely not transcriptional activators of Odf2 and largely rule them out as regulators of ciliation. A knockdown will therefore most likely have no effect on cilia formation. Instead, we found significant transcriptional activation of Odf2 by FOXA1 and therefore focused on this TF. The mammalian FOXA TFs were first identified in the rat liver and hence named hepatocyte nuclear factor 3 (HNF3) α, β, and γ, respectively FOXA1, 2, and 3 62 . Expression of FOXA1, FOXA2, and FOXA3 during development and in adult tissues exhibit overlapping but also distinct patterns 31 . Furthermore, FOXA1 is more widely expressed in adult tissues than FOXA2 31 . Foxa1-deficient mice survive until after birth but die between P2 (postnatal day 2) and P12 due to hypoglycemia and defects in kidney function 63,64 . FOXA2 is essential for node and notochord formation causing defects in the dorsal-ventral patterning of the neural tube and embryonic lethality when absent 65,66 . FOXA1 and FOXA2 regulate positively and negatively Sonic hedgehog (Shh)-signalling, via transcriptional regulation of the downstream effector Gli2, to specify the ventral midbrain progenitor identity 67 . FOXA3 is specifically expressed in the testis, in Leydig cells, and spermatids, and FOXA3deficiency affected male fertility in mice 68 . Deletion of either one of these FOXA-factors in mice revealed that FOXA1 and FOXA2 can compensate for each other and that even FOXA3 may compensate for the deletion of both, FOXA1 and FOXA2, in late gestation in mice 69 . The observed phenotypes of the knockout mice strongly remind of ciliopathies. However, whether cilia are indeed affected has not been investigated and might also be hampered by the lethality of the mutants.
The FOXA TFs enable the access of tissue-specific TFs to their binding site and have, therefore, been denominated as 'pioneer factors' 69,70 . Furthermore, they bookmark temporarily shut-off genes for easy reactivation 71 . Their function as pioneer factors has been annotated to the structural similarity of the forkhead box to histones H1 and H5 72,73 . Our reporter gene assays indicated a cross-talk between FOXA1 and cJUN, especially MEKK1activated cJUN, in the transcriptional activation of Odf2. However, we could not find a direct interaction between FOXA1 and cJUN. Thus, the binding of FOXA1 to the Odf2 promoter might enable concurrent binding of cJUN or phosphorylated cJUN to increase the expression of Odf2 corroborating its function as a pioneer factor.
To the known tasks of FOX TFs, our data add the novel function in the regulation of primary cilia formation (Fig. 10). We have shown that FOXA1 binds to its consensus sequence in the mouse Odf2 promoter and activates the expression of Odf2/ODF2. Knockdown of FOXA1 not only downregulated Odf2 transcripts and ODF2 www.nature.com/scientificreports/ proteins but also inhibited primary cilia formation. We found a significant reduction of Foxa1 (to ~ 0.5x) as well as Odf2 transcripts (to ~ 0.6x) after transfection of Foxa1 siRNA that correlated with a 0.6-fold decrease in ODF2 proteins. Since Foxa1 siRNA caused only a knockdown of FOXA1 but could not completely abolish it, FOXA1 is still present and, in addition, could also be compensated by FOXA2 that contributed to the continued expression of ODF2. Foxa2 is also transcribed in NIH3T3 cells as was observed by nested RT-PCR ( Supplementary Fig. S1) but due to a lack of validated siRNAs for the mouse FOXA2, its impact on primary ciliation could not be investigated. Reduced expression of ODF2 by FOXA1 knockdown provoked a decline of ciliated cells up to 0.55-0.75x www.nature.com/scientificreports/ ( Fig. 8) compared to their reduction to 0.3-0.47x when Odf2 was knocked down (all related to their respective controls) (Fig. 1). Our results, thus, demonstrate that Odf2 is a target gene of FOXA1 that activates its transcription. Consequently, based on the availability of the ODF2 protein and further ciliary proteins, the generation of primary cilia is regulated by FOXA1. The ChIP-seq dataset from the ENCODE Transcription Factor Targets Datasets lists several ciliary genes as FOXA1 target genes, in between the 'Gold Standard' ciliary genes including Odf2, CCP110/CP110, Cep290, Cep250, Cep164, Cep135, and the Bardet-Biedl-Syndrome genes BBS1, BBS4, BBS9, BBS10, and BBS12, and many more ciliary genes 38,74,75 . These data indicate that FOXA1 is a TF for several ciliary genes and thus a regulator of ciliogenesis. To further corroborate, we demonstrated that CP110 is also affected by FOXA1 knockdown, which is, therefore, a FOXA1 target gene as indicated in the ChIP-seq dataset. FOXA TFs are widely expressed during mouse embryogenesis and are required for the normal development of the nervous system and endoderm-derived organs. Their expression is even maintained in several adult tissues 64 . FOXA1 and FOXA2 TFs contribute to the maintenance of epithelial cell identity and their deregulated expression is associated with cancer formation 76,77 . FOXA1/2 are important for Sonic hedgehog (SHH) signalling by restricting the expression of the transcriptional mediators, Gli1 and Gli2 67,78,79 . The hedgehog pathway is essential for vertebrate development, and aberrant activation of the pathway is associated with cancer formation 80 . Furthermore, the hedgehog signal transduction pathway is strictly dependent on the primary cilium 81 . The primary cilium is involved in both the promotion and inhibition of tumorigenesis, as has also been described for deregulated FOXA expression, but is generally absent in cancer cells 82,83 . The data, thus, indicated a link between FOXA1 TF, and hedgehog signalling, finally culminating on the primary cilium. Our data indicated that FOXA1 regulates primary ciliogenesis. We propose, therefore, that the deregulated expression of FOXA1 in cancer cells might affect the primary cilium, causing eventually aberrant hedgehog signalling.
For immune-cytology, cells were reseeded at a density of 2 × 10 5 cells per well of a 6-well plate on glass coverslips. Cells were fixed in 3.7% paraformaldehyde (PFA) for 20 min at 4 °C, permeabilized with 0.3% Triton X-100 in PBS (phosphate-buffered saline) for 10 min at room temperature, followed by blocking non-specific binding sites by incubation in PBS containing 1% bovine serum albumin (BSA) and 0.5% Tween-20 for at least 1 h. Samples were incubated with the primary antibodies anti-acetylated α-Tubulin (clone 6-11B-1; Santa Cruz Biotechnology, Inc.  According to the ChIP-seq dataset from the ENCODE Transcription Factor Targets Datasets, FOXA1 targets many more essential ciliary genes, including CP110, which we have shown is indeed regulated by FOXA1. The finally generated ODF2 protein, which is mandatory for the generation of primary cilia, eventually promotes cilia formation together with other ciliary proteins. The structure of the ODF2 protein (Mus musculus) was predicted by AlphaFold (AlphaFold DB version 2022-11-01, created with the AlphaFold Monomer v2.0 pipeline, licence CC-BY-4.0), and made available by EMBL-EBI (Wellcome Genome Campus, Hinxton, UK). The primary cilium was detected by immunological decoration of both, acetylated α-Tubulin (green) and ARL13B (red), shown here as merged image (yellow), and the nucleus stained with DAPI (blue) (inset).
The quantitative real-time PCR (qRT-PCR) was performed on CFX96TM Real-Time System (Bio-Rad) using BlazeTaq SYBR Green qPCR mix 2.0 (GeneCopoeia, Rockville, MD). Primer efficiency was validated for all primer pairs and the specificity of the amplification reaction was verified by melting curve analyses. The following primer pairs were used for qRT-PCR: Foxa1_f3 (gacgccaagacattcaagcg)/Foxa1_r3 (atcgtgccaccttgacgaaa), Odf2_na-f4 (accatgaaggaccgctcttc)/Odf2 na-r4 (cgcacattcacagtgtcccc), and mHPRT-for2/mHPRT-rev2 and mGapdhf/mGapdhr as above. Three technical replicates were used for each analysis. The relative expression in each probe was calculated by ΔCt using the average Ct values of both housekeeping genes as reference. The relative expression in the experimental condition compared to the control was finally calculated as 2 −ΔΔCt using the ΔΔCt method. For the T-test, the averages of the relative expression of the technical replicates were used.
Reporter gene assay. NIH3T3 cells were seeded at a density of 1 × 10 5 cells per well of a 12 well plate. 24 h later cells were co-transfected with the reporter vector (1 µg/well), the internal control vector phRL-SV40 (Promega, Madison, USA; 10-100 ng/well), and expression plasmids encoding transcription factors (each at 100 ng/well). As reporter vector either 2.2-pGL3 or one of the truncated Odf2-promoter vectors (#1, 22.1, 7.6, 7.1, 1.5, A1, 0.5) were used, in which part of the promoter region was cloned upstream of the firefly luciferase reporter pGL3 47  To investigate the effect of FOXA1 knockdown on reporter gene activity, the Foxa1 expression plasmid (Origene, MR225487) (100 ng/well), and either one of the Foxa1 siRNA duplexes (Origene, SR415184A, SR415184B, or SR415184C, all used at a final concentration of 20 nM), or a universal scrambled negative control siRNA duplex (Origene, SR30004; final concentration 25 nM) were co-transfected. Cells were either cultivated in standard medium for 24 h post-transfection (cycling cells), or the medium was exchanged for serum starvation medium 24 h post-transfection, and cells cultivated for another 48 h (serum-starved cells). The Dual-Glo Luciferase Assay System (Promega, USA) was used for measuring firefly and Renilla luciferase activity using the Centro LB 960 luminometer (Berthold Technologies, Germany). Fold changes were calculated based on the relative luminescence (firefly luminescence/Renilla luminescence). Each experiment was performed in triplicates and repeated up to six times. Proteins were separated on denaturing SDS-gels and transferred to Hybond ECL. Blot membranes were blocked for 1 h in 5% dry milk in TBST (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.05% Tween20), and incubated with the primary antibodies (rabbit anti-GFP, self-made, and mouse monoclonal anti-cJUN, proteintech 66313-1-Ig) overnight at 4 °C. For quantitative Western blots anti-ODF2 (ESAP15572, antibodies-online, diluted 1:1000), and anti-CP110 (Proteintech #12780-1-AP, diluted 1:3000) antibodies were used, and as the internal standard anti-ß-Actin antibody (proteintech #20536-1-AP). Primary antibodies were detected with the fluorescent-labelled secondary antibodies IRDye800CW goat anti-mouse IgG (LI-COR, #925-32210), and IRDye680RD goat anti-rabbit IgG (LI-COR, #925-68071), or with IRDye800CW goat anti-rabbit IgG (LI-COR, #925-32211) and IRDye680RD goat anti-mouse IgG (LI-COR, #925-68070). Images were captured with LI-COR Odyssey CLX and analysed using Image Studio Lite (LI-COR). Quantification was performed by calculating the ratio between the target protein and the internal standard (ß-Actin) in the same lane. The relative quantities were then related to the average relative quantity in the control, giving the fold change of target protein expression.
Chromatin-immune-precipitation. NIH3T3 were transfected either with the Foxa1 expression plasmid and the Odf2 reporter vector 2.2-pGL3 or with 2.2-pGL3 exclusively to investigate the binding of the endogenous FOXA1. The chromatin-immune-precipitation (ChIP) protocol was a modification of Denissov et al. 85 . Briefly, cells were crosslinked with 1.1% formaldehyde in PBS (phosphate-buffered saline) containing 0.07 mM EDTA, 0.035 mM EGTA, and 3.5 mM Hepes for 30 min at room temperature, quenched by adding glycine to 0.125 M final concentration for 5 min at room temperature, and afterward washed twice with cold PBS. Cells were lysed in Hepes-buffer B (20 mM Hepes, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) for 10 min at 4 °C, followed by scraping, and cell collection by centrifugation. Cells were washed once in cold Hepes-buffer C (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 0.15 M NaCl), and finally resuspended in incubation-buffer (20 mM Hepes, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100 with proteinase inhibitor mix; ProteoBlock Protease inhibitor Cocktail, Fermentas R1321). Probes were sonified using 12 cycles (30 s on/30 s off) in the Bioruptor (Diagenode), and clarified by centrifugation.
Binding site occupancy was investigated by qPCR using Foxa1 primers flanking the consensus binding site in the Odf2 promoter (Foxa1-7.6-for gaattctgagattatagctatg / Foxa1-7.6-rev gccttcagatgtatgtgtgc), or primers flanking a putative DREAM binding-site of sequence TTT GAA found at position + 27 to + 33 related to the transcription start (DREAM-E1-for ctcgtgacccagaagtgg / DREAM-E1-rev cggcagctcgcccattgg). Primers were validated first. The Ct-values obtained by either FOXA1-or control IgG-precipitation were adjusted to their respective input Ct (ΔCt), and the enrichment of the binding site sequence was calculated by adjustment to the control IgG (ΔΔCt).
Statistical analyses. Data were processed and analysed using Excel. The box in the boxplots represents the 25-75th percentile. The median is given as a line, the mean by a cross. The whiskers show the minimum and maximum values inside the range given by Q1-1.5 × interquartile range (IQR) and Q3 + 1.5 × IQR. Data were analysed by Student's T-test, p < 0.05*, p < 0.01**, p < 0.001***, p < 0.0001****.

Data availability
There are no datasets generated during and/or analysed during the current study.