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. Author manuscript; available in PMC: 2026 Aug 1.
Published in final edited form as: Dev Biol. 2025 May 17;524:210–218. doi: 10.1016/j.ydbio.2025.05.013

Generation of Hoxa11–3XFLAG and Hoxd11–3XFLAG alleles to investigate Hox11 genome-wide binding

Martha L Echevarría-Andino a, Jane Y Song b,1, Paul van Ginkel a, Shuyang Chen c, Corey GK Flynn a,2, Sunduz Keles c,d, Benjamin L Allen e, Deneen M Wellik a,*
PMCID: PMC12189421  NIHMSID: NIHMS2085274  PMID: 40389054

Abstract

Hox genes encode for evolutionary conserved transcription factors that direct the proper development of the body plan. Despite decades of research, little is known regarding their downstream target genes, especially in vertebrates. The strong evolutionary conservation of their DNA-binding homeodomain, their generic AT-rich binding sites, and the lack of specific antibodies has precluded rigorous examination. To circumvent these limitations, we have generated two mouse models in which a 3XFLAG epitope tag has been inserted into the 5’ end of the coding sequence of both Hoxa11 and Hoxd11 loci via Cas9/CRISPR. The alleles have been validated by sequencing, PCR genotyping, western blotting, and protein expression analyses, demonstrating proper targeting and expression. Breeding these alleles in combination produces viable and fertile Hoxa11FLAG/FLAG; Hoxd11FLAG/FLAG animals, with no overt patterning defects unlike Hoxa11/Hoxd11 mutants that are infertile and have severe kidney and limb defects. By performing CUT&RUN and CUT&Tag analyses, we have confirmed DNA binding to a known Six2 enhancer in the developing kidney. These novel alleles will allow characterization of the genome-wide binding profile of HoxPG11 proteins in vivo.

Keywords: Hox genes, HoxPG11, epitope-tagged alleles, 3XFLAG

Graphical Abstract

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Introduction

Hox genes encode for a family of highly evolutionary conserved transcription factors that were initially discovered in the fruit fly, Drosophila (Lewis, 1978). Mammals possess 39 Hox genes organized in four clusters (A, B, C and D) and can be further subdivided in 13 paralogous groups (Krumlauf, 1994; Scott, 1992). These genes are expressed with distinct anterior-posterior (AP) boundaries along the body axis and along the proximodistal (PD) axis of the limb (reviewed by (Mallo et al., 2010; Wellik, 2024)). Colinear Hox expression mirrors their organization along the chromosome and reflects their temporal activation and expression in an organism. Hox genes function in directing cell fate and regulating tissue and organ patterning specific to each body region. Loss or gain of Hox function disrupts patterning, leading to body structures with defects or transformations (Kaufman et al., 1990; Lewis, 1978; McIntyre et al., 2007; Schneuwly et al., 1987; van den Akker et al., 2001; Wellik and Capecchi, 2003; Zhao and Potter, 2001, 2002). More recent studies have shown continued Hox expression at postnatal and adult stages, revealing roles in organ-specific stem cells and in tissue repair and regeneration throughout the life of an organism (Bradaschia-Correa et al., 2019; Hrycaj et al., 2018; Leclerc et al., 2023; Pineault et al., 2019; Rux et al., 2017; Rux et al., 2016; Song et al., 2020).

Structurally, all Hox transcription factors have a highly conserved, 60 amino acid DNA-binding domain, the homeodomain, which binds to a four base (ATTA) core consensus binding motif (Desplan et al., 1985; Gehring et al., 1990; McGinnis et al., 1984). Through this domain, Hox proteins bind DNA to regulate gene expression. However, the homeodomains for all 39 Hox proteins are highly similar and have little to no binding specificity alone to elicit unique downstream gene regulatory networks (reviewed by (Mann et al., 2009)). Hox specificity for target DNA-binding sites can be potentiated by protein cofactor interactions. One well-characterized interaction is through the hexapeptide motif, which binds Pbx, Prep and Meis proteins of the TALE family of homeodomain proteins (reviewed by (De Kumar and Darland, 2021)). Additional sequences within domains C- or N- terminal to the homeodomain have also been proposed to confer binding specificity though this has been explored less (Joshi et al., 2007; Liu et al., 2008; Papadopoulos et al., 2010).

There is lack of reliable commercial Hox antibodies with demonstrated specificity, likely due to the strong evolutionary conservation of Hox protein sequences. To address this limitation, several studies have utilized different techniques such as biochemical isolations, overexpression of tagged Hox proteins, and in silico analyses (Gong et al., 2007; Kutejova et al., 2008; Lei et al., 2005; Schwab et al., 2006; Tomotsune et al., 1993; Williams et al., 2005). However, to ascertain genome-wide binding in vivo in a wild-type context, it is critical to assess endogenous protein expression.

HoxPG11 genes play essential roles in multiple processes, including kidney formation, patterning of the axial skeleton and limbs, skeletal stem/progenitor cell differentiation, and development of the urogenital and reproductive tracts (Davis and Capecchi, 1994; Gendron et al., 1997; Hsieh-Li et al., 1995; Small and Potter, 1993; Song et al., 2020; Wellik and Capecchi, 2003; Wellik et al., 2002; Wong et al., 2004). However, which downstream target genes are regulated by HoxPG11 to perform these patterning processes remains largely unknown. To circumvent technical limitations and permit dissection of HoxPG11 genome-wide binding in vivo, we generated two endogenously 3XFLAG-tagged alleles via Cas9/CRISPR gene editing for two Hox11 paralogs, Hoxa11 and Hoxd11. This epitope-tag targeting strategy has been previously utilized successfully to perform DNA-protein interaction analyses in different contexts such as the limb bud and craniofacial structures (Elliott et al., 2020; Lex et al., 2020; Lex et al., 2022; Lopez-Rios et al., 2014). Proper targeting of both alleles (Hoxa11–3XFLAG and Hoxd11–3XFLAG) was validated by sequencing, PCR genotyping, western blotting, and immunofluorescence, demonstrating correct placement, insertion and expression. Breeding these alleles in combination, generating Hoxa11FL/FL; Hoxd11FL/FL animals results in viable and fertile animals with no overt patterning defects. Further, to demonstrate that these alleles are suitable for DNA-protein interaction analyses, we confirmed Hoxa11–3XFLAG/Hoxd11–3XFLAG binding to the previously reported Six2 enhancer (Gong et al., 2007; Yallowitz et al., 2009) in the developing kidney using CUT&RUN and CUT&Tag. Taken together, we have generated a reliable in vivo model to examine Hox11 genome-wide binding, and the identification of its downstream target genes in different contexts.

Materials and Methods

Mouse models

To generate Hoxa11–3XFLAG and Hoxd11–3XFLAG mouse lines we utilized the Cas9/CRISPR gene editing at the University of Michigan Transgenic Animal Core. All guide sequences were cloned into the pT7-Guide Vector (Blue Heron Biotech, LLC). The MEGAshortscript T7 kit (Life Technologies) was used to generate in vitro transcribed sgRNA’s from the pT7-Guide Vector and products were subsequently purified using the MEGAclear kit (Life Technologies). Using the pT7-Cas9-Nuclease vector (gift from Dr. Moises Mallo), Cas9 mRNA was in vitro transcribed using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies) and purified using the MEGAclear kit (Life Technologies). Donor oligos were purchased from Integrated DNA Technologies (IDT) as Megamer® Single-stranded DNA fragments. The total length of the donor oligo was kept at 200bp. Thus, the homology sequence on either side was approximately 50bp. Zygote injections were performed as previously described with minor modifications (Wu et al., 2013). C57BL/6 female mice were super-ovulated and mated with C57Bl/6 male mice to collect embryos of one-cell stage for microinjection. CRISPR reagents were microinjected at the following concentrations: Cas9 mRNA (100ng/μL), sgRNA (50ng/μL), and donor oligo (25ng/μL). Freshly injected eggs were transferred into pseudopregnant females and resulting progeny were initially screened for potential recombination events via PCR.

PCR confirmation of Hoxa11–3XFLAG targeting

85 live births were recovered from the microinjections and initial screening for 3XFLAG targeting was performed by PCR genotyping using the following primers: Forward: 5’ – GTC ACA TGA CCA GCA CCT CC – 3’ and Reverse: 5’ – AGT ATG TCA TTG GGC GCG AA – 3’, generating a wildtype 293bp band and a 3XFLAG targeted 371bp band.

PCR confirmation of Hoxd11–3XFLAG targeting

61 live births were recovered from the microinjections and initial screening for 3XFLAG targeting was performed by PCR genotyping using the following primers: Forward: 5’ – ACG TGA CAT AAT TAC CAC CAG AA – 3’ and Reverse: 5’ – CAG GCC GTA GTC GCG AAA – 3’, generating a wildtype 369bp band and a 3XFLAG targeted 450bp band.

Sequence confirmation of Hoxa11–3XFLAG and Hoxd11–3XFLAG targeting

Correct targeting was confirmed by Sanger sequencing using the same primers used for PCR genotyping indicated above. Mice with correct Hoxa11–3XFLAG and Hoxd11–3XFLAG targeting were bred to homozygosity, and the alleles were successfully transmitted through the germline. Through breeding of the two newly generated mouse lines, we generated double homozygous animals (Hoxa11FL/FL;Hoxd11 FL/FL) and maintained them in a C57Bl/6;CD-1 mixed background.

For embryonic dissections, noon of the day on which a vaginal plug was detected was considered as E0.5 All the experiments were performed following protocols approved by the Institutional Animal Care and Use Committee (IACUC) guidelines at the University of Wisconsin-Madison or at the University of Michigan.

Western Blot analysis

Forelimb buds were micro-dissected from E12.5 embryos and lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH7.2, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA) containing a protease inhibitor cocktail (Roche, 11836170001) and extracts were cleared by centrifugation at 20,000× g for 30 min at 4°C. Total protein concentration was determined with the Pierce BCA protein assay kit (Thermo Fisher Scientific, 23225) and separated by SDS/PAGE. Proteins were transferred onto a low-fluorescence polyvinylidene fluoride membrane (Cytiva, 10600022), blocked in Intercept blocking buffer (Licor, 927–60001), and probed with ANTI-FLAG M2 primary antibody (Sigma, F3165, 1:1,000) and Goat anti-Mouse IgG secondary antibody (Licor, IRDye 800CW926–32210, 1:15,000). Fluorescence signals were detected on a LI-COR Odyssey Fc imaging system.

Immunofluorescence

Embryos (E12.5-E16.5) were dissected in PBS (pH 7.4) and fixed in 4% paraformaldehyde on ice for 25 min, followed by 3×5min washes with PBS (pH 7.4) and cryoprotected for 24h in 30% sucrose in PBS. Embryos were embedded in O.C.T. compound (Fisher Scientific, 23–730-571) and sectioned on a Leica CM3050 S cryostat (10–12 μm thick sections). For FLAG staining the slides were fixed in 100% methanol for 15 min, followed by 2×2min washes in PBSTr (PBS + 0.1% Triton X-100), then the sections were immediately placed in sodium citrate buffer (10mM sodium citrate, 0.05% Tween-20, pH 6.0) in an Antigen Retriever (EMS, 62700–20). After antigen retrieval slides were cooled down to room temperature and washed 2×2min in PBSTr. To block endogenous mouse immunoglobulins in tissue sections we used the Mouse on Mouse® (M.O.M.) Blocking Reagent (Vector laboratories, MKB-2213–1) according to the manufacturer’s instructions with the following modifications: Briefly, sections were blocked in M.O.M. Mouse IgG Blocking Reagent for 1hour, followed by 2 min washes in PBS, PBSTw (PBS + 0.1% Tween-20) and PBS. Next, slides were incubated in M.O.M. diluent for 5 min. ANTI-FLAG M2 primary antibody (Sigma, F3165) was diluted (1:500) in M.O.M. diluent and incubated for 30 min at room temperature, followed by 2 min washes in PBS, PBSTw and PBS. FLAG signal was amplified by staining with Biotinylated anti-Mouse IgG (diluted in M.O.M. diluent), followed by 2 min washes in PBS, PBSTw and in PBS. Next the sections were incubated with Alexa Fluor® 594 Streptavidin (Jackson ImmunoResearch AF594, (diluted (1:500) in M.O.M. diluent), followed by 2 min washes in PBS, PBSTw and PBS. Nuclei were labeled with DAPI (Thermo Fisher Scientific, 62248, 1:2,000) for 10 min at room temperature, followed by 3×2min washes with PBS. Slides were mounted with coverslips using Epredia™ Immu-Mount™. Sections were visualized on a Keyence BZ-X800 microscope. Adobe Photoshop was utilized to adjust the brightness and contrast of the images and channel overlay.

Fertility assessment

Sexually mature females of the indicated genotype were paired with Hoxa11FL/FL;Hoxd11FL/FL males to expand the line and determine fertility. Mating cages consisted of one male and one female. Breeding tests for each pair were scored for at least two litters. The litter size was recorded and genotyped.

Skeletal Preparations

Postnatal day 0 (P0) pups were dissected and immersed in a 65°C - 70°C water bath for 1 min and subsequently skinned and eviscerated. Specimens were fixed in 95% ethanol for 4 days at room temperature. To visualize cartilage, specimens were stained with Alcian Blue (0.15 mg/mL Alcian Blue 8GX (Sigma, A5268), 80% ethanol and 20% glacial acetic acid) for 2 days at 37°C. Next, the skeletons were rinsed 2×1hour in 95% ethanol and cleared in 1% potassium hydroxide for 5 hours at room temperature. Bone was stained with Alizarin Red (50mg/mL Alizarin Red S (Sigma, 5533), 2% potassium hydroxide) overnight. The tissue was cleared in 1% potassium hydroxide and progressively increasing concentrations of glycerol (20%, 50% and 100%). For long-term storage skeletons were stored in 100% glycerol.

Zeugopod Bone Length Quantitation

Bone length quantitation was performed as previously described in (Echevarria-Andino et al., 2023). Briefly, forelimbs were dissected from postnatal day 0 skeletons and photographed in 100% glycerol using a Leica MZ125 microscope. Zeugopod length was measured in Adobe Illustrator utilizing the pencil tool to measure the entire length of the bone. All the data are represented as mean±SD. Statistical analyses were performed with GraphPad Prism (www.graphpad.com). Statistical significance was determined using a one-way ANOVA with the Bonferroni correction. A minimum of three embryos of each genotype were examined and each limb was considered an n.

CUT&RUN Experiments

Experiments were performed with EpiCypher’s CUT&RUN kit (SKU: 14–1048 according to manufacturer’s protocol with some modifications). Briefly, E13.5 Hoxa11FL/FL;Hoxd11FL/FL or Hoxa11FL/+;Hoxd11FL/FL kidneys were micro-dissected from embryos and pooled to utilize 300,000 cells/reaction. To obtain a single cell solution, kidneys were dissociated with Liberase (100ug/mL, Roche 05401119001) in PBS at 37°C for 5 min, followed by gentle pipetting to disrupt the remaining tissue. Cells were bound to Concanavalin A-coated paramagnetic activated beads and permeabilized with digitonin (0.025%). Cells were incubated O/N at 4°C with the following antibodies: ANTI-FLAG M2 primary antibody (Sigma, F3165, 1:100) or IgG (EpiCypher, 13–0042k, 1:100) on a nutator. The next day the samples were incubated with secondary antibody (Guinea Pig anti-Rabbit IgG (Antibodies Online, ABIN101961) or Rabbit anti-Mouse IgG (Abcam, ab46540, 1:100) for 30 min at room temperature, followed by 2x washes in Digitonin wash buffer. Next, pAG-MNase was added to the samples and incubated for 10 min at room temperature for binding. For targeted chromatin digestion, 100mM CaCl2 was added to each sample and the MNase reaction was performed for 2 hours at 4°C on a nutator. Chromatin release was performed for 10 min at 37°C. DNA was purified via phenol:chloroform extraction and followed by ethanol precipitation. Libraries were generated using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, E7645S0 and NEBNext Multiplex Oligos for Illumina (Unique Dual Index Primer Pairs, New England Biolabs, E6440S) following the manufacturer’s instructions with 14 cycles of amplification and a 1:25 dilution for the adapter. Libraries were cleaned-up to remove adapters using AMPure XP beads (Beckman Coulter, a63880). Library quality control and fragment size was assessed in a 4200 Tapestation system (Agilent). Samples were sequenced on an NovaSeq6000 (Illumina) sequencing system using a paired-end 150bp sequencing run at a sequencing depth of 5 million reads/sample.

CUT&Tag Experiments

Experiments were performed as described (Li et al., 2021). Briefly, E13.5 Hoxa11FL/FL;Hoxd11FL/FL kidneys were micro-dissected from embryos and pooled to utilize 200,000 cells/reaction. To obtain a single cell solution, kidneys were dissociated as described above for CUT&RUN experiments. Cells were bound to activated Concanavalin A-coated paramagnetic beads and permeabilized with digitonin (0.05%). Next, cells were incubated O/N at 4°C with the following antibodies: ANTI-FLAG M2 primary antibody (Sigma, F3165, 1:100 or IgG (Diagenode, #C15410206, 1:100) on a nutator. The next day the samples were incubated with secondary antibody (Guinea Pig anti-Rabbit IgG (Antibodies Online, ABIN101961) or Rabbit anti-Mouse IgG (Abcam, ab46540), 1:100) for 1 hour at room temperature, followed by several washes to remove unbound antibody. Tn5 transposase mediated tagmentation was then initiated by the addition of MgCl2 for 1 hour. Chromatin release was performed for 1 hour min at 55°C. DNA was purified via phenol:chloroform extraction and followed by ethanol precipitation. Tagmented DNA was utilized to perform a qPCR to determine the number of cycles required for library preparation. Libraries were generated with NEB Next High Fidelity 2x PCR Master Mix and different i5 and i7 primers as previously described by (Kaya-Okur et al., 2019). Library quality control and fragment size was assessed with a Bioanalyzer system (Agilent), and samples were sequenced on an NovaSeq6000 (Illumina) sequencing system using a paired-end 100bp sequencing run at a sequencing depth of 10 million reads/sample.

CUT&RUN and CUT&Tag Analysis

CUT&RUN and CUT&Tag analysis was conducted using a previously published pipeline (https://www.protocols.io/view/cut-amp-tag-data-processing-and-analysis-tutorial-e6nvw93×7gmk/v1) as described in (Ye Zheng, 2020). Sequencing data quality was assessed using MultiQC, and trimming was performed with Trimmomatic using the following parameters: ILLUMINACLIP (2:30:10), LEADING (3), TRAILING (3), SLIDINGWINDOW (4:15), and MINLEN (36). Trimmed reads were aligned to the mouse reference genome (mm39) using Bowtie2 with the parameters: --local, --very-sensitive, --no-mixed, --no-discordant, -I 10, -X 700. For the IgG control sample, duplicates were handled using Picard tools. SAM files were sorted by coordinate with SortSam, and duplicates were marked and removed using MarkDuplicates. Peak calling for FLAG, was performed with SEACR which calls enriched regions in target data using IgG control track with stringent threshold. Peak annotation was performed with HOMER v4.11.1. To assess peak reproducibility, Irreproducible Discovery Rate (IDR) analysis was performed on FLAG replicates from CUT&Tag. Stringent peaks from FLAG-1 and FLAG-2 replicates were analyzed using IDR v2.0.4 with a threshold of 0.01, ranking peaks by signal intensity to identify high-confidence, reproducible peaks. GO enrichment analysis was performed using clusterProfiler. EnrichGO was applied with org.Mm.eg.db to assess enrichment across all three subontologies provided with significance determined by Benjamini-Hochberg (BH) adjusted p-values (p-adjust < 0.05) and q-values (q < 0.05).

Results and Discussion

To generate epitope-tagged alleles for Hoxa11 and Hoxd11 we used Cas9/CRISPR genetic engineering to insert a 3X FLAG epitope tag into the 5’ end of both loci. We selected guide RNAs (gRNA) that would cut close to, but 3’ of, the translational start site (ATG), generating the minimum number of amino acids between the ATG and insertion site (Figure 1A). The donor sequence was designed so that the 3XFLAG sequence would be inserted between the gRNA cut site and downstream PAM site, eliminating the possibility of unwanted Cas9 nuclease targeting to the donor sequence or recombined allele. This editing strategy was designed to prevent disruption of the surrounding sequence and to keep all endogenous regulatory elements intact. Further, a Gly-Ser-Gly bridge was designed on the 3’ end of the 22-amino-acid 3XFLAG peptide sequence to relieve potential conformational strain that might result from the FLAG peptide in the final protein product (Figure 1B). Utilizing this targeting strategy, we recovered seven correctly gene-edited lines out of 85 live births for the Hoxa11–3XFLAG and ten correctly gene-edited lines out of 61 live births for the Hoxd11–3XFLAG alleles as assessed by PCR genotyping. Founder animals (F0s) were confirmed to produce the correct size band for 3XFLAG insertion as assessed by PCR genotyping (Supplemental Figure 1A, B). Correct in-frame targeting was further assessed by sequencing. Three of the seven founders for the Hoxa11–3XFLAG (FLAG peptide inserted one amino acid after the start codon) and four out of the ten for the Hoxd11–3XFLAG (FLAG peptide inserted three amino acids after the start codon) founders were confirmed to have perfectly targeted, in-frame insertions of the FLAG peptide (Figure 1C). One founder from each line was bred to establish colonies. The founders were crossed to C57/Bl6 wildtype animals for two generations to generate heterozygotes and subsequently homozygotes, using PCR genotyping primers designed to discern heterozygous and homozygous animals from wild-type animals (Figure 1A). The newly generated lines were maintained as homozygous males and females, which are viable and fertile and do not display any overt defects. This is in contrast to both male and female Hoxa11 single mutant mice, which are infertile, and male Hoxd11 single mutant mice which are also infertile (Davis and Capecchi, 1994; Gendron et al., 1997; Hsieh-Li et al., 1995).

Figure 1: Generation of Hoxa11–3XFLAG and Hoxd11–3XFLAG alleles via Cas9/CRISPR genetic engineering.

Figure 1:

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(A) Schematic of the Hoxa11 (top) and Hoxd11 (bottom) locus and approximate position of target guide RNA (gRNA) for 3XFLAG insertion. Green underline highlights translation start site, gray box denotes the PAM sequence of the gRNA, and pink arrowheads denote PCR genotyping primers (P) (P1 and P2 for Hoxa11-3XFLAG and P3 and P4 for Hoxd11-3XFLAG) that are upstream and downstream from the gRNA. PCR genotyping analysis of wildtype, heterozygous, homozygous Hoxa11-3XFLAG and Hoxd11-3XFLAG animals, respectively. Note that the primers detect the expected band sizes for a 3XFLAG tag insertion in Hoxa11 (wildtype band (293bp) and 3XFLAG band (371bp)) and Hoxd11 (wildtype band (369bp) and 3XFLAG band (450bp)). (B) Schematic of the HOXA11 (top) and HOXD11 (bottom) proteins with the 3XFLAG insertion. (C) Representative chromatogram of Sanger sequencing results from animals with the correct in-frame insertion of the 3XFLAG tag into the Hoxa11 (top) Hoxd11 (bottom) loci. Green box denotes the translation start site (ATG), magenta box highlights 3XFLAG tag sequence and orange box denotes the Gly-Ser-Gly flexible bridge sequence.

To assess whether both Hoxa11–3XFLAG and Hoxd11–3XFLAG alleles could produce fidelitous protein products, we examined protein expression using a monoclonal ANTI-FLAG M2 antibody (Sigma F3165). Western blot analysis of embryonic (E) 12.5 Hoxa11–3XFLAG and Hoxd11–3XFLAG homozygous (hereafter referred to as Hoxa11FL/FL and Hoxd11FL/FL, respectively) forelimbs revealed a single FLAG band in both alleles (Figure 2A). The predicted protein molecular weight for Hoxa11 and Hoxd11 proteins is ~34 and 35 kDa, respectively. However, both alleles generated bands with a higher molecular weight (Figure 2A and Supplemental Figure 2A-B (uncropped western blot)). We speculate that this discrepancy in molecular weight size arises from post-translational modifications. Previous studies have identified several post-translational modifications in different regions of the proteins such as phosphorylation, acetylation and ubiquitination of Hoxa11 and phosphorylation, ubiquitination and methylation of Hoxd11 (Draime et al., 2018).

Figure 2: FLAG expression and distribution in E12.5 forelimbs.

Figure 2:

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(A) Western blot analysis of FLAG tag expression in the forelimbs of E12.5 Hoxa11FL/FL, Hoxd11FL/FL and wildtype embryos. Immunofluorescent detection of FLAG tag in E12.5 Hoxa11FL/FL (B) and Hoxd11FL/FL (C) longitudinal forelimb sections (magenta; B-C). Immunofluorescent detection of FLAG tag in E14.5 (D, F), E16.5 (E) Hoxa11FL/FL;Hoxd11FL/FL forelimb longitudinal (D-E) and transverse kidney (F) sections (magenta; D-F). Nuclei are identified with DAPI (white; B-F). Abbreviations: r, radius and u, ulna. Scale bars, (B-F) 100 μm.

Next, we verified FLAG expression in E12.5 Hoxa11FL/FL and Hoxd11FL/FL forelimb tissue sections by immunofluorescence. Consistent with previous studies using reporter alleles or in situ hybridization, FLAG expression was detected in the stroma surrounding the zeugopod skeletal elements (Figure 2B-C) (Boulet and Capecchi, 2004; Haack and Gruss, 1993; Nelson et al., 2008). FLAG expression was also detected at E14.5 (Figure 2D) and E16.5 (Figure 2E) in the zeugopod stroma and the perichondrium. Additionally, FLAG expression was also observed in other tissues including the nephrogenic cap mesenchyme of the developing kidney (Figure 2F). These data indicate that both Hoxa11–3XFLAG and Hoxd11–3XFLAG alleles produce similar expression patterns that recapitulate endogenous expression.

To determine whether the insertion of the FLAG peptide permits the tagged HoxPG11 proteins to remain functional, Hoxa11FL/FL and Hoxd11FL/FL animals were bred together to generate Hoxa11FL/FL; Hoxd11FL/FL animals. Hoxa11/Hoxd11 double mutant embryos die at birth and have severe kidney defects and highly truncated radius and ulnar skeletons. Single Hoxa11 or Hoxd11 null mutants have fertility defects (Davis and Capecchi, 1994; Gendron et al., 1997; Hsieh-Li et al., 1995). In contrast, Hoxa11FL/FL;Hoxd11FL/FL males are fully viable and fertile, while females are viable but produce smaller litter sizes than Hoxa11FL/+;Hoxd11FL/FL females (Supplemental Figure 2C). Examination of tissues patterned by HoxPG11 shows normal zeugopod (radius and ulna) skeleton (Figure 3A-C) and normal kidneys as compared to Hoxa11/Hoxd11 mutants (Figure 3F-H). Furthermore, quantitation of the length of the forelimb zeugopod bones revealed that Hoxa11FL/FL; Hoxd11FL/FL animals do not show zeugopod bone length reduction when compared to wildtype embryos (Figure 3D-E). Thus, the insertion of the FLAG peptide does not disrupt the function of the epitope-tagged Hoxa11/Hoxd11 factors. Moreover, maintaining these alleles as Hoxa11FL/FL;Hoxd11FL/FL animals should permit rigorous examination of HoxPG11 genome-wide binding.

Figure 3: Hoxa11FL/FL, Hoxd11FL/FL mice display normal zeugopod and kidney patterning.

Figure 3:

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(A-C) Analysis of forelimb zeugopod patterning in postnatal day 0 animals stained with Alcian Blue and Alizarin Red. Skeletal preparations of forelimbs of wildtype (A), Hox11aadd (B) and Hoxa11FL/FL, Hoxd11FL/FL (C). Black bracket denotes zeugopod length. (D-E) Quantitation of zeugopod length of postnatal day 0 limbs. Quantitation of radius (D) and ulna (E) length from wildtype (n=7), Hox11aadd (n=6) and Hoxa11FL/FL;Hoxd11FL/FL forelimbs (n=7). (F-G) Analysis of kidney gross morphology in postnatal day 0 animals. Whole-mount brightfield images of wildtype (F), Hox11aadd (G) and Hoxa11FL/FL;Hoxd11FL/FL kidneys (H). Arrow cross in (A) specifies the limb axes, A, anterior, Po, posterior, Pr, proximal and D, distal. Yellow dotted lines outline kidneys (F-G). Abbreviations: a, adrenal glands and k, kidneys. P-values (p) were determined by a one-way ANOVA with the Bonferroni correction, non-significant (n.s; p ≥ 0.05) and significant (p ≤ 0.05). Scale bars, (A-C and F-H) 500 μm.

To directly investigate use of these alleles to detect Hoxa11 and Hoxd11 DNA binding, we probed the embryonic kidney, to examine whether these combined alleles could lead to binding confirmation of a known HoxPG11 target, Six2 (Gong et al., 2007; Yallowitz et al., 2009). Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and Cleavage Under Targets and Tagmentation (CUT&Tag) (Kaya-Okur et al., 2019; Skene and Henikoff, 2017) we detected FLAG binding to the Six2 promoter in kidney cells isolated from E13.5 kidneys expressing Hoxa11–3XFLAG and Hoxd11–3XFLAG (Figure 4A-B). To perform this technique, we utilized the same M2 antibody that we used for immunofluorescence; this antibody has been previously utilized to detect DNA-protein interactions via ChIP-Seq (Lex et al., 2020; Lex et al., 2022). Our experiments revealed 25,639 and 7,189 HoxPG11 peaks in the CUT&RUN and CUT&Tag datasets, respectively. In both datasets, these peaks were distributed throughout the mouse genome, with the majority of the peaks enriched in intronic, promoter, and intergenic regions Figure 4C). This peak distribution has been previously observed in studies that have performed ChIP-Seq for HOX binding (Apfelbaum et al., 2022; Desanlis et al., 2020; Sheth et al., 2016). Moreover, gene ontology (GO) analysis revealed that HoxPG11 binding sites are significantly associated to embryonic skeletal system development, renal system development, skeletal system morphogenesis, and kidney development GO terms, which are biological processes regulated by Hox11 patterning functions (Supplemental Figure 3A). Among the most enriched genes in HoxPG11 binding sites associated to renal system and kidney development GO terms we found Six2. Critically, there is a strong enrichment of Hoxa11–3XFLAG/Hoxd11–3XFLAG compared to the IgG control at the previously characterized Six2 enhancer (Figure 4D). Strikingly, we were able to detect enrichment of this interaction even though our CUT&RUN and CUT&Tag experiments were performed on a mixed population of kidney cells, where the HoxPG11-expressing cells are a minority of total cells (Nelson et al., 2008). We also observed strong enrichment in other genes associated with the renal system and kidney development such as Gas1, Spry1 and Wt1 (Supplemental Figure 3B) (Franks and Allen, 2024; Kann et al., 2015; Kreidberg et al., 1993). Together, these data validate and establish Hoxa11–3XFLAG and Hoxd11–3XFLAG alleles as reliable models to assess HoxPG11 genome-wide binding in vivo in mice.

Figure 4: Hoxa11–3XFLAG/Hoxd11–3XFLAG binding to Six2 enhancer in the developing kidney.

Figure 4:

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(A) Schematic of CUT&RUN experiment for HoxPG11 binding validation. (B) Heatmap of FLAG signal (magenta) with CUT&RUN (left) and CUT&Tag (right). (C) Pie chart showing the genomic distribution of FLAG binding sites with CUT&RUN (left) and CUT&Tag (right). Abbreviations: TSS, transcription start site and TTS, transcription termination site. (D) Representative Integrative Genomics Viewer (IGV) of CUT&RUN tracks (top left panel) of IgG (black track) and FLAG (magenta track) binding at the Six2 enhancer and representative IGV CUT&Tag tracks (top right panel) of IgG (black track) and FLAG (magenta track) binding at the Six2 enhancer. Blue shading highlights Hoxa11–3XFLAG/Hoxd11–3XFLAG enrichment at the Six2 enhancer. Bottom panel in (D): schematic of called peaks and binding sites, represented by vertical lines for peak calling (blue), Hox binding sites (magenta) and Pax2 binding site (orange).

Conclusion

Despite decades of research, the downstream target genes of Hox transcription factors remain largely unknown. Here we describe the generation and the validation of two epitope-tagged alleles, Hoxa11–3XFLAG and Hoxd11–3XFLAG. Specifically, we demonstrate that these two novel mouse models display proper genetic targeting, no overt phenotypes and are viable and fertile. Additionally, these alleles recapitulate the proper expression and distribution of Hoxa11 and Hoxd11. Addition of the FLAG peptide does not alter the proper function of the proteins or endogenous binding to a previously validated downstream target. Therefore, these alleles are ideal for the identification of Hox11PG downstream targets in mice.

Supplementary Material

1

KEY RESOURCES TABLE

Reagent or resource Source Identifier
Antibodies
ANTI-FLAG M2 primary antibody Sigma Cat#F3165, RRID:AB_259529
CUTANA™ IgG Negative Control Antibody for CUT&RUN and CUT&Tag EpiCypher Cat#13–0042k
Rabbit IgG Negative Control Diagenode Cat#C15410206, RRID:AB_2722554
Bacterial and Virus Strains
Biological Samples
Chemicals, Peptides, and Recombinant Proteins
Liberase Roche Cat#05401119001
Critical Commercial Assays
MEGAshortscript T7 kit Life technologies
Invitrogen™ 		Cat#AM1354
MEGAclear™ Transcription Clean-Up kit Life technologies
Invitrogen™ 		Cat#AM1908
mMESSAGE mMACHINE™ T7 ULTRA Transcription kit Life technologies
Invitrogen™ 		Cat#AM1345
(M.O.M.) Blocking Reagent (Vector laboratories, MKB-2213–1) Vector Laboratories Cat#MKB-2213–1
CUT&RUN kit EpiCypher’s Cat#SKU: 14–1048
NEBNext Ultra II DNA Library Prep Kit New England
Biolabs		 Cat#E7645S0
NEBNext Multiplex Oligos for Illumina (Unique Dual Index Primer Pairs) New England Biolabs Cat#E6440S
CUTANA™ pAG-Tn5 for CUT&Tag EpiCypher Cat#SKU: 15–1017
Next High Fidelity 2x PCR Master Mix New England
Biolabs		 Cat#M0541S
Deposited Data
Raw and processed CUT&RUN data This paper
Raw and processed CUT&Tag data This paper
Experimental Models: Cell Lines
Experimental Models: Organisms/Strains
Hoxa11–3XFLAG This paper
Hoxd11–3XFLAG This paper
Oligonucleotides
Hoxa11 sgRNA 5’-AGCCCAAGGTAGCCCAATGATGG-3’ This paper
Hoxd11 sgRNA 5’-ATGAACGACTTTGACGAGTGCGG-3’ This paper
Hoxa11 F: 5’ – GTC ACA TGA CCA GCA CCT CC – 3’ This paper
Hoxa11 R: 5’ – AGT ATG TCA TTG GGC GCG AA – 3’ This paper
Hoxd11 F: 5’ – ACG TGA CAT AAT TAC CAC CAG AA – 3’ This paper
Hoxd11 R: 5’ – CAG GCC GTA GTC GCG AAA – 3’ This paper
Recombinant DNA
pT7-Guide IVT Blue Heron Biotech, LLC (Commercially available from OriGene) Cat# SKU GE100025
pT7-Cas9-Nuclease A gift from Dr. Moises Mallo (Commercially available from OriGene) Cat# SKU GE100014
Software and Algorithms
GraphPad Prism GraphPad RRID:SCR_002798
CUT&Tag Data Processing and Analysis Tutorial Ye Zheng, 2020 https://www.protocols.io/view/cut-amp-tag-data-processing-and-analysis-tutorial-e6nvw93x7gmk/v1
MultiQC Ewels et al., 2016) https://www.bioinformatics.babraham.ac.uk/projects/download.html
Trimmomatic (Bolger et al., 2014) http://www.usadellab.org/cms/?page=trimmomatic
Samtools (Li et al., 2009) http://samtools.sourceforge.net/
Bedtools (Quinlan and Hall, 2010) https://bedtools.readthedocs.io/en/latest/
Picard tools Broad Institute, 2012) https://broadinstitute.github.io/picard/
Bowtie2 (Langmead and Salzberg, 2012) http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
SEACR (Meers, et al., 2019) https://seacr.fredhutch.org/
clusterProfiler (Yu, G, et al., 2012) https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html
Other
Antigen Retriever EMS Cat#62700–20
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Highlights.

  • Generation of epitope-tagged alleles for Hoxa11 and Hoxd11.

  • Insertion of a 3XFLAG tag into Hoxa11, Hoxd11 alleles, preserves protein function.

  • Hoxa11–3XFLAG and Hoxd11–3XFLAG are useful for examining genome-wide binding.

Acknowledgments

We thank former and present members of the Wellik lab for discussions and experimental suggestions. We also thank Drs. Steven Vokes (University of Texas at Austin), Rachel Lex (Fred Hutchinson Cancer Research Center), F. Jeffrey Dilworth (University of Wisconsin-Madison) and Marjorie Brand (University of Wisconsin-Madison) for providing valuable technical advice for CUT&RUN and CUT&Tag experiments, Wei Xu (University of Wisconsin-Madison) for access to equipment. Finally, we acknowledge the University of Michigan Transgenic Animal Model Core, the University of Wisconsin-Madison Next Generation Sequencing Core and the Molecular Biology and Genomics Core facility of the Institut de Recherche Clinique de Montreal, Montreal, QC, Canada.

Funding

This work was supported by the National Institutes of Health [5R37AR061402, to D.M.W.], the University of Michigan Rogel Cancer Center Innovation Grant [to D.M.W. and B.L.A.] and the Stem Cell and Regenerative Medicine Center Postdoctoral Training Fellowship [University of Wisconsin-Madison, awarded to M.L.E.-A.].

Footnotes

Declarations of Interests: none

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Data Availability

The genomic CUT&RUN and CUT&Tag datasets generated in this study are available at the GEO repository, accession number GSE294463.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2085274-supplement-1.pdf (376KB, pdf)

Data Availability Statement

The genomic CUT&RUN and CUT&Tag datasets generated in this study are available at the GEO repository, accession number GSE294463.

RESOURCES