A FOXA1-binding enhancer regulates Hoxb13 expression in the prostate gland

Ryan P. McMullina, Albert Dobib, Laura N. Muttona, András Orosza,1, Shilpi Maheshwarib, Cooduvalli S. Shashikantc, and Charles J. Biebericha,2

aDepartment of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250; bCenter for Prostate Disease Research, Department of Surgery, Uniformed Services University, Rockville, MD 20852; and cDepartment of Dairy and Animal Science, College of Agricultural Sciences, The Pennsylvania State University, University Park, PA 16802

Edited by Francis H. Ruddle, Yale University, New Haven, CT, and approved November 13, 2009 (received for review May 1, 2009) Hoxb13 is robustly transcribed in derivatives of posterior endoderm Reporter approaches in transgenic mice have been instru- including the colon, rectum, and the prostate gland. Transcriptional mental in the identification of prostate-restricted transcriptional activity in the prostate persists unabated under conditions of andro- control regions (11–18). However, these analyses have focused ex- gen deprivation and throughout the course of disease progression clusively on androgen-stimulated . Recently, a large Hoxb13 in a mouse prostate cancer model. To elucidate the molecular basis reporter gene capable of recapitulating the androgen-independent, of prostate-restricted transcriptional activation of Hoxb13, a bacte- prostate-restricted transcriptional activity of the endogenous Hoxb13 rial artificial (BAC)-based reporter gene deletion anal- gene has been reported (10). To shed light on the molecular mech- ysis was performed in transgenic mice. Two regions downstream of anisms that mediate this pattern, we performed a the Hoxb13 coding region were found to be required to support detailed analysis of the regulatory regions that confer prostate spe- transcriptional activity in the prostate but were completely dispen- cificity to Hoxb13. Using a combination of bioinformatic, bio- sable for expression in the colon and rectum. Bioinformatic analyses chemical, and transgenic analyses, we scanned a 218-kb genomic of one region identified a 37-bp element conserved in mammals. region near the Hoxb13 and functionally characterized a 37-bp This element, which bears two potential binding sites for Forkhead enhancer module required for Hoxb13 transcriptional activity in the

class transcription factors, is occupied by FOXA1 in a human prostate prostate. We further demonstrate that the Forkhead winged helix BIOLOGY cancer cell line. Precise replacement of this enhancer with an ex- FOXA1 binds this module in the LNCaP pros- DEVELOPMENTAL tended LoxP site in the context of a 218,555-bp BAC reporter nearly tate cancer cell line. These data directly implicate FOXA in extinguished Hoxb13-mediated transcriptional activity in the the regulation of HOXB13 and provide new insights into the mo- mouse prostate. These data demonstrate that FOXA1 directly regu- lecular basis of prostate-restricted gene expression. lates HOXB13 in human prostate epithelial cells, and show that this prostate-specific regulatory mechanism is conserved in mice. Results The B13/LZ218kb transgene, which contains a lacZ reporter gene androgen independent | recombineering | prostate-specific | | under the control of mouse Hoxb13 regulatory elements within a Forkhead 218-kb bacterial artificial chromosome (BAC) (Table 1; Fig. S1), was capable of robustly recapitulating the Hoxb13 expression omeobox genes are major regulators of a diverse array of pattern in the urogenital and gastrointestinal tracts (10). Re- Hdevelopmental processes (1). Within the past decade, a porter gene expression did not induce phenotypic changes. significant body of evidence has accumulated indicating that To localize prostate-specific regulatory elements contained within homeobox genes play important roles during prostate morpho- the 218,555-bp reporter construct, truncations were made in B13/ 218kb genesis (2). Prominent among these are Nkx3.1, Hoxa10, and the LZ by recombineering (19) (Table 1; Fig. S1). Deletion of 96 kb from the 5′ end (with respect to the transcriptional orientation of group 13 paralogs Hoxa13, Hoxb13, and Hoxd13. Null mutations 218kb Δ96kb5′ in these homeobox genes result in defects in branching mor- Hoxb13)ofB13/LZ (designated B13/LZ ) did not affect phogenesis and/or changes in gene expression that suggest al- reporter gene expression in the prostate (Fig. 1A). Whole-mount X- – gal analyses of six independent transgenic lines carrying the B13/ tered identity of certain prostatic lobes (3 7). Δ96kb5′ During embryogenesis, Hoxb13 is robustly expressed in the LZ transgene showed strong, uniform reporter gene ex- posterior axial skeleton as well as the posterior endoderm that pression in the adult prostate that paralleled levels observed for the original B13/LZ218kb transgene (Fig. 1 A–C). The cellular distri- gives rise to the most distal regions of the gastrointestinal tract Δ96kb5′ and the urogentital sinus (8). In adult mice, Hoxb13 expression is bution of reporter gene expression in B13/LZ transgenic pr- restricted to the prostate, descending colon, and rectum, which ostates (Fig. 1G) was indistinguishable from that reported for the B13/LZ218kb transgene (10). In contrast, deletion of ∼45 kb from the are all derived from posterior endoderm (9). Prostatic expression Δ ′ 3′ end of B13/LZ218kb (designated B13/LZ 45kb3 )resultedinasig- is highest in the ventral prostate lobe epithelium, with decreasing fi levels in the lateral, dorsal, and anterior lobe epithelia (9). An ni cant reduction in reporter gene activity (Fig. 1D). This construct retains 14 kb downstream of the Hoxb13 start of translation. Analyses intriguing feature of Hoxb13 expression in the prostate is its Δ ′ performed in four independent lines carrying B13/LZ 45kb3 dem- persistence under androgen deprivation conditions (9). In marked contrast to virtually all other genes with prostate epi- thelial-restricted expression, castration does not diminish Author contributions: R.P.M. and C.J.B. designed research; R.P.M., A.D., L.N.M., A.O., C.S.S., Hoxb13 mRNA accumulation, nor does it reduce expression of a and C.J.B. performed research; R.P.M., A.D., S.M., C.S.S., and C.J.B. analyzed data; and R.P.M. reporter gene driven by Hoxb13 regulatory elements (10). This and C.J.B. wrote the paper. unusual expression pattern makes Hoxb13 an excellent paradigm The authors declare no conflict of interest. to dissect regulatory mechanisms that can confer transcriptional This article is a PNAS Direct Submission. activity in prostate epithelial cells under in vivo androgen de- 1Present address: Division of Metabolism and Health Effects, National Institute on Alcohol pletion conditions. Understanding mechanisms that drive pros- Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892. tate-specific, androgen-independent gene expression is critical to 2To whom correspondence should be addressed. E-mail: [email protected]. support ongoing efforts to develop new strategies to treat lethal This article contains supporting information online at www.pnas.org/cgi/content/full/ androgen-independent prostate cancer. 0902001107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0902001107 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Table 1. Compendium of BAC and transgene data. The table indicates the number of independent transgenic strains surveyed, sequence either upstream or downstream of Hoxb13, and intensity of X-gal staining in prostate or colon decreasing from (+++++)to(+), with (+/−) indicating highly variegated staining and (−) indicating no staining. BAC Designation Transgene Name # of Strains Upstream Downstream Prostate Colon

RP23-335O22Hoxb13-lacZ B13/LZ218kb 6 158,454 60,101 +++++ +++++ RP23-335O22Hoxb13-lacZ{Δ96kb-5′} B13/LZΔ96kb5′ 6 62,205 60,101 +++++ +++++ Δ ′ RP23-335O22Hoxb13lacZ{Δ45kb-3′} B13/LZ 45kb3 4 158,454 15,306 + +++++ Δ ′ RP23-335O22Hoxb13-lacZ{Δ51kb-3′} B13/LZ 51kb3 5 158,454 9,245 − +++++ RP23-335O22Hoxb13-lacZ{R37bpLoxP} B13/LZR37bpLoxP 4 154,454 60,101 +/− +++++

onstrated that reporter gene expression was reduced by greater than lobe, albeit at a reduced level (Fig. 1D), indicating that cis elements 95% in neonatal anterior, dorsal, and lateral lobes, although a that enable Hoxb13 expression in prostate epithelial cells moderate level of expression was retained in the ventral lobes. In lie elsewhere. Δ ′ adult B13/LZ 45kb3 mice, reporter gene expression was no longer In an attempt to locate elements important for supporting detectable above the level of endogenous β-galactosidase activity in prostate-specific Hoxb13 transcriptional activity expression the anterior, dorsal, and lateral lobes. Within the adult ventral lobe, within the ∼15-kb downstream region that was retained in Δ ′ staining did not appear uniform, giving rise to a variegated appear- B13/LZ 45kb3 , the VISTA browser was used to search for ance; however, reporter gene expression remained restricted to ep- noncoding regions conserved between the mouse and human ithelial cells (Fig. 1 D and H). These data demonstrate that enhancer genomes (20). An ∼6-kb region located between 9 and 15 kb elements required to support transcriptional activity of the Hoxb13 downstream of the Hoxb13 coding sequence showed clear locus in the prostate gland reside between 15 and 60 kb downstream evidence of conservation (Fig. 2A). Δ ′ of the coding region. However, the B13/LZ 45kb3 transgene retained To determine whether the ∼6-kb region contained elements the ability to drive detectable reporter gene expression in the ventral required for prostate expression of the reporter gene, the region

Fig. 1. Reporter gene expression in B13/LZ transgenic mice. (A) Partial urogenital tract from a nontransgenic negative control FVB male (left) and a B13/ LZΔ96kb5′ transgenic male (right) after X-gal staining. a, anterior prostate lobe; b, bladder; d, dorsal prostate lobe; l, lateral prostate lobe; u, urethra; v, ventral Δ ′ prostate lobe. (B) B13/LZ218kb transgenic ventral lobe (left) and nontransgenic FVB negative control (right). (C) B13/LZ 96kb5 transgenic ventral lobes. (D) B13/ Δ ′ ′ Δ ′ LZ 45kb3 ventral lobes. (E) B13/LZ51kb3 ventral lobes. (F) B13/LZR37bpLoxP ventral prostate lobes. (G) Nuclear fast red (NFR)-stained section of a B13/LZ 96kb5 Δ ′ Δ ′ ventral lobe duct after whole-mount X-gal staining. (H) NFR-stained section of a ventral lobe duct from a B13/LZ 96kb5 / 45kb3 male. The 3′ deletion in this Δ ′ Δ ′ construct is the same as that in B13/LZ 45kb3 , and staining is indistinguishable from that in B13/LZ 45kb3 mice. (I) NFR-stained section of a ventral lobe duct from a B13/LZR37bpLoxP male. (J) B13/LZΔ96kb5′ colon segment. (K) B13/LZR37bpLoxP colon segment. (L) Immunohistochemical detection of Hoxb13 in the ventral prostate lobe. (M) Immunohistochemical detection of Foxa1 in the ventral prostate lobe.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.0902001107 McMullin et al. Downloaded by guest on September 30, 2021 between +9,245 bp and +15,485 bp downstream of Hoxb13, bioinformatic analyses were used to search this region for conserved composite model matches (21). The FrameWorker module within the GEMS Launcher software (http://www. genomatix.de) (22) was employed to define common models of matching composite sites that are conserved in sequence, order, and spacing between the mouse 6,420-bp region of interest and the corresponding region of the . FrameWorker defined a composite model within 37 bp (≈3 DNA helical turns) (23). The central element of this model consists of two Forkhead domain factor family (V$FKHD) FOXA class matrices (24, 25). The V$FKHD modules are in opposite orientations and separated by 12 bp, such that a complete DNA helical turn could position bound FOXA transcription factors to the same DNA helical surface. Fur- thermore, the position of an adjacent TALE (V$TALE) matrix match was also conserved within the 37-bp enhancer of mouse and human sequences (26–28). Using the core DNA sequences of the mouse 37-bp enhancer defined by the composite model match, we searched the Hoxb13 loci (−15,000 to +30,000 bp) of rhesus monkey (Macaca mu- latta), rat (Rattus norvegicus), dog (Canis lupus familiaris), and zebrafish (Danio rerio) vertebrate species by using the NCBI Blast Blt2seq program. The search identified DNA sequences highly similar to the mouse and human 37-bp enhancer in the rhesus monkey and rat genomes (Fig. 2B). In contrast, the search did not indicate similarity within the zebrafish Hoxb13A locus. BIOLOGY

FrameWorker analysis of the identified homologous sequences DEVELOPMENTAL in human, mouse, rhesus monkey, rat, and dog confirmed the conservation of FKHD and TALE matrices. The presence of two Foxa binding sites in a region demonstrated to regulate urogenital sinus and prostate expression, coupled with similar reported expression patterns between Foxa1 and Hoxb13 in the urogenital sinus and prostate (29, 30), suggests that Foxa1 may directly regulate Hoxb13 expression. To determine whether FOXA1 was recruited to the 37-bp Fig. 2. Analyses of the 37-bp conserved region. (A) VISTA Browser 2.0 plot of sequence conservation between the ∼9 kb and ∼15 kb region downstream of conserved sequence in vivo, we employed the immu- Hoxb13 aligned to the human genome. The calculation window is set at 50 bp, noprecipitation (ChIP) technique. The human prostate cancer with the threshold set at 70% conservation identity over a 50-bp minimum cell line LNCaP was chosen, as it has high levels of FOXA1 conservation width and is indicated by shaded regions. y axis minimum and protein (31) and also expresses HOXB13 (32). An anti-FOXA1 maximum are set at 50% and 100% conservation identity, respectively. The 37- goat polyclonal antibody enriched the immunoprecipitated re- bp conserved region is indicated by the black triangle in the shaded region. (B) gion of interest 115-fold over a nonspecific IgG im- The FKHD_TALE composite model defines the conserved 37-bp Hoxb13 en- − + munoprecipitation control when detected by quantitative PCR. hancer. The Hoxb13 gene 15,000 and 30,000 region of rhesus monkey (3), rat In a comparison that demonstrates antibody specificity and ef- (4), and dog (5) species was searched for homology to the mouse (1) and human fi (2) 37-bp sequences. The model was defined by taking into account sequence ciency of chromatin shearing, a region 2 kb distal that is not variations (bold characters) between the analyzed species. Vertebrate (V$) predicted to be bound by FOXA1 was only enriched 1.8-fold FKHD and TALE matrices of the FKHD_TALE model are marked by boxes. (C) over the IgG control (Fig. 2C). These data demonstrate that Graph of the fold enrichment for human α-FOXA1 goat polyclonal antibody FOXA1 is bound to the 37-bp HOXB13 enhancer in human immunoprecipitated genomic regions over the goat IgG immunoprecipitated prostate epithelial cancer cells. control. The quantitative PCR amplicons were a region containing the con- To compare the cellular distribution of Foxa1 and Hoxb13 in served 37-bp sequence and a region 2 kb distal to the conserved sequence. the mouse prostate gland, immunohistochemical analyses were performed on adjacent sections of adult mouse prostates. Both downstream of Hoxb13 in B13/LZ218kb was reduced from 60,101 proteins were detected in a nearly uniform pattern in luminal Δ51kb3′ L M bp to 9,245 bp. This transgene was designated B13/LZ . epithelial cell nuclei (Fig. 1 and ); however, Hoxb13 was also Δ ′ detected in a subset of basal cells in a manner reminiscent of the Whole-mount analyses of adult B13/LZ 51kb3 prostates revealed distribution of Nkx3.1 (16, 33). the complete absence of X-gal staining above background in all To evaluate the role of the predicted 37-bp enhancer in vivo, the lobes (Fig. 1E, ventral lobe shown). Remarkably, lacZ expression galK positive/negative selection-based BAC recombineering meth- in the colon remained robust and uniform at levels comparable to 218kb od (34) was employed to precisely replace the 37-bp enhancer that of B13/LZ colon (Fig. 1 J and K) (10). These data indicate contained in B13/LZ218kb with a 34-bp LoxP site and three addi- + + that cis-regulatory elements in the region between 9,245 bp and tional base pairs. The LoxP sequence was chosen because the 15,485 bp are required for Hoxb13 transcriptional activity in the commonly used Cre recombination technique leaves a LoxP scar ventral lobe. Furthermore, these data demonstrate that the cis- sequence in the genome which has not been reported to have effects regulatory elements for prostatic expression are completely sep- on gene expression. The resulting recombinant BAC, RP23- arable from the elements for colorectal expression. 335O22Hoxb13-lacZ{R37bpLoxP}, was sequenced across the recombi- Having identified a 6,420-bp sequence containing cis-reg- nation junctions to confirm correct replacement and subjected to ulatory elements required for ventral lobe expression located restriction fragment length polymorphism analysis to verify BAC

McMullin et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 integrity. This transgene construct (B13/LZR37bpLoxP)wasthenused temporal and spatial patterns of transcriptional activity is partic- to generate transgenic mice for reporter gene analysis. ularly challenging, and requires labor-intensive analyses in whole Whole-mount microscopic analyses of X-gal-stained tissue from transgenic animals. The development of efficient methods to B13/LZR37bpLoxP transgenic mice revealed that replacement of the modify large DNA molecules with nucleotide precision combined 37-bp sequence within the context of the full-length 218,555-bp with the availability of powerful informatic tools for genome sequence resulted in a dramatic reduction of β-gal activity in the analysis provides new opportunities to facilitate this arduous prostate (Fig. 1F) without altering cellular distribution (Fig. 1I), process. By applying these approaches to a canonical deletion whereas strong staining in the colon was observed at comparable analysis, we have been able to scan a large region of the Hoxb13 Δ ′ levels to those in mice carrying B13/LZ 96kb5 (Fig. 1K). The 37-bp locus to functionally identify a 37-bp module that is necessary for enhancer replacement transgene phenocopied the loss of reporter prostate-restricted transcriptional activity. Δ ′ gene activity observed in all lobes in B13/LZ 45kb3 transgenic Deletion analyses localized elements required to support mice, demonstrating that multiple downstream enhancers are re- robust transcriptional activity of Hoxb13 in the prostate within quired for robust Hoxb13 prostate-specific transcriptional activity. the region spanning +15 to +61 kb. Extending the deletion to However, the 37-bp enhancer replacement alone also nearly encompass the region spanning +9to+61 kb completely abol- eliminated reporter gene expression in the ventral lobe, indicating ished transcriptional activity exclusively in the prostate, demon- that this element plays a key role in mediating Hoxb13 transcrip- strating that one or more key elements lie within a 6-kb sequence tional activity in the adult prostate. downstream of Hoxb13. Using comparative genomics of this ∼6- Both Foxa1 and Hoxb13 are expressed throughout the entire kb region between mice and humans, a conserved 37-bp region urogenital sinus epithelium before prostate development (29). Be- was recognized composed of two inversely orientated FKHD cause two predicted Foxa binding sites are present in the 37-bp elements with preferential cognate sites for Foxa transcription enhancer, reporter gene analyses of the B13/LZR37bpLoxP transgene factors. To enhance the specificity of this model, we examined were performed to determine the importance of the enhancer in the the evolutionary conservation of this 37-bp sequence by search- newly partitioned urogenital sinus of 12.5 days post coitum (dpc) ing the corresponding Hoxb13-containing regions of the rhesus fi embryos. Robust lacZ expression is found throughout the urogenital monkey, rat, dog, and zebra sh genomes for . sinus in B13/LZ218kb transgenic embryos (Fig. 3A). A profound Conserved sequences were found in all three mammalian ge- fi decrease in lacZ expression in the urogenital sinus was observed in nomes, but not in zebra sh (35). These data are consistent with B13/LZR37bpLoxP 12.5 dpc embryos; however, reporter gene expr- the fact that the prostate gland is a mammalian innovation, and ession in the hindgut remained strong (Fig. 3B). In contrast to the raise the possibility that this regulatory circuit evolved in concert uniform reporter gene activity observed in the urogenital sinus of with the prostate gland. B13/LZ218kb transgenic embryos, expression in B13/LZR37bpLoxP The location of the 37-bp enhancer 11.7 kb downstream of Hoxb13 and the dramatically reduced reporter gene expression embryos was limited to scattered cells within the central third of the R37bpLoxP urogenital sinus. These data indicate that replacement of the 37-bp in B13/LZ transgenic prostates are consistent with module interferes with maintenance of reporter gene activity in the predicted mechanisms of Foxa1-regulated gene activation. The emerging urogenital sinus after septation of the cloaca. transcription factor FOXA1 contributes to gene regulation by its We next sought to determine whether the 37-bp enhancer was ability to act as a that binds to nucleosomal DNA fi (36–41). In a recent report, it was demonstrated that FOXA1 suf cient to direct gene expression to the mouse prostatic epi- fi thelium. To this end, an 843-bp region of the Hoxb13 locus en- cell-type-speci c functions rely primarily on differential recruit- compassing ∼500 bp upstream and 300 bp downstream of the ment to chromatin predominantly at distant enhancers rather enhancer was cloned upstream of the mouse hsp68 promoter than at proximal promoters in the LNCaP and MCF-7 cell lines (42). This differential recruitment leads to cell-type-specific linked to a lacZ reporter gene. Analysis using FrameWorker changes in chromatin structure and functional collaboration with confirmed that the FKHD module was the only conserved ele- lineage-specific transcription factors. The 37-bp enhancer may ment within this region. Four independent founder strains car- act to confer a chromatin structure to the region that is per- rying this transgene were derived. Reporter gene analyses of missive for the binding of transcription factors that confer male urogenital tissues did not detect β-gal activity in the pros- prostate-specific Hoxb13 expression by acting in concert with tate gland; however, one strain showed transgene expression in a other elements in the region between +15 and +61 kb down- few muscle fibers in the bladder, demonstrating that the con- stream. This interpretation is consistent with the fact that the 37- C struct was functional (Fig. S1 ). bp enhancer alone was not sufficient to drive expression of a Discussion heterologous promoter in the prostate. Loss of these 37 bp may result in a generally restrictive chromatin structure over a - Identifying genomic cis-regulatory regions that mediate tissue-re- tively broad region, but with some limited potential for coordi- stricted gene expression functionally underpins efforts to under- nating transcriptional activity to confer the low level of variable stand the molecular basis of animal development and homeostasis. X-gal staining observed. In addition, these elements enable the development of tools to FOXA1 is an important regulator of prostate-specific expres- manipulate gene expression in model systems and, potentially, in a sion. It has been demonstrated to interact with the androgen therapeutic setting in human disease patients. Discerning the lo- on the promoters of PSA and Probasin and was essential cation and function of distant elements that modulate complex for gene expression (14). Interestingly, in the absence of an- drogen stimulation, FOXA1 was still capable of binding the promoter regions but alone was unable to transactivate (14). FOXA1 may interact with a cofactor other than the on the 37-bp HOXB13 regulatory element to activate expression independent of androgen stimulation. Interestingly, there are several parallels between Foxa1 and Hoxb13 expression and the consequences of their deletion in the mouse. Both Foxa1 and Hoxb13 are detected throughout the Fig. 3. Reporter gene expression in the urogenital sinus and hindgut of 12.5 urogenital sinus epithelium at the beginning of prostate devel- dpc transgenic embryos. (A) B13/LZ218kb urogenital sinus (us) and hindgut opment by immunohistochemistry (10, 29). Both are expressed (hg). (B) B13/LZR37bpLoxP urogenital sinus and hindgut. strongly in the prostate buds, and their expression continues

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.0902001107 McMullin et al. Downloaded by guest on September 30, 2021 through development and into adulthood (29). Foxa1 and RP23-335O22Hoxb13lacZ{Δ45kb-3′}, 5′-tggccccgggtgcctttgccctttgattacattccgcg- Hoxb13 are expressed highest in the ventral lobes, at a lower ttgaggaggaactctagacgtcaggtggcac-3′ and 5′-acatttaacaggtataacaggagggtc- aggagttaaaggggttttggcagctcacgttaagggattttggtc-3′; RP23-335O22Hoxb13-lacZ- level in the dorsal and lateral lobes, and weakest in the anterior Δ ′ ′ lobes (9, 10, 29, 43). During prostate carcinogenesis in the hu- { 51kb-3 }, 5 -tggccccgggtgcctttgccctttgattacattccgcgttgaggaggaactctagacg- tcaggtggcac-3′ and 5′-cggggaacccgggcgaagtgcgtctttcccagagatctgctgagagcc- man and mouse, studies suggest that expression of both genes is gactcacgttaagggattttggtc-3′. maintained (31, 44, 45). In the homozygous knockout of either RP23-335O22Hoxb13-lacZ{R37bpLoxP} used the protocol as described (34), Hoxb13 or Foxa1, defects are observed in luminal cell differ- with the addition of 50 mg/mL kanamycin to the selection plates. Primers entiation and secretory function, with a loss of some of the same used for amplification of galK were 5′-tccctttgcctcccctcccCCTGTTGACAATT- major secretory products (6, 30). AATCATCGGCA-3′ and 5′-gctcaccccttgctagggTCAGCACTGTCCTGCTCCTT-3′; Despite the similarities in Foxa1 and Hoxb13 expression and primers used for the replacement of the LoxP sequence were 5′-tccctt- the in vivo evidence of FOXA1 binding to the 37-bp enhancer, it tgcctcccctccctctccctctcctttcccgtctctatcataacttcgtataat-3′ and 5′-gctcaccccttgc- ′ is possible that FOXA2 may also have the ability to bind to the tagggtgttatctgtgctttgtagagaccgataacttcgtatagcat-3 . enhancer and regulate HOXB13 expression. Their binding sites fi Transgenic Mouse Generation and Genotyping. BACs were linearized with PI- are highly conserved, and several prostate-speci c promoters Sce I (New England Biolabs), and then purified by sucrose gradient cen- have been demonstrated to recruit either factor (46). Both Foxa1 trifugation and dialyzed as described (50). The fragments were injected into and Foxa2 are expressed during the initial budding of the pros- FVB single-cell embryos, and transgenic mice were generated as described tate (29) and may have redundant activities early in prostate (51). Founders or transgenic offspring were identified by Southern blot development. In human and mouse prostate cancer, FOXA1 analyses. Genomic DNA was isolated using the Gentra Puregene Mouse Tail expression persists in early- and late-stage cancer, whereas kit (Qiagen). Ten micrograms of extracted DNA was digested with EcoRI and FOXA2 is only detected in neuroendocrine small-cell carcino- EcoRV, electrophoresed on a 0.8% agarose gel, and then transferred to a mas and in some high Gleason score adenocarcinomas (31). Nytran-N membrane (Whatman). The vector pLZKAN-Hoxb13 (10) was lin- earized with SacII, and then the SP6 universal primer was used to create a Interestingly, both factors have been demonstrated to interact fi probe for Southern blot with the Ambion Strip-EZ PCR kit (Applied Bio- with androgen receptor and, on prostate-speci c promoters, systems). Blot hybridization was performed using Ambion ULTRAhyb sol- FOXA2 was demonstrated to activate gene expression in an ution (Applied Biosystems), was washed according to the manufacturer’s androgen-independent manner (46). instructions, and exposed to X-ray film. FOXA1 regulation of HOXB13 may also have important im- All animal procedures were approved by the University of Maryland, plications in . FOXA1 has been demonstrated to be Baltimore County Institutional Animal Care and Use Committee. BIOLOGY

an important cofactor of the in regulating gene DEVELOPMENTAL networks in the breast (36). HOXB13 expression is repressed by Detection of β-Gal Activity. Embryos or tissues were dissected and kept in cold fi – estrogen signaling in human breast cancer cell lines and also in PBS until xed with 4% paraformaldehyde for 30 60 min. The staining was performed as described (50) until the desired intensity was achieved as the neonatal estrogenized rat prostate (47, 48). However, compared to a nontransgenic negative control. HOXB13 is expressed in a subset of estrogen-receptor-positive breast cancer patients who are unresponsive to tamoxifen mon- Chromatin Immunoprecipitation Assay. This assay was adapted from Carroll otherapy (49). When estrogenic signaling is incapable of re- et al. (36), with the following modifications: LNCaP cells were grown to 80% pressing HOXB13 expression during breast carcinogenesis, confluence, harvested, fixed with 37% formaldehyde (Sigma), and lysed. Cell FOXA1 may invoke a mechanism similar to that used in the lysates were sonicated with 8 pulses for 30 s each at power level 4 on a prostate to specifically activate HOXB13 expression. As de- Branson Sonifier, model 450. Chromatin was evaluated for shearing effi- – regulated expression of HOXB13 in breast cancer is associated ciency on a 2.0% agarose gel, with optimal shearing consisting of a 200 with tumor aggressiveness, tamoxifen resistance, and poor 1000 bp smear. Immunoprecipitation, reverse cross-linking, and DNA puri- fication were performed as described (36), using an α-FOXA1 goat polyclonal prognosis, the mechanisms regulating HOXB13 expression in antibody (Abcam, ab5089) and IgG (Jackson Immunoresearch, 005-000-003). breast cancer warrant investigation (49). The in vivo analyses of Hoxb13 regulatory elements reported Quantitative PCR. Quantitative PCR was performed using Sybr Green PCR here identify a 37-bp element distant from the coding region that Master Mix (Applied Biosystems) on a Bio-Rad iCycler IQ Real-time PCR is necessary but not sufficient to support robust transcriptional machine. Primers were designed using Primer3 software (http://primer3. activity of Hoxb13 in the prostate. Additionally, we have dem- sourceforge.net) as follows: 37-bp enhancer, 5′-CCCACCCATCATCAC- onstrated that FOXA1, a protein known to act as a pioneer TAACTG-3′ and 5′-GGATTGATTCACCCCTTCCT-3′; distal region, 5′-CCCAGG- ′ ′ ′ factor in regulating gene expression, binds to a similar conserved GACTCCTCTATTGTC-3 and 5 -GTGATCTCAGCTGGGTGACTC-3 . The presence of a single amplicon was systemically verified by dissociation curve analysis. region of the human genome, and is likely to enable transcription fi fi PCR ampli cations for a region containing the 37-bp enhancer and a region factors that confer prostate-speci c expression of HOXB13. 2 kb distal were conducted in quadruplicate and the calculated thresholds

These factors may interact with FOXA1 directly to form a (CT) were averaged (Avg CT), removing outliers with >1 cycle difference. The unique complex that activates HOXB13 expression in the ab- nonspecific background, as detected by a negative control goat IgG im- IgG sence of androgens, or may gain access to nearby cis-regulatory munoprecipitation, was used to derive (Avg CT ) for both regions. The elements through FOXA1-mediated changes in chromatin change in cycle threshold for each region was normalized using the formula, = α-FOXA1 − IgG structure. Elucidation of additional factors involved in HOXB13 dCT (Avg CT Avg CT ). The fold enrichment for both regions was = expression and their mechanisms of action will further our un- calculated using the formula, fold enrichment 2^ dCT. derstanding of prostate-specific gene regulation, and may have Computational Analysis of Regulatory Elements. The experimentally confirmed implications for understanding the emergence of androgen-in- +9,245 bp to +15,485 bp DNA sequence of mouse Hoxb13 (chromosome 11, dependent prostate cancer. absolute positions: 96064905–96071145, NCBI build 36) was queried in the GenomeVISTA program to plot DNA conservation identity to the aligned Materials and Methods human HOXB13 downstream sequences under conditions indicated in Fig. 2A Construction of Transgenes. Construction of the B13/LZ218kb transgene (for- (20). The experimentally confirmed +9,245 bp to +15,485 bp DNA sequence of merly designated RP23-335O22Hoxb13-lacZ) has been described (10). Trunca- mouse Hoxb13 (chromosome 11, absolute positions: 96064905–96071145, tions of B13/LZR37bpLoxP were generated using the standard DY380 NCBI build 36) and the human HOXB13 downstream (, abso- recombineering protocol (19). The ampicillin-targeting cassettes to create lute positions 44144888–44148592, NCBI build 37) homologous sequences truncations were generated by standard PCR amplifications from pTAMP (19), were searched for conserved composite models by FrameWorker 4.4.2 (2005) using the primers as follows: RP23-335O22Hoxb13-lacZ{Δ96-5′}, 5′-aatgggattca- software with 1–10 bp distance between adjacent elements and allowing as ccggcatgtccaaagaagttgagacagaaagtaaaattatcttagacgtcaggtggcac-3′ and 5′- many as five elements in the composite models. MatInspector Matrix Family acatcattttaaccatcttggcaccttctttctgcccacataatatatcactcacgttaagggattttggtc-3′; Library Version 5.0 (February 2005) was used for the matrix family search. All

McMullin et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 vertebrate matrix families were queried with 0.75 core and “optimized” ma- mulatta (rhesus monkey), Rattus norvegicus (rat), Canis lupus familiaris (dog), trix similarity settings applying the “Urogenital Systems” cocitation filter and Danio rerio (zebrafish) species for homology by using the NCBI Blast (http://www.genomatix.de). To meet the DNA sequence length requirements (Blt2seq) program. Homologous sequences of human, rhesus monkey, dog, of the software (<3000 bp), the actual input sequence lengths were kept under mouse, and rat 37-bp region with 15-bp flanks were further analyzed by 3000 bp, assuring overlaps for exhaustive search. Only one composite model FrameWorker (5.4.3.3) for model definition using the Matrix Family Library reached the maximum (five-element) complexity that was composed of ver- 7.1, core/matrix similarity 0.75/optimized setting allowing 10-bp variance in + + − tebrate matrix TALE (strand , matrix sim. 1.00) and FKHD (strands / , matrix distance between adjacent matrices. FrameWorker confirmed a single – − sim. 0.91 0.98), and two minor matrices (V$) MYT1 (strand , matrix sim. 0.93) FKHD_TALE model [FKHD(−), matrix sim. 0.99, TALE(+), matrix sim. 1.00]. and ETS (strand −, matrix sim. 0.85), respectively, reaching the maximum total FrameWork score of 1.00/1.00 (http://www.genomatix.de). However, a high ACKNOWLEDGMENTS. This work was supported by grant DK59152 from the degree of sequence conservation and the low number of input sequences may National Institutes of Health, and grant DAMD17-98-1-8477 from the result in model overfitting, preventing the recognition of composite model Congressionally Directed Medical Research Program. The authors gratefully matches in divergent species. Therefore, by using the 37-bp core sequences acknowledge Carlise R. Bethel for technical assistance with Foxa1 immuno- and 15-bp flanks of the mouse and human sequences, the Hoxb13 gene up- histochemistry, Albert Zhou for assistance analyzing R37pb-hsp68-lacZ trans- stream −15,000 and downstream +30,000 sequences were searched in Macaca genic mice, and Sandra Tickle for animal care.

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