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1
Kitamura, K., H. Miura, S. Miyagawa-Tomita, M. Yanazawa, Y. Katoh-Fukui, R. Suzuki, H. Ohuchi, et al. "Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism." Development 126, no. 24 (December 15, 1999): 5749–58. http://dx.doi.org/10.1242/dev.126.24.5749.
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Pitx2, a bicoid-related homeobox gene, is involved in Rieger's syndrome and the left-right (L-R) asymmetrical pattern formation in body plan. In order to define the genomic structure and roles of Pitx2, we analyzed the genomic structure and generated Pitx2-deficient mice with the lacZ gene in the homeobox-containing exon of Pitx2. We were able to show that among three isoforms of Pitx2, Pitx2c shows asymmetrical expression whereas Pitx2a, Pitx2b and Pitx2c show symmetrical expression. In Pitx2(−)(/)(−) embryos there was an increase in mesodermal cells in the distal end of the left lateral body wall and an amnion continuous with the lateral body wall thickened in its mesodermal layer. These changes resulted in a failure of ventral body wall closure. In lung and heart in which Pitx2 is expressed asymmetrically, right pulmonary isomerism, atrioventricular canals with prominent swelling, and juxtaposition of the atrium were detected. The hearts failed to develop tricuspid and mitral valves and a common atrioventricular valve forms. Further, dysgenesis of the Pitx2(−)(/)(−) extraocular muscle and thickening of the mesothelial layer of cornea were observed in the ocular system where Pitx2 is expressed symmetrically, and these resulted in enophthalmos. The present study shows that Pitx2 expressed in various sites participates in morphogenesis through three types of actions: the involvement of asymmetric Pitx2 expression in the entire morphogenetic process of L-R asymmetric organs; the involvement of asymmetric Pitx2 expression in the regional morphogenesis of asymmetric organs; and finally the involvement of symmetric Pitx2 expression in the regional morphogenesis of symmetric organs.
2
Yu, X., T. R. St Amand, S. Wang, G. Li, Y. Zhang, Y. P. Hu, L. Nguyen, M. S. Qiu, and Y. P. Chen. "Differential expression and functional analysis of Pitx2 isoforms in regulation of heart looping in the chick." Development 128, no. 6 (March 15, 2001): 1005–13. http://dx.doi.org/10.1242/dev.128.6.1005.
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Pitx2, a bicoid-related homeobox gene, plays a crucial role in the left-right axis determination and dextral looping of the vertebrate developing heart. We have examined the differential expression and function of two Pitx2 isoforms (Pitx2a and Pitx2c) that differ in the region 5′ to the homeodomain, in early chick embryogenesis. Northern blot and RT-PCR analyses indicated the existence of Pitx2a and Pitx2c but not Pitx2b in the developing chick embryos. In situ hybridization demonstrated a restricted expression of Pitx2c in the left lateral plate mesoderm (LPM), left half of heart tube and head mesoderm, but its absence in the extra-embryonic tissues where vasculogenesis occurs. RT-PCR experiments revealed that Pitx2a is absent in the left LPM, but is present in the head and extra-embryonic mesoderm. However, ectopic expression of either Pitx2c or Pitx2a via retroviral infection to the right LMP equally randomized heart looping direction. Mapping of the transcriptional activation function to the C terminus that is identical in both isoforms explained the similar results obtained by the gain-of-function approach. In contrast, elimination of Pitx2c expression from the left LMP by antisense oligonucleotide resulted in a randomization of heart looping, while treatment of embryos with antisense oligonucleotide specific to Pitx2a failed to generate similar effect. We further constructed RCAS retroviral vectors expressing dominant negative Pitx2 isoforms in which the C-terminal transcriptional activation domain was replaced by the repressor domain of the Drosophila Engrailed protein (En(r)). Ectopic expression of Pitx2c-En(r), but not Pitx2a-En(r), to the left LPM randomized the heart looping. The results thus demonstrate that Pitx2c plays a crucial role in the left-right axis determination and rightward heart looping during chick embryogenesis.
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Lamba, Pankaj, Vishal Khivansara, Ana C. D'Alessio, Michelle M. Santos, and Daniel J. Bernard. "Paired-Like Homeodomain Transcription Factors 1 and 2 Regulate Follicle-Stimulating Hormone β-Subunit Transcription through a Conserved cis-Element." Endocrinology 149, no. 6 (March 13, 2008): 3095–108. http://dx.doi.org/10.1210/en.2007-0425.
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Paired-like homeodomain transcription factors (PITX) regulate the activity of pituitary hormone-encoding genes. Here, we examined mechanisms through which the family of PITX proteins control murine FSH β-subunit (Fshb) transcription. We observed that endogenous PITX1 and PITX2 isoforms from murine LβT2 gonadotrope cells could bind a highly conserved proximal cis-element. Transfection of PITX1 or PITX2C in heterologous cells stimulated both murine and human Fshb/FSHB promoter-reporter activities, and in both cases, mutation of the critical cis-element abrogated these effects. In homologous LβT2 cells, the same mutation decreased basal reporter activity and greatly reduced activin A-stimulated transcription from murine and human promoter-reporters. Transfecting dominant-negative forms of PITX1 or PITX2C or knocking down PITX1 or -2 expression by RNA interference in LβT2 cells inhibited murine Fshb transcription, confirming roles for endogenous PITX proteins. Both PITX1 and PITX2C interacted with Smad3 (an effector of the activin signaling cascade in these cells) in coprecipitation experiments, and the PITX binding site mutation greatly inhibited Smad2/3/4-stimulated Fshb transcription. In summary, both PITX1 and PITX2C regulate murine and human Fshb/FSHB transcription through a conserved cis-element in the proximal promoter. Furthermore, the data indicate both common and distinct mechanisms of PITX1 and PITX2C action.
4
Essner, J. J., W. W. Branford, J. Zhang, and H. J. Yost. "Mesendoderm and left-right brain, heart and gut development are differentially regulated by pitx2 isoforms." Development 127, no. 5 (March 1, 2000): 1081–93. http://dx.doi.org/10.1242/dev.127.5.1081.
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The pitx2 gene is a member of the bicoid-homeodomain class of transcription factors that has been implicated in the control of left-right asymmetry during organogenesis. Here we demonstrate that in zebrafish there are two pitx2 isoforms, pitx2a and pitx2c, which show distinct expression patterns and have non-overlapping functions during mesendoderm and asymmetric organ development. pitx2c is expressed symmetrically in presumptive mesendoderm during late blastula stages and in the prechordal plate during late gastrulation. pitx2a expression is first detected at bud stage in the anterior prechordal plate. The regulation of early mesendoderm pitx2c expression is dependent on one-eyed pinhead (EGF-CFC-related gene) and spadetail (tbx-transcription factor) and can be induced by ectopic goosecoid expression. Maintenance of pitx2c midline expression is dependent on cyclops (nodal) and schmalspur, but not no tail (brachyury). Ectopic expression of pitx2 isoforms results in distinct morphological and molecular phenotypes, indicating that pitx2a and pitx2c have divergent regulatory functions. Both isoforms downregulate goosecoid on the dorsal side, but in contrast to earlier reports that nodal and lefty are upstream of pitx2, ectopic pitx2c in other regions induces cyclops, lefty2 and goosecoid expression. Asymmetric isoform expression occurs in non-overlapping domains, with pitx2c in left dorsal diencephalon and developing gut and pitx2a in left heart primordium. Targeted asymmetric expression in Xenopus shows that both isoforms can alter left-right development, but pitx2a has a slightly stronger effect on heart laterality. Our results indicate that distinct genetic pathways regulate pitx2a and pitx2c isoform expression, and each isoform regulates different downstream pathways during mesendoderm and asymmetric organ development.
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Liu, Chengyu, Wei Liu, Mei-Fang Lu, Nigel A. Brown, and James F. Martin. "Regulation of left-right asymmetry by thresholds of Pitx2c activity." Development 128, no. 11 (June 1, 2001): 2039–48. http://dx.doi.org/10.1242/dev.128.11.2039.
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Although much progress has been made in understanding the molecular mechanisms regulating left-right asymmetry, the final events of asymmetric organ morphogenesis remain poorly understood. The phenotypes of human heterotaxia syndromes, in which organ morphogenesis is uncoupled, have suggested that the early and late events of left-right asymmetry are separable. The Pitx2 homeobox gene plays an important role in the final stages of asymmetry. We have used two new Pitx2 alleles that encode progressively higher levels of Pitx2c in the absence of Pitx2a and Pitx2b, to show that different organs have distinct requirements for Pitx2c dosage. The cardiac atria required low Pitx2c levels, while the duodenum and lungs used higher Pitx2c doses for normal development. As Pitx2c levels were elevated, the duodenum progressed from arrested rotation to randomization, reversal and finally normal morphogenesis. In addition, abnormal duodenal morphogenesis was correlated with bilateral expression of Pitx2c. These data reveal an organ-intrinsic mechanism, dependent upon dosage of Pitx2c, that governs asymmetric organ morphogenesis. They also provide insight into the molecular events that lead to the discordant organ morphogenesis of heterotaxia.
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Toro, Rafael, Irfan Saadi, Adisa Kuburas, Mona Nemer, and Andrew F. Russo. "Cell-specific Activation of the Atrial Natriuretic Factor Promoter by PITX2 and MEF2A." Journal of Biological Chemistry 279, no. 50 (October 1, 2004): 52087–94. http://dx.doi.org/10.1074/jbc.m404802200.
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The PITX2 homeodomain protein is mutated in patients with Axenfeld-Rieger syndrome and is involved in the development of multiple organ systems, including the heart. We have examined the interaction of PITX2 isoforms with myocyte-enhancing factor 2A (MEF2A), which is a known regulator of cardiac development. A direct interaction between PITX2a and MEF2A was demonstrated using yeast two-hybrid and GST pull-down assays. To study the functional significance of this interaction, we used the atrial natriuretic factor (ANF) promoter. Coexpression of MEF2A and PITX2a or Pitx2c resulted in a strong synergistic activation of the ANF promoter in LS8 oral epithelial cells but not in other cell lines (NIH/3T3, Chinese hamster ovary, or C2C12). The synergism was dependent on promoter context, because it required MEF2 binding sites and was not seen with two other PITX2 target promoters. DNA binding by MEF2A was required but not sufficient for synergism. Upstream activators of p38 MAP kinases, MKK3 and MKK6, increased PITX2a and Pitx2c activity to yield up to 90-fold activation of the ANF promoter in LS8 cells. Because Axenfeld-Rieger syndrome is autosomal dominant and affects development of the oral epithelium, we tested one of the known PITX2 mutants. The PITX2a-K88E mutant protein suppressed wild type PITX2a synergism with MEF2A. These results demonstrate a promoter- and cell-specific functional interaction between PITX2 and MEF2A and suggest the possibility of coordinate control by these factors in the oral epithelium.
7
Suh, Hoonkyo, Philip J. Gage, Jacques Drouin, and Sally A. Camper. "Pitx2is required at multiple stages of pituitary organogenesis: pituitary primordium formation and cell specification." Development 129, no. 2 (January 15, 2002): 329–37. http://dx.doi.org/10.1242/dev.129.2.329.
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Analysis of an allelic series in mice revealed that the Pitx2 homeobox gene is required at multiple stages of pituitary development. It is necessary for initiating expansion of Rathke’s pouch and maintaining expression of the fetal-specific transcription factors Hesx1 and Prop1. At later stages Pitx2 is necessary for specification and expansion of the gonadotropes and Pit1 lineage within the ventral and caudomedial anterior pituitary. Mechanistically, this is due to the dependence of several critical lineage-specific transcription factors, Pit1, Gata2, Egr1 and Sf1, on a threshold level of PITX2. The related Pitx1 gene has a role in hormone gene transcription, and it is important late in ontogeny for the final expansion of the differentiated cell types. Pitx1 and Pitx2 have overlapping functions in the expansion of Rathke’s pouch, revealing the sensitivity of pituitary organogenesis to the dosage of the PITX family. The model developed for PITX gene function in pituitary development provides a better understanding of the etiology of Rieger syndrome and may extend to other PITX-sensitive developmental processes.
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Schweickert, Axel, Marina Campione, Herbert Steinbeisser, and Martin Blum. "Pitx2 isoforms: involvement of Pitx2c but not Pitx2a or Pitx2b in vertebrate left–right asymmetry." Mechanisms of Development 90, no. 1 (January 2000): 41–51. http://dx.doi.org/10.1016/s0925-4773(99)00227-0.
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Zhang, Min, Matthew C. Hill, Zachary A. Kadow, Ji Ho Suh, Nathan R. Tucker, Amelia W. Hall, Tien T. Tran, et al. "Long-range Pitx2c enhancer–promoter interactions prevent predisposition to atrial fibrillation." Proceedings of the National Academy of Sciences 116, no. 45 (October 21, 2019): 22692–98. http://dx.doi.org/10.1073/pnas.1907418116.
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Genome-wide association studies found that increased risk for atrial fibrillation (AF), the most common human heart arrhythmia, is associated with noncoding sequence variants located in proximity to PITX2. Cardiomyocyte-specific epigenomic and comparative genomics uncovered 2 AF-associated enhancers neighboring PITX2 with varying conservation in mice. Chromosome conformation capture experiments in mice revealed that the Pitx2c promoter directly contacted the AF-associated enhancer regions. CRISPR/Cas9-mediated deletion of a 20-kb topologically engaged enhancer led to reduced Pitx2c transcription and AF predisposition. Allele-specific chromatin immunoprecipitation sequencing on hybrid heterozygous enhancer knockout mice revealed that long-range interaction of an AF-associated region with the Pitx2c promoter was required for maintenance of the Pitx2c promoter chromatin state. Long-range looping was mediated by CCCTC-binding factor (CTCF), since genetic disruption of the intronic CTCF-binding site caused reduced Pitx2c expression, AF predisposition, and diminished active chromatin marks on Pitx2. AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory functions directed at PITX2.
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Herraiz-Martínez, Adela, Carmen Tarifa, Estefanía Lozano-Velasco, Verónica Jiménez-Sábado, Sergi Casabella, Francisco Hernández-Torres, Houria Daimi, et al. "Novel PITX2 Homeodomain-Contained Mutations from ATRIAL Fibrillation Patients Deteriorate Calcium Homeostasis." Hearts 2, no. 2 (May 5, 2021): 251–71. http://dx.doi.org/10.3390/hearts2020020.
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Atrial fibrillation (AF) is the most common cardiac arrhythmia in the human population, with an estimated incidence of 1–2% in young adults but increasing to more than 10% in 80+ years patients. Pituitary Homeobox 2, Paired Like Homeodomain 2 (PITX2c) loss-of-function in mice revealed that this homeodomain (HD)-containing transcription factor plays a pivotal role in atrial electrophysiology and calcium homeostasis and point to PITX2 as a candidate gene for AF. To address this issue, we recruited 31 AF patients for genetic analyses of both the known risk alleles and PITX2c open reading frame (ORF) re-sequencing. We found two-point mutations in the homedomain of PITX2 and three other variants in the 5’untranslated region. A 65 years old male patient without 4q25 risk variants but with recurrent AF displayed two distinct HD-mutations, NM_000325.5:c.309G>C (Gln103His) and NM_000325.5:c.370G>A (Glu124Lys), which both resulted in a change within a highly conserved amino acid position. To address the functional impact of the PITX2 HD mutations, we generated plasmid constructs with mutated version of each nucleotide variant (MD4 and MD5, respectively) as well as a dominant negative control construct in which the PITX2 HD was lacking (DN). Functional analyses demonstrated PITX2c MD4 and PITX2c MD5 decreased Nppa-luciferase transactivation by 50% and 40%, respectively, similar to the PITX2c DN (50%), while Shox2 promoter repression was also impaired. Co-transactivation with other cardiac-enriched co-factors, such as Gata4 and Nkx2.5, was similarly impaired, further supporting the pivotal role of these mutations for correct PITX2c function. Furthermore, when expressed in HL1 cardiomyocyte cultures, the PITX2 mutants impaired endogenous expression of calcium regulatory proteins and induced alterations in sarcoplasmic reticulum (SR) calcium accumulation. This favored alternating and irregular calcium transient amplitudes, causing deterioration of the beat-to-beat stability upon elevation of the stimulation frequency. Overall this data demonstrate that these novel PITX2c HD-mutations might be causative of atrial fibrillation in the carrier.
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Liu, Chengyu, Wei Liu, Jennifer Palie, Mei Fang Lu, Nigel A. Brown, and James F. Martin. "Pitx2cpatterns anterior myocardium and aortic arch vessels and is required for local cell movement into atrioventricular cushions." Development 129, no. 21 (November 1, 2002): 5081–91. http://dx.doi.org/10.1242/dev.129.21.5081.
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Inactivation of the left-right asymmetry gene Pitx2 has been shown, in mice, to result in right isomerism with associated defects that are similar to that found in humans. We show that the Pitx2c isoform is expressed asymmetrically in a presumptive secondary heart field within the branchial arch and splanchnic mesoderm that contributes to the aortic sac and conotruncal myocardium. Pitx2c was expressed in left aortic sac mesothelium and in left splanchnic and branchial arch mesoderm near the junction of the aortic sac and branchial arch arteries. Mice with an isoform-specific deletion of Pitx2c had defects in asymmetric remodeling of the aortic arch vessels. Fatemapping studies using a Pitx2 cre recombinase knock-in allele showed that daughters ofPitx2-expressing cells populated the right and left ventricles,atrioventricular cushions and valves and pulmonary veins. In Pitx2mutant embryos, descendents of Pitx2-expressing cells failed to contribute to the atrioventricular cushions and valves and the pulmonary vein,resulting in abnormal morphogenesis of these structures. Our data provide functional evidence that the presumptive secondary heart field, derived from branchial arch and splanchnic mesoderm, patterns the forming outflow tract and reveal a role for Pitx2c in aortic arch remodeling. Moreover, our findings suggest that a major function of the Pitx2-mediated left right asymmetry pathway is to pattern the aortic arches, outflow tract and atrioventricular valves and cushions.
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Nagel, Stefan, Letizia Venturini, Grzegorz K. Przybylski, Piotr Grabarczyk, Björn Schneider, Corinna Meyer, Maren Kaufmann, et al. "Activation of Paired-Homeobox Gene PITX1 by Del(5)(q31) In T-Cell Acute Lymphoblastic Leukemia (T-ALL)." Blood 116, no. 21 (November 19, 2010): 4644. http://dx.doi.org/10.1182/blood.v116.21.4644.4644.
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Abstract Abstract 4644 Aberrant activation of various oncogenes, including homeobox genes encoding fundamental transcription factors, during T-cell development contributes to T-cell acute lymphoblastic leukemia (T-ALL). Neoplastic chromosome rearrangements are known to deregulate Antennapedia-class homeobox genes of the NKL-family (TLX1, TLX3, NKX2-5) and HOXA-cluster genes of the Extended-Hox-family. Following analysis of T-ALL cell lines and primary cells, we describe leukemogenic involvement of a third homeobox gene group, the Paired (PRD)-class. Ascertainment was performed in an early stage-arrested T-ALL cell line (LOUCY) which revealed chromosomal deletion at 5q31, removing the downstream regulatory region of the PRD-homeobox gene PITX1. Comparative expression analysis confirmed ectopic PITX1 expression, consistent with aberrant activation by del(5)(q31) which removes a STAT1 binding site. STAT1 mediates repressive IL2-STAT1 signaling, implicating IL2-pathway avoidance as a possible activation mechanism. Furthermore, we detected expression of the physiologically similar PITX2 in 11/24 (46%) T-ALL cell lines, 5 with genomic PITX2 gains. Among primary T-ALL samples, 2/22 (9%) – both pediatric pre-T-ALL - ectopically expressed PITX1 but not PITX2. Forced expression of PITX1 by lentiviral transduction of JURKAT cells and subsequent analysis by expression profiling, prompted upregulation of RUNX2 and JUN and inhibition of RUNX1 and NKX3-1, indicating impaired T-cell differentiation. Taken together, our data show leukemic activation of PITX1, a novice PRD-class homeobox gene in the T-ALL repertoire, which may promote leukemogenesis by inhibiting differentiation. Disclosures: No relevant conflicts of interest to declare.
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Wang, Y., H. Zhao, X. Zhang, and H. Feng. "Novel Identification of a Four-base-pair Deletion Mutation in PITX2 in a Rieger Syndrome Family." Journal of Dental Research 82, no. 12 (December 2003): 1008–12. http://dx.doi.org/10.1177/154405910308201214.
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Rieger syndrome is one of the most serious causes of tooth agenesis. Mutations in the PITX2, FOXC1, and PAX6 genes have been associated with Rieger syndrome. We have studied a three-generation Chinese family affected with Rieger syndrome and showing prominent dental abnormalities. Mutational screening and sequence analysis of the PITX2 gene revealed a previously unidentified four-base-pair deletion of nucleotides 717-720 in exon 5 in all affected members. The mutation causes a frame shift after Thr44, the 7th amino acid of the homeo-domain, and introduces a premature stop codon in the gene sequence. This deletion is the first unquestionable loss-of-function mutation, deleting all the functionally important parts of the protein. Our novel discovery indicates that the oligodontia and other phenotypes of Rieger syndrome observed in this family are due to this PITX2 mutation, and these data further support the critical role of PIXT2 in tooth morphogenesis.
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Poelmann, Robert E., Monique R. M. Jongbloed, and Adriana C. Gittenberger-de Groot. "Pitx2." Circulation Research 102, no. 7 (April 11, 2008): 749–51. http://dx.doi.org/10.1161/circresaha.108.174847.
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Saadi, Irfan, Adisa Kuburas, Jamison J. Engle, and Andrew F. Russo. "Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome." Molecular and Cellular Biology 23, no. 6 (March 15, 2003): 1968–82. http://dx.doi.org/10.1128/mcb.23.6.1968-1982.2003.
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ABSTRACT Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.
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Kieusseian, Aurélie, Jalila Chagraoui, Cécile Kerdudo, Philippe-Emmanuel Mangeot, Philip J. Gage, Nicole Navarro, Brigitte Izac, Georges Uzan, Bernard G. Forget, and Anne Dubart-Kupperschmitt. "Expression of Pitx2 in stromal cells is required for normal hematopoiesis." Blood 107, no. 2 (January 15, 2006): 492–500. http://dx.doi.org/10.1182/blood-2005-02-0529.
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AbstractAlthough the expression of Pitx2, a bicoid family homeodomain transcription factor, is highly regulated during hematopoiesis, its function during this process was not documented; we thus studied hematopoiesis in Pitx2-null mice. We found that Pitx2–/– embryos display hypoplastic livers with reduced numbers of hematopoietic cells, but these cells had normal hematopoietic potential, as evidenced by colony-forming assays, immature progenitor cell assays, and long-term repopulation assays. Because the microenvironment is also crucial to the development of normal hematopoiesis, we established Pitx2–/– and Pitx2+/+ stromas from fetal liver and studied their hematopoietic supportive capacity. We showed that the frequency of cobblestone area-forming cells was 4-fold decreased when using Pitx2–/– stromal cells compared with Pitx2+/+ stromal cells, whatever the Pitx2 genotype of hematopoietic cells tested in this assay. This defect was rescued by expression of Pitx2 into Pitx2–/– fetal liver stromal cells, demonstrating a major and direct role of Pitx2 in the hematopoietic supportive capacity of fetal liver stroma. Finally, we showed a reduced capacity of MS5 stromal cells expressing Pitx2 RNAi to support human hematopoiesis. Altogether these data showed that Pitx2 has major functions in the hematopoietic supportive capacity of fetal liver and adult bone marrow stromal cells.
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Tuerxun, Kebinuer, Shufang Zhang, and Yuexin Zhang. "Downregulation of PITX2 inhibits the proliferation and migration of liver cancer cells and induces cell apoptosis." Open Life Sciences 16, no. 1 (January 1, 2021): 1322–29. http://dx.doi.org/10.1515/biol-2021-0133.
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Abstract Paired-like homeodomain 2 (PITX2) functions as a transcription factor to participate in vertebrate embryogenesis, and dysregulated PITX2 expression was associated with the progression of various cancers. The functional role of PITX2 in tumorigenesis of liver cancer remains unknown. Western blot analysis showed that expression levels of PITX2 were enhanced in the liver cancer tissues and cells. siRNAs targeting PITX2 induced downregulation of PITX2 in liver cancer cells. siRNA-induced knockdown of PITX2 decreased liver cancer cell viability and proliferation, while promoting cell apoptosis by increasing cleaved-PARP, cleaved caspase 3, and cleaved caspase 9. The knockdown of PITX2 repressed liver cancer cell migration and invasion. In conclusion, elevated PITX2 expression was associated with liver cancer progression through repression of cell apoptosis and promoting cell proliferation and metastasis, and silencing of PITX2 might serve as a potential therapeutic strategy for the treatment of liver cancer.
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Amen, Melanie, Xiaoming Liu, Usha Vadlamudi, Gabriela Elizondo, Evan Diamond, John F. Engelhardt, and Brad A. Amendt. "PITX2 and β-Catenin Interactions Regulate Lef-1 Isoform Expression." Molecular and Cellular Biology 27, no. 21 (September 4, 2007): 7560–73. http://dx.doi.org/10.1128/mcb.00315-07.
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ABSTRACT Lef-1 and PITX2 function in the Wnt signaling pathway by recruiting and interacting with β-catenin to activate target genes. Chromatin immunoprecipitation (ChIP) assays identified the Lef-1 promoter as a PITX2 downstream target. Transgenic mice expressing LacZ driven by the 2.5-kb LEF-1 promoter demonstrated expression in the tooth epithelium correlated with endogenous Lef-1 FL epithelial expression. PITX2 isoforms regulate the LEF-1 promoter, and β-catenin synergistically enhanced activation of the LEF-1 promoter in combination with PITX2 and Lef-1 isoforms. PITX2 enhances endogenous expression of the full-length β-catenin-dependent Lef-1 isoform (Lef-1 FL) while decreasing expression of the N-terminally truncated β-catenin-independent isoform. Our research revealed a novel interaction between PITX2, Lef-1, and β-catenin in which the Lef-1 β-catenin binding domain is dispensable for its interaction with PITX2. PITX2 interacts with two sites within the Lef-1 protein. Furthermore, β-catenin interacts with the PITX2 homeodomain and Lef-1 interacts with the PITX2 C-terminal tail. Lef-1 and β-catenin interact simultaneously and independently with PITX2 through two different sites to regulate PITX2 transcriptional activity. These data support a role for PITX2 in cell proliferation, migration, and cell division through differential Lef-1 isoform expression and interactions with Lef-1 and β-catenin.
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Hjalt, Tord A., Brad A. Amendt, and Jeffrey C. Murray. "Pitx2 Regulates Procollagen Lysyl Hydroxylase (Plod) Gene Expression." Journal of Cell Biology 152, no. 3 (February 5, 2001): 545–52. http://dx.doi.org/10.1083/jcb.152.3.545.
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The Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1–luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI.
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Jiang, Qi, Mixue Xie, Mengye He, Feifei Yan, Ming Chen, Suzhen Xu, Xiaochen Zhang, and Peng Shen. "PITX2 methylation." Medicine 98, no. 1 (January 2019): e13820. http://dx.doi.org/10.1097/md.0000000000013820.
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Amendt, Brad A., Lillian B. Sutherland, and Andrew F. Russo. "Multifunctional Role of the Pitx2 Homeodomain Protein C-Terminal Tail." Molecular and Cellular Biology 19, no. 10 (October 1, 1999): 7001–10. http://dx.doi.org/10.1128/mcb.19.10.7001.
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ABSTRACT Pitx2 is a newly described bicoid-like homeodomain transcription factor that is defective in Rieger syndrome and shows a striking leftward developmental asymmetry. We have previously shown that Pitx2 (also called Ptx2 and RIEG) transactivates a reporter gene containing abicoid enhancer and synergistically transactivates the prolactin promoter in the presence of the POU homeodomain protein Pit-1. In this report, we focused on the C-terminal region which is mutated in some Rieger patients and contains a highly conserved 14-amino-acid element. Deletion analysis of Pitx2 revealed that the C-terminal 39-amino-acid tail represses DNA binding activity and is required for Pitx2-Pit-1 interaction and Pit-1 synergism. Pit-1 interaction with the Pitx2 C terminus masks the inhibitory effect and promotes increased DNA binding activity. Interestingly, cotransfection of an expression vector encoding the C-terminal 39 amino acids of Pitx2 specifically inhibits Pitx2 transactivation activity. In contrast, the C-terminal 39-amino-acid peptide interacts with Pitx2 to increase its DNA binding activity. These data suggest that the C-terminal tail intrinsically inhibits the Pitx2 protein and that this inhibition can be overcome by interaction with other transcription factors to allow activation during development.
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Okubo, Toru, Ryuhei Hayashi, Shun Shibata, Yuji Kudo, Yuki Ishikawa, Saki Inoue, Yuki Kobayashi, et al. "Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells." Journal of Biological Chemistry 295, no. 11 (February 7, 2020): 3456–65. http://dx.doi.org/10.1074/jbc.ra119.010713.
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PITX2 (Paired-like homeodomain transcription factor 2) plays important roles in asymmetric development of the internal organs and symmetric development of eye tissues. During eye development, cranial neural crest cells migrate from the neural tube and form the periocular mesenchyme (POM). POM cells differentiate into several ocular cell types, such as corneal endothelial cells, keratocytes, and some ocular mesenchymal cells. In this study, we used transcription activator–like effector nuclease technology to establish a human induced pluripotent stem cell (hiPSC) line expressing a fluorescent reporter gene from the PITX2 promoter. Using homologous recombination, we heterozygously inserted a PITX2–IRES2–EGFP sequence downstream of the stop codon in exon 8 of PITX2. Cellular pluripotency was monitored with alkaline phosphatase and immunofluorescence staining of pluripotency markers, and the hiPSC line formed normal self-formed ectodermal autonomous multizones. Using a combination of previously reported methods, we induced PITX2 in the hiPSC line and observed simultaneous EGFP and PITX2 expression, as indicated by immunoblotting and immunofluorescence staining. PITX2 mRNA levels were increased in EGFP-positive cells, which were collected by cell sorting, and marker gene expression analysis of EGFP-positive cells induced in self-formed ectodermal autonomous multizones revealed that they were genuine POM cells. Moreover, after 2 days of culture, EGFP-positive cells expressed the PITX2 protein, which co-localized with forkhead box C1 (FOXC1) protein in the nucleus. We anticipate that the PITX2–EGFP hiPSC reporter cell line established and validated here can be utilized to isolate POM cells and to analyze PITX2 expression during POM cell induction.
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Castinetti, F., M. L. Brinkmeier, D. F. Gordon, K. R. Vella, J. M. Kerr, A. H. Mortensen, A. Hollenberg, T. Brue, E. C. Ridgway, and S. A. Camper. "PITX2 AND PITX1 Regulate Thyrotroph Function and Response to Hypothyroidism." Molecular Endocrinology 25, no. 11 (November 1, 2011): 1950–60. http://dx.doi.org/10.1210/me.2010-0388.
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Marcil, A. "Pitx1 and Pitx2 are required for development of hindlimb buds." Development 130, no. 1 (January 1, 2003): 45–55. http://dx.doi.org/10.1242/dev.00192.
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Zhang, Feng, Lusi Zhang, Li He, Mengdan Cao, Yuting Yang, Xuanchu Duan, Jingming Shi, and Ke Liu. "A PITX2 splice-site mutation in a family with Axenfeld-Rieger syndrome leads to decreased expression of nuclear PITX2 protein." International Ophthalmology 41, no. 4 (January 25, 2021): 1503–11. http://dx.doi.org/10.1007/s10792-021-01704-5.
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Abstract Purpose Axenfeld-Rieger syndrome (ARS) is an autosomal dominant disorder characterized by ocular anterior segment abnormalities. In the current study, we describe clinical and genetic findings in a Chinese ARS pedigree. Methods An ARS pedigree was recruited and patients were given comprehensive ophthalmic examinations and general physical examinations. DNA from the proband II:2 was used for exome sequencing. Sanger sequencing was utilized to identify and validate PITX2 variations. qPCR and western blotting were performed to detect PITX2 expression in immortalized peripheral blood lymphocytes. Results All affected family members showed typical ocular abnormalities, including iris atrophy, corectopia, shallow anterior chamber, complete or partial angle closure, and advanced glaucoma. They also exhibited systemic anomalies, such as microdontia, hypodontia, and redundant periumbilical skin. A heterozygous splice-site variation c.390 + 1G > A in PITX2, which might lead to a truncated PITX2 protein (p.Val131IlefsX127), was found in the proband. Sanger sequencing validated that the variation completely co-segregated with the ARS phenotype within this family and was absent in 100 unrelated controls. Western blotting revealed that the nuclear PITX2 protein was significantly decreased in patients compared with controls. Nonetheless, there was no significant difference in the total PITX2 protein level, consistent with qPCR results showing no alteration in PITX2 mRNA levels in the patient group. Conclusions PITX2 c.390 + 1G > A (p.Val131IlefsX127) was a novel genetic etiology of the ARS pedigree. The mutation leads to decreased nuclear PITX2, indicating lower transcriptional activity.
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Campione, M., H. Steinbeisser, A. Schweickert, K. Deissler, F. van Bebber, L. A. Lowe, S. Nowotschin, et al. "The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping." Development 126, no. 6 (March 15, 1999): 1225–34. http://dx.doi.org/10.1242/dev.126.6.1225.
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Left-right asymmetry in vertebrates is controlled by activities emanating from the left lateral plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2. It is asymmetrically expressed in the left lateral plate mesoderm, tubular heart and early gut tube. Localized Pitx2 expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1 in the right lateral plate induces Pitx2 transcription in Xenopus. Misexpression of Pitx2 affects situs and morphology of organs. These experiments suggest a role for Pitx2 in promoting looping of the linear heart and gut.
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Huang, Liqin, Yong Meng, and Xiangming Guo. "Novel PITX2 Mutations including a Mutation Causing an Unusual Ophthalmic Phenotype of Axenfeld-Rieger Syndrome." Journal of Ophthalmology 2019 (July 1, 2019): 1–10. http://dx.doi.org/10.1155/2019/5642126.
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Purpose. The aims of this study were to examine novel mutations in PITX2 and FOXC1 in Chinese patients with anterior segment dysgenesis (ASD) and to compare the clinical presentations of these mutations with previously reported associated phenotypes. Methods. Twenty-six unrelated patients with different forms of ASD were enrolled from our paediatric and genetic eye clinic. The ocular manifestations of both eyes of each patient were recorded. Genomic DNA was prepared from venous leukocytes. All coding exons of PITX2 and FOXC1 were amplified by polymerase chain reaction (PCR) from genomic DNA and subjected to direct DNA sequencing. Analysis of mutations in control subjects was performed by heteroduplex single-strand conformation polymorphism (SSCP) analysis. Results. Sequence analysis of the PITX2 gene revealed four mutations, including c.475_476delCT (P.L159VfsX39), c.64C > T (P.Q22X), c.296delG (P.R99PfsX56), and c.206G > A (P.R69H). The first three mutations were found to be novel. The c.475_476delCT (P.L159VfsX39) mutation, located at the 3′ end of the PITX2-coding region, was identified in a Chinese Axenfeld-Rieger syndrome (ARS) patient who presented with an unusual severe phenotype of bilateral aniridia. The clinical characteristics, including the severity and manifestations of the patient’s phenotype, were compared with reported PITX2-associated aniridia phenotypes of ARS in the literature. Conclusions. These results expand the mutation spectrum of the PITX2 gene in patients with ARS. The PITX2 gene may be responsible for a significant portion of ARS with additional systemic defects in the Chinese population. This is the first reported case of a mutation at the 3′ end of the PITX2-coding region extending the phenotypic consequences to bilateral aniridia. The traits of ARS could display tremendous variability in severity and manifestations due to the dominant-negative effect of PITX2. Our results further emphasize the importance of careful clinical and genetic analysis in determining mutation-disease associations and may lead to a better understanding of the role of PITX2 in ocular development.
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Zhang, Hui Z., Svetlana Rogulina, Wendy Chen, Barbara A. Degar, and Bernard G. Forget. "Effects of the Homeodomain Gene Pitx2 on Self-Renewal of Hematopoietic Stem Cells." Blood 110, no. 11 (November 16, 2007): 95. http://dx.doi.org/10.1182/blood.v110.11.95.95.
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Abstract Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, is also a downstream target of genes important for hematopoiesis such as MLL and Wnt/Dvl/β-Catenin. We have previously reported that Pitx2 null hematopoietic stem cells (HSCs) can contribute to multi-lineage hematopoiesis under physiologic conditions. We have now carried out serial bone marrow transplantation experiments and demonstrated that after the 3rd round of serial transplantation, Pitx2 null cells reconstituted only 28.6% of the recipient hematopoietic cells as compared to 60% in the case of wild type cells (P<0.001). There were no Pitx2 null donor-derived cells in recipient mice after the 4th round of transplantation, whereas donor-derived chimerism was 57% with wild type cells (P<0.001), and 26% with Pitx2 +/− cells (P<0.001). Therefore, Pitx2 null HSCs have decreased self renewal capacity. To further study the function of Pitx2 in HSC, we constitutively overexpressed the Pitx2 gene in murine bone marrow cells following transduction using a MSCV/IRES/GFP retroviral vector, and analyzed the effects on hematopoiesis in vitro and in vivo. Bone marrow cells overexpressing Pitx2 were isolated on the basis of their GFP expression and analyzed for their colony forming ability in vitro. Retrovirally transduced bone marrow cells were also transplanted into lethally irradiated mice, and the transplanted mice were observed for long-term reconstitution. Colony-forming unit assays showed that Pitx2 overexpressing bone marrow cells, compared to control cells transduced with vector only, had increased numbers of GM colony forming units and reduced numbers of megakaryocytic colony forming units. Pitx2-overexpressing cells continued to form GM colonies after more than eight serial replatings. When these cells were cultured in liquid medium containing SCF, IL-3 and IL-6, they gave rise to cells that stained positively either for alpha naphthyl butyrate, indicating monocytic differentiation, or for peroxidase, indicating neutrophilic differentiation. The ability of these GM-colony forming cells to cause leukemia is currently under investigation. Long-term reconstitution of hematopoiesis in mice by Pitx2 over-expressing HSCs was demonstrated by identifying GFP positive multi-lineage peripheral blood cells four months following transplantation. One of these mice manifested leukemia at this time, as evidenced by a markedly elevated WBC count and other hematologic abnormalities. The leukemic WBCs had very high levels of GFP and Pitx2 expression and were shown to contain two retroviral integration sites, neither of which involved a known oncogene or overexpression of the gene at the integration site. Immunophenotyping by flow cytometry demonstrated that the majority of the leukemic cells were c-kit positive and expressed the megakaryocytic marker CD41, as well as the common myeloid progenitor marker, CD16/32. Some of the cells expressed the erythroid marker Ter119. The leukemic cells did not express any lymphoid markers, including CD3ε, B220, CD19, and IL7R3. This Pitx2-overexpression-associated leukemia was transplantable. Experiments are under way to characterize the leukemia initiating cells. Taken together, our results provide evidence that the homeodomain gene Pitx2 plays a role in the self-renewal of hematopoietic stem/progenitor cells.
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Gage, P. J., H. Suh, and S. A. Camper. "Dosage requirement of Pitx2 for development of multiple organs." Development 126, no. 20 (October 15, 1999): 4643–51. http://dx.doi.org/10.1242/dev.126.20.4643.
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Pitx2 is a homeodomain transcription factor that is mutated in Rieger syndrome, a haploinsufficiency disorder affecting eyes and teeth. Pitx2 also has a postulated role in left-right axis determination. We assessed the requirements for Pitx2 directly by generating hypomorphic and null alleles. Heterozygotes for either allele have eye abnormalities consistent with Rieger syndrome. The ventral body wall fails to close in embryos homozygous for the null allele, leaving the heart and abdominal organs externalized and the body axis contorted. In homozygotes for either allele, the heart tube undergoes normal, rightward looping and the stomach is positioned normally. In contrast, homozygotes for both alleles exhibit right isomerization of the lungs. Thus, Pitx2 is required for left-right asymmetry of the lungs but not other organs. Homozygotes for either allele exhibit septal and valve defects, and null homozygotes have a single atrium proving that a threshold level of Pitx2 is required for normal heart development. Null homozygotes exhibit arrest of pituitary gland development at the committed Rathke pouch stage and eye defects including optic nerve coloboma and absence of ocular muscles. This allelic series establishes that Pitx2 is required for the development of mulitple organs in a dosage-sensitive manner.
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Ai, Di, Jun Wang, Melanie Amen, Mei-Fang Lu, Brad A. Amendt, and James F. Martin. "Nuclear Factor 1 and T-Cell Factor/LEF Recognition Elements Regulate Pitx2 Transcription in Pituitary Development." Molecular and Cellular Biology 27, no. 16 (June 11, 2007): 5765–75. http://dx.doi.org/10.1128/mcb.01848-06.
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ABSTRACT Pitx2, a paired-related homeobox gene that is mutated in Rieger syndrome I, is the earliest known marker of oral ectoderm. Pitx2 was previously shown to be required for tooth, palate, and pituitary development in mice; however, the mechanisms regulating Pitx2 transcription in the oral ectoderm are poorly understood. Here we used an in vivo transgenic approach to investigate the mechanisms regulating Pitx2 transcription. We identified a 7-kb fragment that directs LacZ expression in oral ectoderm and in many of its derivatives. Deletion analysis of transgenic embryos reduced this fragment to a 520-bp region that directed LacZ activity to Rathke's pouch. A comparison of the mouse and human sequences revealed a conserved nuclear factor 1 (NF-1) recognition element near a consensus T-cell factor (TCF)/LEF binding site. The mutation of either site individually abolished LacZ activity in transgenic embryos, identifying Pitx2 as a direct target of Wnt signaling in pituitary development. These findings uncover a requirement for NF-1 and TCF factors in Pitx2 transcriptional regulation in the pituitary and provide insight into the mechanisms controlling region-specific transcription in the oral ectoderm and its derivatives.
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L’honoré, Aurore, Pierre-Henri Commère, Jean-François Ouimette, Didier Montarras, Jacques Drouin, and Margaret Buckingham. "Redox Regulation by Pitx2 and Pitx3 Is Critical for Fetal Myogenesis." Developmental Cell 29, no. 4 (May 2014): 392–405. http://dx.doi.org/10.1016/j.devcel.2014.04.006.
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L'honoré, Aurore, Pierre-Henri Commère, Jean-François Ouimette, Didier Montarras, Jacques Drouin, and Margaret Buckingham. "Redox Regulation by Pitx2 and Pitx3 Is Critical for Fetal Myogenesis." Developmental Cell 39, no. 6 (December 2016): 756. http://dx.doi.org/10.1016/j.devcel.2016.12.011.
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Shang, Yueting, Tadashi Yoshida, Brad A. Amendt, James F. Martin, and Gary K. Owens. "Pitx2 is functionally important in the early stages of vascular smooth muscle cell differentiation." Journal of Cell Biology 181, no. 3 (May 5, 2008): 461–73. http://dx.doi.org/10.1083/jcb.200711145.
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Mechanisms that control vascular smooth muscle cell (SMC) differentiation are poorly understood. We identify Pitx2 as a previously unknown homeodomain transcription factor that is rapidly induced in an in vitro model of SMC differentiation from multipotent stem cells. Pitx2 induces expression of multiple SMC differentiation marker genes by binding to a TAATC(C/T) cis-element, by interacting with serum response factor, and by increasing histone acetylation levels within the promoters of SMC differentiation marker genes. Suppression of Pitx2 reduces expression of SMC differentiation marker genes in the early stages of SMC differentiation in vitro, whereas Prx1, another homeodomain protein, regulates SMC differentiation marker genes in fully differentiated SMCs. Pitx2, but not Prx1, knockout mouse embryos exhibit impaired induction of SMC differentiation markers in the dorsal aorta and branchial arch arteries. Our results demonstrate that Pitx2 functions to regulate the early stages of SMC differentiation.
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Heiden, E., G. Weiss, L. Banez, S. Freedland, L. Sun, A. Hartmann, G. van Leenders, C. Bangma, M. Ittmann, and T. Wheeler. "PITX2 methylation and biochemical recurrence in postradical prostatectomy prostate cancer patients." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 5124. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.5124.
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5124 Background: PITX2 is a bicoid-related transcription factor induced by the Wnt pathway and required for effective cell-type-specific proliferation during development. We previously reported prognostic potential of PITX2 gene promoter methylation for outcome prediction in breast and prostate cancer (PC) patients. Radical prostatectomy (RP) is potentially curative in patients with clinically localized PC. However, biochemical recurrence (BCR) affects 15–30% of patients undergoing RP. In the current study, we validate PITX2 methylation status as a predictor of BCR following RP. Methods: PITX2 methylation status was assessed in formalin-fixed paraffin-embedded RP tumor tissue samples from 476 patients from four different institutions in USA and Europe using customized microarrays. Associations between PITX2 methylation and BCR were assessed using log-rank test and Cox regression controlling for PC features. Results: In multivariate analysis, patients with a high methylation status were at significantly higher risk for BCR compared to patients with low methylation status (HR = 3.0; 95%CI = 2.0–4.5; p < 10-5). BCR-free survival at five years after surgery was 85% and 61% for patients in the low and high methylation group, respectively. In patients with pathological Gleason 7 tumors, the relative risk of suffering BCR was twice as high for a patient with high PITX2 methylation relative to patients with low PITX2 methylation (HR = 2.0; 95%CI = 1.2–3.3; p = 0.005). Moreover, PITX2 methylation status was significant in the group of patients with tumor involvement of the surgical margins in the prostatectomy specimen. (HR = 3.36, 95% CI: 2.24–5.06, p = 0.001). Conclusions: PITX2 methylation status identifies PC patients most likely to experience BCR. This test independently adds to prognostic information provided by standard clinico-pathological analyses improving stratification of RP patients into high- or low-risk for BCR. This new clinical tool would be of particular benefit in assessment of intermediate-risk patients (Gleason 7) or patients with positive surgical margins wherein risk stratification remains a challenge. [Table: see text]
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Hjalt, Tord A., and Elena V. Semina. "Current molecular understanding of Axenfeld–Rieger syndrome." Expert Reviews in Molecular Medicine 7, no. 25 (November 8, 2005): 1–17. http://dx.doi.org/10.1017/s1462399405010082.
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Axenfeld–Rieger syndrome (ARS) is a rare autosomal dominant inherited disorder affecting the development of the eyes, teeth and abdomen. The syndrome is characterised by complete penetrance but variable expressivity. The ocular component of the ARS phenotype has acquired most clinical attention and has been dissected into a spectrum of developmental eye disorders, of which open-angle glaucoma represents the main challenge in terms of treatment. Mutations in several chromosomal loci have been implicated in ARS, including PITX2, FOXC1 and PAX6. Full-spectrum ARS is caused primarily by mutations in the PITX2 gene. The homeobox transcription factor PITX2 is produced as at least four different transcriptional and splicing isoforms, with different biological properties. Intriguingly, PITX2 is also involved in left–right polarity determination, although asymmetry defects are not a feature of ARS. In experimental animal models and in cell culture experiments using PITX2, abundant evidence indicates that a narrow window of expression level of this gene is vital for its correct function.
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Smoczer, Cristine, Lara Hooker, Sarah Brode, Marian Wolanski, Farhad KhosrowShahian, and Michael Crawford. "The Xenopus homeobox gene pitx3 impinges upon somitogenesis and laterality." Biochemistry and Cell Biology 91, no. 2 (April 2013): 79–87. http://dx.doi.org/10.1139/bcb-2012-0057.
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Pitx3 has been identified as the causative locus in a developmental eye mutation associated with mammalian anterior segment dysgenesis, congenital cataracts, and aphakia. In recent studies of frog eye development we discovered that pitx3 expresses symmetrically in the somites and lateral plate mesoderm and asymmetrically during cardiac and gut looping. We report that disruption of pitx3 activity on one side of an embryo relative to the other, either by over- or underexpression of pitx3, elicits a crooked dorsal axis in embryos that is a consequence of a retarded progression through somitogenesis. Unlike in amniotes, Xenopus somites form as cohorts of presomitic cells that rotate perpendicular to the dorsal axis. Since no vertebral anomalies have been reported in mouse and human Pitx3 mutants, we attempt to distinguish whether the segmentation clock is uniquely affected in frog or if the pitx3 perturbation inhibits the cellular changes that are necessary to rotation of presomitic cells. In Xenopus, pitx3 appears to inhibit the rotation of presomitic cell cohorts and to be necessary to the bilaterally symmetric expression of pitx2 in somites.
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Bai, Jieyun, Yijie Zhu, Andy Lo, Meng Gao, Yaosheng Lu, Jichao Zhao, and Henggui Zhang. "In Silico Assessment of Class I Antiarrhythmic Drug Effects on Pitx2-Induced Atrial Fibrillation: Insights from Populations of Electrophysiological Models of Human Atrial Cells and Tissues." International Journal of Molecular Sciences 22, no. 3 (January 27, 2021): 1265. http://dx.doi.org/10.3390/ijms22031265.
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Electrical remodelling as a result of homeodomain transcription factor 2 (Pitx2)-dependent gene regulation was linked to atrial fibrillation (AF) and AF patients with single nucleotide polymorphisms at chromosome 4q25 responded favorably to class I antiarrhythmic drugs (AADs). The possible reasons behind this remain elusive. The purpose of this study was to assess the efficacy of the AADs disopyramide, quinidine, and propafenone on human atrial arrhythmias mediated by Pitx2-induced remodelling, from a single cell to the tissue level, using drug binding models with multi-channel pharmacology. Experimentally calibrated populations of human atrial action po-tential (AP) models in both sinus rhythm (SR) and Pitx2-induced AF conditions were constructed by using two distinct models to represent morphological subtypes of AP. Multi-channel pharmaco-logical effects of disopyramide, quinidine, and propafenone on ionic currents were considered. Simulated results showed that Pitx2-induced remodelling increased maximum upstroke velocity (dVdtmax), and decreased AP duration (APD), conduction velocity (CV), and wavelength (WL). At the concentrations tested in this study, these AADs decreased dVdtmax and CV and prolonged APD in the setting of Pitx2-induced AF. Our findings of alterations in WL indicated that disopyramide may be more effective against Pitx2-induced AF than propafenone and quinidine by prolonging WL.
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Bai, Jieyun, Yaosheng Lu, Yijie Zhu, Huijin Wang, Dechun Yin, Henggui Zhang, Diego Franco, and Jichao Zhao. "Understanding PITX2-Dependent Atrial Fibrillation Mechanisms through Computational Models." International Journal of Molecular Sciences 22, no. 14 (July 19, 2021): 7681. http://dx.doi.org/10.3390/ijms22147681.
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Atrial fibrillation (AF) is a common arrhythmia. Better prevention and treatment of AF are needed to reduce AF-associated morbidity and mortality. Several major mechanisms cause AF in patients, including genetic predispositions to AF development. Genome-wide association studies have identified a number of genetic variants in association with AF populations, with the strongest hits clustering on chromosome 4q25, close to the gene for the homeobox transcription PITX2. Because of the inherent complexity of the human heart, experimental and basic research is insufficient for understanding the functional impacts of PITX2 variants on AF. Linking PITX2 properties to ion channels, cells, tissues, atriums and the whole heart, computational models provide a supplementary tool for achieving a quantitative understanding of the functional role of PITX2 in remodelling atrial structure and function to predispose to AF. It is hoped that computational approaches incorporating all we know about PITX2-related structural and electrical remodelling would provide better understanding into its proarrhythmic effects leading to development of improved anti-AF therapies. In the present review, we discuss advances in atrial modelling and focus on the mechanistic links between PITX2 and AF. Challenges in applying models for improving patient health are described, as well as a summary of future perspectives.
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Dawson, Timothy, Joe Iwanaga, Binghao Zou, Muralidharan Anbalagan, Aaron S. Dumont, Marios Loukas, Brian G. Rowan, and R. Shane Tubbs. "Transcription factor support for the dual embryological origin of the sternocleidomastoid and trapezius muscles." Clinical Anatomy 37, no. 1 (December 6, 2023): 147–52. http://dx.doi.org/10.1002/ca.24124.
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AbstractThe embryological origin of the trapezius and sternocleidomastoid muscles has been debated for over a century. To shed light on this issue, the present anatomical study was performed. Five fresh frozen human cadavers, three males and two females, were used for this study. Samples from each specimen's trapezius and sternocleidomastoid were fixed in 10% formalin and placed in paraffin blocks. As Paired like homeodomain 2 (Pitx2) and T‐box factor 1(Tbx1) have been implicated in the region and muscle type regulation, we performed Tbx1 and Pitx2 Immunohistochemistry (IHC) on these muscle tissue samples to identify the origin of the trapezius and sternocleidomastoid muscles. We have used the latest version of QuPath, v0.4.3, software to quantify the Tbx and Pitx2 staining. For the sternocleidomastoid muscle, for evaluated samples, the average amount of positively stained Tbx1 and Pitx2 was 25% (range 16%–30%) and 18% (range 12%–23%), respectively. For the trapezius muscles, for evaluated samples, the average amount of positively stained Tbx1 and Pitx2 parts of the samples was 17% (range 15%–20%) and 15% (14%–17%), respectively. Our anatomical findings suggest dual origins of both the trapezius and sternocleidomastoid muscles. Additionally, as neither Pitx2 nor Tbx1 made up all the staining observed for each muscle, other contributions to these structures are likely. Future studies with larger samples are now necessary to confirm these findings.
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Harbeck, Nadia, Inko Nimmrich, Arndt Hartmann, Jeffrey S. Ross, Tanja Cufer, Robert Grützmann, Glen Kristiansen, et al. "Multicenter Study Using Paraffin-Embedded Tumor Tissue Testing PITX2 DNA Methylation As a Marker for Outcome Prediction in Tamoxifen-Treated, Node-Negative Breast Cancer Patients." Journal of Clinical Oncology 26, no. 31 (November 1, 2008): 5036–42. http://dx.doi.org/10.1200/jco.2007.14.1697.
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Purpose We recently reported DNA methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene to be strongly correlated with increased risk of recurrence in node-negative, hormone receptor–positive, tamoxifen-treated breast cancer patients using fresh frozen specimens. Aims of the present study were to establish determination of PITX2 methylation for routine analysis in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue and to test PITX2 DNA methylation as a biomarker for outcome prediction in an independent patient cohort. Patients and Methods Real-time polymerase chain reaction (PCR) technology was validated for FFPE tissue by comparing methylation measurements in FFPE specimens with those in fresh frozen specimens from the same tumor. The impact of PITX2 methylation on time to distant metastasis was then evaluated in FFPE specimens from hormone receptor–positive, node-negative breast cancer patients (n = 399, adjuvant tamoxifen monotherapy). Results Reproducibility of the PCR assay in replicate measurements (rs ≥ 0.95; n = 150) and concordant measurements between fresh frozen and FFPE tissues (rs = 0.81; n = 89) were demonstrated. In a multivariate model, PITX2 methylation added significant information (hazard ratio = 2.35; 95% CI, 1.20 to 4.60) to established prognostic factors (tumor size, grade, and age). Conclusion PITX2 methylation can be reliably assessed by real-time PCR technology in FFPE tissue. Together with our earlier studies, we have accumulated substantial evidence that PITX2 methylation analysis holds promise as a practical assay for routine clinical use to predict outcome in node-negative, tamoxifen-treated breast cancer, which might allow, based on future validation studies, the identification of low-risk patients who may be treated by tamoxifen alone.
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L'Honoré, Aurore, Vincent Coulon, Alexandre Marcil, Mélanie Lebel, Julien Lafrance-Vanasse, Philip Gage, Sally Camper, and Jacques Drouin. "Sequential expression and redundancy of Pitx2 and Pitx3 genes during muscle development." Developmental Biology 307, no. 2 (July 2007): 421–33. http://dx.doi.org/10.1016/j.ydbio.2007.04.034.
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Rodriguez-Leon, J., C. R. Esteban, M. Marti, B. Santiago-Josefat, I. Dubova, X. Rubiralta, and J. C. I. Belmonte. "Pitx2 regulates gonad morphogenesis." Proceedings of the National Academy of Sciences 105, no. 32 (August 4, 2008): 11242–47. http://dx.doi.org/10.1073/pnas.0804904105.
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Clauss, Sebastian, and Stefan Kääb. "Is Pitx2 Growing Up?" Circulation: Cardiovascular Genetics 4, no. 2 (April 2011): 105–7. http://dx.doi.org/10.1161/circgenetics.111.959791.
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Basu, Moitri, Satinath Mukhopadhyay, Uttara Chatterjee, and Sib Sankar Roy. "FGF16 Promotes Invasive Behavior of SKOV-3 Ovarian Cancer Cells through Activation of Mitogen-activated Protein Kinase (MAPK) Signaling Pathway." Journal of Biological Chemistry 289, no. 3 (November 19, 2013): 1415–28. http://dx.doi.org/10.1074/jbc.m113.535427.
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Uncontrolled cell growth and tissue invasion define the characteristic features of cancer. Several growth factors regulate these processes by inducing specific signaling pathways. We show that FGF16, a novel factor, is expressed in human ovary, and its expression is markedly increased in ovarian tumors. This finding indicated possible involvement of FGF16 in ovarian cancer progression. We observed that FGF16 stimulates the proliferation of human ovarian adenocarcinoma cells, SKOV-3 and OAW-42. Furthermore, through the activation of FGF receptor-mediated intracellular MAPK pathway, FGF16 regulates the expression of MMP2, MMP9, SNAI1, and CDH1 and thus facilitates cellular invasion. Inhibition of FGFR as well as MAPK pathway reduces the proliferative and invasive behavior of ovarian cancer cells. Moreover, ovarian tumors with up-regulated PITX2 expression also showed activation of Wnt/β-catenin pathway that prompted us to investigate possible interaction among FGF16, PITX2, and Wnt pathway. We identified that PITX2 homeodomain transcription factor interacts with and regulates FGF16 expression. Furthermore, activation of the Wnt/β-catenin pathway induces FGF16 expression. Moreover, FGF16 promoter possesses the binding elements of PITX2 as well as T-cell factor (Wnt-responsive), in close proximity, where PITX2 and β-catenin binds to and synergistically activates the same. A detail study showed that both PITX2 and T-cell factor elements and the interaction with their binding partners are necessary for target gene expression. Taken together, our findings indicate that FGF16 in conjunction with Wnt pathway contributes to the cancer phenotype of ovarian cells and suggests that modulation of its expression in ovarian cells might be a promising therapeutic strategy for the treatment of invasive ovarian cancers.
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Burdine, Rebecca D., and Daniel T. Grimes. "Antagonistic interactions in the zebrafish midline prior to the emergence of asymmetric gene expression are important for left–right patterning." Philosophical Transactions of the Royal Society B: Biological Sciences 371, no. 1710 (December 19, 2016): 20150402. http://dx.doi.org/10.1098/rstb.2015.0402.
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Left–right (L-R) asymmetry of the internal organs of vertebrates is presaged by domains of asymmetric gene expression in the lateral plate mesoderm (LPM) during somitogenesis. Ciliated L-R coordinators (LRCs) are critical for biasing the initiation of asymmetrically expressed genes, such as nodal and pitx2 , to the left LPM. Other midline structures, including the notochord and floorplate, are then required to maintain these asymmetries. Here we report an unexpected role for the zebrafish EGF-CFC gene one-eyed pinhead ( oep ) in the midline to promote pitx2 expression in the LPM. Late zygotic oep (LZ oep ) mutants have strongly reduced or absent pitx2 expression in the LPM, but this expression can be rescued to strong levels by restoring oep in midline structures only. Furthermore, removing midline structures from LZ oep embryos can rescue pitx2 expression in the LPM, suggesting the midline is a source of an LPM pitx2 repressor that is itself inhibited by oep . Reducing lefty1 activity in LZ oep embryos mimics removal of the midline, implicating lefty1 in the midline-derived repression. Together, this suggests a model where Oep in the midline functions to overcome a midline-derived repressor, involving lefty1 , to allow for the expression of left side-specific genes in the LPM. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’.
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Cao, Haiyue, Wei Zhou, Yuge Tan, Xiuli Xu, Haiguang Mao, Xinyang Dong, Ningying Xu, and Zhaozheng Yin. "Chronological Expression of PITX2 and SIX1 Genes and the Association between Their Polymorphisms and Chicken Meat Quality Traits." Animals 11, no. 2 (February 8, 2021): 445. http://dx.doi.org/10.3390/ani11020445.
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Meat quality is closely related to the development of skeletal muscle, in which PITX2 and SIX1 genes play important regulatory roles. The present study firstly provided the data of chronological expression files of PITX2 and SIX1 genes in the post-hatching pectoral muscle and analyzed the association of their polymorphisms with the meat quality traits of Wuliang Mountain Black-bone (WLMB) chickens. The results showed that both PITX2 and SIX1 genes were weakly expressed in the second and third weeks, and then increased significantly from the third week to the fourth week. Furthermore, there was a significant positive correlation between the expression levels of the two genes. Twelve and one SNPs were detected in the chicken PITX2 and SIX1 genes, respectively, of which four SNPs (g.9830C > T, g.10073C > T, g.13335G > A, g.13726A > G) of the PITX2 gene and one SNP (g.564G > A) of the SIX1 gene were significantly associated with chicken meat quality traits. For the PITX2 gene, chickens with the CT genotype of g.9830C > T showed the highest meat color L*, shear force (SF), pH, and the lowest electrical conductivity (EC), and drip loss (DL) (p < 0.05 or p < 0.01); chickens with the CC genotype of g.10073C > T had the lowest L*, pH, and the highest DL (p < 0.01). For the SIX1 gene, chickens with the GG genotype of g.564G > A had the highest (p < 0.05) SF and pH. Furthermore, pH had a significant correlation with all the other meat quality traits. The current study could contribute to the research of regulatory mechanisms of meat quality and lay the foundation for improving meat quality based on marker-assisted selection in chickens.
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Wei, Qize, and Robert S. Adelstein. "Pitx2a Expression Alters Actin-Myosin Cytoskeleton and Migration of HeLa Cells through Rho GTPase Signaling." Molecular Biology of the Cell 13, no. 2 (February 2002): 683–97. http://dx.doi.org/10.1091/mbc.01-07-0358.
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We ectopically expressed the transcription factor Pitx2a, one of the Pitx2 isoforms, in HeLa cells by using a tetracycline-inducible expression system and examined whether Pitx2a was capable of modulating Rho GTPase signaling and altering the cell's cytoskeleton. Ectopic expression of Pitx2a induced actin-myosin reorganization, leading to increased cell spreading, suppression of cell migration, and the strengthening of cell-cell adhesion, marked by the accumulation and localization of β-catenin and N-cadherin to the sites of cell-cell contacts. Moreover, Pitx2a expression resulted in activation of the Rho GTPases Rac1 and RhoA, and the dominant negative Rac1 mutant N17Rac1 inhibited cell spreading and disrupted localization of β-catenin to the sites of cell-cell contacts. Both reorganization of actin-myosin and cell spreading require phosphatidylinositol 3-kinase activity, which is also necessary for activation of the Rho GTPase proteins. Pitx2a induced the expression of Trio, a guanine nucleotide exchange factor for Rac1 and RhoA, which preceded cell spreading, and the expression of Trio protein was down-regulated after the changes in cell spreading and cell morphology were initiated. In addition, Pitx2a also induces cell cycle arrest at G0/G1, most likely due to the accumulation of the tumor suppressor proteins p53 and p21. Our data indicate that the transcriptional activities initiated in the nucleus by Pitx2a result in profound changes in HeLa cell morphology, migration, and proliferation.
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Cao, Haiyue, Xinyang Dong, Haiguang Mao, Ningying Xu, and Zhaozheng Yin. "Expression Analysis of the PITX2 Gene and Associations between Its Polymorphisms and Body Size and Carcass Traits in Chickens." Animals 9, no. 12 (November 20, 2019): 1001. http://dx.doi.org/10.3390/ani9121001.
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PITX2 is expressed in and plays an important role in myocytes of mice, and it has effects on late myogenic differentiation in chickens. However, the expression profile and polymorphisms of PITX2 remain unclear in chickens. Therefore, the aim of the present study was to detect its expression and investigate single nucleotide polymorphisms (SNPs) within its exons and then to evaluate whether these polymorphisms affect body size as well as carcass traits in chickens. The expression analysis showed that the expression level of chicken PITX2 mRNA in the leg muscle and hypophysis was significantly higher (p < 0.01) than those in other tissues. The results of polymorphisms analysis identified two SNPs (i.e., g.9830C > T and g.10073C > T) in exon 1 and 10 SNPs (i.e., g.12713C > T, g.12755C > T, g.12938G > A, g. 3164C > T, g.13019G > A, g.13079G > A, g.13285G > A, g.13335G > A, g.13726A > G and g.13856C > T) in exon 3, including four novel SNPs (i.e., g.9830C > T, g.12713C > T, g.12938G > A and g.13856C > T). In the loci of g.10073C > T and g.12713C > T, chickens with the CT genotype had the highest (p < 0.05 or p < 0.01) breast depth and breast angle, respectively. For the locus of g.13335G > A, chickens with the GG genotype had the highest (p < 0.05 or p < 0.01) breast angle and shank circumference. For the locus of g.13726A > G, chickens with the GG genotype had the highest breast width, fossil keel bone length and shank circumference. The locus of g.12713A > G had significant effects on the PITX2 mRNA expression level in leg muscle. The H1H7 diplotype showed the highest shank circumference, and the H2H8 diplotype showed the highest breast muscle rate. The present research suggested that polymorphisms of the exons of the PITX2 gene were significantly associated with the body size and carcass traits of Wuliang Mountain Black-bone chickens and the PITX2 gene could be a potential candidate gene for molecular marker-aided selection in Wuliang Mountain Black-bone chickens and other chicken breeds.
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Kum, Su Jung, Hye Won Lee, Soon Gu Kim, Hyungsik Park, Ilseon Hwang, and Sang Pyo Kim. "Association of PTTG1 expression with invasiveness of non-functioning pituitary adenomas." Journal of Pathology and Translational Medicine 56, no. 1 (January 15, 2022): 22–31. http://dx.doi.org/10.4132/jptm.2021.08.31.
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Background: Pituitary tumor transforming gene 1 (PTTG1), paired-like homeodomain 2 (PITX2), and galectin-3 have been widely studied as predictive biomarkers for various tumors and are involved in tumorigenesis and tumor progression. We evaluated the usefulness of PTTG1, PITX2, and galectin-3 as predictive biomarkers for invasive non-functioning pituitary adenomas (NFPAs) by determining the relationship between the expressions of these three proteins and the invasiveness of the NFPAs. We also investigated whether PTTG1, E-cadherin, and Ki-67, which are known to be related to each other, show a correlation with NFPA features. Methods: A retrospective study was conducted on 87 patients with NPFAs who underwent surgical removal. The NFPAs were classified into three groups based on magnetic resonance imaging findings of suprasellar extension and cavernous sinus invasion. Immunohistochemical staining for PTTG1, PITX2, galectin-3, E-cadherin, and Ki-67 was performed on tissue microarrays. Results: PTTG1 expression showed a statistically significant correlation with the invasiveness of NFPAs, whereas PITX2 and galectin-3 did not have a relationship with the invasiveness of NFPAs. Moreover, there was no association among PTTG1, E-cadherin, and Ki-67 expression. Conclusions: PTTG1 has the potential to serve as a predictive biomarker for invasive NFPA. Furthermore, this study may serve as a reference for the development of PTTG1-targeted therapeutic agents.
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Schmitt, Manfred, Olaf G. Wilhelm, Aurelia Noske, Gabriele Schricker, Rudolph Napieralski, Martina Vetter, Michaela Aubele, et al. "Clinical Validation of PITX2 DNA Methylation to Predict Outcome in High-Risk Breast Cancer Patients Treated with Anthracycline-Based Chemotherapy." Breast Care 13, no. 6 (2018): 425–33. http://dx.doi.org/10.1159/000493016.
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Background: Breast cancer patients at high risk for recurrence are treated with anthracycline-based chemotherapy, but not all patients do equally benefit from such a regimen. To further improve therapy decision-making, biomarkers predicting outcome are of high unmet medical need. Methods: The percent DNA methylation ratio (PMR) of the promoter gene coding for the Paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time polymerase chain reaction (PCR) test. The multicenter study was conducted in routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue from 205 lymph node-positive breast cancer patients treated with adjuvant anthracycline-based chemotherapy. Results: The cut-off for the PITX2 methylation status (PMR = 12) was confirmed in a randomly selected cohort (n = 60) and validated (n = 145) prospectively with disease-free survival (DFS) at the 10-year follow-up. DFS was significantly different between the PMR ≤ 12 versus the PMR > 12 group with a hazard ratio (HR) of 2.74 (p < 0.001) in the validation cohort and also for the patient subgroup treated additionally with endocrine therapy (HR 2.47; p = 0.001). Conclusions: Early-stage lymph node-positive breast cancer patients with low PITX2 methylation do benefit from adjuvant anthracycline-based chemotherapy. Patients with a high PITX2 DNA methylation ratio, approximately 30%, show poor outcome and should thus be considered for alternative chemotherapy regimens.