Homeoprotein Family Research Articles

Dynamic Expression of the Homeobox Factor PBX1 during Mouse Testis Development

Members of the pre-B-cell leukemia transcription factor (PBX) family of homeoproteins are mainly known for their involvement in hematopoietic cell differentiation and in the development of leukemia. The four PBX proteins, PBX1, PBX2, PBX3 and PBX4, belong to the three amino acid loop extension (TALE) superfamily of homeoproteins which are important transcriptional cofactors in several developmental processes involving homeobox (HOX) factors. Mutations in the human PBX1 gene are responsible for cases of gonadal dysgenesis with absence of male sex differentiation while Pbx1 inactivation in the mouse causes a failure in Leydig cell differentiation and function. However, no data is available regarding the expression profile of this transcription factor in the testis. To fill this knowledge gap, we have characterized PBX1 expression during mouse testicular development. Real time PCRs and Western blots confirmed the presence Pbx1 mRNA and PBX1 protein in different Leydig and Sertoli cell lines. The cellular localization of the PBX1 protein was determined by immunohistochemistry and immunofluorescence on mouse testis sections at different embryonic and postnatal developmental stages. PBX1 was detected in interstitial cells and in peritubular myoid cells from embryonic life until puberty. Most interstitial cells expressing PBX1 do not express the Leydig cell marker CYP17A1, indicating that they are not differentiated and steroidogenically active Leydig cells. In adults, PBX1 was mainly detected in Sertoli cells. The presence of PBX1 in different somatic cell populations during testicular development further supports a direct role for this transcription factor in testis cell differentiation and in male reproductive function.

Homeobox C4 promotes hepatocellular carcinoma progression by the transactivation of Snail.

Homeobox C4 (HOXC4) belongs to the homeoprotein family of transcription factors, which play a critical role in morphogenesis and differentiation during embryonic development. Aberrant expression of HOXC4 has been reported in several types of cancers. However, the role of HOXC4 in hepatocellular carcinoma (HCC) remains unknown. Here, we reported that HOXC4 is upregulated in HCC tissues and predicts a poor outcome in patients with HCC. HOXC4 promotes HCC progression and induces an EMT-like phenotype both in vitro and in vivo. Furthermore, we demonstrated that the EMT-related transcription factor Snail is a transcriptional target of HOXC4 and HOXC4 regulates EMT by regulation of transforming growth factor β (TGF-β) signaling in HCC. Together, our study suggests that HOXC4 as a novel potential therapeutic target for HCC therapy.

The Physiology of Homeoprotein Transduction.

The homeoprotein family comprises ~300 transcription factors and was long seen as primarily involved in developmental programs through cell autonomous regulation. However, recent evidence reveals that many of these factors are also expressed in the adult where they exert physiological functions not yet fully deciphered. Furthermore, the DNA-binding domain of most homeoproteins contains two signal sequences allowing their secretion and internalization, thus intercellular transfer. This review focuses on this new-found signaling in cell migration, axon guidance, and cerebral cortex physiological homeostasis and speculates on how it may play important roles in early arealization of the neuroepithelium. It also describes the use of homeoproteins as therapeutic proteins in mouse models of diseases affecting the central nervous system, in particular Parkinson disease and glaucoma.

The Eya phosphatase: Its unique role in cancer.

The Eya proteins were originally identified as essential transcriptional co-activators of the Six family of homeoproteins. Subsequently, the highly conserved C-terminal domains of the Eya proteins were discovered to act as a Mg2+-dependent Tyr phosphatases, making Eyas the first transcriptional activators to harbor intrinsic phosphatase activity. Only two direct targets of the Eya Tyr phosphatase have been identified: H2AX, whose dephosphorylation directs cells to the DNA repair instead of the apoptotic pathway upon DNA damage, and ERβ, whose dephosphorylation inhibits its anti-tumor transcriptional activity. The Eya Tyr phosphatase mediates breast cancer cell transformation, migration, invasion, as well as metastasis, through targets not yet identified. Intriguingly, the N-terminal domain of Eya contains a separate Ser/Thr phosphatase activity implicated in innate immunity and in regulating c-Myc stability. Thus, Eya proteins are highly complex, containing two separable phosphatase domains and a transcriptional activation domain, thereby influencing tumor progression through multiple mechanisms.

Spleen hypoplasia leads to abnormal stress hematopoiesis in mice with loss of Pbx homeoproteins in splenic mesenchyme.

The spleen plays critical roles in immunity and also provides a permissive microenvironment for hematopoiesis. Previous studies have reported that the TALE-class homeodomain transcription factor Pbx1 is essential in hematopoietic stem and progenitor cells (HSPCs) for stem cell maintenance and progenitor expansion. However, the role of Pbx1 in the hematopoietic niche has not been investigated. Here we explored the effects that genetic perturbation of the splenic mesenchymal niche has on hematopoiesis upon loss of members of the Pbx family of homeoproteins. Splenic mesenchyme-specific inactivation of Pbx1 (SKO) on a Pbx2- or Pbx3-deficient genetic background (DKO) resulted in abnormal development of the spleen, which is dysmorphic and severely hypoplastic. This phenotype, in turn, affected the number of HSPCs in the fetal and adult spleen at steady state, as well as markedly impairing the kinetics of hematopoietic regeneration in adult mice after sub-lethal and lethal myelosuppressive irradiation. Spleens of mice with compound Pyx deficiency 8days following sublethal irradiation displayed significant downregulation of multiple cytokine-encoding genes, including KitL/SCF, Cxcl12/SDF-1, IL-3, IL-4, GM-CSF/Csf2 IL-10, and Igf-1, compared with controls. KitL/SCF and Cxcl12/SDF-1 were recently shown to play key roles in the splenic niche in response to various haematopoietic stresses such as myeloablation, blood loss, or pregnancy. Our results demonstrate that, in addition to their intrinsic roles in HSPCs, non-cell autonomous functions of Pbx factors within the splenic niche contribute to the regulation of hematopoiesis, at least in part via the control of KitL/SCF and Cxcl12/SDF-1. Furthermore, our study establishes that abnormal spleen development and hypoplasia have deleterious effects on the efficiency of hematopoietic recovery after bone marrow injury.

Investigating the functional interplay of IRX1 and IRX2 homeoprotein family members and MLL fusion proteins

Investigating the functional interplay of IRX1 and IRX2 homeoprotein family members and MLL fusion proteins

Local homeoprotein diffusion can stabilize boundaries generated by graded positional cues.

Boundary formation in the developing neuroepithelium decides on the position and size of compartments in the adult nervous system. In this study, we start from the French Flag model proposed by Lewis Wolpert, in which boundaries are formed through the combination of morphogen diffusion and of thresholds in cell responses. In contemporary terms, a response is characterized by the expression of cell-autonomous transcription factors, very often of the homeoprotein family. Theoretical studies suggest that this sole mechanism results in the formation of boundaries of imprecise shapes and positions. Alan Turing, on the other hand, proposed a model whereby two morphogens that exhibit self-activation and reciprocal inhibition, and are uniformly distributed and diffuse at different rates lead to the formation of territories of unpredictable shapes and positions but with sharp boundaries (the 'leopard spots'). Here, we have combined the two models and compared the stability of boundaries when the hypothesis of local homeoprotein intercellular diffusion is, or is not, introduced in the equations. We find that the addition of homeoprotein local diffusion leads to a dramatic stabilization of the positioning of the boundary, even when other parameters are significantly modified. This novel Turing/Wolpert combined model has thus important theoretical consequences for our understanding of the role of the intercellular diffusion of homeoproteins in the developmental robustness of and the changes that take place in the course of evolution.

Investigating the interplay between the Iroquois homeoprotein family and MLL or derivatives thereof

The Iroquois 1 gene has been identified as top 1 upregulated gene in t(4;11) leukemia patients that display a transcriptional downregulation of HOXA genes, a phenomenon that was correlated with an even worser outcome (Trentin et al., 2009; Stam et al., 2010.). Here we present first data about Iroquois 1 and 2, both influencing the transcriptional properties of key players assumed to be important for the leukemogenic process in MLL-rearranged leukemia cell. From our data we can conclude that Iroquois proteins seem to counteract the molecular actions deriving from the MLL-AF4 protein.

Epigenetic silencing of Aristaless‐like homeobox‐4, a potential tumor suppressor gene associated with lung cancer

Using genome-wide methylation screening, we found Aristaless-like homeobox-4 (ALX4) preferentially methylated in lung cancer. ALX4 is a putative transcription factor that belongs to the family of paired-class homeoproteins involved in epithelial development. However, the role of ALX4 in tumorigenesis remains largely unclear. Here, we analyzed its epigenetic regulation, biological functions and related molecular mechanisms in lung cancer. CpG island methylation and expression of ALX4 were evaluated by methylation-specific polymerase chain reaction (PCR), bisulfite genomic sequencing, reverse-transcription PCR and Western blotting. ALX4 functions were determined by cell viability, colony formation, flow cytometry and in vivo tumorigenicity assays. ALX4 hypermethylation was detected in 55% (54/98) of primary lung cancers compared to none (0/20) of the normal lung tissue samples tested (p < 0.01). ALX4 was readily expressed in normal lung tissues with an unmethylated status, but downregulated or silenced in 90% (9/10) of lung cancer cell lines with a hypermethylation status. Demethylation experiments further confirmed that loss of ALX4 expression was regulated by CpG island hypermethylation. Re-expression of ALX4 in lung cancer cell lines suppressed cell viability, colony formation and migration, whereas it induced apoptosis and G1/S arrest and restrained the tumorigenicity in nude mice. These effects were associated with upregulation of proapoptotic proteins caspase-7, -8 and -9, and downregulation of Bcl-2. On the other hand, knockdown of ALX4 expression by siRNA increased cell viability and proliferation, whereas it inhibited apoptosis and cell cycle arrest. In conclusion, our results suggest that ALX4 is a novel putative tumor suppressor with epigenetic silencing in lung carcinogenesis.

HOXC6 Is Deregulated in Human Head and Neck Squamous Cell Carcinoma and Modulates Bcl-2 Expression

Homeobox C6 (HOXC6) genes belong to the homeoprotein family of transcription factors, which play an important role in morphogenesis and cellular differentiation during embryonic development. The aim of this study was to explore the role of HOXC6 in the regulation of Bcl-2 in human head and neck squamous cell carcinoma (HNSCC). The HOXC6 and Bcl-2 gene were identified as being overexpressed in HNSCC tissue and cell lines. Transfection assays demonstrated that HOXC6 increased the levels of Bcl-2 mRNA and protein. A luciferase reporter assay suggested that HOXC6 induced activity of the Bcl-2 promoter. A series of Bcl-2 promoter deletion mutants were examined and the minimal HOXC6-responsive region was identified to be in the TAAT motif (-420 bp) of the Bcl-2 promoter. Interestingly, the inhibition of HOXC6 using siRNA led to the repression of Bcl-2 expression and induced caspase-3-dependent apoptosis; overexpression of HOXC6 in HNSCC cells increased the resistance to paclitaxel-induced apoptosis. Together, our findings suggest that HOXC6 is an important mechanism of the anti-apoptotic pathway via regulation of Bcl-2 expression.

Abstract 5337: The role of Eya3 as a target of EWS/Fli1 in Ewing's sarcoma

Abstract Ewing's sarcoma is an aggressive pediatric bone cancer most commonly affecting patients between 10 and 20 years of age. The vast majority of Ewing's sarcomas harbor a characteristic translocation between chromosomes 11 and 22, encoding the EWS/Fli1 fusion protein. Fli1 is an Ets transcription factor with a DNA-binding domain, whereas the EWS portion of the fusion contributes a transcriptional activation domain. EWS/Fli1 acts as a potent oncogenic transcription factor that is critical in the development of Ewing's sarcoma. EWS/Fli1 knockdown and rescue microarray data suggest that Eya3 may be a downstream target of EWS/Fli1, which we have validated using EWS/Fli1 overexpression and knockdown systems. Eya3 is important in DNA repair, and can interact with the Six family of homeoproteins as a bipartite transcription factor. The Six-Eya complex promotes proliferation and survival to facilitate the proper development of many tissues, but these features can also lead to aggressive cancers when re-expressed in adult tissues. We have shown that Eya3 knockdown in the human tumor-derived Ewing's sarcoma cells line, A673, decreases the rate of proliferation of these cells. Additionally Eya3 knockdown sensitizes Ewing's sarcoma cells to chemotherapeutics currently used to treat Ewing's sarcoma: doxorubicin, etoposide, and vincristine. Furthermore, clonal isolates of A673 Eya3 knockdown cells, expressing very low levels of Eya3, morphologically resemble their presumed cell of origin, the mesenchymal stem cell. These data suggest that Eya3 is an important target of EWS/Fli1 that may serve as a clinically significant novel therapeutic target for the treatment of Ewing's sarcoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5337. doi:10.1158/1538-7445.AM2011-5337

YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein

Precisely controlled cellular differentiation is essential for the proper development of vertebrate embryo and deregulated differentiation is a major cause of many human congenital diseases as well as cancer. Msx1 is a member of the homeoprotein family implicated in these processes, which inhibits the differentiation of skeletal muscle and other cell types, presumably by regulating transcription of target genes through interaction with other cellular factors. We presently show that YB1/p32, a nuclear Y-box binding protein 1, interacts with Msx1 homeoprotein and functions as a regulator of C2C12 myoblast differentiation. We demonstrate that YB1/p32 functionally interacts with Msx1 through its N-terminal region and colocalizes with Msx1 at the nuclear periphery. Moreover, we find that YB1/p32 is competent for inhibition of C2C12 myoblast differentiation, which is correlated with its activity as a negative regulator of MyoD gene expression and binding to the MyoD core enhancer region (CER). Furthermore, YB1/p32 cooperates with Msx1 in transcriptional repression and knocking down the expression of endogenous YB1 attenuates the effects of Msx1. Taken together, our study has uncovered a new function of YB1/p32, a regulator of skeletal muscle differentiation.

Expression of Six1 homeobox gene during development of the mouse submandibular salivary gland

Members of the Six family of homeoproteins are expressed in numerous tissues during vertebrate embryogenesis, and are critical regulators of both cell proliferation and survival. Here we report the temporal and spatial expression of Six1 during maturation of the mouse submandibular salivary gland (SSG) from embryonic day 18.5 (E18.5) to postnatal day 28. Additionally, we examine the role of Six1 during SSG development using Six1-deficient mice. Six1 expression was assessed by reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence. Proliferation was measured by bromodeoxyuridine (BrdU) incorporation index, and apoptosis was evaluated by TUNEL assay. Six1 mRNA and protein levels are high in the epithelial SSG cells at E18.5 and decrease progressively in the postnatal maturing SSG. Although SSGs from Six1(-/-) embryos are significantly smaller than wild type SSGs, the histological structures of the SSG acini and ducts are similar. Six1(-/-) salivary epithelial cells exhibit an intrinsic defect in cell proliferation accompanied by a significant reduction in the Six1 target gene cyclin A1, previously shown to be a critical mediator of Six1-induced proliferation. Our results suggest that the reduction in size of Six1(-/-) SSGs is result of a decrease in cell proliferation during development/maturation.

Biochemical and Functional Characterization of Six SIX1 Branchio-oto-renal Syndrome Mutations

Branchio-oto-renal syndrome (BOR) is an autosomal dominant developmental disorder characterized by hearing loss, branchial arch defects, and renal anomalies. Recently, eight mutations in the SIX1 homeobox gene were discovered in BOR patients. To characterize the effect of SIX1 BOR mutations on the EYA-SIX1-DNA complex, we expressed and purified six of the eight mutants in Escherichia coli. We demonstrate that only the most N-terminal mutation in SIX1 (V17E) completely abolishes SIX1-EYA complex formation, whereas all of the other mutants are able to form a stable complex with EYA. We further show that only the V17E mutant fails to localize EYA to the nucleus and cannot be stabilized by EYA in the cell. The remaining five SIX1 mutants are instead all deficient in DNA binding. In contrast, V17E alone has a DNA binding affinity similar to that of wild type SIX1 in complex with the EYA co-factor. Finally, we show that all SIX1 BOR mutants are defective in transcriptional activation using luciferase reporter assays. Taken together, our experiments demonstrate that the SIX1 BOR mutations contribute to the pathology of the disease through at least two different mechanisms that involve: 1) abolishing the formation of the SIX1-EYA complex or 2) diminishing the ability of SIX1 to bind DNA. Furthermore, our data demonstrate for the first time that EYA: 1) requires the N-terminal region of the SIX1 Six domain for its interaction, 2) increases the level of the SIX1 protein within the cell, and 3) increases the DNA binding affinity of SIX1.

DLX5 overexpression impairs osteogenic differentiation of human bone marrow stromal cells

The transcription factor DLX5 belongs to a family of homeoproteins required for craniofacial morphogenesis and forebrain development. DLX5 is expressed during formation of several skeletal elements such as cartilage, teeth and bone, and its knockout causes severe craniofacial malformations with a delay in the ossification process. Bone marrow contains mesenchymal progenitor cells which may differentiate along multiple pathways, therefore representing an interesting in vitro and in vivo model to study the mesodermal lineage differentiation. Here we report the effect of DLX5 overexpression in ex vivo expanded human bone marrow stromal cells by retroviral infection on the osteogenic lineage differentiation. A reduced mineral deposition was observed in DLX5-transduced cells upon osteogenic induction in culture. When DLX5-transduced cells were implanted in immunodeficient mice, a 60% reduction in bone matrix deposition was observed, whereas the in vitro chondrogenic potential was unaffected. A quantitative gene expression study indicated that DLX5 overexpression does not affect the early osteogenic commitment of bone marrow stromal cells but prevents their terminal differentiation. This block may be mediated by the observed persistent expression of SOX2, a transcription factor known to inhibit osteogenic differentiation.

UNC-4 represses CEH-12/HB9 to specify synaptic inputs to VA motor neurons in C. elegans

In Caenorhabditis elegans, VA and VB motor neurons arise as lineal sisters but synapse with different interneurons to regulate locomotion. VA-specific inputs are defined by the UNC-4 homeoprotein and its transcriptional corepressor, UNC-37/Groucho, which function in the VAs to block the creation of chemical synapses and gap junctions with interneurons normally reserved for VBs. To reveal downstream genes that control this choice, we have employed a cell-specific microarray strategy that has now identified unc-4-regulated transcripts. One of these genes, ceh-12, a member of the HB9 family of homeoproteins, is normally restricted to VBs. We show that expression of CEH-12/HB9 in VA motor neurons in unc-4 mutants imposes VB-type inputs. Thus, this work reveals a developmental switch in which motor neuron input is defined by differential expression of transcription factors that select alternative presynaptic partners. The conservation of UNC-4, HB9, and Groucho expression in the vertebrate motor circuit argues that similar mechanisms may regulate synaptic specificity in the spinal cord.

Amino terminal tyrosine phosphorylation of human MIXL1

Seven members of the Mix family of paired-type homeoproteins regulate mesoderm/endoderm differentiation in amphibians. In mammals, the MIXL1 (Mix. 1 homeobox [Xenopus laevis]-like gene 1) gene is the sole representative of this family. Unlike the amphibian Mix genes that encode an open reading frame of >300 amino acids, mammalian MIXL1 encodes a smaller protein (~230aa). However, mammalian MIXL1 contains a unique proline-rich domain (PRD) with a potential to interact with signal transducing Src homolgy 3 (SH3) domains. Notably, human MIXL1 also contains a unique tyrosine residue Tyr20 that is amino-terminal to the PRD. Here we report that mammalian MIXL1 protein is phosphorylated at Tyr20 and the phosphorylation is dramatically reduced in the absence of PRD. Our findings are consistent with Tyr20 phosphorylation of MIXL1 being a potential regulatory mechanism that governs its activity.

Control of ACAT2 liver expression by HNF1

ACAT catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acids. There are two known genes encoding the two ACAT enzymes, ACAT1 and ACAT2 (also known as Soat1 and Soat2). In adult humans, ACAT1 is present in most tissues, whereas ACAT2 is localized to enterocytes and hepatocytes. In this report, we elucidate the mechanisms that control the liver-specific expression of the human ACAT2 gene. We identified hepatic nuclear factor 1 (HNF1) as an important liver-specific trans-acting element for the human ACAT2 gene using the human hepatocellular carcinoma cell lines HuH7 and HepG2. Targeted deletion of the HNF1 binding site in the DNA sequence abolished not only the basal promoter function in HepG2 and HuH7 cells but also the induction of the ACAT2 promoter by HNF1. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the transcription factors HNF1alpha and HNF1beta interact with this region in the human ACAT2 gene in vitro and in vivo. These data indicate that a) the identified HNF1 binding site serves as a positive regulator sequence, b) the binding site is functionally active both in vivo and in vitro, and c) the transcription factors HNF1alpha and HNF1beta, which bind to this site, play an important part in the regulation of the human ACAT2 promoter.

Muscular Restrictions

In metazoa, homeoproteins are involved in many different developmental processes. Lee et al. (see the Perspective by Cirillo and Zaret) describe how the Msx family of homeoproteins regulate myogenic gene expression during Xenopus embryogenesis. Msx1 homeoprotein associates with the linker histone H1b, which is involved in the structural assembly of chromatin fibers. Msx1 and H1b repress the myogenic regulator MyoD and can inhibit myogenic differentiation in cell culture and in Xenopus animal caps. H. Lee, R. Habas, C. Abate-Shen, Msx1 cooperates with histone H1b for inhibition of transcription and myogenesis. Science 304 , 1675-1678. [Abstract] [Full Text] L. Cirillo, K. Zaret, A linker histone restricts muscle development. Science 304 , 1607-1609 (2004). [Summary] [Full Text]

MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis.

During embryogenesis, differentiation of skeletal muscle is regulated by transcription factors that include members of the Msx homeoprotein family. By investigating Msx1 function in repression of myogenic gene expression, we identified a physical interaction between Msx1 and H1b, a specific isoform of mouse histone H1. We found that Msx1 and H1b bind to a key regulatory element of MyoD, a central regulator of skeletal muscle differentiation, where they induce repressed chromatin. Moreover, Msx1 and H1b cooperate to inhibit muscle differentiation in cell culture and in Xenopus animal caps. Our findings define a previously unknown function for "linker" histones in gene-specific transcriptional regulation.