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Idiopathic intestinal varicosis is a developmental disorder defined by dilated and convoluted submucosal veins in the colon or small bowel. A limited number of families with idiopathic intestinal varices has been reported, but the genetic cause has not yet been identified. We performed whole exome and targeted Sanger sequencing of candidate genes in five intestinal varicosis families. In four families mutations in the RPSA gene were found, a gene previously linked to congenital asplenia. Individuals in these pedigrees had intestinal varicose veins and angiodysplasias, often in combination with asplenia. In a further four generation pedigree that only showed intestinal varicosities, the RPSA gene was normal. Instead, a nonsense mutation in the homeobox gene NKX2-3 was detected which co-segregated with the disease in this large family with a LOD score of 3.3. NKX2-3 is a component of a molecular pathway underlying spleen and gut vasculature development in mice. Our results provide a molecular basis for familial idiopathic intestinal varices. We provide evidence for a relationship between the molecular pathways underlying the development of the spleen and intestinal mucosal vasculature that is conserved between humans and mice. We propose that clinical management of intestinal varices, should include assessment of a functional spleen. This article is protected by copyright. All rights reserved.

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3&minus, /&minus, ) the spleen&rsquo, s histological structure is fundamentally changed, therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3&minus, mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3&minus, and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3&minus, mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3&minus, mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures, in the sera, we found less anti-CCP-IgG2a, IL-17 and IFN&gamma, but more IL-1&beta, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3&minus, mice showed decreased intracellular Ca2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation.

Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX. Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA., Highlighted Article: Analysis of the expression pattern and targets of the human transcription factor LEUTX suggests a two-stage model for embryonic genome activation, also involving DRPX.

The development of peripheral lymphoid tissues from the mesoderm is the result of a complex convergence combining lymphohematopoietic differentiation with the local specification of nonhematopoietic mesenchymal components. Although the various transcriptional regulators with fate-determining effects in diversifying the mobile leukocyte subsets have been thoroughly studied and identified, the tissue-specific determinants promoting the regional differentiation of resident mesenchyme are less understood. Of these factors, various members of the NK-class Nkx paralogues have emerged as key regulators for the organogenesis of spleen and mucosal lymphoid tissues, and recent data have also indicated their involvement in various pathological events, including gut inflammation and hematopoietic malignancies. Here, we summarize available data on the roles of Nkx2-3 in lymphoid tissue development and discuss its possible value as a developmental marker and disease-associated pathogenic trait. The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Z.K. is supported by the ÚNKP-17-4-I New National Excellence Program of the Ministry of Human Capacities and the postdoctoral research grant of the Faculty of Medicine, University of Pécs. This work was supported by OTKA K108429, GINOP-232-15-2016-00050, and EFOP-361-16-2016- 00004 research funds. Sí

International audience; On the basis of phylogenetic analyses, we uncovered a variant of the CDX2 homeobox gene, a major regulator of the development and homeostasis of the gut epithelium, also involved in cancer. This variant, miniCDX2, is generated by alternative splicing coupled to alternative translation initiation, and contains the DNA-binding homeodomain but is devoid of transactivation domain. It is predominantly expressed in crypt cells, whereas the CDX2 protein is present in crypt cells but also in differentiated villous cells. Functional studies revealed a dominant-negative effect exerted by miniCDX2 on the transcriptional activity of CDX2, and conversely similar effects regarding several transcription-independent functions of CDX2. In addition, a regulatory role played by the CDX2 and miniCDX2 homeoproteins on their pre-mRNA splicing is displayed, through interactions with splicing factors. Overexpression of miniCDX2 in the duodenal Brunner glands leads to the expansion of the territory of these glands and ultimately to brunneroma. As a whole, this study characterized a new and original variant of the CDX2 homeobox gene. The production of this variant represents not only a novel level of regulation of this gene, but also a novel way to fine-tune its biological activity through the versatile functions exerted by the truncated variant compared to the full-length homeoprotein. This study highlights the relevance of generating protein diversity through alternative splicing in the gut and its diseases. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Homeodomain proteins are evolutionary conserved proteinspresent in the entire eukaryote kingdom. They execute functions that areessential for life, both in developing and adult organisms. Mosthomeodomain proteins act as transcription factors and bind DNA to controlthe activity of other genes. In contrast to their similar DNA bindingspecificity, homeodomain proteins execute highly diverse and contextdependentfunctions. Several factors, including genome accessibility, DNAshape, combinatorial binding and the ability to interact with manytranscriptional partners, diversify the activity of homeodomain proteinsand culminate in the activation of highly dynamic, context-specifictranscriptional programs. Clarifying how homeodomain transcriptionfactors work is central to our understanding of development, disease andevolution.

PREP1 and PBX1 are homeodomain (HD) transcription factors that play crucial roles in embryonic development. Here, we present the first biophysical characterization of a PREP1 HD, and the NMR spectroscopic study of its DNA binding pocket. The data show that residues flanking the HD participate in DNA binding. The kinetic parameters for DNA binding of individual PREP1 and PBX1 HDs, and of their combination, show that isolated PREP1 and PBX1 HDs bind to DNA in a cooperative manner. A novel PREP1 motif, flanking the HD at the C-terminus, is required for cooperativity.

PTPN2 is a risk gene for Crohn's disease (CD). We investigated whether PTPN2 genetic variants (rs2542151 and rs2542152) were associated with CD in a familial IBD registry. Both rs2542151 and rs2542152 are associated with CD, but not ulcerative colitis (UC). mRNA expression levels of PTPN2 were significantly increased in intestinal tissues (p=0.0493), and nearly significantly increased in B cells (p=0.0889) from CD patients, but not significantly altered in UC. cDNA microarray results found that PTPN2 was down-regulated by NKX2-3 knockdown in human cells. We confirmed this observation by RT-PCR analyses in NKX2-3 knockdown in B cells from IBD patients and human intestinal microvascular endothelial cells (HIMEC). In addition, we found that mRNA expression of another IBD-associated gene, NKX2-3, was increased in intestinal tissues and B cells from CD patients, but not significantly increased in UC patients. A positive correlation was observed between mRNA expression of PTPN2 and NKX2-3 in B cells and in intestinal tissues from both CD and UC patients. These results suggest that PTPN2 may have an important role in CD pathogenesis and may represent a potential diagnostic and therapeutic target for IBD.

Here we used predictive gene expression signatures within a multi-species framework to identify the genes that underlie cardiac cell fate decisions in differentiating embryonic stem cells. We show that the overlapping orthologous mouse and human genes are the most accurate candidate cardiogenic genes as these genes identified the most conserved developmental pathways that characterize the cardiac lineage. An RNAi-based screen of the candidate genes in Drosophila uncovered numerous novel cardiogenic genes. shRNA knockdown combined with transcriptome profiling of the newly-identified transcription factors zinc finger protein 503 and zinc finger E-box binding homeobox 2 and the well-known cardiac regulatory factor NK2 homeobox 5 revealed that zinc finger E-box binding homeobox 2 activates terminal differentiation genes required for cardiomyocyte structure and function whereas zinc finger protein 503 and NK2 homeobox 5 are required for specification of the cardiac lineage. We further demonstrated that an essential role of NK2 homeobox 5 and zinc finger protein 503 in specification of the cardiac lineage is the repression of gene expression programs characteristic of alternative cell fates. Collectively, these results show that orthologous gene expression signatures can be used to identify conserved cardiogenic pathways.

Homeobox genes play crucial roles for the development of multicellular eukaryotes. We have generated a revised list of all homeobox genes for Caenorhabditis elegans and provide a nomenclature for the previously unnamed ones. We show that, out of 103 homeobox genes, 70 are co-orthologous to human homeobox genes. 14 are highly divergent, lacking an obvious ortholog even in other Caenorhabditis species. One of these homeobox genes encodes 12 homeodomains, while three other highly divergent homeobox genes encode a novel type of double homeodomain, termed HOCHOB. To understand how transcription factors regulate cell fate during development, precise spatio-temporal expression data need to be obtained. Using a new imaging framework that we developed, Endrov, we have generated spatio-temporal expression profiles during embryogenesis of over 60 homeobox genes, as well as a number of other developmental control genes using GFP reporters. We used dynamic feedback during recording to automatically adjust the camera exposure time in order to increase the dynamic range beyond the limitations of the camera. We have applied the new framework to examine homeobox gene expression patterns and provide an analysis of these patterns. The methods we developed to analyze and quantify expression data are not only suitable for C. elegans, but can be applied to other model systems or even to tissue culture systems.

Maize KNOTTED (KN1) homeodomain (HD) protein is a well-known mobile transcription factor crucial for stem cell maintenance. Recent studies have revealed that the trihelical HD of KNOTTED1-like homeobox (KNOX) proteins is necessary and sufficient for selective cell-to-cell trafficking. Also, the efficient trafficking ability for HD is likely to be acquired during the evolution of early nonvascular land plants. Here, using the point-mutated HD of KN1 and SHOOT MERISTEMLESS (STM) in the trichome rescue system, together with molecular structure modeling, we have found the evolutionarily conserved amino acid residues, such as arginine in helix α1 and leucine in helix α3, which are essential for intercellular trafficking. Our studies provided important clues for the 3-dimensional protein structure required for cell-to-cell movement of non-cell-autonomous transcription factors.

The homeodomain-containing transcription factor Pitx2 (pituitary homeobox protein 2) is present in many developing embryonic tissues, including the heart. Its homeodomain is responsible for the recognition and binding to target DNA sequences and thus constitutes a major functional unit in the Pitx2 protein. Nuclear magnetic resonance techniques were employed to determine the solution structure of the native Pitx2 homeodomain and a R24H mutant that causes autosomal dominantly inherited ring dermoid of the cornea syndrome. The structures reveal that both isoforms possess the canonical homeodomain fold. However, the R24H mutation results in a 2-fold increase in DNA binding affinity and a 5 °C decrease in thermal stability, while changing the dynamic environment of the homeodomain only locally. When introduced into full-length Pitx2c, the mutation results in an only 25% loss of transactivation activity. Our data correlate well with clinical observations suggesting a milder deficiency for the R24H mutation compared to those of other Pitx2 homeodomain mutations.

Background Many transcription factors control gene expression by binding to specific DNA sequences at or near the genes that they regulate. However, some transcription factors play more global roles in the control of gene expression by altering the architecture of sections of chromatin or even the whole genome. The ability to form oligomeric protein assemblies allows many of these proteins to manipulate extensive segments of DNA or chromatin via the formation of structures such as DNA loops or protein-DNA fibres. Principal findings Here we show that the proline rich homeodomain protein PRH/Hhex forms predominantly octameric and/or hexadecameric species in solution as well as larger assemblies. We show that these assemblies are highly stable resisting denaturation by temperature and chemical denaturants. Conclusion These data indicate that PRH is functionally and structurally related to the Lrp/AsnC family of proteins, a group of proteins that are known to act globally to control gene expression in bacteria and archaea.

Homeobox genes encode transcription factors impacting key developmental processes including embryogenesis, organogenesis, and cell differentiation. Reflecting their tight transcriptional control, homeobox genes are often embedded in large non-coding, cis-regulatory regions, containing tissue specific elements. In T-cell acute lymphoblastic leukemia (T-ALL) homeobox genes are frequently deregulated by chromosomal aberrations, notably translocations adding T-cell specific activatory elements. NKX3-1 is a prostate specific homeobox gene activated in T-ALL patients expressing oncogenic TAL1 or displaying immature T-cell characteristics. After investigating regulation of NKX3-1 in primary cells and cell lines, we report its ectopic expression in T-ALL cells independent of chromosomal rearrangements. Using siRNAs and expression profiling, we exploited NKX3-1 positive T-ALL cell lines as tools to investigate aberrant activatory mechanisms. Our data confirmed NKX3-1 activation by TAL1/GATA3/LMO and identified LYL1 as an alternative activator in immature T-ALL cells devoid of GATA3. Moreover, we showed that NKX3-1 is directly activated by early T-cell homeodomain factor MSX2. These activators were regulated by MLL and/or by IL7-, BMP4- and IGF2-signalling. Finally, we demonstrated homeobox gene SIX6 as a direct leukemic target of NKX3-1 in T-ALL. In conclusion, we identified three major mechanisms of NKX3-1 regulation in T-ALL cell lines which are represented by activators TAL1, LYL1 and MSX2, corresponding to particular T-ALL subtypes described in patients. These results may contribute to the understanding of leukemic transcriptional networks underlying disturbed T-cell differentiation in T-ALL.

Homeobox genes encode a large superclass of transcription factors with widespread roles in animal development. Within chordates there are over 100 homeobox genes in the invertebrate cephalochordate amphioxus and over 200 in humans. Set against this general trend of increasing gene number in vertebrate evolution, some ancient homeobox genes that were present in the last common ancestor of chordates have been lost from vertebrates. Here, we describe the embryonic expression of four amphioxus descendants of these genes—AmphiNedxa, AmphiNedxb, AmphiMsxlxandAmphiNKx7. All four genes are expressed with a striking asymmetry about the left–right axis in the pharyngeal region of neurula embryos, mirroring the pronounced asymmetry of amphioxus embryogenesis.AmphiMsxlxandAmphiNKx7are also transiently expressed in an anterior neural tube region destined to become the cerebral vesicle. These findings suggest significant rewiring of developmental gene regulatory networks occurred during chordate evolution, coincident with homeobox gene loss. We propose that loss of otherwise widely conserved genes is possible when these genes function in a confined role in development that is subsequently lost or significantly modified during evolution. In the case of these homeobox genes, we propose that this has occurred in relation to the evolution of the chordate pharynx and brain.

Background Pancreas organogenesis is the result of well-orchestrated and balanced activities of transcription factors. The homeobox transcription factor PDX-1 plays a crucial role in the development and function of the pancreas, both in the maintenance of progenitor cells and in determination and maintenance of differentiated endocrine cells. However, the activity of homeobox transcription factors requires coordination with co-factors, such as PBX and MEIS proteins. PBX and MEIS proteins belong to the family of three amino acid loop extension (TALE) homeodomain proteins. In a previous study we found that PDX-1 negatively regulates the transcriptional activity of the ductal specific keratin 19 (Krt19). In this study, we investigate the role of different domains of PDX-1 and elucidate the functional interplay of PDX-1 and MEIS1 necessary for Krt19 regulation. Methodology/Principal Findings Here, we demonstrate that PDX-1 exerts a dual manner of regulation of Krt19 transcriptional activity. Deletion studies highlight that the NH2-terminus of PDX-1 is functionally relevant for the down-regulation of Krt19, as it is required for DNA binding of PDX-1 to the Krt19 promoter. Moreover, this effect occurs independently of PBX. Second, we provide insight on how PDX-1 regulates the Hox co-factor MEIS1 post-transcriptionally. We find specific binding of MEIS1 and MEIS2 to the Krt19 promoter using IP-EMSA, and siRNA mediated silencing of Meis1, but not Meis2, reduces transcriptional activation of Krt19 in primary pancreatic ductal cells. Over-expression of PDX-1 leads to a decreased level of MEIS1 protein, and this decrease is prevented by inhibition of the proteasome. Conclusions/Significance Taken together, our data provide evidence for a dual mechanism of how PDX-1 negatively regulates Krt19 ductal specific gene expression. These findings imply that transcription factors may efficiently regulate target gene expression through diverse, non-redundant mechanisms.

In the bipolar basidiomycete Pholiota microspora , a pair of homeodomain protein genes located at the A -mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. microspora to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene ( A3-hox1 or A3-hox2 ) from the A3 monokaryon strain was transformed into the A4 monokaryon strain, the transformants produced many pseudoclamps but very few clamps. When two homeodomain protein genes ( A3-hox1 and A3-hox2 ) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamplike cells in the transformants was significantly increased to ca. 50%. We therefore concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 ( A4 ), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12-163 × NGW19-6). The results of real-time reverse transcription-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. microspora. Thus, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A -mating-type genes alone is sufficient to drive true clamp formation.

The homeobox genes comprise a large and diverse gene superfamily, many of which encode transcription factors with pivotal roles in the embryonic development of animals. We searched the assembled draft genome sequence of an amphioxus, Branchiostoma floridae, for genes possessing homeobox sequences. Phylogenetic analysis was used to divide these into gene families and classes. The 133 amphioxus homeobox genes comprise 60 ANTP class genes, 29 PRD genes (excluding Pon and Pax1/9), nine TALE genes, seven POU genes, seven LIM genes, five ZF genes, four CUT genes, four HNF genes, three SINE genes, one CERS gene, one PROS gene, and three unclassified genes. Ten of the 11 homeobox gene classes are less diverse in amphioxus than humans, as a result of gene duplication on the vertebrate lineage. Amphioxus possesses at least one member for all of the 96 homeobox gene families inferred to be present in the common ancestor of chordates, including representatives of the Msxlx, Bari, Abox, Nk7, Ro, and Repo gene families that have been lost from tunicates and vertebrates. We find duplication of several homeobox genes in the cephalochordate lineage (Mnx, Evx, Emx, Vent, Nk1, Nedx, Uncx, Lhx2/9, Hmbox, Pou3, and Irx) and several divergent genes that probably originated by extensive sequence divergence (Hx, Ankx, Lcx, Acut, Atale, Azfh, Ahbx, Muxa, Muxb, Aprd1-6, and Ahnf). The analysis reveals not only the repertoire of amphioxus homeobox genes but also gives insight into the evolution of chordate homeobox genes.

Several tissue-specific regulatory genes have been found to play essential roles in both organogenesis and carcinogenesis. In the prostate, the Nkx3.1 homeobox gene plays an important role in normal differentiation of the prostatic epithelium while its loss of function is an initiating event in prostate carcinogenesis in both mouse models and human patients. Thus, the Nkx3.1 homeobox gene provides a paradigm for understanding the relationship between normal differentiation and cancer, as well as studying the roles of homeobox genes in these processes. Here, we review recent findings concerning the roles of Nkx3.1 in development and discuss how its normal function is disrupted in processes of early prostate carcinogenesis.

Background Members of the Paired-like homeodomain transcription factor (PITX) gene family, particularly PITX1 and PITX2, play important roles in normal development and in differentiated cell functions. Three major isoforms of PITX2 were previously reported to be produced through both alternative mRNA splicing (PITX2A and PITX2B) and alternative promoter usage (PITX2C). The proteins derived from these mRNAs contain identical homeodomain and carboxyl termini. Differences in the amino-termini of the proteins may confer functional differences in some contexts. Results Here, we report the identification of two novel PITX2 isoforms. First, we demonstrate that the Pitx2c mRNA generates two protein products, PITX2Cα and PITX2Cβ, via alternative translation initiation. Second, we identified a novel mRNA splice variant, Pitx2b2, which uses the same 5' splice donor in intron 2 as Pitx2b (hereafter referred to as Pitx2b1), but employs an alternative 3' splice acceptor, leading to an in-frame deletion of 39 base pairs relative to Pitx2b1. Pitx2b2 mRNA is expressed in both murine and human pituitary. The data show that in a murine gonadotrope cell line and adult murine pituitary what was previously thought to be PITX2B1 is actually PITX2Cβ, or perhaps PITX2B2. PITX2B1 is expressed at lower levels than previously thought. PITX2Cβ and PITX2B2 activate gonadotrope-specific gene promoter-reporters similarly to known PITX2 isoforms. Conclusion We have identified and characterized two novel isoforms of PITX2, generated by alternative translation initiation (PITX2Cβ) and alternative mRNA splicing (PITX2B2). These proteins show similar DNA binding and trans-activation functions as other PITX2 isoforms in vitro, though their conservation across species suggests that they may play distinct, as yet unidentified, roles in vivo.

The transcription efficiency of an adhesion protein gene, ap65 - 1 , in Trichomonas vaginalis varies with changes in the iron supply and with the growth stage. In the present study, two Myb recognition elements, MRE-1/MRE-2r and MRE-2f, were found to play antagonistic roles in regulating the iron-inducible activity of an ap65 - 1 reporter gene. Intriguingly, either of these elements was shown to be sufficient to repress basal activity, but together they were also shown to activate growth-related activity of the reporter gene in iron-depleted cells. A myb1 gene which encodes a 24-kDa protein containing a Myb-like R2R3 DNA binding domain was identified from Southwestern screening of MRE-2f-binding proteins. The Myb1 protein was detected as a major 35-kDa protein which exhibited variations in nuclear concentration with changes in the iron supply. A recombinant Myb1 protein was shown to differentially interact with MRE-1/MRE-2r and MRE-2f in vitro. Overexpression of hemagglutinin-tagged Myb1 in T. vaginalis resulted in repression or activation of ap65 - 1 transcription in iron-depleted cells at an early and a late stage of cell growth, respectively, while iron-inducible ap65 - 1 transcription was constitutively repressed. The hemagglutinin-tagged Myb1 protein was found to constantly occupy the chromosomal ap65 - 1 promoter at a proximal site, but it also selected two more distal sites only at the late growth stage. Together, these observations suggest that Myb1 critically regulates multifarious ap65 - 1 transcription, possibly via differential selection of multiple promoter sites upon environmental changes.

Cardiac development requires the action of transcription factors, which control the specification and differentiation of cardiac cell types. One of these factors, encoded by the homeobox gene tinman (tin), is essential for the specification of all cardiac cells in Drosophila. An increasing number of examples show that protein-protein interactions can be important for determining the specific transcriptional activities of homeodomain proteins, in addition to their binding to specific DNA target sites. Here, we show that Tin and Bagpipe (Bap), another homeodomain protein, form homo- and heterodimeric complexes. We demonstrate that homo- and heterodimerization of Tin is mediated through its homeodomain and that the region required for this interaction corresponds to the first two helices that are also necessary for DNA binding. We further show that, in the yeast system, the homeodomain can function as a transcriptional repressor domain. These findings suggest that protein-protein interactions of Tin play a role in its transcriptional and developmental functions.

The homeodomain (encoded by the homeobox) is the DNA-binding domain of a large variety of transcriptional regulators involved in controlling cell fate decisions and development. Mutations of homeobox-containing genes cause several diseases in humans. A variety of missense mutations giving rise to human diseases have been described. These mutations are an excellent model to better understand homeodomain molecular functions. To this end, homeobox missense mutations giving rise to human diseases are reviewed. Seventy-four independent homeobox mutations have been observed in 17 different genes. In the same genes, 30 missense mutations outside the homeobox have been observed, indicating that the homeodomain is more easily affected by single amino acids changes than the rest of the protein. Most missense mutations have dominant effects. Several data indicate that dominance is mostly due to haploinsufficiency. Among proteins having the homeodomain as the only DNA-binding domain, three “hot spot” regions can be delineated: 1) at codon encoding for Arg5; 2) at codon encoding for Arg31; and 3) at codons encoding for amino acids of recognition helix. In the latter, mutations at codons encoding for Arg residues at positions 52 and 53 are prevalent. In the recognition helix, Arg residues at positions 52 and 53 establish contacts with phosphates in the DNA backbone. Missense mutations of amino acids that contribute to sequence discrimination (such as those at positions 50 and 54) are present only in a minority of cases. Similar data have been obtained when missense mutations of proteins possessing an additional DNA-binding domain have been analyzed. The only exception is observed in the POU1F1 (PIT1) homeodomain, in which Arg58 is a “hot spot” for mutations, but is not involved in DNA recognition. Hum Mutat 18:361–374, 2001. © 2001 Wiley-Liss, Inc.

PCR-based methods and mobility shift competition assays were used to determine the basic biochemical features of the homeodomain transcription factor Distal-less 3 (Dlx3), including an optimal DNA binding site, the binding constant and dissociation rates of this protein. Expression of Dlx3 protein in either HeLa cells or Xenopus embryos resulted in strong activation of a model target gene construct containing three tandem copies of the Dlx3 binding site upstream from the TATA element. In addition, deletion analysis revealed that transcriptional activation by Dlx3 depends on two subdomains located on either side of the homeobox: removal of either subdomain resulted in complete loss of Dlx3 function. These observations provide new insight regarding the function of Dlx3 in vertebrate development and tissue differentiation and also suggest a mechanism for the dominant inheritance pattern of a hereditary disease resulting from mutation of the DLX3 gene in human.

Chromosomal translocations in acute leukemias frequently involve activation of genes encoding proteins involved in transcription (2, 18, 19). The HOX11 homeobox gene was first identified by cloning the breakpoints of t(7;10)(q35;q24) and t(10;14)(q24;q11) chromosomal translocations, found in 5 to 10% of patients with acute T-cell leukemia (5, 9, 13, 15). These translocations result in the HOX11 gene, which is normally found on chromosome 10, band q24, being placed within the transcriptional control region of the T-cell receptor δ gene on chromosome 14 or the T-cell receptor δ gene on chromosome 7q35 (13, 29). The abnormal expression of HOX11 in T cells as a result of these chromosomal translocations in humans is thought to be a key step in the progression toward malignancy (5, 9, 13, 15), a view reinforced by the demonstration of oncogenic activity of HOX11 in transplant recipients of MSCV-HOX11-transduced bone marrow cells (10, 11). The HOX11 gene belongs to a family of homeodomain-encoding genes which includes the related Hox11L1 and Hox11L2 genes first characterized in the mouse (4). The homeodomain is conserved within this family, and, like other homeodomain proteins, the HOX11 family has been implicated in the regulation of cellular growth and differentiation. In mice, Hox11 is essential for generation of the spleen (3, 21), promoting survival of the splenic precursor cells (3), and null mutation of the Hox11L1 gene results in myenteric neuronal hyperplasia and megacolon (25). Since the homeodomain is a sequence-specific DNA-binding element, most homeodomain-containing proteins are believed to function through trans regulation (direct or indirect) of specific target genes (16). Consistent with this, HOX11 is a nuclear protein and can bind to DNA in vitro (4, 26) and a fusion protein consisting of HOX11 fused to the GAL4 DNA-binding domain (GAL4-DBD) was found to transactivate various promoters in both yeast and mammalian cells (28). Three distinct domains of HOX11 were found to be necessary for transactivation by this fusion protein (28), namely, the glycine-proline-rich region at the NH2 terminus, the glutamine-rich region at the COOH terminus, and the homeodomain itself. However, the only data which pertain to functional characteristics in non-artificial reporter assays are that the NH2- and COOH-terminal regions of HOX11 appear dispensable for transforming function (11) and that introduction of HOX11 into NIH 3T3 cells results in transcription of a HOX11-dependent gene (denoted Hdg-1 [8]). Therefore, we have assessed regions of HOX11 required for optimal in vivo transactivation of the endogenous Hdg-1 gene. In this stringent assay, we show that deletions in the HOX11 homeodomain (in either the NH2-terminal arm or the third helix) prevent induction of Hdg-1 by HOX11. We also show that the NH2-terminal 50- amino-acid stretch is crucial for optimal function of the HOX11 protein in vivo.

A subfamily of bicoid -related homeodomain factors was recently discovered through its involvement in transcription of pituitary-specific genes. We isolated the first member of this family, Ptx1 (pituitary homeobox 1), through its DNA binding properties whereas a second related gene, Ptx2 (RIEG), was identified by positional cloning as the causative gene for Rieger's syndrome. The mechanisms of Ptx action on its target genes as well as its putative roles during development are reviewed with particular emphasis on its role in pituitary function.

The homeodomain containing thyroid transcription factor 1 (TTF-1) is a lung- and thyroid-enriched protein implicated in the regulation of a number of pulmonary specific genes. Within the lung TTF-1 is expressed within the epithelial cells. Although the molecular mechanisms that govern this tight cell-type-specific distribution are unclear, transient transfection studies have suggested that tissue specificity is conferred in part by regions of the proximal promoter. Further studies have shown that two functionally important regions (BS1 and BS2) are sites for activation of the TTF-1 gene by the homeodomain protein HoxB3, raising the possibility that Hox proteins might function in the regulation of TTF-1 in vivo. The different cellular distributions of the two proteins within the lung suggest, however, that proteins distinct from HoxB3 might be the mediators of expression through these sites. In the present study we have used gel-mobility-shift experiments to show that in a pulmonary adenocarcinoma cell line (NCI-H441) that expresses TTF-1, the same single protein binds to both of these sites. The binding of this protein is competed for specifically by the addition of oligonucleotides containing a range of octamer-binding sites but not by a variety of non-related binding sites. Using specific antiserum we have identified this protein as being the ubiquitously expressed POU-domain protein Oct-1. Reverse transcriptase-PCR performed with degenerated primers suggests that Oct-1 is the major POU-domain-containing protein expressed in H441 cells. These results suggest that BS1 and BS2 are functional octamer sites and might therefore be implicated in the basal rather than the tissue-restricted expression of the TTF-1 gene.

even-skipped is a homeobox gene important in controlling segment patterning in the embryonic fruit fly. Its homeobox encodes a DNA binding domain which binds with similar affinities to two DNA consensus sequences, one AT-rich, the other GC-rich. We describe a crystallographic analysis of the Even-skipped homeodomain complexed to an AT-rich oligonucleotide at 2.0 A resolution. The structure reveals a novel arrangement of two homeodomains bound to one 10 bp DNA sequence in a tandem fashion. This arrangement suggests a mechanism for the homeoproteins' regulatory specificity. In addition, the functionally important residue Gln50 is observed in multiple conformations making direct and water-mediated hydrogen bonds with the DNA bases.

The A mating type genes of the mushroom Coprinus cinereus encode two classes of putative transcription factor with distinctive homeodomain motifs (HD1 and HD2). A successful mating brings together different allelic forms of these genes and this triggers part of a developmental sequence required for sexual reproduction. In this report we provide evidence that this developmental programme is promoted by a physical interaction between the two classes of homeodomain protein. Rare dominant mutations conferring self-compatibility map to the A locus and result in constitutive operation of the A-regulated developmental pathway. Our molecular analysis of one of these mutations shows that it has generated a chimeric gene by inframe fusion of an HD2 and an HD1 gene. Fusion has overcome the normal incompatibility between two proteins coded by genes of the same A locus and generated a protein that is sufficient to promote development in the absence of any other active A mating type genes. The fusion protein retains most of the HD2 sequence, but only the C-terminal part of the HD1 protein. It has only the HD2 homeodomain motif as a potential DNA binding domain fused to an essential C-terminal region of the HD1 protein, which in a normal HD1-HD2 protein complex may be the major activation domain.

Background Many homeobox genes show remarkable conservation between divergent animal phyla. In contrast, the ARGFX (Arginine-fifty homeobox) homeobox locus was identified in the human genome but is not present in mouse or invertebrates. Here we ask when and how this locus originated and examine its pattern of molecular evolution. Results Phylogenetic and phylogenomic analyses suggest that ARGFX originated by gene duplication from Otx1, Otx2 or Crx during early mammalian evolution, most likely on the stem lineage of the eutherians. ARGFX diverged extensively from its progenitor homeobox gene and its exons have been functional and subject to purifying selection through much of placental mammal radiation. Surprisingly, the coding region is disrupted in most mammalian genomes analysed, with human being the only mammal identified in which the full open reading frame is retained. Indeed, we describe a transcript from human testis that has the potential to encode the full deduced protein. Conclusions The unusual pattern of evolution suggests that the ARGFX gene may encode a functional RNA or alternatively it may have 'flickered' between functional and non-functional states in the evolutionary history of mammals, particularly in the period when many mammalian lineages diverged within a relatively short time span.

A new maize homeobox gene was isolated by screening a lambda gt11 expression library with the 26 bp Shrunken feedback control element. Zmhox1a (Zea mays homeobox) is an unidentified maize gene mapping to the long arm of chromosome 8. It is a member of a new class of maize homeobox genes only distantly related to the Knotted class. The 3.1 kb Zmhox1a transcript can be detected in different maize tissues and encodes a polypeptide of 719 amino acids. Western blotting experiments detect the native 112 or 115 kDa protein in nuclear protein extracts, the nuclear localization being compatible with a function in transcriptional control. No Zmhox1a protein is detected in maize roots despite the presence of the Zmhox1a transcript; this may indicate a post-transcriptional control mechanism. A highly acidic central region of the Zmhox1a polypeptide implies a transcriptional activator function. The carboxy-terminal part of the maize homeodomain protein is related to the human Oct2 transcription factor, but homology to the POU specific domain is restricted to the POU-B subdomain. It was confirmed by DNase I footprinting experiments that DNA binding of the Zmhox1a homeodomain was at three sites flanking the TATA-box of the Shrunken promoter.

This paper reports the cloning and sequencing of a quail homeobox-containing gene, Quox-1, and its expression pattern in embryos from 3 to 6 days (E3 to E6) of development as determined by in situ hybridization. The opening reading frame in cDNA clone g11 corresponds to a predicted protein of 242 amino acids. Quox-1 protein displays high sequence similarity to the Antennapedia family, especially to the mouse homeodomain-containing protein Hox-1.1 (100% identity in the homeobox region, 77% at the 5' end beyond the homeobox). However, the carboxyl-terminal domain of the postulated protein has no significant homology with other known homeoproteins, including Hox-1.1. In situ hybridization experiments showed that Quox-1 is widely expressed in the developing central nervous system including the entire brain and the spinal cord. Outside the central nervous system, transcription of Quox-1 was mainly detected in the endoderm-derived epithelium of esophagus, trachea, and other digestive organs, as well as in the sensory epithelium of the olfactory region and perichondrium of the vertebrae. Thus, Quox-1 transcripts have a remarkably wide distribution that, unlike the other vertebrate homeobox genes examined to date, encompasses the rostral part of the developing nervous system, including the forebrain.

The cDNA for TTF-1, a thyroid nuclear factor that binds to the promoter of thyroid specific genes, has been cloned. The protein encoded by the cDNA shows binding properties indistinguishable from those of TTF-1 present in nuclear extracts of differentiated rat thyroid cells. The DNA binding domain of TTF-1 is a novel mammalian homeodomain that shows considerable sequence homology to the Drosophila NK-2 homeodomain. TTF-1 mRNA and corresponding binding activity are detected in thyroid and lung. The chromosomal localization of the TTF-1 gene has been determined in humans and mice and corresponds to chromosomes 14 and 12, respectively, demonstrating that the TTF-1 gene is not located within previously described clusters of homeobox-containing genes.