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The first TALE homeodomain transcription factor gene to be described in plants was maize knotted1 (kn1). Dominant mutations in kn1 disrupt leaf development, with abnormal knots of tissue forming in the leaf blade. kn1 was found to be expressed in the shoot meristem but not in a peripheral region that gives rise to leaves. Furthermore, KN1 and closely related proteins were excluded from initiating and developing leaves. These findings were a prelude to a large body of work wherein TALE homeodomain proteins have been identified as vital regulators of meristem homeostasis and organ development in plants. KN1 homologues are widely represented across land plant taxa. Thus, studying the regulation and mechanistic action of this gene class has allowed investigations into the evolution of diverse plant morphologies. This review will focus on the function of TALE homeodomain transcription factors in leaf development in eudicots. Here, we discuss how TALE homeodomain proteins contribute to a spectrum of leaf forms, from the simple leaves of Arabidopsis thaliana to the compound leaves of Cardamine hirsuta and species beyond the Brassicaceae. [ABSTRACT FROM AUTHOR]

Floral organ abscission in Arabidopsis thaliana is regulated by the putative ligand-receptor system comprising the signaling peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and the two receptor-like kinases HAESA and HAESA-LIKE2. The IDA signaling pathway presumably activates a MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade to induce separation between abscission zone (AZ) cells. Misexpression of IDA effectuates precocious floral abscission and ectopic cell separation in latent AZ cell regions, which suggests that negative regulators are in place to prevent unrestricted and untimely AZ cell separation. Through a screen for mutations that restore floral organ abscission in ida mutants, we identified three new mutant alleles of the KNOTTED - LIKE HOMEOBOX gene BREVIPEDICELLUS (BP)/ KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1). Here, we show that bp mutants, in addition to shedding their floral organs prematurely, have phenotypic commonalities with plants misexpressing IDA , such as enlarged AZ cells. We propose that BP / KNAT1 inhibits floral organ cell separation by restricting AZ cell size and number and put forward a model whereby IDA signaling suppresses BP/KNAT1, which in turn allows KNAT2 and KNAT6 to induce floral organ abscission. [ABSTRACT FROM AUTHOR]

Brassinosteroid (BR) phytohormones play crucial roles in regulating internode elongation in rice (Oryza sativa). However, the underlying mechanism remains largely unclear. The dwarf and low-tillering (dlt) mutant is a mild BR-signaling-defective mutant. Here, we identify two dlt enhancers that show more severe shortening of the lower internodes compared to the uppermost internode (IN1). Both mutants carry alleles of ORYZA SATIVA HOMEOBOX 15 (OSH15), the founding gene for dwarf6-type mutants, which have shortened lower internodes but not IN1. Consistent with the mutant phenotype, OSH15 expression is much stronger in lower internodes, particularly in IN2, than IN1. The osh15 single mutants have impaired BR sensitivity accompanied by enhanced BR synthesis in seedlings. DLT physically interacts with OSH15 to co-regulate many genes in seedlings and internodes. OSH15 targets and promotes the expression of the BR receptor gene BR INSENSITIVE1 (OsBRI1), and DLT facilitates this regulation in a dosage-dependent manner. In osh15, dlt, and osh15 dlt, BR levels are higher in seedlings and panicles, but unexpectedly lower in internodes compared with the wild-type. Taken together, our results suggest that DLT interacts with OSH15, which functions in the lower internodes, to modulate rice internode elongation via orchestrating BR signaling and metabolism. [ABSTRACT FROM AUTHOR]

It compares proteins from krueppel C2H2-type zinc-finger protein family (upper sequences) with proteins from other families (bottom sequences) HT

Shoot branching is an important factor that influences the architecture of apple trees and cytokinin is known to promote axillary bud outgrowth. The cultivar 'Fuji', which is grown on ~75% of the apple-producing area in China, exhibits poor natural branching. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes BRANCHED1/2 (BRC1/2) are involved in integrating diverse factors that function locally to inhibit shoot branching; however, the molecular mechanism underlying the cytokinin-mediated promotion of branching that involves the repression of BRC1/2 remains unclear. In this study, we found that apple WUSCHEL2 (MdWUS2), which interacts with the co-repressor TOPLESS-RELATED9 (MdTPR9), is activated by cytokinin and regulates branching by inhibiting the activity of MdTCP12 (a BRC2 homolog). Overexpressing MdWUS2 in Arabidopsis or Nicotiana benthamiana resulted in enhanced branching. Overexpression of MdTCP12 inhibited axillary bud outgrowth in Arabidopsis , indicating that it contributes to the regulation of branching. In addition, we found that MdWUS2 interacted with MdTCP12 in vivo and in vitro and suppressed the ability of MdTCP12 to activate the transcription of its target gene, HOMEOBOX PROTEIN 53b (MdHB53b). Our results therefore suggest that MdWUS2 is involved in the cytokinin-mediated inhibition of MdTCP12 that controls bud outgrowth, and hence provide new insights into the regulation of shoot branching by cytokinin. [ABSTRACT FROM AUTHOR]

LEUTX is a homeodomain transcription factor expressed in the very early embryo with a function around embryonic genome activation. The LEUTX gene is found only in eutherian mammals including humans but, unlike the majority of homeobox genes, the encoded amino acid sequence is very different between divergent mammalian species. However, whether dynamic evolution has also occurred between closely related mammalian species remains unclear. In this work, we perform a comparative genomics study of LEUTX within the primates, revealing dramatic evolutionary sequence change between closely related species. Positive selection has acted on sites in the LEUTX protein, including six sites within the homeodomain; this suggests that selection has driven changes in the set of downstream targets. Transfection into cell culture followed by transcriptomic analysis reveals small functional differences between human and marmoset LEUTX, suggesting rapid sequence evolution has fine-tuned the role of this homeodomain protein within the primates. [ABSTRACT FROM AUTHOR]

In plants, the stem cells that form the shoot system reside within the shoot apical meristem (SAM), which is regulated by feedback signaling between the WUSCHEL (WUS) homeobox protein and CLAVATA (CLV) peptides and receptors. WUS–CLV feedback signaling can be modulated by various endogenous or exogenous factors, such as chromatin state, hormone signaling, reactive oxygen species (ROS) signaling and nutrition, leading to a dynamic control of SAM size corresponding to meristem activity. Despite these insights, however, the knowledge of genes that control SAM size is still limited, and in particular, the regulation by ROS signaling is only beginning to be comprehended. In this study, we report a new function in maintenance of SAM size, encoded by the OKINA KUKI1 (OKI1) gene. OKI1 is expressed in the SAM and encodes a mitochondrial aspartyl tRNA synthetase (AspRS). oki1 mutants display enlarged SAMs with abnormal expression of WUS and CLV3 and overaccumulation of ROS in the meristem. Our findings support the importance of normal AspRS function in the maintenance of the WUS–CLV3 feedback loop and SAM size. [ABSTRACT FROM AUTHOR]

Prostate cancer is the most common cancer among men, with the most common metastatic sites in bone, regional lymph nodes, liver and thorax. It is most commonly diagnosed in the early stages with clinical findings of enlarged prostate on digital rectal exam and positive prostate specific antigen. Distant metastases associated with prostate cancer commonly occur to bone. It is imperative to be cautious in assuming primary breast, lung or head and neck malignancy in patients presenting with lymphadenopathy in the upper aerodigestive pathways. Cervical lymphadenopathy due to prostate cancer is becoming more prevalent since previously reported. Here we present a case of prostate cancer recurrence found through metastasis to supraclavicular lymph nodes and we also highlight homeobox protein CDX2 as a potential clinico-pathological marker in metastatic prostate cancer. [ABSTRACT FROM AUTHOR]

Paired-homeodomain transcription factor 4 (PAX4) gene encodes a transcription factor which plays an important role in the generation, differentiation, development, and survival of insulin-producing b-cells during mammalian pancreas development. PAX4 is a key diabetes mellitus (DM) susceptibility gene, which is associated with many different types of DM, including T1DM, T2DM, maturity onset diabetes of the young 9 (MODY9) and ketosis prone diabetes. In this study, a novel PAX4 gene knockout (KO) model was generated through co-injection of clustered regularly interspaced short palindromic repeats (CRISPR)- associated protein 9 (Cas9) mRNA/sgRNA into rabbit zygotes. Typical phenotypes of growth retardation, persistent hyperglycemia, decreased number of insulin-producing b cells and increased number of glucagon- producing a cells were observed in the homozygous PAX4 KO rabbits. Furthermore, DM associated phenotypes including diabetic nephropathy, hepatopathy, myopathy and cardiomyopathy were also observed in the homozygous PAX4 KO rabbits but not in the wild type (WT) controls and the heterozygous PAX4 KO rabbits. In summary, this is the first PAX4 gene KO rabbit model generated by CRISPR/Cas9 system. This novel rabbit model may provide a new platform for function study of PAX4 gene in rabbit and gene therapy of human DM in clinical trails [ABSTRACT FROM AUTHOR]

Eutherian Totipotent Cell Homeobox (ETCHbox) genes are mammalian-specific PRD-class homeobox genes with conserved expression in the preimplantation embryo but fast-evolving and highly divergent sequences. Here, we exploit an ectopic expression approach to examine the role of bovine ETCHbox genes and show that ARGFX and LEUTX homeodomain proteins upregulate genes normally expressed in the blastocyst; the identities of the regulated genes suggest that, in vivo, the ETCHbox genes play a role in coordinating the physical formation of the blastocyst structure. Both genes also downregulate genes expressed earlier during development and genes associated with an undifferentiated cell state, possibly via the JAK/STAT pathway. We find evidence that bovine ARGFX and LEUTX have overlapping functions, in contrast to their antagonistic roles in humans. Finally, we characterize a mutant bovine ARGFX allele which eliminates the homeodomain and show that homozygous mutants are viable. These data support the hypothesis of functional overlap between ETCHbox genes within a species, roles for ETCHbox genes in blastocyst formation and the change of their functions over evolutionary time. [ABSTRACT FROM AUTHOR]

We investigated the role of hematopoietically expressed homeobox protein (Hhex) in osteoclast development. Trimethylation of lysine 27 of histone H3 at the cis‐regulatory element of Hhex was maintained and that of lysine 4 was reduced during receptor activator of nuclear factor κB ligand (RANKL)‐induced osteoclastogenesis, which was associated with a reduction of Hhex expression. Overexpression of Hhex in bone marrow–derived macrophages inhibited, whereas Hhex suppression promoted, RANKL‐induced osteoclastogenesis in vitro. Conditional deletion of Hhex in osteoclast‐lineage cells promoted osteoclastogenesis and reduced cancellous bone volume in mice, confirming the negative regulatory role of Hhex in osteoclast differentiation. Expression of cyclin‐dependent kinase inhibitors such as Cdkn2a and Cdkn1b in osteoclast precursors was negatively regulated by Hhex, and Hhex deletion increased the ratio of cells at the G1 phase of the cell cycle. In conclusion, Hhex is an inhibitor of osteoclast differentiation that is regulated in an epigenetic manner and regulates the cell cycle of osteoclast precursors and the skeletal homeostasis. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Class 1 KNOTTED1-LIKE HOMEOBOX (KNOXI) genes encode transcription factors that are expressed in the shoot apical meristem (SAM) and are essential for SAM maintenance. In some species with compound leaves, including tomato (Solanum lycopersicum), KNOXI genes are also expressed during leaf development and affect leaf morphology. To dissect the role of KNOXI proteins in leaf patterning, we expressed in tomato leaves a fusion of the tomato KNOXI gene Tkn2 with a sequence encoding a repressor domain, expected to repress common targets of tomato KNOXI proteins. This resulted in the formation of small, narrow, and simple leaves due to accelerated differentiation. Overexpression of the wild-type form of Tkn1 or Tkn2 in young leaves also resulted in narrow and simple leaves, but in this case, leaf development was blocked at the initiation stage. Expression of Tkn1 or Tkn2 during a series of spatial and temporal windows in leaf development identified leaf initiation and primary morphogenesis as specific developmental contexts at which the tomato leaf is responsive to KNOXI activity. Arabidopsis thaliana leaves responded to overexpression of Arabidopsis or tomato KNOXI genes during the morphogenetic stage but were largely insensitive to their overexpression during leaf initiation. These results imply that KNOXI proteins act at specific stages within the compound-leaf development program to delay maturation and enable leaflet formation, rather than set the compound leaf route. [ABSTRACT FROM AUTHOR]

Hypomyelinating leukodystrophies are genetically heterogeneous disorders with overlapping clinical and neuroimaging features reflecting variable abnormalities in myelin formation. We report on the identification of biallelic inactivating mutations in NKX6-2, a gene encoding a transcription factor regulating multiple developmental processes with a main role in oligodendrocyte differentiation and regulation of myelin-specific gene expression, as the cause underlying a previously unrecognized severe variant of hypomyelinating leukodystrophy. Five affected subjects (three unrelated families) were documented to share biallelic inactivating mutations affecting the NKX6-2 homeobox domain. A trio-based whole exome sequencing analysis in the first family detected a homozygous frameshift change [c.606delinsTA; p.(Lys202Asnfs*?)]. In the second family, homozygosity mapping coupled to whole exome sequencing identified a homozygous nucleotide substitution (c.565G>T) introducing a premature stop codon (p.Glu189*). In the third family, whole exome sequencing established compound heterozygosity for a non-conservative missense change affecting a key residue participating in DNA binding (c.599G>A; p.Arg200Gln) and a nonsense substitution (c.589C>T; p.Gln197*), in both affected siblings. The clinical presentation was homogeneous, with four subjects having severe motor delays, nystagmus and absent head control, and one individual showing gross motor delay at the age of 6 months. All exhibited neuroimaging that was consistent with hypomyelination. These findings define a novel, severe form of leukodystrophy caused by impaired NKX6-2 function. [ABSTRACT FROM AUTHOR]

Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS domain protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA binding specificity of floral MADS domain proteins. Differential DNA binding of MADS domain protein complexes plays a role in the specificity of target gene regulation. [ABSTRACT FROM AUTHOR]

Shoot-borne crown roots are the major root system in cereals. Previous work has shown that the Wuschel-related homeobox gene WOX11 is necessary and sufficient to promote rice (Oryza sativa) crown root emergence and elongation. Here, we show that WOX11 recruits the ADA2-GCN5 histone acetyltransferase module to activate downstream target genes in crown root meristem. Rice ADA2 and GCN5 genes are highly expressed in root meristem and are shown to be essential for cell division and growth. WOX11 and ADA2-GCN5 commonly target and regulate a set of root-specific genes involved in energy metabolism, cell wall biosynthesis, and hormone response, some of which are known to be important for root development. The results indicate that the recruitment of ADA2-GCN5 by WOX11 establishes gene expression programs of crown root meristem cell division and suggest that permissive chromatin modification involving histone acetylation is a strategy for WOX11 to stimulate root meristem development. [ABSTRACT FROM AUTHOR]

Study Question: Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility?Summary Answer: MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples.What Is Known Already: Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility.Study Design, Size Duration: MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and β-catenin in epithelial cells.Participants/materials, Setting Methods: MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases.Main Results and the Role Of Chance: MSX1 transcript increased 5-fold (P < 0.05) between the late-proliferative and early secretory phase and was then down-regulated (P < 0.05) prior to receptivity for implantation. In fertile patients, MSX1 protein displayed strong nuclear localization in the luminal epithelium and glands, while it was weakly expressed in nuclei of the stroma. MSX1 protein levels accumulated throughout the secretory phase in all endometrial cellular compartments. MSX1 protein decreased (P < 0.05) in the glands between mid- and late-secretory phases. However, infertile patients demonstrated a broad reduction (P < 0.001) of MSX1 accumulation in all cell types throughout the secretory phase that was most pronounced (∼3-fold) in stroma and glands. Infertility was associated with persistent co-localization of E-cadherin and β-catenin in epithelial cell junctions in the mid- and late-secretory phases.Limitations, Reasons For Caution: Details of the infertility diagnoses and other patient demographic data were not available. Therefore, patients with uterine abnormalities (Mullerian) could not be distinguished from other sources of infertility. Antibody against human MSX2 is not available, limiting the study to MSX1. However, both RNAs in the human endometrium are similarly regulated. In mice, Msx1 and Msx2 are imperative for murine embryo implantation, with Msx2 compensating for genetic ablation of Msx1 through its up-regulation in a knockout model.Wider Implications Of the Findings: This investigation establishes that the MSX1 homeobox protein accumulation is associated with the secretory phase in endometrium of fertile couples, and is widely disrupted in infertile patients. It is the first study to examine MSX1 protein localization in the human endometrium, and supported by genetic findings in mice, suggests that genes regulated by MSX1 are linked to the loss of epithelial cell polarity required for uterine receptivity during implantation.Study Funding/competing Interests: This research was supported by the NICHD National Cooperative Reproductive Medicine Network grant HD039005 (M.P.D.), NIH grants HD068524 (S.K.D.), HD071408 (D.R.A., M.P.D.), and HL128628 (S.D.), the Intramural Research Program of the NICHD, March of Dimes (S.K.D., S.D.) and JSPS KAKENHI grant 26112506 (Y.H.). There were no conflicts or competing interests. [ABSTRACT FROM AUTHOR]

Aims Atrial fibrillation (AF) produces rapid changes in the electrical properties of the atria (electrical remodelling) that promote its own recurrence. In chronic AF (CAF) patients, up-regulation of the slow delayed rectifier K+ current (Iks) and down-regulation of the voltage-gated Ca2+ current (ICa,L) are hallmarks of electrical remodelling and critically contribute to the abbreviation of action potential duration and atrial refractory period. Recent evidences suggested that Pitx2c, a bicoid-related homeodomain transcription factor involved in directing cardiac asymmetric morphogenesis, could play a role in atrial remodelling. However, its effects on Iks and ICa,L are unknown. Methods and results Real-time quantitative polymerase chain reaction analysis showed that Pitx2c mRNA expression was significantly higher in human atrial myocytes from CAF patients than those from sinus rhythm patients. The expression of Pitx2c was positively and negatively correlated with Iks and ICa,L densities, respectively. Expression of Pitx2c in HL-1 cells increased Iks density and reduced ICa,L density. Luciferase assays demonstrated that Pitx2c increased transcriptional activity of KCNQ1 and KCNE1 genes. Conversely, its effects on ICa,L could be mediated by the atrial natriuretic peptide. Conclusion Our results demonstrated for the first time that CAF increases Pitx2c expression in isolated human atrial myocytes and suggested that this transcription factor could contribute to the CAF-induced Iks increase and ICa,L reduction observed in humans. [ABSTRACT FROM AUTHOR]

Floral patterning is regulated by intricate networks of floral identity genes. The peculiar MADS32 subfamily genes, absent in eudicots but prevalent in monocots, control floral organ identity. However, how the MADS32 family genes interact with other floral homeotic genes during flower development is mostly unknown. We show here that the rice homeotic transcription factor OsMADS32 regulates floral patterning by interacting synergistically with E class protein OsMADS6 in a dosage-dependent manner. Furthermore, our results indicate important roles for OsMADS32 in defining stamen, pistil, and ovule development through physical and genetic interactions with OsMADS1 , OsMADS58 , and OsMADS13 , and in specifying floral meristem identity with OsMADS6 , OsMADS3 , and OsMADS58 , respectively. Our findings suggest that OsMADS32 is an important factor for floral meristem identity maintenance and that it integrates the action of other MADS-box homeotic proteins to sustain floral organ specification and development in rice. Given that OsMADS32 is an orphan gene and absent in eudicots, our data substantially expand our understanding of flower development in plants. [ABSTRACT FROM AUTHOR]

Glandular secreting trichomes (GSTs) synthesize and secrete large quantities of secondary metabolites, some of which have well-established commercial value. An example is the anti-malarial compound artemisinin, which is synthesized in the GSTs of Artemisia annua. Accordingly, there is considerable interest in understanding the processes that regulate GST density as a strategy to increase artemisinin production. In this study, we identified a GST-specific WRKY transcription factor from A. annua , AaGSW2, which is positively regulated by the direct binding of the homeodomain proteins AaHD1 and AaHD8 to the L1-box of the AaGSW2 promoter. Overexpression of AaGSW2 in A. annua significantly increased GST density, while AaGSW2 knockdown lines showed impaired GST initiation. Ectopic expression of AaGSW2 homologs from two mint cultivars, Mentha spicata and Mentha haplocalyx , in A. annua also induced GST formation. These results reveal a molecular mechanism involving homeodomain and WRKY proteins that controls glandular trichome initiation, at least part of which is shared by A. annua and mint. [ABSTRACT FROM AUTHOR]

Flowering is an important trait in Chinese cabbage, because premature flowering reduces yield and quality of the harvested products. Water deficit, caused by drought or other environmental conditions, induces early flowering. Drought resistance involves global reprogramming of transcription, hormone signaling, and chromatin modification. We show that a histone H4 protein, BrHIS4.A04, physically interacts with a homeodomain protein BrVIN3.1, which was selected during the domestication of late-bolting Chinese cabbage. Over-expression of BrHIS4.A04 resulted in premature flowering under normal growth conditions, but prevented further premature bolting in response to drought. We show that the expression of key abscisic acid (ABA) signaling genes, and also photoperiodic flowering genes was attenuated in BrHIS4.A04 -overexpressing (BrHIS4.A04 OE ) plants under drought conditions. Furthermore, the relative change in H4-acetylation at these gene loci was reduced in BrHIS4.A04 OE plants. We suggest that BrHIS4.A04 prevents premature bolting by attenuating the expression of photoperiodic flowering genes under drought conditions, through the ABA signaling pathway. Since BrHIS4.A04 OE plants displayed no phenotype related to vegetative or reproductive development under laboratory-induced drought conditions, our findings contribute to the potential fine-tuning of flowering time in crops through genetic engineering without any growth penalty, although more data are necessary under field drought conditions. [ABSTRACT FROM AUTHOR]

Cotton (Gossypium) stem trichomes are mostly single cells that arise from stem epidermal cells. In this study, a homeodomainleucine zipper gene (HD1) was found to cosegregate with the dominant trichome locus previously designated as T1 and mapped to chromosome 6. Characterization of HD1 orthologs revealed that the absence of stem trichomes in modern Gossypium barbadense varieties is linked to a large retrotransposon insertion in the ninth exon, 2565 bp downstream from the initial codon in the At subgenome HD1 gene (At-GbHD1). In both the At and Dt subgenomes, reduced transcription of GbHD1 genes is caused by this insertion. The disruption of At-HD1 further affects the expression of downstream GbMYB25 and GbHOX3 genes. Analyses of primitive cultivated accessions identified another retrotransposon insertion event in the sixth exon of At-GbHD1 that might predate the previously identified retrotransposon in modern varieties. Although both retrotransposon insertions results in similar phenotypic changes, the timing of these two retrotransposon insertion events fits well with our current understanding of the history of cotton speciation and dispersal. Taken together, the results of genetics mapping, gene expression and association analyses suggest that GbHD1 is an important component that controls stem trichome development and is a promising candidate gene for the T1 locus. The interspecific phenotypic difference in stem trichome traits also may be attributable to HD1 inactivation associated with retrotransposon insertion. [ABSTRACT FROM AUTHOR]

A gene encoding a KNOTTED1-LIKE HOMEOBOX PROTEIN1 (KD1) is highly expressed in both leaf and flower abscission zones. Reducing the abundance of transcripts of this gene in tomato (Solanum lycopersicum) by both virus-induced gene silencing and stable transformation with a silencing construct driven by an abscission-specific promoter resulted in a striking retardation of pedicel and petiole abscission. In contrast, Petroselinum, a semidominant KD1 mutant, showed accelerated pedicel and petiole abscission. Complementary DNA microarray and quantitative reverse transcription-polymerase chain reaction analysis indicated that regulation of abscission by KD1 was associated with changed abundance of genes related to auxin transporters and signaling components. Measurement of auxin content and activity of a DR5::β-glucuronidase auxin reporter assay showed that changes in KD1 expression modulated the auxin concentration and response gradient in the abscission zone. [ABSTRACT FROM AUTHOR]

The TALE homeodomain transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is part of a regulatory network governing the commitment to secondary cell wall biosynthesis of Arabidopsis thaliana , where it contributes to negative regulation of this process. Here, we report that BLH6, a BELL1-LIKE HOMEODOMAIN protein, specifically interacts with KNAT7, and this interaction influences secondary cell wall development. BLH6 is a transcriptional repressor, and BLH6-KNAT7 physical interaction enhances KNAT7 and BLH6 repression activities. The overlapping expression patterns of BLH6 and KNAT7 and phenotypes of blh6 , knat7 , and blh6 knat7 loss-of-function mutants are consistent with the existence of a BLH6-KNAT7 heterodimer that represses commitment to secondary cell wall biosynthesis in interfascicular fibers. BLH6 and KNAT7 overexpression results in thinner interfascicular fiber secondary cell walls, phenotypes that are dependent on the interacting partner. A major impact of the loss of BLH6 and KNAT7 function is enhanced expression of the homeodomain-leucine zipper transcription factor REVOLUTA / INTERFASCICULAR FIBERLESS1 (REV / IFL1). BLH6 and KNAT7 bind to the REV promoter and repress REV expression, while blh6 and knat7 interfascicular fiber secondary cell wall phenotypes are suppressed in blh6 rev and knat7 rev double mutants, suggesting that BLH6/KNAT7 signaling acts through REV as a direct target. [ABSTRACT FROM AUTHOR]

Tomato (Solanum lycopersicum) is a model plant for studying fruit development and ripening. In this study, we found that down-regulation of a tomato bell-like homeodomain 4 (SlBL4) resulted in a slightly darker-green fruit phenotype and increased accumulation of starch, fructose, and glucose. Analysis of chlorophyll content and TEM observations was consistent with these phenotypes, indicating that SlBL4 was involved in chlorophyll accumulation and chloroplast formation. Ripened fruit of SlBL4 -RNAi plants had noticeably decreased firmness, larger intercellular spaces, and thinner cell walls than the wild-type. RNA-seq identified differentially expressed genes involved in chlorophyll metabolism, chloroplast development, cell wall metabolism, and carotenoid metabolism. ChIP-seq identified (G/A) GCCCA (A/T/C) and (C/A/T) (C/A/T) AAAAA (G/A/T) (G/A) motifs. SlBL4 directly inhibited the expression of protoporphyrinogen oxidase (SlPPO), magnesium chelatase H subunit (SlCHLD), pectinesterase (SlPE), protochlorophyllide reductase (SlPOR), chlorophyll a/b binding protein 3B (SlCAB -3B), and homeobox protein knotted 2 (TKN2). In contrast, it positively regulated the expression of squamosa promoter binding protein-like colorless non-ripening (LeSPL-CNR). Our results indicate that SlBL4 is involved in chlorophyll accumulation, chloroplast development, cell wall metabolism, and the accumulation of carotenoids during tomato fruit ripening, and provide new insights for the transcriptional regulation mechanism of BELL-mediated fruit growth and ripening. [ABSTRACT FROM AUTHOR]

Oligodontia, which is the congenital absence of six or more permanent teeth, excluding the third molars, may contribute to masticatory dysfunction, speech alteration, aesthetic problems and malocclusion. Msh homeobox 1 (MSX1) was the first gene identified as causing non-syndromic oligodontia. In this study, we identified a novel heterozygous non-stop mutation (c.910_911dupTA, p.*304Tyrext*48) in MSX1 in a Chinese family with autosomal dominant non-syndromic oligodontia. This novel mutation substitutes the stop codon with a tyrosine residue, potentially adding 48 amino acids to the C-terminus of MSX1. Further in vitro study found that mutant MSX1 could be expressed but had lost its ability to enter the nucleus. This is the first report indicating that a non-stop mutation in MSX1 is responsible for oligodontia. This study broadens the mutation spectrum for MSX1 and provides a new way to clarify the mechanism of MSX1 in tooth agenesis. [ABSTRACT FROM AUTHOR]

The article presents a study which examined the role of homeodomain-leucine zipper (HD-Zip) proteins in cold stress responses and development in tomato, Solanum lycopersicum. The methodology includes the extraction of the sequence of Arabidopsis HD-Zip proteins, analysis of phylogeny, conserved motifs and exon-intron structures, and determination of chromosomal location and gene duplication. A discussion on key features and classifications of homeobox (HB) proteins is offered.

Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines. [ABSTRACT FROM AUTHOR]

In plants, dioecy characterizes species that carry male and female flowers on separate plants and it occurs in about 6% of angiosperms; however, the molecular mechanisms that underlie dioecy are essentially unknown. The ability for sex-reversal by hormone application raises the hypothesis that the genes required for the expression of both sexes are potentially functional but are regulated by epigenetic means. In this study, proteomic analysis of nuclear proteins isolated from flower buds of females, males, and feminized males of the dioecious plant Mercurialis annua revealed differential expression of nuclear proteins that are implicated in chromatin structure and function, including floral homeotic proteins. Focusing on floral genes, we found that class B genes were mainly expressed in male flowers, while class D genes, as well as SUPERMAN -like genes, were mainly expressed in female flowers. Cytokinin-induced feminization of male plants was associated with down-regulation of male-specific genes concomitantly with up-regulation of female-specific genes. No correlation was found between the expression of class B and D genes and the changes in DNA methylation or chromatin conformation of these genes. Thus, we could not confirm DNA methylation or chromatin conformation of floral genes to be the major determinant regulating sexual dimorphisms. Instead, determination of sex in M. annua might be controlled upstream of floral genes by one or more sex-specific factors that affect hormonal homeostasis. A comprehensive model is proposed for sex-determination in M. annua. [ABSTRACT FROM AUTHOR]

The Arabidopsis thaliana MADS box transcription factors APETALA3 (AP3) and PISTILLATA (PI) heterodimerize and are required to specify petal identity, yet many details of how this regulatory process is effected are unclear. We have identified three related genes, BHLH136/BANQUO1 (BNQ1), BHLH134/BANQUO2 (BNQ2), and BHLH161/BANQUO3 (BNQ3), as being directly and negatively regulated by AP3 and PI in petals. BNQ1 , BNQ2 , and BNQ3 encode products belonging to a family of atypical non-DNA binding basic helix-loop-helix (bHLH) proteins that heterodimerize with and negatively regulate bHLH transcription factors. We show that bnq3 mutants have pale-green sepals and carpels and decreased chlorophyll levels, suggesting that BNQ3 has a role in regulating light responses. The ap3 bnq3 double mutant displays pale second-whorl organs, supporting the hypothesis that BNQ3 is downstream of AP3. Consistent with a role in light response, we show that the BNQ gene products regulate the function of HFR1 (for LONG HYPOCOTYL IN FAR-RED1), which encodes a bHLH protein that regulates photomorphogenesis through modulating phytochrome and cryptochrome signaling. The BNQ genes also are required for appropriate regulation of flowering time. Our results suggest that petal identity is specified in part through downregulation of BNQ -dependent photomorphogenic and developmental signaling pathways. [ABSTRACT FROM AUTHOR]

Germline fate determination is a critical event in sexual reproduction. Unlike animals, plants specify the germline by reprogramming somatic cells at the late stages of their development. However, the genetic basis of germline fate determination and how it evolved during the land plant evolution are still poorly understood. Here, we report that the plant homeodomain finger protein GERMLINE IDENTITY DETERMINANT (GLID) is a key regulator of the germline specification in liverwort, Marchantia polymorpha. Loss of the MpGLID function causes failure of germline initiation, leading to the absence of sperm and egg cells. Remarkably, the overexpression of MpGLID in M. polymorpha induces the ectopic formation of cells with male germline cell features exclusively in male thalli. We further show that MpBONOBO (BNB), with an evolutionarily conserved function, can induce the formation of male germ cell-like cells through the activation of MpGLID by directly binding to its promoter. The Arabidopsis (Arabidopsis thaliana) MpGLID ortholog, MALE STERILITY1 (AtMS1), fails to replace the germline specification function of MpGLID in M. polymorpha, demonstrating that a derived function of MpGLID orthologs has been restricted to tapetum development in flowering plants. Collectively, our findings suggest the presence of the BNB–GLID module in complex ancestral land plants that has been retained in bryophytes, but rewired in flowering plants for male germline fate determination. The genetic module underlying germline fate determination has been rewired during the land plant evolution. [ABSTRACT FROM AUTHOR]

Drought and salinity are the two major environmental constraints that severely affect global agricultural productivity. Plant-specific HD-Zip transcription factors are involved in plant growth, development and stress responses. In the present study, we explored the functional characteristics and regulation of a novel HD-Zip (I) gene from chickpea, CaHDZ12, in response to water-deficit and salt-stress conditions. Transgenic tobacco lines over-expressing CaHDZ12 exhibited improved tolerance to osmotic stresses and increased sensitivity to abscisic acid (ABA). Physiological compatibility of transgenic lines was found to be more robust compared to the wild-type plants under drought and salinity stress. Additionally, expression of several stress-responsive genes was significantly induced in CaHDZ12 transgenic plants. On the other hand, silencing of CaHDZ12 in chickpea resulted in increased sensitivity to salt and drought stresses. Analysis of different promoter deletion mutants identified CaWRKY70 transcription factor as a transcriptional regulator of CaHDZ12 expression. In vivo and in vitro interaction studies detected an association between CaWRKY70 and CaHDZ12 promoter during stress responses. Epigenetic modifications underlying histone acetylation at the CaHDZ12 promoter region play a significant role in stressinduced activation of this gene. Collectively, our study describes a crucial and unique mechanistic link between two distinct transcription factors in regulating plant adaptive stress response. [ABSTRACT FROM AUTHOR]

Transcription factors (TFs) regulate gene expression through binding to specific target DNA sites. Accurate annotation oftranscription factor binding sites (TFBSs) at genome scale represents an essential step toward our understanding of gene regulation networks. In this article, we present a structure-based method for computational prediction of TFBSs using a novel, integrative energy (IE) function. The new energy function combines a multibody (MB) knowledge-based potential and two atomic energy terms (hydrogen bond and p interaction) that might not be accurately captured by the knowledge-based potential owing to the mean force nature and low count problem. We applied the new energy function to the TFBS prediction using a non-redundant dataset that consists of TFs from 12 different families. Our results show that the new IE function improves the prediction accuracy over the knowledge-based, statistical potentials, especially for homeodomain TFs, the second largest TF family in mammals. [ABSTRACT FROM AUTHOR]

Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout ( ph8 -/Y) model, we found that deletion of phf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type ( ph8 +/Y) and ph8 -/Y ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown of pmaip1 mimicked the phenotype of ph8 -/Y by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8 -/Y ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. S tem C ells 2016;34:1527-1540 [ABSTRACT FROM AUTHOR]

Aims Atrial fibrillation (AF) produces rapid changes in the electrical properties of the atria (electrical remodelling) that promote its own recurrence. In chronic AF (CAF) patients, up-regulation of the slow delayed rectifier K+ current (Iks) and down-regulation of the voltage-gated Ca2+ current (ICa,L) are hallmarks of electrical remodelling and critically contribute to the abbreviation of action potential duration and atrial refractory period. Recent evidences suggested that Pitx2c, a bicoid-related homeodomain transcription factor involved in directing cardiac asymmetric morphogenesis, could play a role in atrial remodelling. However, its effects on Iks and ICa,L are unknown. Methods and results Real-time quantitative polymerase chain reaction analysis showed that Pitx2c mRNA expression was significantly higher in human atrial myocytes from CAF patients than those from sinus rhythm patients. The expression of Pitx2c was positively and negatively correlated with Iks and ICa,L densities, respectively. Expression of Pitx2c in HL-1 cells increased Iks density and reduced ICa,L density. Luciferase assays demonstrated that Pitx2c increased transcriptional activity of KCNQ1 and KCNE1 genes. Conversely, its effects on ICa,L could be mediated by the atrial natriuretic peptide. Conclusion Our results demonstrated for the first time that CAF increases Pitx2c expression in isolated human atrial myocytes and suggested that this transcription factor could contribute to the CAF-induced Iks increase and ICa,L reduction observed in humans. [ABSTRACT FROM AUTHOR]

Plant vascular tissues, xylem and phloem, differentiate in distinct patterns from procambial cells as an integral transport system for water, sugars, and signaling molecules. Procambium formation is promoted by high auxin levels activating class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs). In the root of Arabidopsis (Arabidopsis thaliana), HD-ZIP III TFs dose-dependently govern the patterning of the xylem axis, with higher levels promoting metaxylem cell identity in the central axis and lower levels promoting protoxylem at its flanks. It is unclear, however, by what mechanisms the HD-ZIP III TFs control xylem axis patterning. Here, we present data suggesting that an important mechanism is their ability to moderate the auxin response. We found that changes in HD-ZIP III TF levels affect the expression of genes encoding core auxin response molecules. We show that one of the HD-ZIP III TFs, PHABULOSA, directly binds the promoter of both MONOPTEROS (MP)/AUXIN RESPONSE FACTOR5, a key factor in vascular formation, and IAA20, encoding an auxin/indole acetic acid protein that is stable in the presence of auxin and able to interact with and repress MP activity. The double mutant of IAA20 and its closest homolog IAA30 forms ectopic protoxylem, while overexpression of IAA30 causes discontinuous protoxylem and occasional ectopic metaxylem, similar to a weak loss-of-function mp mutant. Our results provide evidence that HD-ZIP III TFs directly affect the auxin response and mediate a feed-forward loop formed by MP and IAA20 that may focus and stabilize the auxin response during vascular patterning and the differentiation of xylem cell types. [ABSTRACT FROM AUTHOR]

Diverse leaf morphology has been observed among accessions of Gossypium hirsutum, including okra leaf, which has advantages and disadvantages in cotton production. The okra leaf locus has been mapped to chromosome 15 of the Dt subgenome, but the underlying gene has yet to be identified. In this study, we used a combination of targeted association analysis, F2 population-based fine mapping, and comparative sequencing of orthologues to identify a candidate gene underlying the okra leaf trait in G. hirsutum. The okra leaf gene identified, GhOKRA, encoded a homeodomain leucine-zipper class I protein, whose closely related genes in several other plant species have been shown to be involved in regulating leaf morphology. The transcript levels of GhOKRA in shoot apices were positively correlated with the phenotypic expression of the okra leaf trait. Of the multiple sequence variations observed in the coding region among GrOKRA of Gossypium raimondii and GhOKRA-Dt of normal and okra/superokra leaf G. hirsutum accessions, a non-synonymous substitution near the N terminus and the variable protein sequences at the C terminus may be related to the leaf shape difference. Our results suggest that both transcription and protein activity of GhOKRA may be involved in regulating leaf shape. Furthermore, we found that non-reciprocal homoeologous recombination, or gene conversion, may have played a role in the origin of the okra leaf allele. Our results provided tools for further investigating and understanding the fundamental biological processes that are responsible for the cotton leaf shape variation and will help in the design of cotton plants with an ideal leaf shape for enhanced cotton production. [ABSTRACT FROM AUTHOR]

Candida albicans is the most common human fungal pathogen and can cause life-threatening infections. Filamentous growth is critical in the pathogenicity of C. albicans, as the transition from yeast to hyphal forms is linked to virulence and is also a pivotal process in fungal biofilm development. Homeodomain-containing transcription factors have been linked to developmental processes in fungi and other eukaryotes. We report here on GRF10, a homeobox transcription factor-encoding gene that plays a role in C. albicans filamentation. Deletion of the GRF10 gene, in both C. albicans SN152 and BWP17 strain backgrounds, results in mutants with strongly decreased hyphal growth. The mutants are defective in chlamydospore and biofilm formation, as well as showing dramatically attenuated virulence in a mouse infection model. Expression of the GRF10 gene is highly induced during stationary phase and filamentation. In summary, our study emphasizes a new role for the homeodomain-containing transcription factor in morphogenesis and pathogenicity of C. albicans. [ABSTRACT FROM AUTHOR]

The canonical Wnt signaling pathway is critical for myogenesis and can induce muscle progenitors to switch from proliferation to differentiation; how Wnt signals integrate with muscle-specific regulatory factors in this process is poorly understood. We previously demonstrated that the Barx2 homeobox protein promotes differentiation in cooperation with the muscle regulatory factor (MRF) MyoD. Pax7, another important muscle homeobox factor, represses differentiation. We now identify Barx2, MyoD, and Pax7 as novel components of the Wnt effector complex, providing a new molecular pathway for regulation of muscle progenitor differentiation. Canonical Wnt signaling induces Barx2 expression in muscle progenitors and perturbation of Barx2 leads to misregulation of Wnt target genes. Barx2 activates two endogenous Wnt target promoters as well as the Wnt reporter gene TOPflash, the latter synergistically with MyoD. Moreover, Barx2 interacts with the core Wnt effectors β-catenin and T cell-factor 4 (TCF4), is recruited to TCF/lymphoid enhancer factor sites, and promotes recruitment of β-catenin. In contrast, Pax7 represses the Wnt reporter gene and antagonizes the activating effect of Barx2. Pax7 also binds β-catenin suggesting that Barx2 and Pax7 may compete for interaction with the core Wnt effector complex. Overall, the data show for the first time that Barx2, Pax7, and MRFs can act as direct transcriptional effectors of Wnt signals in myoblasts and that Barx2 and Wnt signaling participate in a regulatory loop. We propose that antagonism between Barx2 and Pax7 in regulation of Wnt signaling may help mediate the switch from myoblast proliferation to differentiation. S tem C ells 2014;32:1661-1673 [ABSTRACT FROM AUTHOR]

Optimal inflorescence architecture is important for plant reproductive success by affecting the ultimate number of lowers that set fruits and for plant competitiveness when interacting with biotic or abiotic conditions. The pedicel is one of the key contributors to inflorescence architecture diversity. To date, knowledge about the molecular mechanisms of pedicel development is derived from Arabidopsis. Not much is known regarding other plants. Here, an SVP family MADS-box gene, NtSVP, in tobacco (Nicotiana tabacum) that is required for pedicel elongation was identified. It is shown that knockdown of NtSVP by RNA interference (RNAi) caused elongated pedicels, while overexpression resulted in compact inflorescences with much shortened pedicels. Moreover, an Arabidopsis BREVIPEDECELLUS/KNAT1 homologue NtBP-Like (NtBPL) was significantly up-regulated in NtSVP-RNAi plants. Disruption of NtBPL decreased pedicel lengths and shortened cortex cells. Consistent with the presence of a CArG-box at the NtBPL promoter, the direct binding of NtSVP to the NtBPL promoter was demonstrated by yeast one-hybrid assay, electrophoretic mobility shift assay, and dual-luciferase assay, in which NtSVP may act as a repressor of NtBPL. Microarray analysis showed that down-regulation of NtBPL resulted in differential expression of genes associated with a number of hormone biogenesis and signalling genes such as those for auxin and gibberellin. These findings together suggest the function of a MADS-box transcription factor in plant pedicel development, probably via negative regulation of a BP-like class I KNOX gene. The present work thus postulates the conservation and divergence of the molecular regulatory pathways underlying the development of plant inflorescence architecture. [ABSTRACT FROM AUTHOR]

The floral meristem (FM), which develops from the inflorescence meristem upon completion of the floral transition, terminates after producing a defined number of floral organs. This is in contrast to the shoot apical meristem, which is active throughout the entire life span of plants. WUSCHEL (WUS) encodes a homeodomain-containing protein and plays a critical role in shoot apical meristem, inflorescence meristem, and FM establishment and maintenance as well as FM determinacy. Although many genes have been implicated in FM determinacy through the regulation of WUS expression, precisely how these genes are coordinated to regulate WUS and consequently dictate FM fate remains unclear. Emerging lines of evidence indicate that epigenetic mechanisms, such as histone modification, chromatin remodeling, noncoding RNAs, and DNA methylation, play vital roles in meristem maintenance and termination. Here, recent findings demonstrating the involvement of the epigenetic network in the regulation of WUS expression in the context of FM determinacy are summarized and discussed. [ABSTRACT FROM AUTHOR]

The homeobox domain transcription factor NANOG, a key regulator of embryonic development and cellular reprogramming, has been reported to be broadly expressed in human cancers. Functional studies have provided strong evidence that NANOG possesses protumorigenic attributes. In addition to promoting self-renewal and long-term proliferative potential of stem-like cancer cells, NANOG-mediated oncogenic reprogramming may underlie clinical manifestations of malignant disease. In this review, we examine the molecular origin, expression, biological activities, and mechanisms of action of NANOG in various malignancies. We also consider clinical implications such as correlations between NANOG expression and cancer prognosis and/or response to therapy. We surmise that NANOG potentiates the molecular circuitry of tumorigenesis, and thus may represent a novel therapeutic target or biomarker for the diagnosis, prognosis, and treatment outcome of cancer. Finally, we present critical pending questions relating NANOG to cancer stem cells and tumor development. S tem C ells 2015;33:2381-2390 [ABSTRACT FROM AUTHOR]

Hhex encodes a homeodomain transcription factor that is widely expressed in hematopoietic stem and progenitor cell populations. Its enforced expression induces T-cell leukemia and we have implicated it as an important oncogene in early T-cell precursor leukemias where it is immediately downstream of an LMO2-associated protein complex. Conventional Hhex knockouts cause embryonic lethality precluding analysis of adult hematopoiesis. Thus, we induced highly efficient conditional knockout (cKO) using vav-Cre transgenic mice. Hhex cKO mice were viable and born at normal litter sizes. At steady state, we observed a defect in B-cell development that we localized to the earliest B-cell precursor, the pro-B-cell stage. Most remarkably, bone marrow transplantation using Hhex cKO donor cells revealed a more profound defect in all hematopoietic lineages. In contrast, sublethal irradiation resulted in normal myeloid cell repopulation of the bone marrow but markedly impaired repopulation of T- and B-cell compartments. We noted that Hhex cKO stem and progenitor cell populations were skewed in their distribution and showed enhanced proliferation compared to WT cells. Our results implicate Hhex in the maintenance of LT-HSCs and in lineage allocation from multipotent progenitors especially in stress hematopoiesis. S tem C ells 2015;33:2628-2641 [ABSTRACT FROM AUTHOR]

Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2 is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. S tem C ells 2015;33:699-712 [ABSTRACT FROM AUTHOR]

Congenital hypothyroidism (CH) is the most common congenital endocrine disorder. The early treatment of CH patients has successfully improved the prognosis and management of this disorder. Optimal treatment and management throughout the patient's life, beginning in the neonatal period, are required to ensure long-term health. Affected patients should be offered assessments of associated medical conditions and provided with accurate information about their condition throughout their lives, but particularly during the transition from pediatric to adult services. This review provides a summary of current knowledge about the long-term outcomes of these patients and appropriate management into early adulthood. We carried out a systematic search of the Medline database to identify relevant articles. Despite major improvements in prognosis, the impact of CH is clearly not uniform, and management should take into account a broader range of relevant indicators, including CH severity, associated comorbid conditions and the adequacy of treatment during childhood and adulthood. The early diagnosis and management of associated medical conditions, and better educational strategies to improve compliance with treatment, should improve the long-term prognosis. Further studies are required to explore changes with aging. [ABSTRACT FROM AUTHOR]

Understanding early differentiation events leading to cardiogenesis is crucial for controlling fate of human pluripotent stem cells and developing protocols that yield sufficient cell numbers for use in regenerative medicine and drug screening. Here, we develop a new tool to visualize patterning of early cardiac mesoderm and cardiomyocyte development in vitro by generating a dual MESP1mCherry/w-NKX2-5eGFP/w reporter line in human embryonic stem cells (hESCs) and using it to examine signals that lead to formation of cardiac progenitors and subsequent differentiation. MESP1 is a pivotal transcription factor for precardiac mesoderm in the embryo, from which the majority of cardiovascular cells arise. Transcription factor NKX2-5 is expressed upon cardiac crescent formation. Induction of cardiac differentiation in this reporter line resulted in transient expression of MESP1-mCherry, followed by continuous expression of NKX2-5-eGFP. MESP1-mCherry cells showed increased expression of mesodermal and epithelial-mesenchymal-transition markers confirming their mesodermal identity. Whole-genome microarray profiling and fluorescence-activated cell sorting analysis of MESP1-mCherry cells showed enrichment for mesodermal progenitor cell surface markers PDGFR-α, CD13, and ROR-2. No enrichment was found for the previously described KDR+PDGFR-α+ progenitors. MESP1-mCherry derivatives contained an enriched percentage of NKX2-5-eGFP and Troponin T expressing cells, indicating preferential cardiac differentiation; this was enhanced by inhibition of the Wnt-pathway. Furthermore, MESP1-mCherry derivatives harbored smooth muscle cells and endothelial cells, demonstrating their cardiac and vascular differentiation potential under appropriate conditions. The MESP1-NKX2-5 hESC reporter line allows us to identify molecular cues crucial for specification and expansion of human cardiac mesoderm and early progenitors and their differentiation to specific cardiovascular derivatives. S tem C ells 2015;33:56-67 [ABSTRACT FROM AUTHOR]

Background Loss of the DNA-binding activity of a transcription factor is detrimental to its function in responsive gene regulation. We diagnosed a Taiwanese family with nail-patella syndrome ( NPS) whose members inherited the mutated LMX1b transcription factor with no DNA-binding homeodomain. The loss-of-function variants cause haploinsufficiency of LMX1b, leading to the clinical manifestation of NPS. The underlying molecular mechanism is unclear. Objectives To test whether the recurrent pathogenic truncated LMX1b-R198X reported in our patients might be a functional protein. Its biochemical properties were explored. Methods The luciferase reporter driven by the human interleukin ( IL)-6 gene promoter was assayed to measure the transcriptional activity of LMX1b. The nuclear localization of different enhanced green fluorescent protein-tagged LMX1b proteins was observed using fluorescence microscopy. Western blotting was employed to evaluate the expression of various transfected LMX1b constructs. Results LMX1b-R198X enhanced the IL-6 promoter activity activated by the wild-type LMX1b and diminished the promoter activity induced by phorbol 12-myristate 13-acetate. LMX1b-R198X carried out its effect differentially in the expression of various human genes. The nuclear localization of the wild-type LMX1b was disrupted by the C-terminus truncation. The protein stability exhibited by LMX1b-R198X appears to be much higher than that of the wild-type protein. Conclusions We demonstrated that loss of function might not be the only way for mutated LMX1b to cause haploinsufficiency as the main pathogenic mechanism for NPS. LMX1b-R198X has less nuclear localization and higher stability than the wild-type protein; consequently, it might function as a competitor to sequester other effectors by protein-protein interaction to interfere with downstream transcriptional events. [ABSTRACT FROM AUTHOR]

We reported the expression of the homeodomain-containing transcription factor Engrailed-2 ( EN2) in prostate cancer and showed that the presence of EN2 protein in the urine was highly predictive of prostate cancer. This study aimed to determine whether patients with prostate cancer have EN2 autoantibodies, what the prevalence of these antibodies is and whether they are associated with disease stage. The spontaneous immunoglobulin ( Ig)G immune response against EN2 and for comparison the tumour antigen New York Esophageal Squamous Cell Carcinoma 1 ( NY-ESO-1), were tested by enzyme-linked immunosorbent assay ( ELISA) in three different cohorts of prostate cancer patients as well as a group of men genetically predisposed to prostate cancer. Thirty-two of 353 (9·1%) of the SUN cohort representing all stages of prostate cancer demonstrated EN2 IgG responses, 12 of 107 patients (11·2%) in the advanced prostate cancer patients showed responses, while only four of 121 patients (3·3%) with castrate-resistant prostate cancer showed EN2 autoantibodies. No significant responses were found in the predisposed group. Anti- EN2 IgG responses were significantly higher in patients with prostate cancer compared to healthy control males and similarly prevalent to anti- NY-ESO-1 responses. While EN2 autoantibodies are not a useful diagnostic or monitoring tool, EN2 immunogenicity provides the rationale to pursue studies using EN2 as an immunotherapeutic target. [ABSTRACT FROM AUTHOR]

A bstract Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate along the retinal lineage in a manner that mimics normal mammalian development. Under certain culture conditions, hiPSCs form optic vesicle-like structures (OVs), which contain proliferating progenitors capable of yielding all neural retina (NR) cell types over time. Such observations imply conserved roles for regulators of retinogenesis in hiPSC-derived cultures and the developing embryo. However, whether and to what extent this assumption holds true has remained largely uninvestigated. We examined the role of a key NR transcription factor, visual system homeobox 2 (VSX2), using hiPSCs derived from a patient with microphthalmia caused by an R200Q mutation in the VSX2 homeodomain region. No differences were noted between (R200Q)VSX2 and sibling control hiPSCs prior to OV generation. Thereafter, (R200Q)VSX2 hiPSC-OVs displayed a significant growth deficit compared to control hiPSC-OVs, as well as increased production of retinal pigmented epithelium at the expense of NR cell derivatives. Furthermore, (R200Q)VSX2 hiPSC-OVs failed to produce bipolar cells, a distinctive feature previously observed in Vsx2 mutant mice. (R200Q)VSX2 hiPSC-OVs also demonstrated delayed photoreceptor maturation, which could be overcome via exogenous expression of wild-type VSX2 at early stages of retinal differentiation. Finally, RNAseq analysis on isolated hiPSC-OVs implicated key transcription factors and extracellular signaling pathways as potential downstream effectors of VSX2-mediated gene regulation. Our results establish hiPSC-OVs as versatile model systems to study retinal development at stages not previously accessible in humans and support the bona fide nature of hiPSC-OV-derived retinal progeny. S tem C ells 2014;32:1480-1492 [ABSTRACT FROM AUTHOR]

Magnesium superoxide dismutase (SOD2) has been shown to cause dysfunction of p53 transcriptional activity, whereas, in turn, SOD2 expression is regulated by p53 to modulate lung tumorigenesis. In this study, we found that the level of SOD2 expression in a panel of lung cancer cells was negatively correlated with that of NK2 homeobox 1 (NKX2-1) but was not associated with p53 status. Mechanistic studies indicated that a decrease in NKX2-1 caused by SOD2-activated IKKβ transcription was achieved by derepression of binding of Sp1 to the IKKβ promoter. Immunoprecipitation, glutathione S-transferase pull-down experiments and electrophoretic mobility shift assays demonstrated a direct interaction between NKX2-1 and Sp1, blocking Sp1-mediated IKKβ transcription. SOD2-mediated nuclear factor-kappaB activation, via elevation of IKKβ transcription, promoted anchorage-independent soft-agar growth, invasion and xenograft tumor formation, because of development of the epithelial-to-mesenchymal transition. The expression level of NKX2-1 messenger RNA was negatively associated with the extent of SOD immunostaining and the IKKβ messenger RNA expression level in lung tumors. The extent of SOD2 immunostaining and IKKβ messenger RNA levels may independently predict overall survival and relapse-free survival in lung adenocarcinoma patients. In summary, we found that SOD2 activates nuclear factor-kappaB signaling by increasing IKKβ transcription, which results in progression of lung adenocarcinoma and poorer patient outcomes. We suggest that IKKβ may potentially be targeted to improve outcomes in patients with SOD2-positive tumors. [ABSTRACT FROM PUBLISHER]

A novel rare mutation, homeobox B13 (HOXB13) G84E, was reported to co-segregate with prostate cancer (PCa) in hereditary PCa families and associate with PCa risk in unrelated cases and controls. In this study, we aim to compare the G84E mutation frequency among subjects of different races/ethnicities from various geographic regions in the world and to assess its risk for developing PCa, in the Reduction by Dutasteride of Prostate Cancer Events (REDUCE) trial. All the 3508 subjects had initial negative prostate biopsy and were biopsied at Year 2 and 4 for detection of PCa. The G84E mutation was detected only in Caucasians, with the highest carrier frequency in Northern Europe (1.06%), followed by Western Europe (0.60%) and North America (0.31%). No mutation carrier was observed in Southern Europe, Eastern Europe, Latin America, Australia and South Africa. In Caucasians, the G84E mutation frequency was 0.99% and 0.24% in positive and negative biopsy subjects, respectively (P = 0.01). In positive biopsy subjects, the frequency was significantly higher in subjects with a positive family history than those without (4.31% versus 0.34%, P = 0.002). In the 4 year follow-up, the PCa detection rate was 53.8% among the 13 mutation carriers and 22.0% among 3186 non-carriers, relative risk = 2.45 (95% confidence interval: 1.48–4.07). All mutation carriers shared a common haplotype, suggesting a founder effect. In Finland, the G84E mutation was estimated to occur in the year 1792 (95% credible interval: 1735–1831). In conclusion, the G84E mutation of HOXB13, a relatively recent mutation that likely occurred in Northern Europe, significantly increases risk for PCa. [ABSTRACT FROM PUBLISHER]