Homeodomain Transcription Factor Research Articles

Schlank orchestrates insect developmental transition by switching H3K27 acetylation to trimethylation in the prothoracic gland

Insect developmental transitions are precisely coordinated by ecdysone and juvenile hormone (JH). We previously revealed that accumulated H3K27 trimethylation (H3K27me3) at the locus encoding JH signal transducer Hairy is involved in the larval-pupal transition in insects, but the underlying mechanism remains to be fully defined. Here, we show in Drosophila and Bombyx that Rpd3-mediated H3K27 deacetylation in the prothoracic gland during the last larval instar promotes ecdysone biosynthesis and the larval-pupal transition by enabling H3K27me3 accumulation at the Hairy locus to induce its transcriptional repression. Importantly, we find that the homeodomain transcription factor Schlank acts to switch active H3K27 acetylation (H3K27ac) to repressive H3K27me3 at the Hairy locus by directly binding to the Hairy promoter and then recruiting the histone deacetylase Rpd3 and the histone methyltransferase PRC2 component Su(z)12 through physical interactions. Moreover, Schlank inhibits Hairy transcription to facilitate the larval-pupal transition, and the Schlank signaling cascade is suppressed by JH but regulated in a positive feedback manner by ecdysone. Together, our data uncover that Schlank mediates epigenetic reprogramming of H3K27 modifications in hormone actions during insect developmental transition.

Neph1 is required for neurite branching and is negatively regulated by the PRRXL1 homeodomain factor in the developing spinal cord dorsal horn

The cell-adhesion molecule NEPH1 is required for maintaining the structural integrity and function of the glomerulus in the kidneys. In the nervous system of Drosophila and C. elegans, it is involved in synaptogenesis and axon branching, which are essential for establishing functional circuits. In the mammalian nervous system, the expression regulation and function of Neph1 has barely been explored. In this study, we provide a spatiotemporal characterization of Neph1 expression in mouse dorsal root ganglia (DRGs) and spinal cord. After the neurogenic phase, Neph1 is broadly expressed in the DRGs and in their putative targets at the dorsal horn of the spinal cord, comprising both GABAergic and glutamatergic neurons. Interestingly, we found that PRRXL1, a homeodomain transcription factor that is required for proper establishment of the DRG-spinal cord circuit, prevents a premature expression of Neph1 in the superficial laminae of the dorsal spinal cord at E14.5, but has no regulatory effect on the DRGs or on either structure at E16.5. By chromatin immunoprecipitation analysis of the dorsal spinal cord, we identified four PRRXL1-bound regions within the Neph1 introns, suggesting that PRRXL1 directly regulates Neph1 transcription. We also showed that Neph1 is required for branching, especially at distal neurites. Together, our work showed that Prrxl1 prevents the early expression of Neph1 in the superficial dorsal horn, suggesting that Neph1 might function as a downstream effector gene for proper assembly of the DRG-spinal nociceptive circuit.

Tissue-Level Integration Overrides Gradations of Differentiating Cell Identity in Beetle Extraembryonic Tissue.

During animal embryogenesis, one of the earliest specification events distinguishes extraembryonic (EE) from embryonic tissue fates: the serosa in the case of the insects. While it is well established that the homeodomain transcription factor Zen1 is the critical determinant of the serosa, the subsequent realization of this tissue's identity has not been investigated. Here, we examine serosal differentiation in the beetle Tribolium castaneum based on the quantification of morphological and morphogenetic features, comparing embryos from a Tc-zen1 RNAi dilution series, where complete knockdown results in amnion-only EE tissue identity. We assess features including cell density, tissue boundary morphology, and nuclear size as dynamic readouts for progressive tissue maturation. While some features exhibit an all-or-nothing outcome, other key features show dose-dependent phenotypic responses with trait-specific thresholds. Collectively, these findings provide nuance beyond the known status of Tc-Zen1 as a selector gene for serosal tissue patterning. Overall, our approach illustrates how the analysis of tissue maturation dynamics from live imaging extends but also challenges interpretations based on gene expression data, refining our understanding of tissue identity and when it is achieved.

Adaptation of turnip mosaic virus to Arabidopsis thaliana involves rewiring of VPg-host proteome interactions.

The outcome of a viral infection depends on a complex interplay between the host physiology and the virus, mediated through numerous protein-protein interactions. In a previous study, we used high-throughput yeast two-hybrid (HT-Y2H) to identify proteins in Arabidopsis thaliana that bind to the proteins encoded by the turnip mosaic virus (TuMV) genome. Furthermore, after experimental evolution of TuMV lineages in plants with mutations in defense-related or proviral genes, most mutations observed in the evolved viruses affected the VPg cistron. Among these mutations, D113G was a convergent mutation selected in many lineages across different plant genotypes, including cpr5-2 with constitutive expression of systemic acquired resistance. In contrast, mutation R118H specifically emerged in the jin1 mutant with affected jasmonate signaling. Using the HT-Y2H system, we analyzed the impact of these two mutations on VPg's interaction with plant proteins. Interestingly, both mutations severely compromised the interaction of VPg with the translation initiation factor eIF(iso)4E, a crucial interactor for potyvirus infection. Moreover, mutation D113G, but not R118H, adversely affected the interaction with RHD1, a zinc-finger homeodomain transcription factor involved in regulating DNA demethylation. Our results suggest that RHD1 enhances plant tolerance to TuMV infection. We also discuss our findings in a broad virus evolution context.

Functional analysis of conserved C. elegans bHLH family members uncovers lifespan control by a peptidergic hub neuron.

Throughout the animal kingdom, several members of the basic helix-loop-helix (bHLH) family act as proneural genes during early steps of nervous system development. Roles of bHLH genes in specifying terminal differentiation of postmitotic neurons have been less extensively studied. We analyze here the function of five C. elegans bHLH genes, falling into three phylogenetically conserved subfamilies, which are continuously expressed in a very small number of postmitotic neurons in the central nervous system. We show (a) that two orthologs of the vertebrate bHLHb4/b5 genes, called hlh-17 and hlh-32, function redundantly to specify the identity of a single head interneuron (AUA), as well as an individual motor neuron (VB2), (b) that the PTF1a ortholog hlh-13 acts as a terminal selector to control terminal differentiation and function of the sole octopaminergic neuron class in C. elegans, RIC, and (c) that the NHLH1/2 ortholog hlh-15 controls terminal differentiation and function of the peptidergic AVK head interneuron class, a known neuropeptidergic signaling hub in the animal. Strikingly, through null mutant analysis and cell-specific rescue experiments, we find that loss of hlh-15/NHLH in the peptidergic AVK neurons and the resulting abrogation of neuropeptide secretion causes a substantially expanded lifespan of the animal, revealing an unanticipated impact of a central, peptidergic hub neuron in regulating lifespan, which we propose to be akin to hypothalamic control of lifespan in vertebrates. Taken together, our functional analysis reveals themes of bHLH gene function during terminal differentiation that are complementary to the earlier lineage specification roles of other bHLH family members. However, such late functions are much more sparsely employed by members of the bHLH transcription factor family, compared to the function of the much more broadly employed homeodomain transcription factor family.

Pan-cancer exploration of oncogenic and clinical impacts revealed that HOXA9 is a diagnostic indicator of tumorigenesis

Homeodomain transcription factor A9 (HOXA9) is a member of the HOX cluster family of transcription factors that are crucially involved in embryo implantation, morphogenesis, body axis development, and endothelial cell differentiation. Despite numerous reports on its aberrant expression in a few malignancies, the molecular and functional complexity of HOXA9 across cancers remains obscure. We aimed to analyze the dynamic role of HOXA9 across cancers by identifying, analyzing, and understanding its multiple modes of regulation and functional implications and identifying possible therapeutic avenues. We conducted a comprehensive analysis to determine the role of HOXA9 across cancers. This approach involved the integration of large-scale datasets from public repositories such as the Genomic Data Commons, specifically the Cancer Genome Atlas (GDC-TCGA), across 33 different cancer types. The multiple modes of HOXA9 regulation by genetic and epigenetic factors were determined using online tools, which comprised experimentally validated observations. Furthermore, downstream pathways were identified by predicting the targets of HOXA9 and by performing functional enrichment analysis. We also assessed the clinical significance of HOXA9 in terms of prognosis and stage stratification. This study evaluated the correlation between HOXA9 and tumor-infiltrating molecules and discussed its association with therapeutically approved antineoplastic drugs. HOXA9 was significantly upregulated in 9 tumors and downregulated in 2 cancers. The deregulation of HOXA9 is primarily attributed to epigenetic factors, including promoter DNA methylation and noncoding RNAs (ncRNAs). The HOXA9 transcription factor interacts with PBX/MEIS cofactors and regulates multiple genes involved in cancer-associated EMT, autophagy, the cell cycle, metabolic pathways, Wnt signaling, TGF-β signaling, the AMPK pathway, PI3K/AKT signaling, and NF-κB signaling, thereby establishing control over downstream mechanisms. Differential expression in various clinical stages across cancers was shown to have prognostic significance and to be correlated with tumor-infiltrating immune molecules. The assessment of the correlation of HOXA9 expression with approved antineoplastic drugs revealed that targeting HOXA9 could be the most reliable strategy for preventing cancer progression. HOXA9 is upregulated in the majority of malignancies and drives cancer progression by regulating multiple signaling mechanisms. Hence, HOXA9 could be a reliable diagnostic indicator and a potential therapeutic candidate for solid cancer types.

ETMR-24. SOX2 INFLUENCES IDENTITY OF GLIA-LIKE PROGENITORS AND TUMORIGENESIS IN CHOROID PLEXUS THROUGH LIM HOMEODOMAIN TRANSCRIPTION FACTORS

Abstract Choroid plexus (CP) tumors encompass a spectrum of rare brain neoplasms ranging from benign papilloma (CPP) to aggressive carcinoma (CPC) that collectively affect the pediatric population. Poor outcomes for high-grade lesions and side effects of current treatment paradigms underscore a critical need to develop safer and more efficacious therapies guided by biological understanding of these tumors. We developed mouse models from common molecular defects found in human disease. These models exploit sustained NOTCH signaling activation in the hindbrain upper rhombic lip to generate CPP and CPC. To delineate cellular composition of CP tumors, we conducted snRNA-seq in murine CP tumors. Similar to adult CP, cells in NOTCH-driven CPP partitioned into clusters identified as epithelial, mesenchymal, endothelial, immune, neuronal, and glial cells. Though tumor cells are confined to epithelial compartment based on inferCNV analysis of snRNA-seq data, they are derived from glia-like progenitors. Tumor cells are in a distinct differentiation state characterized by a lack of epithelial maturation, and aberrant expression of markers of neural progenitor/stem-like cells including SRY-Box Transcription Factor 2 (SOX2). Consistently, SOX2 inactivation decreased tumor cell proliferation and blocked NOTCH-driven CP tumor formation. ChIP-seq studies revealed SOX2 binding to promoters of LIM homeodomain transcription factors Lmx1a and Lmx1b. Enforced SOX2 expression activated the expression Lmx1a and Lmx1b, whereas Sox2 loss reduced their expressions in tumor cells. Importantly, knockdown of Lmx1a or Lmx1b decreased tumor cell proliferation, whereas overexpression of Lmx1a/b rescued proliferation of tumor cells subjected to SOX2 suppression. Contextualizing these results in CP development, SOX2 critically maintains progenitor cell identity through regulating Lmx1a/b expression in the rhombic lip. Finally, analysis of human CP tumor samples revealed abnormal expressions of SOX2 and LMX1A. Together, these results indicate that oncogenic signaling subverts SOX2 transcriptional programs that specify normal progenitor identity and lock tumor cells in unique differentiation state to facilitate CP tumor growth.

Cooperative Gsx2-DNA binding requires DNA bending and a novel Gsx2 homeodomain interface.

The conserved Gsx homeodomain (HD) transcription factors specify neural cell fates in animals from flies to mammals. Like many HD proteins, Gsx factors bind A/T-rich DNA sequences prompting the following question: How do HD factors that bind similar DNA sequences in vitro regulate specific target genes in vivo? Prior studies revealed that Gsx factors bind DNA both as a monomer on individual A/T-rich sites and as a cooperative homodimer to two sites spaced precisely 7 bp apart. However, the mechanistic basis for Gsx-DNA binding and cooperativity is poorly understood. Here, we used biochemical, biophysical, structural and modeling approaches to (i) show that Gsx factors are monomers in solution and require DNA for cooperative complex formation, (ii) define the affinity and thermodynamic binding parameters of Gsx2/DNA interactions, (iii) solve a high-resolution monomer/DNA structure that reveals that Gsx2 induces a 20° bend in DNA, (iv) identify a Gsx2 protein-protein interface required for cooperative DNA binding and (v) determine that flexible spacer DNA sequences enhance Gsx2 cooperativity on dimer sites. Altogether, our results provide a mechanistic basis for understanding the protein and DNA structural determinants that underlie cooperative DNA binding by Gsx factors.

40 years of homeodomain transcription factors in the Drosophila nervous system.

Drosophila nervous system development progresses through a series of well-characterized steps in which homeodomain transcription factors (HDTFs) play key roles during most, if not all, phases. Strikingly, although some HDTFs have only one role, many others are involved in multiple steps of the developmental process. Most Drosophila HDTFs engaged in nervous system development are conserved in vertebrates and often play similar roles during vertebrate development. In this Spotlight, we focus on the role of HDTFs during embryogenesis, where they were first characterized.

The peptidase DA1 cleaves and destabilizes WUSCHEL to control shoot apical meristem size

Stem cells in plants and animals are the source of new tissues and organs. In plants, stem cells are maintained in the central zone (CZ) of multicellular meristems, and large shoot meristems with an increased stem cell population hold promise for enhancing yield. The mobile homeodomain transcription factor WUSCHEL (WUS) is a central regulator of stem cell function in plant shoot meristems. Despite its central importance, the factors that directly modulate WUS protein stability have been a long-standing question. Here, we show that the peptidase DA1 physically interacts with and cleaves the WUS protein, leading to its destabilization. Furthermore, our results reveal that cytokinin signaling represses the level of DA1 protein in the shoot apical meristem, thereby increasing the accumulation of WUS protein. Consistent with these observations, loss of DA1 function results in larger shoot apical meristems with an increased stem cell population and also influences cytokinin-induced enlargement of shoot apical meristem. Collectively, our findings uncover a previously unrecognized mechanism by which the repression of DA1 by cytokinin signaling stabilizes WUS, resulting in the enlarged shoot apical meristems with the increased stem cell number during plant growth and development.

Loss of PBX1 function in Leydig cells causes testicular dysgenesis and male sterility

Leydig cells are essential components of testicular interstitial tissue and serve as a primary source of androgen in males. A functional deficiency in Leydig cells often causes severe reproductive disorders; however, the transcriptional programs underlying the fate decisions and steroidogenesis of these cells have not been fully defined. In this study, we report that the homeodomain transcription factor PBX1 is a master regulator of Leydig cell differentiation and testosterone production in mice. PBX1 was highly expressed in Leydig cells and peritubular myoid cells in the adult testis. Conditional deletion of Pbx1 in Leydig cells caused spermatogenic defects and complete sterility. Histological examinations revealed that Pbx1 deletion impaired testicular structure and led to disorganization of the seminiferous tubules. Single-cell RNA-seq analysis revealed that loss of Pbx1 function affected the fate decisions of progenitor Leydig cells and altered the transcription of genes associated with testosterone synthesis in the adult testis. Pbx1 directly regulates the transcription of genes that play important roles in steroidogenesis (Prlr, Nr2f2 and Nedd4). Further analysis demonstrated that deletion of Pbx1 leads to a significant decrease in testosterone levels, accompanied by increases in pregnenolone, androstenedione and luteinizing hormone. Collectively, our data revealed that PBX1 is indispensable for maintaining Leydig cell function. These findings provide insights into testicular dysgenesis and the regulation of hormone secretion in Leydig cells.Graphical abstract

TAD boundary deletion causes PITX2-related cardiac electrical and structural defects

While 3D chromatin organization in topologically associating domains (TADs) and loops mediating regulatory element-promoter interactions is crucial for tissue-specific gene regulation, the extent of their involvement in human Mendelian disease is largely unknown. Here, we identify 7 families presenting a new cardiac entity associated with a heterozygous deletion of 2 CTCF binding sites on 4q25, inducing TAD fusion and chromatin conformation remodeling. The CTCF binding sites are located in a gene desert at 1 Mb from the Paired-like homeodomain transcription factor 2 gene (PITX2). By introducing the ortholog of the human deletion in the mouse genome, we recapitulate the patient phenotype and characterize an opposite dysregulation of PITX2 expression in the sinoatrial node (ectopic activation) and ventricle (reduction), respectively. Chromatin conformation assay performed in human induced pluripotent stem cell-derived cardiomyocytes harboring the minimal deletion identified in family#1 reveals a conformation remodeling and fusion of TADs. We conclude that TAD remodeling mediated by deletion of CTCF binding sites causes a new autosomal dominant Mendelian cardiac disorder.

Genetic mechanism regulating diversity in the placement of eyes on the head of animals

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

Role of moss and Arabidopsis zinc-finger homeodomain transcription factors in regulating plant architecture

Role of moss and Arabidopsis zinc-finger homeodomain transcription factors in regulating plant architecture

Interaction among homeodomain transcription factors mediates ethylene biosynthesis during pear fruit ripening.

Fruit ripening is manipulated by the plant phytohormone ethylene in climacteric fruits. While the transcription factors (TFs) involved in ethylene biosynthesis and fruit ripening have been extensively studied in tomato, their identification in pear remains limited. In this study, we identified and characterized a HOMEODOMAIN TF, PbHB.G7.2, through transcriptome analysis. PbHB.G7.2 could directly bind to the promoter of the ethylene biosynthetic gene, 1-aminocyclopropane-1-carboxylic acid synthase (PbACS1b), thereby enhancing its activity and resulting in increased ethylene production during pear fruit ripening. Yeast-two-hybrid screening revealed that PbHB.G7.2 interacted with PbHB.G1 and PbHB.G2.1. Notably, these interactions disrupted the transcriptional activation of PbHB.G7.2. Interestingly, PbHB.G1 and PbHB.G2.1 also bind to the PbACS1b promoter, albeit different regions from those bound by PbHB.G7.2. Moreover, the regions of PbHB.G1 and PbHB.G2.1 involved in their interaction with PbHB.G7.2 differ from the regions responsible for binding to the PbACS1b promoter. Nonetheless, these interactions also disrupt the transcriptional activation of PbHB.G1 and PbHB.G2.1. These findings offer a new mechanism of ethylene biosynthesis during climacteric fruit ripening.

Shaping leaves through TALE homeodomain transcription factors.

The first TALE homeodomain transcription factor 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.

Spatial enhancer activation influences inhibitory neuron identity during mouse embryonic development.

The mammalian telencephalon contains distinct GABAergic projection neuron and interneuron types, originating in the germinal zone of the embryonic basal ganglia. How genetic information in the germinal zone determines cell types is unclear. Here we use a combination of in vivo CRISPR perturbation, lineage tracing and ChIP-sequencing analyses and show that the transcription factor MEIS2 favors the development of projection neurons by binding enhancer regions in projection-neuron-specific genes during mouse embryonic development. MEIS2 requires the presence of the homeodomain transcription factor DLX5 to direct its functional activity toward the appropriate binding sites. In interneuron precursors, the transcription factor LHX6 represses the MEIS2-DLX5-dependent activation of projection-neuron-specific enhancers. Mutations of Meis2 result in decreased activation of regulatory enhancers, affecting GABAergic differentiation. We propose a differential binding model where the binding of transcription factors at cis-regulatory elements determines differential gene expression programs regulating cell fate specification in the mouse ganglionic eminence.

Cdx1 and Gsc distinctly regulate the transcription of BMP4 target gene ventx3.2 by directly binding to the proximal promoter region in Xenopus gastrulae

A comprehensive regulatory network of transcription factors controls the dorsoventral patterning of the body axis in developing vertebrate embryos. Bone morphogenetic protein signaling is essential for activating the Ventx family of homeodomain transcription factors, which regulates embryonic patterning and germ layer identity during Xenopus gastrulation. Although Ventx1.1 and Ventx2.1 of the Xenopus Ventx family have been extensively investigated, Ventx3.2 remains largely understudied. Therefore, this study aimed to investigate the transcriptional regulation of ventx3.2 during the embryonic development of Xenopus. We used goosecoid (Gsc) genome-wide chromatin immunoprecipitation-sequencing data to isolate and replicate the promoter region of ventx3.2. Serial deletion and site-directed mutagenesis were used to identify the cis-acting elements for Gsc and caudal type homeobox 1 (Cdx1) within the ventx3.2 promoter. Cdx1 and Gsc differentially regulated ventx3.2 transcription in this study. Additionally, positive cis-acting and negative response elements were observed for Cdx1 and Gsc, respectively, within the 5' flanking region of the ventx3.2 promoter. This result was corroborated by mapping the active Cdx1 response element (CRE) and Gsc response element (GRE). Moreover, a point mutation within the CRE and GRE completely abolished the activator and repressive activities of Cdx1 and Gsc, respectively. Furthermore, the chromatin immunoprecipitation-polymerase chain reaction confirmed the direct binding of Cdx1 and Gsc to the CRE and GRE, respectively. Inhibition of Cdx1 and Gsc activities at their respective functional regions, namely, the ventral marginal zone and dorsal marginal zone, reversed their effects on ventx3.2 transcription. These results indicate that Cdx1 and Gsc modulate ventx3.2 transcription in the ventral marginal zone and dorsal marginal zone by directly binding to the promoter region during Xenopus gastrulation.

Abstract 207: Tumor suppressive role of NKX2-1 in advanced thyroid cancer progression

Abstract NKX2-1 is a homeodomain transcription factor, known as a master regulator of thyroid development. NKX2-1 is also crucial for lung epithelium differentiation, and the involvement of NKX2-1 in lung carcinogenesis and cancer progression has been intensely investigated. However, the role of NKX2-1 in thyroid cancer progression remains elusive. The Cancer Genome Atlas (TCGA) dataset analysis demonstrated that papillary thyroid cancer (PTC) patients with low NKX2-1 mRNA expression had significantly poorer prognosis than those with a combination of intermediate and high mRNA expression of NKX2-1. Lentivirus-transduced tetracycline (Tet)-inducible system was used to achieve NKX2-1 overexpression in advanced follicular thyroid cancer (FTC) cell lines, FTC-238 and WRO, and an anaplastic thyroid cancer (ATC) cell line 8305C, in which NKX2-1 expression is barely/not detected. All these thyroid cancer cell lines showed significant suppression of cell proliferation with NKX2-1 overexpression. In addition, NKX2-1 overexpression inhibited migration of FTC-238 cells. The levels of mRNA and protein expression of mesenchymal markers, matrix metalloproteinase-2 (MMP-2) and fibronectin, were decreased by NKX2-1 overexpression in FTC-238 cells, suggesting a possibility that NKX2-1 partially inhibits epithelial-mesenchymal transition (EMT). SNAIL and SLUG are essential EMT transcription factors involved in metastasis in cancer progression. NKX2-1 mRNA expression was significantly lower in high SNAI1 (encoding SNAIL) expressing PTC in TCGA database. Indeed, suppression of SNAIL protein expression was found upon overexpression of NKX2-1 in FTC-238 cells which naturally express high level of SNAIL. These findings suggest that NKX2-1 may inhibit the migration of advanced thyroid cancer cells through SNAIL-induced EMT. Similarly, suppression of SLUG protein expression by overexpression of NKX2-1 was observed in WRO and 8305C cells which express high level of SLUG protein. Whether NKX2-1 overexpression inhibits migration and invasion of WRO and 8305C cells through SLUG-induced EMT is currently under examination. The Tet-inducible NKX2-1 expressing thyroid cancer cells will be used for in vivo evaluation of their metastatic ability using an immunocompromised mouse model. The findings so far suggest that NKX2-1 exerts a tumor suppressive activity in advanced thyroid cancers through suppression of cell proliferation, and cell migration presumably via suppression of EMT transcription factors. Citation Format: Yo-Taro Shirai, Shioko Kimura. Tumor suppressive role of NKX2-1 in advanced thyroid cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 207.

Conserved Noncoding Elements Evolve Around the Same Genes Throughout Metazoan Evolution.

Conserved noncoding elements (CNEs) are DNA sequences located outside of protein-coding genes that can remain under purifying selection for up to hundreds of millions of years. Studies in vertebrate genomes have revealed that most CNEs carry out regulatory functions. Notably, many of them are enhancers that control the expression of homeodomain transcription factors and other genes that play crucial roles in embryonic development. To further our knowledge of CNEs in other parts of the animal tree, we conducted a large-scale characterization of CNEs in more than 50 genomes from three of the main branches of the metazoan tree: Cnidaria, Mollusca, and Arthropoda. We identified hundreds of thousands of CNEs and reconstructed the temporal dynamics of their appearance in each lineage, as well as determining their spatial distribution across genomes. We show that CNEs evolve repeatedly around the same genes across the Metazoa, including around homeodomain genes and other transcription factors; they also evolve repeatedly around genes involved in neural development. We also show that transposons are a major source of CNEs, confirming previous observations from vertebrates and suggesting that they have played a major role in wiring developmental gene regulatory mechanisms since the dawn of animal evolution.