TALE Homeodomain Transcription Factors Research Articles

Molecular basis of facilitated target search and sequence discrimination of TALE homeodomain transcription factor Meis1

Transcription factors specifically bind to their consensus sequence motifs and regulate transcription efficiency. Transcription factors are also able to non-specifically contact the phosphate backbone of DNA through electrostatic interaction. The homeodomain of Meis1 TALE human transcription factor (Meis1-HD) recognizes its target DNA sequences via two DNA contact regions, the L1-α1 region and the α3 helix (specific binding mode). This study demonstrates that the non-specific binding mode of Meis1-HD is the energetically favored process during DNA binding, achieved by the interaction of the L1-α1 region with the phosphate backbone. An NMR dynamics study suggests that non-specific binding might set up an intermediate structure which can then rapidly and easily find the consensus region on a long section of genomic DNA in a facilitated binding process. Structural analysis using NMR and molecular dynamics shows that key structural distortions in the Meis1-HD–DNA complex are induced by various single nucleotide mutations in the consensus sequence, resulting in decreased DNA binding affinity. Collectively, our results elucidate the detailed molecular mechanism of how Meis1-HD recognizes single nucleotide mutations within its consensus sequence: (i) through the conformational features of the α3 helix; and (ii) by the dynamic features (rigid or flexible) of the L1 loop and the α3 helix. These findings enhance our understanding of how single nucleotide mutations in transcription factor consensus sequences lead to dysfunctional transcription and, ultimately, human disease.

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.

Touch receptor end-organ innervation and function require sensory neuron expression of the transcription factor Meis2

Touch sensation is primarily encoded by mechanoreceptors, called low-threshold mechanoreceptors (LTMRs), with their cell bodies in the dorsal root ganglia. Because of their great diversity in terms of molecular signature, terminal endings morphology, and electrophysiological properties, mirroring the complexity of tactile experience, LTMRs are a model of choice to study the molecular cues differentially controlling neuronal diversification. While the transcriptional codes that define different LTMR subtypes have been extensively studied, the molecular players that participate in their late maturation and in particular in the striking diversity of their end-organ morphological specialization are largely unknown. Here we identified the TALE homeodomain transcription factor Meis2 as a key regulator of LTMRs target-field innervation in mice. Meis2 is specifically expressed in cutaneous LTMRs, and its expression depends on target-derived signals. While LTMRs lacking Meis2 survived and are normally specified, their end-organ innervations, electrophysiological properties, and transcriptome are differentially and markedly affected, resulting in impaired sensory-evoked behavioral responses. These data establish Meis2 as a major transcriptional regulator controlling the orderly formation of sensory neurons innervating peripheral end organs required for light touch.

Touch receptor end-organ innervation and function require sensory neuron expression of the transcription factor Meis2.

Touch sensation is primarily encoded by mechanoreceptors, called low-threshold mechanoreceptors (LTMRs), with their cell bodies in the dorsal root ganglia. Because of their great diversity in terms of molecular signature, terminal endings morphology, and electrophysiological properties, mirroring the complexity of tactile experience, LTMRs are a model of choice to study the molecular cues differentially controlling neuronal diversification. While the transcriptional codes that define different LTMR subtypes have been extensively studied, the molecular players that participate in their late maturation and in particular in the striking diversity of their end-organ morphological specialization are largely unknown. Here we identified the TALE homeodomain transcription factor Meis2 as a key regulator of LTMRs target-field innervation in mice. Meis2 is specifically expressed in cutaneous LTMRs, and its expression depends on target-derived signals. While LTMRs lacking Meis2 survived and are normally specified, their end-organ innervations, electrophysiological properties, and transcriptome are differentially and markedly affected, resulting in impaired sensory-evoked behavioral responses. These data establish Meis2 as a major transcriptional regulator controlling the orderly formation of sensory neurons innervating peripheral end organs required for light touch.

Transcriptional cooperation of PBX1 and PAX6 in adult neural progenitor cells

PAX6 is a highly conserved transcription factor and key regulator of several neurogenic processes, including the continuous generation of dopaminergic/GABAergic interneurons in the adult ventricular-subventricular (V-SVZ) neurogenic system in mice. Here we report that PAX6 cooperates with the TALE-homeodomain transcription factor PBX1 in this context. Chromatin-immunoprecipitation showed that PBX1 and PAX6 co-occupy shared genomic binding sites in adult V-SVZ stem- and progenitor cell cultures and mouse embryonic stem cells, while depletion of Pbx1 revealed that association of PAX6 with these sites requires the presence of PBX1. Expression profiling together with viral overexpression or knockdown of Pax6 or Pbx1 identified novel PBX1-PAX6 co-regulated genes, including several transcription factors. Computational modeling of genome wide expression identified novel cross-regulatory networks among these very transcription factors. Taken together, the results presented here highlight the intimate link that exists between PAX6 and TALE-HD family proteins and contribute novel insights into how the orchestrated activity of transcription factors shapes adult V-SVZ neurogenesis.

Biochemical characteristics of a diffusible factor that induces gametophyte to sporophyte switching in the brown alga Ectocarpus.

The haploid-diploid life cycle of the filamentous brown alga Ectocarpus involves alternation between two independent and morphologically distinct multicellular generations, the sporophyte and the gametophyte. Deployment of the sporophyte developmental program requires two TALE homeodomain transcription factors OUROBOROS and SAMSARA. In addition, the sporophyte generation has been shown to secrete a diffusible factor that can induce uni-spores to switch from the gametophyte to the sporophyte developmental program. Here, we determine optimal conditions for production, storage, and detection of this diffusible factor and show that it is a heat-resistant, high molecular weight molecule. Based on a combined approach involving proteomic analysis of sporophyte-conditioned medium and the use of biochemical tools to characterize arabinogalactan proteins, we present evidence that sporophyte-conditioned medium contains AGP epitopes and suggest that the diffusible factor may belong to this family of glycoproteins.

Tumorigenic and Antiproliferative Properties of the TALE-Transcription Factors MEIS2D and MEIS2A in Neuroblastoma.

Neuroblastoma is one of only a few human cancers that can spontaneously regress even after extensive dissemination, a poorly understood phenomenon that occurs in as many as 10% of patients. In this study, we identify the TALE-homeodomain transcription factor MEIS2 as a key contributor to this phenomenon. We identified MEIS2 as a MYCN-independent factor in neuroblastoma and showed that in this setting the alternatively spliced isoforms MEIS2A and MEIS2D exert antagonistic functions. Specifically, expression of MEIS2A was low in aggressive stage 4 neuroblastoma but high in spontaneously regressing stage 4S neuroblastoma. Moderate elevation of MEIS2A expression reduced proliferation of MYCN-amplified human neuroblastoma cells, induced neuronal differentiation and impaired the ability of these cells to form tumors in mice. In contrast, MEIS2A silencing or MEIS2D upregulation enhanced the aggressiveness of the tumor phenotype. Mechanistically, MEIS2A uncoupled a negative feedback loop that restricts accumulation of cellular retinoic acid, an effective agent in neuroblastoma treatment. Overall, our results illuminate the basis for spontaneous regression in neuroblastoma and identify an MEIS2A-specific signaling network as a potential therapeutic target in this common pediatric malignancy.Significance: This study illuminates the basis for spontaneous regressions that can occur in a common pediatric tumor, with implications for the development of new treatment strategies. Cancer Res; 78(8); 1935-47. ©2018 AACR.

Abstract B15: Targeting PBX1 signaling to sensitize carboplatin cytotoxicity in ovarian cancer

Abstract Introduction: Resistance to chemotherapy represents a major obstacle to achieving long-term remission in ovarian cancer patients. Thereby, there is an unmet need to understand the pathogenesis of chemoresistance and to develop effective strategy to overcome drug resistance. To better understand the molecular etiology of drug resistance, we previously studied ovarian high-grade serous carcinoma (HGSC) to identify the genes and pathways they controlled in the development of chemoresistance. Toward this goal, we have demonstrated Notch3 pathway as one of the molecular contributors to carboplatin resistance in ovarian cancer cells. We further identified that PBX1, a TALE homeodomain transcription factor and proto-oncogene, mediates the functions of Notch3 signaling. This finding together with the fact that PBX1 is involved in several tumor-promoting functions raises the possibility that PBX1 may play an important role in chemoresistance. Thus, in this study, we aim to determine the roles of PBX1 in the development of carboplatin resistance in ovarian HGSC. Experimental procedures: Western blot and immunohistochemistry were performed to determine the expression levels of PBX1 in primary and recurrent/chemoresistant ovarian HGSC tissues and cell lines. To assess the functional roles of PBX1, PBX1 siRNA or cDNA was transfected into ovarian cancer cell lines. Stem cell-like characteristics were measured using an ALDH1 staining kit and flow cytometry to identify verapamil-sensitive side populations. A small molecule inhibitor targeting the binding between PBX1 and DNA was employed in mouse ovarian cancer xenograft models. Results: Immunohistochemistry demonstrated that PBX1 protein was upregulated in recurrent post-chemotherapy HGSCs as compared to the matched primary tumors. Western blot analysis showed that carboplatin-resistant (CR) ovarian cancer cell lines expressed higher protein levels of PBX1 than both parental cells and paclitaxel resistant cells. Gene knockdown of PBX1 using siRNA significantly suppressed growth in CR cell lines, indicating the dependence of CR cells on PBX1 expression. Since ALDH1, a stem cell marker, was also upregulated in CR cells as compared to the parental cells, we determined whether ectopic expression of PBX1 in PBX1-low ovarian cancer cells enhanced stem-cell phenotypes. Indeed, the fraction of verapamil-sensitive side population and the number of ALDH1 positive cells both increased in ovarian cells transfected with the PBX1-expressing plasmids, as were the stem cell associated markers including ABCB1 and GPR160. Moreover, we designed a novel small molecule inhibitor that disrupted the Pbx1/DNA complex and this PBX1 inhibitor potently interrupted the binding of PBX1 to the consensus DNA motif responsible for PBX1 binding. We observed an anti-proliferative effect of this inhibitor in CR ovarian cancer lines and an anti-tumorigenic effect in ovarian cancer mouse xenografts. Conclusion: This study demonstrates that PBX1 is a marker for recurrent post-chemotherapy HGSCs. Inactivating PBX1 using gene silencing and small molecule inhibitors enhances cytotoxicity of CR-resistant ovarian cancer cells. The above findings should have significant implications in cancer biology, and promise a novel strategy to combat ovarian cancer patients suffering from chemoresistance. Citation Format: Jin G. Jung, Joon T. Park, Alexader Stoeck, Tasmeen Hussain, Ren-Chin Wu, Ie-Ming Shih, Tian-Li Wang. Targeting PBX1 signaling to sensitize carboplatin cytotoxicity in ovarian cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B15.

BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 interact and regulate secondary cell wall formation via repression of REVOLUTA.

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.

351: The TALE homeodomain transcription factor MEIS1 activates the pro-metastatic melanoma cell adhesion molecule Mcam to promote migration of pancreatic cancer cells

351: The TALE homeodomain transcription factor MEIS1 activates the pro-metastatic melanoma cell adhesion molecule Mcam to promote migration of pancreatic cancer cells

Patterning of sexually dimorphic neurogenesis in the Caenorhabditis elegans ventral cord by Hox and TALE homeodomain transcription factors

COVER PHOTOGRAPH: Pseudocolored array of C. elegans L1 larvae overexpressing the midbody Hox reporter, lin‐39::lacZ. From Patterning of sexually dimorphic neurogenesis in the Caenorhabditis elegans ventral cord by Hox and TALE homeodomain transcription factors; Kalis, et al, Developmental Dynamics 243:159–171.

Patterning of sexually dimorphic neurogenesis in the Caenorhabditis elegans ventral cord by Hox and TALE homeodomain transcription factors

Reproduction in animals requires development of distinct neurons in each sex. In C. elegans, most ventral cord neurons (VCNs) are present in both sexes, with the exception of six hermaphrodite-specific neurons (VCs) and nine pairs of male-specific neurons (CAs and CPs) that arise from analogous precursor cells. How are the activities of sexual regulators and mediators of neuronal survival, division, and fate coordinated to generate sex-specificity in VCNs? To address this, we have developed a toolkit of VCN markers that allows us to examine sex-specific neurogenesis, asymmetric fates of daughters of a neuroblast division, and regional specification on the anteroposterior axis. Here, we describe the roles of the Hox transcription factors LIN-39 and MAB-5 in promoting survival, differentiation, and regionalization of VCNs. We also find that the TALE class homeodomain proteins CEH-20 and UNC-62 contribute to specification of neurotransmitter fate in males. Furthermore, we identify that VCN sex is determined during the L1 larval stage. These findings, combined with future analyses made possible by the suite of VCN markers described here, will elucidate how Hox-mediated cell fate decisions and sex determination intersect to influence development of neuronal sex differences.

Identification of PBX1 Target Genes in Cancer Cells by Global Mapping of PBX1 Binding Sites

PBX1 is a TALE homeodomain transcription factor involved in organogenesis and tumorigenesis. Although it has been shown that ovarian, breast, and melanoma cancer cells depend on PBX1 for cell growth and survival, the molecular mechanism of how PBX1 promotes tumorigenesis remains unclear. Here, we applied an integrated approach by overlapping PBX1 ChIP-chip targets with the PBX1-regulated transcriptome in ovarian cancer cells to identify genes whose transcription was directly regulated by PBX1. We further determined if PBX1 target genes identified in ovarian cancer cells were co-overexpressed with PBX1 in carcinoma tissues. By analyzing TCGA gene expression microarray datasets from ovarian serous carcinomas, we found co-upregulation of PBX1 and a significant number of its direct target genes. Among the PBX1 target genes, a homeodomain protein MEOX1 whose DNA binding motif was enriched in PBX1-immunoprecipicated DNA sequences was selected for functional analysis. We demonstrated that MEOX1 protein interacts with PBX1 protein and inhibition of MEOX1 yields a similar growth inhibitory phenotype as PBX1 suppression. Furthermore, ectopically expressed MEOX1 functionally rescued the PBX1-withdrawn effect, suggesting MEOX1 mediates the cellular growth signal of PBX1. These results demonstrate that MEOX1 is a critical target gene and cofactor of PBX1 in ovarian cancers.

Out of step: The function of TALE homeodomain transcription factors that regulate shoot meristem maintenance and meristem identity

The indeterminate growth pattern displayed by shoots is mediated by the proper maintenance of the shoot meristem. Meristem maintenance is dependent upon the balance of stem cell perpetuation in the central zone (CZ) and organogenesis in the peripheral zone (PZ). Although the mechanisms that coordinate CZ and PZ function is not understood, meristem cell fate is likely achieved by the spatial interplay between gene regulatory networks and hormone signaling pathways. During shoot maturation, the identity of the shoot meristem as well as the lateral organs are transformed during the vegetative and reproductive transitions. Studies in model plant systems indicate that three amino acid extension (TALE) homeodomain proteins integrate signaling events that transform the identity of the shoot meristem and establish reproductive patterns of growth. This review will highlight the function of TALE homeodomain transcription factors that regulate shoot meristem cell fate and also function with phase specific regulators to maintain shoot meristem identity.

TALE homeodomain proteins regulate site-specific terminal differentiation, LCE genes and epidermal barrier

The epidermal barrier varies over the body surface to accommodate regional environmental stresses. Regional skin barrier variation is produced by site-dependent epidermal differentiation from common keratinocyte precursors and often manifests as site-specific skin disease or irritation. There is strong evidence for body-site-dependent dermal programming of epidermal differentiation in which the epidermis responds by altering expression of key barrier proteins, but the underlying mechanisms have not been defined. The LCE multigene cluster encodes barrier proteins that are differentially expressed over the body surface, and perturbation of LCE cluster expression is linked to the common regional skin disease psoriasis. LCE subclusters comprise genes expressed variably in either external barrier-forming epithelia (e.g. skin) or in internal epithelia with less stringent barriers (e.g. tongue). We demonstrate here that a complex of TALE homeobox transcription factors PBX1, PBX2 and Pknox (homologues of Drosophila Extradenticle and Homothorax) preferentially regulate external rather than internal LCE gene expression, competitively binding with SP1 and SP3. Perturbation of TALE protein expression in stratified squamous epithelia in mice produces external but not internal barrier abnormalities. We conclude that epidermal barrier genes, such as the LCE multigene cluster, are regulated by TALE homeodomain transcription factors to produce regional epidermal barriers.

TALE homeodomain proteins regulate site-specific terminal differentiation,LCEgenes and epidermal barrier

The epidermal barrier varies over the body surface to accommodate regional environmental stresses. Regional skin barrier variation is produced by site-dependent epidermal differentiation from common keratinocyte precursors and often manifests as site-specific skin disease or irritation. There is strong evidence for body-site-dependent dermal programming of epidermal differentiation in which the epidermis responds by altering expression of key barrier proteins, but the underlying mechanisms have not been defined. The LCE multigene cluster encodes barrier proteins that are differentially expressed over the body surface, and perturbation of LCE cluster expression is linked to the common regional skin disease psoriasis. LCE subclusters comprise genes expressed variably in either external barrier-forming epithelia (e.g. skin) or in internal epithelia with less stringent barriers (e.g. tongue). We demonstrate here that a complex of TALE homeobox transcription factors PBX1, PBX2 and Pknox (homologues of Drosophila Extradenticle and Homothorax) preferentially regulate external rather than internal LCE gene expression, competitively binding with SP1 and SP3. Perturbation of TALE protein expression in stratified squamous epithelia in mice produces external but not internal barrier abnormalities. We conclude that epidermal barrier genes, such as the LCE multigene cluster, are regulated by TALE homeodomain transcription factors to produce regional epidermal barriers.

Meis1regulatescyclin D1andc-mycexpression, and controls the proliferation of the multipotent cells in the early developing zebrafish eye

During eye development, retinal progenitors are drawn from a multipotent, proliferative cell population. In Drosophila the maintenance of this cell population requires the function of the TALE-homeodomain transcription factor Hth, although its mechanisms of action are still unknown. Here we investigate whether members of the Meis gene family, the vertebrate homologs of hth, are also involved in early stages of eye development in the zebrafish. We show that meis1 is initially expressed throughout the eye primordium. Later, meis1 becomes repressed as neurogenesis is initiated, and its expression is confined to the ciliary margin, where the retinal stem population resides. Knocking down meis1 function through morpholino injection causes a delay in the G1-to-S phase transition of the eye cells, and results in severely reduced eyes. This role in cell cycle control is mediated by meis1 regulating cyclin D1 and c-myc transcription. The forced maintenance of meis1 expression in cell clones is incompatible with the normal differentiation of the meis1-expressing cells, which in turn tend to reside in undifferentiated regions of the retinal neuroepithelium, such as the ciliary margin. Together, these results implicate meis1 as a positive cell cycle regulator in early retinal cells, and provide evidence of an evolutionary conserved function for Hth/Meis genes in the maintenance of the proliferative, multipotent cell state during early eye development.

Pbx1 regulates nephrogenesis and ureteric branching in the developing kidney

Pbx1 encodes a TALE homeodomain transcription factor that regulates developmental gene expression in a variety of tissues. Loss-of-function studies have demonstrated a critical role for Pbx1 in cellular proliferation and patterning and suggest its involvement in numerous regulatory pathways. In this study, examination of metanephric development in Pbx1 −/− embryos was conducted to further elucidate Pbx1-dependent processes during organogenesis. Prior to death at E15.5, Pbx1 −/− embryos displayed kidneys that were reduced in size, axially mispositioned, and in more severe cases, exhibited unilateral agenesis. Analysis with molecular markers revealed the effective induction of tubulogenic mesenchyme; however, Pbx1 −/− kidneys contained fewer nephrons and were characterized by expanded regions of mesenchymal condensates in the nephrogenic zone. Despite the restricted expression of Pbx1 in metanephric mesenchyme, developing nephrons, and stroma, decreased branching and elongation of the ureter were also observed. Moreover, heterologous recombination studies with explant cultures verified that Pbx1 −/− renal defects arose exclusively from mesenchymal dysfunction. Taken together, these data establish a role for Pbx1 in mesenchymal–epithelial signaling and demonstrate that Pbx1 is an essential regulator of mesenchymal function during renal morphogenesis.