Control of limb initiation and antero-posterior patterning by cooperation between Meis and Tbx transcription factors [ChIP-seq]
ABSTRACT: Meis1 and Meis2 are highly similar homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Overexpression experiments have suggested an essential role for Meis factors in limb proximo-distal patterning; however, loss-of-function experiments supporting this notion have not been reported. Meis1 and Meis2 are coexpressed during limb development, first in the lateral plate mesoderm, before limb induction, and then they become restricted to a proximal domain of the growing limb bud. Here, we report that complete double conditional Meis1/2 inactivation in the lateral plate mesoderm leads to limb agenesis. Meis factors cooperate with Tbx factors in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles develop a complete PD set of limb skeletal elements, but show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior skeletal element specification reveals an early role of Meis factors in establishing the limb AP prepattern required for Shh activation at later stages. Our results uncover novel roles for Meis transcription factors in early limb development and identify their involvement in new molecular interaction networks that regulate organogenesis Overall design: ChIPSeq was used to determine Meis genome occupancy in WT E10.5 (36 somites approximately) forelimbs and hindlimbs.
Project description:<h4>Background</h4>Pancreas organogenesis is the result of well-orchestrated and balanced activities of transcription factors. The homeobox transcription factor PDX-1 plays a crucial role in the development and function of the pancreas, both in the maintenance of progenitor cells and in determination and maintenance of differentiated endocrine cells. However, the activity of homeobox transcription factors requires coordination with co-factors, such as PBX and MEIS proteins. PBX and MEIS proteins belong to the family of three amino acid loop extension (TALE) homeodomain proteins. In a previous study we found that PDX-1 negatively regulates the transcriptional activity of the ductal specific keratin 19 (Krt19). In this study, we investigate the role of different domains of PDX-1 and elucidate the functional interplay of PDX-1 and MEIS1 necessary for Krt19 regulation.<h4>Methodology/principal findings</h4>Here, we demonstrate that PDX-1 exerts a dual manner of regulation of Krt19 transcriptional activity. Deletion studies highlight that the NH(2)-terminus of PDX-1 is functionally relevant for the down-regulation of Krt19, as it is required for DNA binding of PDX-1 to the Krt19 promoter. Moreover, this effect occurs independently of PBX. Second, we provide insight on how PDX-1 regulates the Hox co-factor MEIS1 post-transcriptionally. We find specific binding of MEIS1 and MEIS2 to the Krt19 promoter using IP-EMSA, and siRNA mediated silencing of Meis1, but not Meis2, reduces transcriptional activation of Krt19 in primary pancreatic ductal cells. Over-expression of PDX-1 leads to a decreased level of MEIS1 protein, and this decrease is prevented by inhibition of the proteasome.<h4>Conclusions/significance</h4>Taken together, our data provide evidence for a dual mechanism of how PDX-1 negatively regulates Krt19 ductal specific gene expression. These findings imply that transcription factors may efficiently regulate target gene expression through diverse, non-redundant mechanisms.
Project description:Pax6 is a pivotal regulator of eye development throughout Metazoa, but the direct upstream regulators of vertebrate Pax6 expression are unknown. In vertebrates, Pax6 is required for formation of the lens placode, an ectodermal thickening that precedes lens development. Here we show that the Meis1 and Meis2 homeoproteins are direct regulators of Pax6 expression in prospective lens ectoderm. In mice, Meis1 and Meis2 are developmentally expressed in a pattern remarkably similar to Pax6 and their expression is Pax6-independent. Biochemical and transgenic experiments reveal that Meis1 and Meis2 bind a specific sequence in the Pax6 lens placode enhancer that is required for its activity. Furthermore, Pax6 and Meis2 exhibit a strong genetic interaction in lens development, and Pax6 expression is elevated in lenses of Meis2-overexpressing transgenic mice. When expressed in embryonic lens ectoderm, dominant-negative forms of Meis down-regulate endogenous Pax6. These results contrast with those in Drosophila, where the single Meis homolog, Homothorax, has been shown to negatively regulate eye formation. Therefore, despite the striking evolutionary conservation of Pax6 function, Pax6 expression in the vertebrate lens is uniquely regulated.
Project description:Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction.
Project description:Vertebrate limbs develop by integrating signals that control patterning along three main orthogonal axes. Flank-produced retinoic acid (RA) is initially required for limb induction and establishment of the apical ectodermal ridge (AER), a distal signaling center that produces fibroblast growth factors (FGFs), which are essential for limb growth and distalization. Once the AER is established, RA:FGF antagonism determines the restricted expression of a set of genes that control limb proximodistal patterning. Essential for this antagonism is the activation by FGF of the RA-degrading enzyme CYP26B1 in the distal limb bud. In addition, sonic hedgehog produced from the zone of polarizing activity (ZPA) is essential for distal limb anteroposterior patterning and contributes to RA reduction by cooperating in CYP26B1 activation. Meis transcription factors are expressed in the proximal limb bud, are activated by RA and can regulate proximodistal limb development; however, the mechanisms underlying their activity remain unknown. Here we studied Meis function in the mouse limb bud through Meis2 conditional overexpression and elimination of Meis1 and Meis2. We found that Meis activity is first required for limb bud initiation and the proper establishment of the AER and ZPA signaling centers, and subsequently for the development of proximal limb structures. Functional genomic analyses reveal that Meis is an important conveyor of the RA:FGF antagonism through the regulation of components of the RA and FGF signaling pathways, including CYP26B1. In addition, Meis regulates a set of proximal limb genes controlling proximodistal patterning and differentiation. Our work reveals a regulatory module essential for limb patterning and potentially co-opted in other patterning processes involving RA:FGF antagonism. Overall design: Examination of gene profile on distal limb bud tips of mice overexpressing Meis2
Project description:Myeloid ecotropic insertion site (Meis)2 is a homeodomain protein containing a conserved homothorax (Hth) domain that is present in all Meis and Prep family proteins and in the Drosophila Hth protein. The Hth domain mediates interaction with Pbx homeodomain proteins, allowing for efficient DNA binding. Here we show that, like Meis1, Meis2 has a strong C-terminal transcriptional activation domain, which is required for full activation of transcription by homeodomain protein complexes composed of Meis2 and Pbx1. We also show that the activity of the activation domain is inhibited by the Hth domain, and that this autoinhibition can be partially relieved by the interaction of Pbx1 with the Hth domain of Meis2. Targeting of the Hth domain to DNA suggests that it is not a portable trans-acting repression domain. However, the Hth domain can inhibit a linked activation domain, and this inhibition is not limited to the Meis2 activation domain. Database searching reveals that the Meis3.2 splice variant, which is found in several vertebrate species, disrupts the Hth domain by removing 17 codons from the 5'-end of exon 6. We show that the equivalent deletion in Meis2 derepresses the C-terminal activation domain and weakens interaction with Pbx1. This work suggests that the transcriptional activity of all members of the Meis/Prep Hth protein family is subject to autoinhibition by their Hth domains, and that the Meis3.2 splice variant encodes a protein that bypasses this autoinhibitory effect.
Project description:Identification of target genes that mediate required functions downstream of transcription factors is hampered by the large number of genes whose expression changes when the factor is removed from a specific tissue and the numerous binding sites for the factor in the genome. Retinoic acid (RA) regulates transcription via RA receptors bound to RA response elements (RAREs) of which there are thousands in vertebrate genomes. Here, we combined chromatin immunoprecipitation sequencing (ChIP-seq) for epigenetic marks and RNA-seq on trunk tissue from wild-type and Aldh1a2-/- embryos lacking RA synthesis that exhibit body axis and forelimb defects. We identified a relatively small number of genes with altered expression when RA is missing that also have nearby RA-regulated deposition of histone H3 K27 acetylation (H3K27ac) (gene activation mark) or histone H3 K27 trimethylation (H3K27me3) (gene repression mark) associated with conserved RAREs, suggesting these genes function downstream of RA. RA-regulated epigenetic marks were identified near RA target genes already known to be required for body axis and limb formation, thus validating our approach; plus, many other candidate RA target genes were found. Nuclear receptor 2f1 (Nr2f1) and nuclear receptor 2f2 (Nr2f2) in addition to Meis homeobox 1 (Meis1) and Meis homeobox 2 (Meis2) gene family members were identified by our approach, and double knockouts of each family demonstrated previously unknown requirements for body axis and/or limb formation. A similar epigenetic approach can be used to determine the target genes for any transcriptional regulator for which a knockout is available.
Project description:Suppression of Meis genes in the distal limb bud is required for Proximal-Distal (PD) specification of the forelimb. Polycomb group (PcG) factors play a role in downregulation of retinoic acid (RA)-related signals in the distal forelimb bud, causing Meis repression. It is, however, not known if downregulation of RA-related signals and PcG-mediated proximal genes repression are functionally linked. Here, we reveal that PcG factors and RA-related signals antagonize each other to polarize Meis2 expression along the PD axis. With mathematical modeling and simulation, we propose that PcG factors are required to adjust the threshold for RA-related signaling to regulate Meis2 expression. Finally, we show that a variant Polycomb repressive complex 1 (PRC1), incorporating PCGF3 and PCGF5, represses Meis2 expression in the distal limb bud. Taken together, we reveal a previously unknown link between PcG proteins and downregulation of RA-related signals to mediate the phase transition of Meis2 transcriptional status during forelimb patterning. Overall design: ChIP-seq analysis of RAR and H3K27me3 in the proximal and distal forelimb buds at mouse E11.5
Project description:Meis1, which belongs to TALE-type class of homeobox gene family, appeared as one of the key regulators of hematopoietic stem cell (HSC) self-renewal and a potential therapeutical target. However, small molecule inhibitors of MEIS1 remained unknown. This led us to develop inhibitors of MEIS1 that could modulate HSC activity. To this end, we have established a library of relevant homeobox family inhibitors and developed a high-throughput in silico screening strategy against homeodomain of MEIS proteins using the AutoDock Vina and PaDEL-ADV platform. We have screened over a million druggable small molecules in silico and selected putative MEIS inhibitors (MEISi) with no predicted cytotoxicity or cardiotoxicity. This was followed by in vitro validation of putative MEIS inhibitors using MEIS dependent luciferase reporter assays and analysis in the ex vivo HSC assays. We have shown that small molecules named MEISi-1 and MEISi-2 significantly inhibit MEIS-luciferase reporters in vitro and induce murine (LSKCD34l°w cells) and human (CD34+, CD133+, and ALDHhi cells) HSC self-renewal ex vivo. In addition, inhibition of MEIS proteins results in downregulation of Meis1 and MEIS1 target gene expression including Hif-1?, Hif-2? and HSC quiescence modulators. MEIS inhibitors are effective in vivo as evident by induced HSC content in the murine bone marrow and downregulation of expression of MEIS target genes. These studies warrant identification of first-in-class MEIS inhibitors as potential pharmaceuticals to be utilized in modulation of HSC activity and bone marrow transplantation studies.