Gene name - runt
Cytological map position - 19E1-2
Function - transcription factor
Symbol - run
Genetic map position - 1-65
Classification - novel
Cellular location - nuclear
|Recent literature||Hang, S. and Gergen, J.P. (2017). Different modes of enhancer-specific regulation by Runt and Even-skipped during Drosophila segmentation. Mol Biol Cell [Epub ahead of print]. PubMed ID: 28077616
The initial metameric expression of the Drosophila sloppy paired 1 (slp1) gene is controlled by two distinct cis-regulatory DNA elements that interact in a non-additive manner to integrate inputs from transcription factors encoded by the pair-rule segmentation genes. This study performed Chromatin Immuno-Precipitation (ChIP) on reporter genes containing these elements in different embryonic genotypes to investigate the mechanism of their regulation. The Distal Early Stripe Element (DESE) mediates both activation and repression by Runt. The differential response of DESE to Runt was found to be due to an inhibitory effect of Fushi tarazu (Ftz) on P-TEFb recruitment and the regulation of RNA Polymerase II (Pol II) pausing. The Proximal Early Stripe Element (PESE) is also repressed by Runt, but in this case Runt prevents PESE-dependent Pol II recruitment and pre-initiation complex (PIC) assembly. PESE is also repressed by Even-skipped (Eve) but interestingly this repression involves regulation of P-TEFb recruitment and promoter-proximal Pol II pausing. These results demonstrate that the mode of slp1 repression by Runt is enhancer-specific whereas the mode of repression of the slp1 PESE enhancer is transcription factor-specific. The study proposes a model based on these differential regulatory interactions that accounts for the non-additive interactions between the PESE and DESE enhancers during Drosophila segmentation.
|Koromila, T. and Stathopoulos, A. (2017). Broadly expressed repressors integrate patterning across orthogonal axes in embryos. Proc Natl Acad Sci U S A. PubMed ID: 28720706
The role of spatially localized repressors in supporting embryonic patterning is well appreciated, but, alternatively, the role ubiquitously expressed repressors play in this process is not well understood. This study investigated the function of two broadly expressed repressors, Runt (Run) and Suppressor of Hairless [Su(H)], in patterning the Drosophila embryo. Previous studies have shown that Run and Su(H) regulate gene expression along anterior-posterior (AP) or dorsal-ventral (DV) axes, respectively, by spatially limiting activator action, but this study characterizes a different role. The data show that broadly expressed repressors silence particular enhancers within cis-regulatory systems, blocking their expression throughout the embryo fully but transiently, and, in this manner, regulate spatiotemporal outputs along both axes. These results suggest that Run and Su(H) regulate the temporal action of enhancers and are not dedicated regulators of one axis but, instead, act coordinately to pattern both axes, AP and DV.
Runt is a novel protein, unrelated to homeodomain, zinc finger or other transcription factors. It has a mammalian homolog that binds to enhancers of retroviruses and polyoma virus, and is involved in T-cell maturation. Like hairy and even-skipped, runt is termed a primary pair rule gene, as opposed to a secondary pair rule gene. Transcription of primary pair rule genes is regulated directly by maternal genes and gap genes, while secondary pair rule genes are regulated by the primary pair rule genes. Arguments can be made as to the validity and ultimate usefulness of primary/secondary pair rule distinctions; the notion is discussed in more detail the fushi tarazu overview.
Runt's effects are felt throughout the developmental hierarchy. Runt can modulate the activity of other pair rule genes, including hairy, even-skipped and ftz. runt possesses gap gene properties as well, altering the expression of giant and hunchback when transiently overexpressed (Tsai, 1994). Runt acts early in sex determination. Its activity is necessary to activate Sex lethal in the soma, but not in the germ line (Garandino, 1993). Runt also interacts with bicoid, restricting bicoid expression in the trunk, although the mechanism for this regulation is not completely understood (Tsai,1994). runt is also involved in neurogenesis, in the specification of neuroblasts (Kania, 1990).
runt is expressed by a subset of the 30 neuroblasts that give rise to each neuromere of the Drosophila embryo. Runt is also expressed in a subset of ganglion mother cells and neurons and its activity has been shown to be necessary for the formation of a subset of even-skipped (eve)-expressing lateral neurons, the EL neurons. There are 8-10 EL neurons per abdominal hemisegment, which originate from neuroblast 3-3. The EL neurons are interneurons that express the zinc-finger transcription factor encoded by eagle. The EL neurons project axons through the anterior commissure across the midline, then turn anteriorly into the longitudinal fascicles. Inactivation of runt during neuroblast delamination, using a temperature-sensitive allele of runt, leads to a loss of eve expression in the EL neurons. Eve expression in the EL neurons is not affected when Runt is inactivated after the neuroblasts have delaminated, suggesting that Runt activity is necessary only at the time of neuroblast delamination for the development of the EL neurons (Dormand, 1998).
To determine which neuroblasts express Runt, embryos were triple labelled with anti-Runt, anti-En and anti-Gsb-d. En is expressed by neuroblasts in row 6 and 7 and neuroblast 1-2; Gsb-d is expressed by neuroblasts in row 5 and 6 and neuroblast 7-1. Contrary to the previously published expression pattern, which showed expression of Runt in the neuroblasts only up to stage 10, it was found that Runt is expressed in neuroblasts throughout neurogenesis. Neuroblasts 2-2, 2-5, 3-1, 3-2, 5-2 and 5-3 express Runt from the time of their delamination (stage 10). By stage 11, Runt is also expressed in neuroblasts 2-3 and 3-3, and expression is lost from neuroblast 2-5. Runt was verified to be expressed in neuroblast 3-3 by double staining for Eagle, which is expressed by neuroblasts 2-4, 3-3, 6-4 and 7-3. A previously published expression pattern of Runt had not shown Runt expression in neuroblast 3-3, which gives rise to the EL neurons. It has been reported that neuroblasts 1-1 and 4-1 also express Runt, but this is not seen in the current study. Therefore, Runt is expressed in five neuroblasts in rows 2 to 3 (neuroblast 2-2, 2-3, 3-1, 3-2 and 3-3) and two neuroblasts in row 5 (neuroblast 5-2 and 5-3) (Dormand, 1998).
Runt is expressed by a large number of GMCs and neurons including the EL neurons. Runt is also expressed in a cluster of two to four cells on the midline and in a pair of neurons one on each side of the midline. By double labelling with anti-Odd, which labels MP1 and dMP2, it was found that the neurons on each side of the midline are the MP1 neurons. Double labelling with antibody to Slit, which labels the midline glia, identifies the cluster of Runt-expressing cells on the midline as the midline glia (Dormand, 1998).
runt is a good candidate for a gene that specifies neuroblast identities. To test this, Runt was ectopically expressed in restricted subsets of neuroblasts. Runt is sufficient to activate even-skipped expression in the progeny of specific neuroblasts. Eve is ectopically induced when runt is mis-expressed in all neuroblasts, using the pan neural driver scabrous-GAL4. The average of 9 EL neurons per hemisegment is increased to an average of 16 eve-expressing lateral cells per hemisegment. Ectopic Runt expression causes a severe disruption of the nerve cord, as shown by the abnormal medial eve expression and severe disorganization of the axons. However, Runt is not sufficient to induce eve expression in the progeny of all the neuroblasts. Neuroblast 6-1 and/or neuroblast 6-2 must express another protein that is essential for Runt to activate eve expression. Using the marker Tau-green fluorescent protein to highlight the axons, it was found that the extra Even-skipped-expressing neurons project axons along the same pathway as the EL neurons. Runt is expressed in neuroblast 3-3, supporting an autonomous role for runt during neuroblast specification (Dormand, 1998).
Proteins expressed both by neuroblast 3-3 and by neuroblasts 6-1 or 6-2 are possible candidates for cofactors acting with Runt to induce EL neurons. Neuroblast 6-1 expresses the steroid receptor superfamily member Seven-up and neuroblast 6-2 expresses the zinc-finger transcription factor Ming (Castor) in common with neuroblast 3-3. Although Eve expression is not affected in castor mutants, it would be interesting to investigate whether either Ming or Seven-up contribute to other aspects of the EL neuron fate (Dormand, 1998).
The Drosophila CNS contains a variety of glia, including highly specialized glia that reside at the CNS midline and functionally resemble the midline floor plate glia of the vertebrate spinal cord. Both insect and vertebrate midline glia play important roles in ensheathing axons that cross the midline and secreting signals that control a variety of developmental processes. The Drosophila midline glia consist of two spatially and functionally distinct populations. The anterior midline glia (AMG) are ensheathing glia that migrate, surround and send processes into the axon commissures. By contrast, the posterior midline glia (PMG) are non-ensheathing glia. Together, the Notch and hedgehog signaling pathways generate AMG and PMG from midline neural precursors. Notch signaling is required for midline glial formation and for transcription of a core set of midline glial-expressed genes. The Hedgehog morphogen is secreted from ectodermal cells adjacent to the CNS midline and directs a subset of midline glia to become PMG. Two transcription factor genes, runt and engrailed, play important roles in AMG and PMG development. The runt gene is expressed in AMG, represses engrailed and maintains AMG gene expression. The engrailed gene is expressed in PMG, represses runt and maintains PMG gene expression. In addition, engrailed can direct midline glia to a PMG-like non-ensheathing fate. Thus, two signaling pathways and runt-engrailed mutual repression initiate and maintain two distinct populations of midline glia that differ functionally in gene expression, glial migration, axon ensheathment, process extension and patterns of apoptosis (Watson, 2011).
This paper describes how the Hh morphogen patterns the midline cells to generate two populations of MG with distinct functional properties. The key output of this signaling is the expression of en that imparts PMG cell fate, in part, by repressing runt. In turn, the runt gene maintains AMG fate by repressing en. Thus, morphogenetic signaling and transcriptional regulation lead to AMG and PMG with divergent molecular, morphological and functional differences (Watson, 2011).
At stage 10, the 16 midline cells per segment consist of three equivalence groups of neural precursors, four to six cells each. Notch signaling directs ten of these 16 cells to become MG; the remainder become MPs and the MNB. Thus, Notch represses neuronal development in MG and activates a core set of MG-expressed genes (e.g., wrapper). MG in the anterior of the segment become AMG; those in the posterior of the segment become PMG. Notch signaling by itself is unlikely to influence different MG fates, as expression of activated Suppressor of Hairless in midline cells drives all cells into a MG fate but does not affect their AMG or PMG patterns of gene expression. Thus, additional factors that can direct AMG and PMG cell fates were sought (Watson, 2011).
Previous work demonstrated that hh can pattern midline cells along the A/P axis, and, indeed, this study demonstrates that hh is required for PMG cell fate. The source of Hh is not in the midline, but in the lateral ectoderm in a stripe of cells, collinear with the pair of midline early en+ cells. Hh signals to midline cells posterior to the early en+ cells, inducing en in an additional six to seven cells. These late en+ cells plus the early en+ cells become about four PMG, as well as MP4-6 and the MNB. Misexpression and mutant analyses indicate that hh is required for all PMG gene expression and for repressing AMG expression. hh signaling probably has multiple target genes because hh is required for en and l(1)sc expression, but en does not regulate l(1)sc. Misexpression of hh can activate en expression in anterior MG, and both hh and en misexpression convert these cells functionally into non-ensheathing MG that resemble PMG, results also consistent with observations that ectopic expression of hh and en in midline cells affects AMG differentiation. However, neither hh nor en can activate all PMG gene expression in anterior MG, because neither activates masquerade (mas) expression in anterior MG. The mas gene is expressed transiently at stage 12 in a subset of PMG, suggesting that functionally distinct classes of PMG might exist. Expression of mas might require other signals in addition to hh that are absent in anterior MG (Watson, 2011).
runt is present in AMG and represses en and PMG-specific gene expression. In runt mutant cells that are runt- en+, expression of three genes expressed in only AMG (CG33275, Fhos and nemy) are absent and wrapper is reduced. This could be due to runt repression of en, repression of other genes or activation by runt. In runt mutant cells that are runt- en-, Fhos and nemy are present, wrapper is at high levels, but CG33275 expression is absent. This suggests that runt does not activate expression of Fhos, nemy and wrapper in AMG, but maintains their AMG levels by repressing en. By contrast, runt is required for expression of CG33275, possibly indicating a positive role for runt in AMG differentiation in addition to its repressive role in AMG maintenance. However, CG33275 is most prominently expressed in a subset of AMG closest to the commissures, and this AMG expression could be dependent on additional signals, perhaps from the developing axon commissure. Thus, absence of CG33275 expression in runt mutant embryos could alternatively be due to an effect of runt on developing axons or CNS development (Watson, 2011).
As most AMG gene expression is not dependent on runt, it is proposed that Notch signaling initially induces an AMG pattern of gene expression in all glia and, either simultaneously or soon after, Hh signaling in the posterior of the segment generates PMG. One important downstream target of Notch signaling is likely to be the sim gene, which encodes a bHLH-PAS protein that functions as a DNA-binding heterodimer with the Tango (Tgo) bHLH-PAS protein. During early development, sim is expressed in all midline primordia and is required for midline cell development. However, later in development, sim is restricted to MG and a subset of midline neurons. Genetically, sim expression is absent in embryos mutant for Notch signaling. The sim gene is likely to be an important aspect of MG transcription, because mutation of Sim-Tgo binding sites in the slit and wrapper MG enhancers results in loss of MG expression, and Sim-Tgo binding sites are present in other identified MG enhancers. The Hh morphogen transforms only posterior MG into PMG. It is unknown why hh does not affect anterior MG, but it is likely to be owing to the presence of unknown factors in these cells that inhibit hh signaling. Since Notch signaling, rather than runt, is primarily required for AMG gene expression, the key role of runt is probably to maintain AMG gene expression by repressing en. Similarly, en functions to maintain PMG gene expression by repressing runt, but also contributes positively to PMG cell fate, as en misexpression confers PMG-like function to AMG (Watson, 2011).
The most striking features of AMG are their ability to migrate around the commissures, ensheath them and extend processes into the axons. The function of PMG is unknown, but they are unable to ensheath the commissures, even though they are in close proximity. One of the major factors influencing AMG-axon interactions is Nrx-IV-Wrapper adhesion. Levels of wrapper expression in AMG are higher than in PMG, and this is likely to be a key determinant of why AMG ensheath commissures, and PMG do not, because loss of wrapper expression results in incomplete migration and ensheathment. Recent work has demonstrated that sim directly regulates wrapper expression, and spitz signaling from axons might also form a positive feedback loop to control wrapper levels and strengthen Nrx-IV-Wrapper interactions. As en genetically reduces wrapper levels in PMG, it will be interesting to determine if this regulation is direct or indirect. Although the control of wrapper levels is likely to be a major factor in AMG-PMG differences and the ability of glia to ensheath axons, other genes whose levels differ between AMG and PMG might also contribute. This illustrates why it will be important to identify target genes and understand better the roles that Notch//Suppressor of Hairless, sim, hh, Ci, en, runt and other MG transcription factors play in regulating MG gene expression and function (Watson, 2011).
Bases in 5' UTR -251
Exons - two
Bases in 3' UTR - 621
The absence of an identifiable transcription factor motif (e.g., homeo box, zinc finger, leucine zipper, or helix-loop-helix) makes Runt different from the other early-acting segmentation proteins. The subcellular location of the protein is in the nucleus (Kania, 1990).
A highly conserved region in the Runt protein is termed the Runt domain. The functional properties of the Runt domain from the D. melanogaster gene and the human AML1 (acute myeloid leukemia 1) gene were compared. The different DNA binding properties of Runt and AML1 are due to differences within their respective Runt domains. Proteins containing the AML1 Runt domain function in Drosophila embryos, but sequences outside of the runt domain are important in vivo (Pepling, 1995).
date revised: 12 December 98
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