Gene name - lilliputian
Synonyms - CG8817
Cytological map position - 23C1--2
Function - transcription factor
Symbol - lilli
FlyBase ID: FBgn0041111
Genetic map position -
Classification - Fragile X mental retardation 2 (Fmr2) family
Cellular location - nuclear
|Recent literature||Yang, H., Basquin, D., Pauli, D. and Oliver, B. (2017). Drosophila melanogaster positive transcriptional elongation factors regulate metabolic and sex-biased expression in adults. BMC Genomics 18(1): 384. PubMed ID: 28521739
Transcriptional elongation is a generic function, but is also regulated to allow rapid transcription responses. Following relatively long initiation and promoter clearance, RNA polymerase II can pause and then rapidly elongate following recruitment of positive elongation factors. Multiple elongation complexes exist, but the role of specific components in adult Drosophila is underexplored. RNA-seq experiments were carried out to analyze the effect of RNAi knockdown of Suppressor of Triplolethal and lilliputian. Similarly the effect of expressing a dominant negative Cyclin-dependent kinase 9 allele was analyzed. Almost half of the genes expressed in adults showed reduced expression, supporting a broad role for the three tested genes in steady-state transcript abundance. Expression profiles following lilliputian and Suppressor of Triplolethal RNAi were nearly identical raising the possibility that they are obligatory co-factors. Genes showing reduced expression due to these RNAi treatments were short and enriched for genes encoding metabolic or enzymatic functions. The dominant-negative Cyclin-dependent kinase 9 profiles showed both overlapping and specific differential expression, suggesting involvement in multiple complexes. Hundreds of genes were observed with sex-biased differential expression following treatment. Thus, transcriptional profiles suggest that Lilliputian and Suppressor of Triplolethal are obligatory cofactors in the adult and that they can also function with Cyclin-dependent kinase 9 at a subset of loci. These results suggest that transcriptional elongation control is especially important for rapidly expressed genes to support digestion and metabolism, many of which have sex-biased function.
Mutations in lilliputian (lilli) have been identified in three separate studies: (1) lilli is a dominant suppressor of the rough eye phenotype caused by constitutive activation of Raf during eye development (Wittwer, 2001); (2) lilli mutation strongly suppresses the rough eye phenotype of ectopically expressed phyllopod (Tang, 2001), and (3) lilli was identified in a screen for genes that enhance the embryonic lethal phenotype of dpp alleles (Su, 2001). Lilli plays a partially redundant function downstream of Raf in cell fate specification in the developing eye. Complete loss of Lilli function leads to a reduction in cell and organ size (Wittwer, 2001). Embryos lacking maternal lilli expression show specific defects in the establishment of a functional cytoskeleton during cellularization, and exhibit a pair-rule segmentation phenotype. These mutant phenotypes correlate with markedly reduced expression of the early zygotic genes serendipity alpha, fushi tarazu and huckebein, which are essential for cellularization and embryonic patterning (Tang, 2001). lilli mutations disrupt the transvection-dependent dpp phenotype and are also dominant maternal enhancers of recessive embryonic lethal alleles of dpp and screw. lilli zygotic mutant embryos exhibit a partially ventralized phenotype similar to dpp embryonic lethal mutations (Su, 2001). The lilli gene encodes a nuclear protein related to the AF4/FMR2 family. In humans, mutations affecting the genes of this family are associated with specific diseases. Loss of FMR2 gene transcription causes mental retardation. Translocations between MLL (a human trithorax-related gene) and AF4 or AF5q31 are involved in acute lymphoblastic leukemia (Wittwer, 2001, Tang, 2001 and Su, 2001).
lilli functions as a maternally provided pair-rule gene that is essential for proper cellularization, gastrulation and segmentation during embryogenesis. Two classes of phenotypes have been observed. Approximately half of the embryos are seen to be missing odd numbered segments, with the remaining denticle belts often fused. The other class of lilli GLC embryos fail to secrete cuticle properly. These two phenotypic classes appear to reflect variation inherent to the lilli loss-of-function phenotype, rather than partial rescue by a paternal copy of lilli, since they are similarly observed whether wild-type or heterozygous lilli males are used. Four lines of evidence support the idea that lilli functions during embryogenesis as a pair-rule gene: lilli germ-line clone (GLC) embryos fail to establish even-number Engrailed stripes; they subsequently lack odd-number segments; they exhibit defects in germband extension, and these phenotypes are not seen in homozygous lilli embryos lacking zygotic Lilli function. Thus, Lilli is required maternally for normal segmentation during embryogenesis (Tang, 2001).
What is the mode of action of lilli in segmentation? Unlike zygotic pair-rule genes that are expressed in the trunk region of the embryo in seven stripes, the expression pattern of lilli transcript is not segmental. Thus, lilli may play a role in the expression, localization or activity of other pair-rule genes. lilli probably does not act through the major gap genes knirps, Krüppel, or giant, because the striped pattern of the primary pair-rule genes eve, h and run are unaffected in lilli GLC embryos. One function of lilli is to regulate ftz transcription, since expression of both endogenous ftz mRNA and a ftz-lacZ transgene are markedly reduced in lilli GLC embryos. Although a low level of ftz mRNA remains in lilli GLC embryos, these transcripts never resolve into seven stripes. It is possible that lilli directly regulates ftz transcription in combination with other transcription and regulatory factors, or that lilli is required for the function or expression of such molecules. A candidate for such a factor is the product of the ftz-f1 gene, which is required maternally for ftz expression (Tang, 2001).
Lilli has a partially redundant function downstream of Ras/MAPK signaling in cell fate specification in the Drosophila eye. Loss-of-function mutations in lilli were identified as dominant suppressors of the specification of supernumerary R7 photoreceptor cells in response to constitutive activation of Raf in the developing eye (Dickson, 1996). However, without constitutive activity of the Ras/MAPK pathway, the normal number of photoreceptor cells is specified in each ommatidium in the complete absence of Lilli function. It is concluded that Lilli has a specific function in regulating the efficiency of signal transduction downstream of Raf. As a putative transcription factor, Lilli may regulate the expression levels of one or multiple components of the Ras/MAPK signaling pathway that become rate-limiting when Ras/MAPK signaling is too high, in cells where it is normally low (as in the case of ectopic activation of Raf in the eye), or when signaling is reduced (Wittwer, 2001).
The ability of lilli loss-of-function mutations to suppress MAPK signaling gain-of-function phenotypes and to enhance dpp loss-of-function phenotypes is very intriguing. lilli is one of the first genes involved in MAPK and TGF-ß signaling pathways in a developmental system. lilli encodes a transcription factor. This fact suggests one hypothesis for lilli's role in MAPK signaling and another hypothesis for a role in Dpp signaling. For MAPK signaling, lilli may be a transcriptional effector of MAPK signal transduction pathways. This hypothesis fits the observation that lilli loss-of-function mutations suppress MAPK signaling gain-of-function phenotypes. For Dpp signaling, lilli may be a maternally supplied transcriptional activator of dpp and/or scw during dorsal-ventral patterning. This hypothesis fits three observations: (1) lilli loss-of-function mutations maternally enhance the recessive lethality of several dorsal-ventral patterning mutations; (2) lilli mutant phenotypes mimic the mutant phenotypes of dorsal-ventral patterning mutations; (3) lilli mutations do not enhance, either maternally or zygotically, the embryonic lethality of genes that encode Dpp signal transduction proteins. Alternatively, lilli could participate in a signaling pathway parallel to the Dpp pathway that is also required for the expression of Dpp target genes (Su, 2001).
To test the hypothesis that lilli is a maternal activator of dpp and/or scw in dorsal-ventral patterning one would examine dpp and scw expression in embryos derived from lilli mutant germline clones. The prediction is that there would be reduced dpp and/or scw expression in these embryos during dorsal-ventral patterning. At this time, maternal activators of zygotic dorsal-ventral patterning genes such as dpp and scw, as opposed to well-known repressors such as Dorsal, are unknown. It is tempting to speculate that a maternal MAPK signal induces lilli to activate dpp in embryonic dorsal-ventral patterning (Su, 2001).
Determining a role for lilli in dpp signaling in adult wings, where lilli mutations enhance the heldout phenotype, is more problematic. There is no a priori reason to believe that lilli plays the same role in dpp signaling during dorsal-ventral patterning and adult appendage formation but it seems a logical place to begin. Thus it is possible that lilli activates dpp expression in wing imaginal discs. This hypothesis fits a report of dpp transcriptional regulation by the heldout cis-regulatory region. Two consensus HMGI binding sites (A/TA/TCAAG) are identified as dTcf (Pangolin) binding sites in the heldout region. The expression of reporter genes containing the dpp heldout region is disrupted when these putative dTcf sites are mutagenized. In addition, dominant negative forms of dTcf expressed in wing imaginal discs eliminate dpp expression. Thus dTcf is required for dpp expression by the heldout cis-regulatory region. However, these data do not preclude the possibility that the HMGI binding sites are actually the target of another HMGI domain protein, such as Lilli. To determine which HMGI domain protein is actually responsible for dpp expression from heldout regulatory sequences, one would examine dpp expression in wing imaginal discs bearing dTcf or lilli somatic clones (Su, 2001).
In addition to specific involvement of Lilli in MAPK and Dpp pathways, Lilli also functions in growth control. Growth is controlled by many different signals and during development it is tightly linked to pattern formation. In Drosophila, the insulin receptor controls growth via the PI3K/PKB pathway without affecting pattern formation. The EGF receptor also controls growth most likely via the Ras/MAPK pathway. Little is known about the nuclear factors that integrate the signals from these two pathways. Could the nuclear factor Lilli be a point of convergence of these two pathways for growth control? Although the cell size phenotype observed in lilli mutants is similar to that of mutants in either of the two pathways, mutations in lilli also display some unexpected phenotypes that are difficult to reconcile with Lilli functioning exclusively as a target of the two pathways. (1) lilli mutant cells do not have a growth disadvantage during early imaginal disc development. (2) The partial suppression of the overgrowth phenotype of Pten mutants by lilli mutants suggests a complex epistatic relationship between lilli and the PI3K/PKB pathway. (3) Both Lilli overexpression and lilli loss-of-function mutations reduce cell size. This is in contrast to components of the PI3K/PKB and Ras/MAPK signaling pathways. Overexpression of PI3K or PKB increases cell size, whereas loss-of-function mutations decrease cell size. Similarly, loss- and gain-of-function mutations of Ras have opposite effects on cell size. Lilli may activate the transcription of genes involved in cell growth and differentiation (Wittwer, 2001).
A fourth function for Lilli is suggested by the fact that significant percentage of embryos fail to properly secrete cuticle. Thus lilli GLC embryos have defects in addition to the patterning defects described above. Cytoskeletal architecture integrity during cellularization was examined. lilli germ-line clone (GLC) embryos exhibit specific defects in the maintenance of the actin network during cellularization. The initial phase of cellularization occurs normally. However, during the second phase of cellularization, specific defects in the maintenance of the contractile actin network are observed, as the actin network begin to contract and the furrow tips move basally. The actin filaments becomes unevenly distributed between nuclei, ranging from abnormally large bundles to regions where the actin network is thin or absent, resulting in multinucleated cells. In addition to the failure in maintaining the actin network, lilli GLC embryos exhibit defects in transport of organelles during cellularization. Living lilli GLC embryos have an abnormal distribution of lipid droplets during cellularization and about 80% of the embryos fail to separate from the central yolk sac shortly after cellularization. lilli does not induce general breakdown of microtubule-based transport, but rather is required specifically for the microtubule-based basal transport of lipid droplets. lilli is also required for the expression of serendipity alpha, a zygotic regulator of the actin cytoskeleton. The cellularization phenotypes of lilli GLC embryos are similar to those observed for mutations in the blastoderm-specific genes nullo and Sry alpha. Tang (2001) relates the insulin pathway-like phenotypes of lilli in the eye disc to the defects in cytoskeleton observed during embryonic development. Tang argues that lilli affects the cell size through a growth-independent and PI3K-independent mechanism. It is suggested that mutations in lilli may affect final cell size by disrupting the cytoskeletal based morphological changes that cells such as rhabdomeres and bristles, which are the specializations of photoreceptor and trochogen cells respectively, undergo during pupal development (Tang, 2001).
How does the function of Lilli in Drosophila correlate with the phenotypes observed in disease conditions associated with mutations in the human homologs AF4, AF5q31 and FMR2? In the case of acute lymphoblastic leukemia, two related genes, AF4 and AF5q31, are involved in translocations to the MLL gene (also known as ALL-1, htrx), which encodes a human homolog of the Drosophila protein Trithorax. Lilli function is essential for increased activity in the Ras/MAPK pathway. Activating mutations in Ras are frequently found in lymphomas but normally absent in leukemias associated with MLL rearrangements. It is possible that expression of the MLL/AF4 fusion gene circumvents the need for Ras activation in transformation. Recently, the targeted inactivation of the Af4 gene in mice has been reported (Isnard, 2000). Af4 mutant mice display an altered development of lymphoid cells and, on a mixed 129/Balb/c background, a subset show a significant reduction in body weight at birth. The reduction in weight may suggest that AF4 and Lilli control cellular growth in mice and Drosophila, respectively (Wittwer, 2001).
In summary, Lilli appears to function in MAPK and Dpp signaling pathways; it appears to affect growth, a function associated with the insulin pathway, and also affects the cytoskeleton early in development. Detailed studies of Lilli function in Drosophila will likely shed light on the wild-type function of human FMR2/LAF4 family members. For example, the functional conservation of dpp signaling pathway components suggests that human homologs of Lilli's transcriptional targets are likely to be targets of human FMR2/LAF4 family members. Given that mutations in these human genes lead to mental retardation or childhood cancer and that information on human developmental genes is difficult to gather directly, studies of Lilli are an important weapon in efforts to combat these human syndromes (Su, 2001).
BLAST searches (Su, 2001) using arbitrarily defined segments of the predicted Lilli protein identified very similar regions in four human proteins. These proteins belong to a multigene family called the FMR2/LAF4 family (Gecz, 1997; Nilson, 1997).
FMR2 was identified via mutations that result in Fragile-X mental retardation syndrome. Fragile X mental retardation syndrome is the most common form of inherited mental retardation in humans. FMR2 is highly expressed in the fetal brain. LAF4 was identified via chromosomal translocations that result in Burkitt's lymphoma. Burkitt's lymphoma is associated with highly malignant tumors and is the most common form of childhood cancer. LAF4 is highly expressed in fetal lymphoid tissue, particularly in preB-cells (Ma, 1996). The other family members, AF4 and AF5, were identified via distinct chromosomal translocations that give rise to infant acute lymphoblastic leukemia (ALL). At this time, ALL is resistant to treatment and invariably fatal. AF5 is highly expressed in fetal heart, lung, and brain while AF4 is highly expressed in fetal heart, liver, and brain (Li, 1998; Taki, 1999). These human proteins are nuclear proteins capable of DNA binding and transcriptional activation (Li, 1998).
Previous studies of this family identified three conserved domains (Gecz, 1997). Near the N terminus there is a conserved domain that includes a high mobility group I (HMGI) DNA-binding motif. In the center there is a conserved transcriptional activation domain with no recognizable motif. At the C terminus there is a highly conserved domain diagnostic for the FMR2/LAF4 family with no recognizable motif and unknown function. BLAST searches showed that Lilli contains segments, in the proper locations, very similar to each of these domains (Su, 2001).
An exhaustive analysis of the D. melanogaster genome database was conducted using the conserved regions of Lilli and the four human FMR2/LAF4 family sequences. A total of 15 different domains were used as query sequences. No additional proteins were identified that contain all 3 conserved domains. No additional proteins were identified with obvious similarity to only the C-terminal domain diagnostic for the FMR2/LAF4 family. At this time, Lilli appears to be the only D. melanogaster member of this multigene family. The same set of exhaustive searches was conducted using the C. elegans genome database. No proteins were identified with all three domains or any with convincing similarity to the C-terminal diagnostic domain (Su, 2001).
An alignment of the C-terminal domain of Lilli with all of the human family members shows extensive amino acid similarity with all of the human proteins. However, the alignment gives the overall impression that the four human family members are more similar to each other than they are to Lilli. The degree of amino acid identity and similarity, calculated from pairwise comparisons between all five sequences for each of the conserved domains show that there is a significant amount of amino acid similarity (greater than 51%) between Lilli and each human protein in all domains. The human proteins show greater than 63% similarity in all domains with most comparisons greater than 72% (Su, 2001).
Data derived from pairwise comparisons were used to construct phylogenetic trees for each domain. A composite tree was also constructed from an alignment consisting of all three domains. Only slight differences were noted between the individual domain trees and the composite tree. The similarity of the trees suggests that the tripartite structure of these proteins predates the divergence of arthropods and vertebrates. The composite tree shows that the human family members are indeed more similar to each other than they are to Lilli (Su, 2001).
lilli encodes a novel nuclear protein related to the mammalian AF4/FMR2 family of transcription factors The predicted lilli open reading frame encodes a protein of 1673 amino acids that has a C-terminal domain (amino acids 1418-1668, C-terminal homology domain (CHD) defined by Nilson, 1997) homologous to members of the AF4/FMR2 family of mammalian proteins. In this region, Lilli is 31%-37% identical to the corresponding sequences of human AF4, FMR2, AF5q13 and LAF4. Interestingly, the position of the intron/exon boundaries within the region encoding the CHD domain are conserved between lilli and the AF4 family members, suggesting that those exons have a common evolutionary origin (Tang, 2001).
Searching the Drosophila genome database does not reveal any sequences in addition to lilli that share homologies with AF4/FMR2 family members. Thus lilli appears to be the only member of this family in Drosophila. FMR2, AF4 and LAF4 are nuclear proteins and these have properties of transcription factors. They possess a putative transactivation domain and have the ability to bind DNA in vitro (Ma, 1996; Gu, 1996; Nilson, 1997; Gecz, 1997; Miller, 2000). Like other family members, Lilli is rich in proline and serine residues (9.0% and 12.7% of all amino acids, respectively). For human AF4 and LAF4, the serine-rich region has been shown to be situated in a part of the protein that has transactivation potential (Ma, 1996). Serines are concentrated in Lilli at the same relative position as in AF4/FMR2 family members (Ser domain, amino acid 860-965 of Lilli). However, domains within the AF4/FMR2 family members that are located N-terminal of the serine-rich region (i.e. NHD domain and ALF domain, defined by Nilson, 1997) are not present in Lilli (Tang, 2001).
In addition to the serine-rich region, two other features of Lilli indicate a possible function in activating transcription: (1) four glutamine-rich stretches in the N-terminal region of Lilli (Q1-Q4: amino acids 3-14, 32-48, 172-180 and 500-521) and (2) a domain (amino acid 245-300) with homology to part of a putative transactivation domain from the POU class III transcription factor. Lilli has two putative bipartite nuclear localization signals (amino acid 889-906 and amino acid 1286-1303, respectively). In addition, Lilli has a sequence (amino acid 836-859) which matches the consensus sequence for the HMG-I(Y) DNA binding motif, termed the AT-hook. This DNA binding domain is not found in other members of the AF4/FMR2 family. The AT-hook motif binds to the minor groove of the DNA double helix at A/T-rich sequences (Tang, 2001).
date revised: 4 March 2001
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