BIOLOGICAL OVERVIEW

The jing zinc-finger transcription factor, identified as a downstream target of slbo required for developmental control of border cell migration (Liu, 2001) also plays an essential role in controlling CNS midline and tracheal cell differentiation. The jing locus ('jing' means 'still' in Chinese) was initially identified in a screen for mutations that cause border cell migration defects in mosaic clones (Liu, 2001). Zygotically jing transcripts and protein accumulate from stage 9 in the CNS midline, trachea and in segmental ectodermal stripes. Jing protein localizes to the nuclei of CNS midline and tracheal cells implying a regulatory role during their development. Loss of jing-lacZ expression in homozygous single-minded (sim) mutants and induction of jing-lacZ by ectopic sim expression establish that jing is part of the CNS midline lineage. Embryonic recessive lethal jing mutations display genetic interactions in the embryonic CNS midline and trachea, with mutations in the bHLH-PAS genes sim and trachealess, and their downstream target genes (slit and breathless). Loss- and gain-of-function jing is associated with defects in CNS axon and tracheal tubule patterning. In jing homozygous mutant embryos, reductions in marker gene expression and inappropriate apoptosis in the CNS midline and trachea establish that jing is essential for the proper differentiation and survival of these lineages. These results establish that jing is a key component of CNS midline and tracheal cell development. Given the similarities between Jing and the vertebrate CCAAT-binding protein AEBP2 (He, 1999), it is proposed that jing regulates transcriptional mechanisms in Drosophila embryos and promotes cellular differentiation in ectodermal derivatives (Sedaghat, 2002a).

The jing expression pattern and gene dose effects in the CNS midline and trachea suggest that jing function may be important for the development of both systems. Therefore, CNS axon and tracheal tubule development was assessed in jing homozygous mutant embryos stained with monoclonal antibodies BP102 and 2A12, respectively. In jing³ homozygous mutant embryos, commissural growth cones are often absent in the midline at stage 12 when compared with wild type. By stage 14, homozygous jing³ mutants show losses of longitudinal connections and reduced commissures compared with wild type. Embryos double mutant for jing and sim display phenotypes similar to those of sim homozygotes. Therefore, the sim embryonic CNS axon phenotype is epistatic to that of jing, implying that jing functions downstream of sim (Sedaghat, 2002a).

The GAL4/UAS system was used to determine the effects of overexpressing jing in the CNS midline. Flies containing P[sim-GAL4] were crossed to flies containing P[jing-UAS] and their progeny stained with BP102 to assess CNS axon formation. Expression of one copy of P[jing-UAS] specifically in the CNS midline is sufficient to inhibit commissural and longitudinal axon formation. Therefore, the jing midline overexpression phenotype is similar to that resulting from jing loss of function, and phenotypes of jing and sim double heterozygotes. These results demonstrate that appropriate jing dose is a requirement for proper CNS axon development in the CNS midline. Interestingly, a similar CNS axon phenotype is observed after overexpression of sim in the CNS midline (Sedaghat, 2002a).

The homozygous jing CNS phenotype suggests an alteration in the mechanisms that guide CNS axons. Fasciclin 2 staining using 1D4 mAb, shows that longitudinal fascicles stall within segment boundaries, causing breaks in the longitudinal tracts in 95% of jing³ mutant segments. A subset of normally ipsilateral axons of the most medial fascicle project instead contralaterally in jing³ mutants. Since ipsilateral fascicles are prevented from crossing the midline in wild-type embryos, these results suggest that midline repulsive mechanisms are perturbed in jing mutant embryos (Sedaghat, 2002a).

jing's involvement in tracheal patterning was assessed. Embryos homozygous for a jing deficiency [Df(2R)ST1] and jing³ mutations are associated with losses of the dorsal trunk, severely disrupted transverse connectives and absences of the visceral branch. Embryos doubly mutant for jing and trh lack all tracheal tubules and display phenotypes identical to trh homozygous mutants. Therefore, trh loss-of-function is epistatic to jing loss-of-function, implying that jing functions downstream of trh (Sedaghat, 2002a).

To determine the effects of overexpressing jing in the trachea, flies containing the P[breathless (btl)-GAL4] driver were crossed to those containing P[jing-UAS]. Progeny from this cross were stained with 2A12 antibody and tracheal tubule development was analyzed by light microscopy. Overexpression of jing in the trachea is associated with defects in dorsal trunk fusion, as well as improper formation of the transverse connective, dorsal branch and visceral branch. Therefore, jing overexpression tracheal phenotypes are similar to jing loss-of-function tracheal phenotypes (Sedaghat, 2002a).

Cell type-specific markers were used to follow CNS midline development in homozygous jing mutant embryos. Midline cells were identified using anti-Sim and the glial-specific marker anti-Slit. Expression of sli was assessed in homozygous jing mutant embryos using the lacZ reporter P[1.0 HV, sli-lacZ]. There are reductions in the number of Sim-positive and sli-lacZ expressing midline cells in homozygous jing³ mutants compared with wild-type embryos during stage 9 and 11, respectively. This clearly demonstrates that the early differentiation of midline lineages requires jing function. By later stages of embryogenesis (stage 15), Sim and Sli immunoreactivity is drastically reduced in jing mutant nerve cords. The presence of Sli-positive cellular profiles in macrophages outside the VNC suggests that midline lineages are lost by cell death. Similar results were obtained using anti-Wrapper as a marker of glial identity (Sedaghat, 2002a).

To address whether midline glia enter apoptotic pathways, jing mutant embryos were double-labeled with anti-Sli and TUNEL, and the occurrence of apoptotic glia was monitored from stages 12 to 15. On average, there are one or two apoptotic midline glia within an entire nerve cord of a stage 12 wild-type embryo. By contrast, every nerve cord segment in jing³ mutant embryos contains apoptotic glia in addition to the presence of more TUNEL-positive profiles in the CNS. The increased occurrence of apoptotic glia correlates with reductions in Sli immunoreactivity in the midline of jing³ mutant embryos and establishes that jing function is required for midline glial survival (Sedaghat, 2002a).

Enhancer traps and antibodies were used to follow the development of individual motoneurons (VUMs, 22C10) and interneurons, such as the midline precursors (MP1, dMP2, vMP2; P223, anti-ODD and 22C10) and the median neuroblast (MNB; anti-Engrailed) in wild-type and homozygous jing³ mutant embryos. jing loss-of-function mutations are associated with reductions in the expression of all neuronal markers tested. There are absences of immunoreactivity in the VUMs, MNB and MP1 neuronal lineages in some VNC segments in jing³ mutant embryos. There is a loss of Odd immunoreactivity as early as stage 10 in MP neurons in homozygous jing³ mutants. Similar reductions in the number of immunoreactive vMP2 and dMP2 are observed by 22C10 staining of stage10 homozygous jing³ mutant embryos (Sedaghat, 2002a).

Within a particular VNC segment in jing³ mutants, there is a loss of Engrailed (EN)-positive neurons while the number of EN-expressing neuroectodermal cells remains equal to that in wild-type embryos. In addition, jing mutant embryos displaying reduced 22C10 staining of the VUMs in the CNS midline do not show any visible defects in peripheral nervous system development. These results strongly suggest that the primary site of jing CNS function is at the midline (Sedaghat, 2002a).

In summary, these results demonstrate that midline neuronal and glial populations do not differentiate without proper jing function and suggest a positive role for jing in promoting CNS midline cell development (Sedaghat, 2002a).

To determine the role of jing during tracheal development, a phenotypic analysis of homozygous jing mutant embryos was performed using antibodies to Trh as a marker of cell identity and to En for identifying the anterior border of the trachea. Initial defects in tracheal morphogenesis occur during tracheal placode stages in embryos homozygous mutant for all jing alleles. This correlates with the nuclear localization of Jing in tracheal placode cells. The number of Trh-positive precursors in stage 10 homozygous jing³ mutant embryos is approximately 22% of the expected number of wild-type cells. The relatively normal pattern of ectodermal segmentation in jing³ mutant embryos, as revealed by En staining, suggests that the improper differentiation of tracheal cells in these mutants is not likely to result from indirect effects of ectodermal patterning. These results also reveal that the positioning of tracheal placodes in jing³ mutants is not altered from that of wild-type embryos (Sedaghat, 2002a).

To determine the fate of tracheal lineages the pattern of cell death by double labeling wild-type and jing³ mutant stage 11 embryos with TUNEL and anti-Trh. Cell death is not common in the tracheal pits of wild-type stage 11 embryos. On average, there is a maximum of three TUNEL- and Trh-positive cells within an entire stage 11 wild-type embryo. By contrast, there is an average of 20 TUNEL- and Trh-positive precursors in stage 11 jing³ mutant embryos. There is also an increase in the number of apoptotic profiles surrounding the tracheal pits in jing³ compared with wild-type embryos. Cell death is observed by TUNEL labeling throughout embryogenesis in all tracheal branches in homozygous jing³ mutant embryos, suggesting that the requirement for jing function is not branch specific (Sedaghat, 2002a).

In jing³ homozygous mutant embryos, tracheal cells invaginate but the tracheal branches do not migrate properly to the anterior across En-positive stripes, as they do in wild-type embryos. In addition, fewer Trh-positive cells express En in homozygous jing³ mutant embryos compared with wild-type at stage 12. By stage 15 in jing³ mutant embryos, parts of the dorsal trunk, the dorsal branch and transverse connectives are missing and correlate with a loss of cells by apoptosis. In addition, the visceral branch does not form in jing³ mutant embryos. Therefore, the EGFR-dependent visceral and dorsal trunk branches appear more severely affected than the Dpp-dependent dorsal and ganglionic branches, as well as the transverse connectives in jing³mutant embryos. Despite the death of tracheal cells in jing mutant embryos, the overall embryonic pattern of cell death is not significantly altered by the end of embryogenesis from that of wild-type embryos. Therefore, the tracheal defects in jing mutants are not likely to result from widespread defects in embryonic differentiation (Sedaghat, 2002a).

The results presented here show that CNS midline neurons and glia do not differentiate properly in homozygous jing mutant embryos. Several lines of evidence support this. The expression of cell-type-specific markers of midline neuronal and glial identity is altered in jing mutants compared with that in wild-type embryos. For example, expression of the sli-lacZ 1.0 HV reporter initiates in six midline glia in each wild-type nerve cord segment during stage 11. By contrast, sli-lacZ 1.0 HV reporter expression in jing mutants initiates in only an average of three midline glia per nerve cord segment by stage 11. In addition, there are reductions in the number of Sim-positive midline cells and ODD-positive/22C10-positive MP neurons by stage 9 in jing³ homozygous mutant embryos, respectively. Therefore, early midline glial and neuronal differentiation is aberrant in homozygous jing mutant embryos. By the end of embryogenesis, many neuronal and glial cell type markers are barely detectable in homozygous jing mutant ventral nerve cords (Sedaghat, 2002a).

The loss of sim, sli, odd and 22C10/futsch expression in jing mutants may reflect improper activation/regulation of gene expression or may be secondary to cell loss. To address this issue, the pattern of cell death was analyzed in the CNS midline of jing mutant embryos. Apoptosis occurs in the midline glial lineage in wild-type embryos and begins during stage 12 to refine the number of cells from six to an average of three per nerve cord segment by the end of embryogenesis. In homozygous jing mutants, however, there are more apoptotic glia during stage 12 than in wild-type embryos and this correlates with the loss of Sli-positive glia. It is, therefore, likely that the loss in CNS midline gene expression in jing mutants results from a loss of cells. In summary, the loss in expression of cell identity markers and inappropriate cell death lead to the conclusion that midline neurons and glia do not differentiate properly in jing mutant embryos (Sedaghat, 2002a).

The arthropod ventral nerve cord is characterized by the ladder-like pattern of the major CNS axon tracts. The nerve cord is segmental and each neuromere is connected by longitudinal axons, which are separated by anterior and posterior commissures. Disruption of this pattern by jing gain-of-function specifically in the CNS midline reveals the requirement for proper jing function within these cells for axon patterning. In addition, homozygous mutant jing embryos display reductions in CNS midline cells while neuroectodermal and peripheral nervous system development is unperturbed. Together, these results show that jing mutations have strong effects on the CNS midline and that jing dosage is crucial for their development (Sedaghat, 2002a).

Genetic analysis of axon patterning in the Drosophila CNS has revealed the important role of neuron-glial function in this process. Mutations leading to reductions in midline neuron numbers correlate with a reduction in the number of commissural tracts, whereas mutations leading to reductions in midline glia numbers show fused commissure phenotypes. These observations are consistent with the hypothesis that midline neurons (such as the VUMs) are required to attract commissural growth cones initially to the CNS midline, whereas midline glia are required subsequently for the organization of commissural axons. Based on these observations, it is proposed that defects in the differentiation of midline neuronal precursors, such as the VUMs, in jing loss-of-function mutants, inhibit the attraction of commissural growth cones to the CNS midline during stage 12. As the attraction of commissural axons to the CNS midline precedes the separation of anterior from posterior commissures, the defects in midline neuronal differentiation and the associated lack of growth cones in the midline of jing mutants probably mask subsequent defects in glial-associated functions. During axon patterning, the MP1 interneurons participate in the formation of specific longitudinal pathways. Therefore, the defects in MP1 neuronal differentiation in jing mutants may account for the inhibition in the formation of the longitudinal connectives (Sedaghat, 2002a).

Signals generated by CNS midline cells control the commissural axon pattern by either guiding growth cones toward the midline or preventing them from crossing the midline. Defects in glial-associated functions occur in the CNS of homozygous jing mutant embryos. Reduced glial numbers and Sli production in jing mutants are consistent with the reduction in midline repulsion of longitudinal pathways as visualized by Fasciclin 2 staining. The remaining Sli protein product in stage 12 jing mutant nerve cords, however, is apparently sufficient to prevent a total collapse of the longitudinal connectives, as observed in homozygous sim and sli mutations (Sedaghat, 2002a).

This work has also identified multiple roles for jing in tracheal morphogenesis. The earliest function of jing is to allocate the correct number of cells to the tracheal placodes. Several lines of evidence support this. The number of tracheal placode cells is significantly reduced from wild-type in homozygous jing mutant embryos. In addition, tracheal precursors die in jing mutant embryos, suggesting that jing is essential for their differentiation. Since Jing localizes to the nuclei of tracheal placode cells and contains potential DNA-binding and transactivation domains, it is possible that it regulates genes essential for the differentiation and survival of tracheal precursors (Sedaghat, 2002a).

Although loss of jing function affects cellular differentiation in all tracheal lineages, it appears to have more severe effects on dorsal trunk and visceral branch development. The dorsal trunk and visceral branches derive from the same position in the tracheal placode and are induced by Epidermal growth factor receptior (Egfr). Egfr is activated in the central portion of the tracheal placodes by the restricted expression of rhomboid (rho). The defects in dorsal trunk and visceral branch formation in homozygous jing mutant embryos are similar to those in embryos homozygous mutant for Egfr signaling. Given that mutations in Egfr pathway genes do not affect tracheal placode cell numbers, it is proposed that jing may function prior to Egfr signaling (Sedaghat, 2002a).

Several lines of evidence suggest that jing functions specifically in tracheal cells: (1) Jing protein is detected within nuclei of tracheal precursors and differentiated lineages; (2) defective placodes in jing mutants are observed in hemisegments with normal en expression patterns indicating that defects in the metamerization process do not cause the jing tracheal phenotype. However, the possibility that Hedgehog signaling in segmental ectodermal stripes is affected by jing mutations cannot be ruled out. hh is required in determining proper tracheal placode numbers in some hemisegments. (3) The most severe defects in tracheal patterning in jing mutant embryos occur in the dorsal trunk and visceral branch, suggesting that there is some specificity to jing tracheal function, and (4) overexpression of jing specifically in the trachea results in defects in tracheal patterning that resemble jing loss-of-function phenotypes (Sedaghat, 2002a).

Based on genetic and phenotypic analyses, a role is proposed for jing downstream of sim and trh during CNS midline and tracheal development, respectively. (1) jing expression is not observed prior to that of either sim or trh in the CNS midline and trachea, respectively. jing expression is detected in the CNS midline during stage 9, which comes after the initiation of sim expression and establishment of midline fates. Jing protein is present in tracheal precursor nuclei, coincident with Trh during stage 10. (2) The CNS axon and tracheal phenotypes of homozygous jing mutations are less severe than those of homozygous sim and trh mutations, respectively. However, it cannot be rule out that maternal Jing may rescue the effects of zygotic jing mutations or that jing functions in a combinatorial fashion and therefore may not display severe phenotypes. (3) jing can be activated by ectopic expression of sim, suggesting that sim may regulate jing. The presence of three E-box ACGTG core sites in the 5' regulatory region of jing suggest that this regulation may be direct. (4) The sim and trh embryonic phenotypes are epistatic to that of jing, as shown by double mutant analysis. (5) jing mutations genetically interact with mutations in bHLH-PAS target genes such as sli and btl. The ventral displacement of midline cells in jing and sli double heterozygotes strongly suggests that jing is required for proper sli regulation (Sedaghat, 2002a).

In addition to its functions in CNS midline and tracheal cell development jing function is required for initiation of border cell migration during oogenesis. Epithelial to mesenchymal transitions and cell migration are important features of embryonic development and tumor metastasis. Expression of the basic-region/leucine zipper transcription factor, C/EBP, is required for the border cells to initiate their migration. jing locus was identified in a screen for mutations that cause border cell migration defects in mosaic clones. The jing mutant phenotype resembles that of slbo mutations, which disrupt the Drosophila C/EBP gene, but is distinct from other classes of border cell migration mutants. Expression of a jing-lacZ reporter in border cells requires C/EBP. Moreover, expression of jing from a heat-inducible promoter rescues the border cell migration defects of hypomorphic slbo mutants. The Jing protein is most closely related to a mouse protein, AEBP2 (He, 1999), which was identified on the basis of its ability to bind a small regulatory sequence within the adipocyte AP2 gene to which mammalian C/EBP also binds (Liu, 2001).

It appears that the border cell epithelial-to-mesenchymal transition requires changes in gene expression, some of which are mediated by C/EBP and Jing. DE-Cadherin is clearly one key downstream target of C/EBP, however it is not the only relevant downstream target. A thorough understanding of all of the changes required to convert a stationary epithelial cell to a migratory cell will require identification of more of the genes required for this process. It is striking that the three genes that have been identified to date in forward genetic screens for mutations that inhibit border cell migration, slbo, jing and taiman, have not been identified in any previous genetic screens. This raises the possibility that the genetic control of the epithelial-to-mesenchymal transition is significantly different from genetic control of pattern formation, eye development and other processes that have been subjected to extensive genetic analysis in Drosophila. However more extensive characterization of genes controlling border cell migration will be required before it is clear how different this process is from the others (Liu, 2001).

What are the similarities between control of border cell migration and adipocyte differentiation? It is proposed that the need to coordinate cell differentiation with nutritional status may be the link between mammalian adipocytes and Drosophila border cells that led to the conservation of C/EBP and AEBP2. Undoubtedly many genes are required for an epithelial cell to become motile; therefore it is striking that the jing locus encodes a protein with such a clear functional connection to C/EBP. Evolution, it seems, has conserved functional networks of transcriptional regulators, rather than individual genes. Mammalian proteins related to Jing and Slbo appear to be involved in the regulation of adipocyte differentiation, a process that bears little obvious resemblance to border cell migration. One similarity between adipocytes and border cells is that both cell types appear to coordinate their differentiation with nutritional status of the organism. In the case of adipocytes, at least two independent transcriptional regulatory pathways appear to be required. One pathway is the C/EBP pathway, which may also require AEBP2. A second pathway requires the activity of PPARgamma, a steroid hormone receptor-like molecule that is activated by circulating fatty acids whose levels depend upon diet. Drosophila ovarian development also responds to nutritional signals. Flies emerge from the pupal case with ovaries that contain only immature egg chambers. Further progression of oogenesis requires that the flies consume a rich diet. Application of lipophilic hormones, such as juvenile hormone or ecdysone, can bypass this requirement. The ecdysone receptor is required in the border cells for their migration. Thus border cell migration, like adipocyte differentiation, requires a hormonal signal, which reflects nutritional status, to be integrated with an intrinsic developmental program. It is this latter program that appears to be mediated by the C/EBP, AEBP2 and the Drosophila proteins Slbo and Jing (Liu, 2001 and references therein).

An interesting parallel between the ovarian and embryonic pathways involving jing is the activation of btl. btl is expressed in embryonic tracheal and midline glial cells, as well as border cells of the ovary, and is essential for their migration. btl is a direct target of C/EBP and Trh::Tgo heterodimers in vitro. Therefore, the strong dominant interactions between jing and btl in the embryonic trachea coupled with the role of jing in border cell migration implies an important link between the maternal and embryonic pathways involving jing. C/EBP is expressed in the embryonic trachea after btl expression begins. Therefore, C/EBP probably does not regulate jing or btl in the trachea, as it does in border cells. Furthermore, C/EBP expression is not sufficient to cause ectopic btl expression and therefore, it is proposed that this transcription factor carries out the gene expression program initiated by other factors. In this way, C/EBP can function in very different pathways, including fat metabolism in adipocytes, long-term memory in Aplysia neurons and cell migration in the ovarian border cells. The transcriptional capabilities of jing have not yet been tested and therefore it is not known whether jing co-operates with Tgo::Trh or Tgo::Sim heterodimers in activation of btl or other targets (Sedaghat, 2002a).

It is proposed that bHLH-PAS heterodimers may activate jing transcription by binding any or all of the three CNS midline elements (CMEs) present in the 5' regulatory region of jing. The initiation or maintenance of jing transcription may also require the function of additional transcription factors such as VVL in the CNS midline and trachea or Fish-hook in the CNS midline. The presence of a Fish-hook DNA-binding site (TACAAT) adjacent to the CMEs in the jing 5' regulatory region suggests this possibility (Sedaghat, 2002a).

Both genetic and phenotypic evidence suggest that jing functions in a different manner from either vvl or fish. vvl and fish have combinatorial regulatory roles in CNS midline or tracheal pathways, and therefore vvl and fish-null embryos do not show strong phenotypes. By contrast, the defects in cell numbers and increased cell death during stage 10 and 11 in jing mutants precede and are different from the defects in homozygous vvl and dfr-fish double mutants in the CNS midline or from dfr homozygotes in the trachea. Therefore, activation of jing transcription in the CNS midline or trachea cannot be controlled exclusively by Fish-hook or VVL (Sedaghat, 2002a).

jing encodes a putative DNA-binding protein with putative transcriptional regulatory domains, and its product can be seen in the nuclei of CNS midline and tracheal cells. Based on jing expression patterns and phenotypes, it is proposed that jing participates in the activation of genes downstream of both Sim::Tgo and Trh::Tgo in the CNS midline and trachea, respectively. Whether jing targets are also regulated by bHLH-PAS, POU or SOX combinatorial transcriptional activities remains to be determined. Nevertheless, Jing function is required to promote the differentiation of CNS midline and tracheal lineages, which, in the absence of jing function, do not differentiate and instead undergo apoptosis (Sedaghat, 2002a).

How does jing promote cellular differentiation? It is proposed that jing regulates the transcription of important survival factors including those of the Egfr pathway. For example, the rho gene product regulates the processing of the Egf receptor ligand Spitz and is expressed at stage 9/10 in the CNS midline and in the center region of the tracheal placodes. Proper function of Egfr pathway genes is required for the survival of cells most highly affected by jing mutations, including the CNS midline glia, the tracheal dorsal trunk and visceral branches. Furthermore, the rho regulatory region is controlled by Tgo:Trh:Dfr interactions and also contains multiple CAAT sites similar to those bound by AEBP2, the protein most related to Jing (He, 1999). This raises the possibility that jing may be involved in a combinatorial fashion in the regulation of bHLH-PAS target genes. However, this hypothesis does not account for the role of jing in EGFR-independent process such as survival of CNS midline neurons and dpp-dependent tracheal branches. One can then argue that jing function in the CNS midline and trachea is generic, and that Jing carries out the gene expression programs initiated by bHLH-PAS, POU domain and SOX transcription factors. If the transcriptional programs are not maintained by jing, cells enter default apoptotic pathways. Alternatively, Jing may be responsible for directly activating unknown survival factors in midline neurons and dpp-dependent tracheal branches (Sedaghat, 2002a).

GENE STRUCTURE

cDNA clone length - 5137

Bases in 5' UTR - 536

Exons - 7

Bases in 3' UTR - 140

PROTEIN STRUCTURE

Amino Acids - 1486

Structural Domains

To construct a transcript map of the jing region, fragments of genomic DNA flanking the sites of P-element insertions were used to probe Northern blots and an embryonic cDNA library. Sequencing of cDNAs isolated from the cDNA library, as well as est clones, have indicated that alternative splicing of the locus produces at least four different classes of mRNAs. However, all of the mRNAs contain the same coding sequence. A major transcript of 6.4 kb and a minor larger transcript hybridizes to probes made from DNA flanking the P-element insertion sites. The jing transcripts contained an open reading frame of 1486 amino acids. A search of the predicted protein sequence using Prosite has revealed the presence of three zinc finger motifs. A BLAST search using the predicted protein sequence has revealed that the Jing protein is most highly related to a mouse transcription factor known as AEBP2, which is also predicted to have three zinc fingers. Jing exhibits 50% amino acid identity with AEBP2 within the zinc finger motifs and 20% identity C-terminal to the zinc fingers (BLAST E value of 1e-34). After AEBP2, the most similar proteins are several members of the GLI family of zinc finger transcription factors. However GLI proteins typically contain five zinc finger motifs. Members of the GLI family of proteins, which includes Drosophila CI, are only 25% identical to Jing within the zinc fingers and do not exhibit homology outside of these motifs (Liu, 2001).

EVOLUTIONARY HOMOLOGS

A novel transcriptional repressor, AEBP2, has been identified that binds to a regulatory sequence (termed AE-1) located in the proximal promoter region of the aP2 gene that encodes the adipose fatty acid-binding protein. Sequence analysis of AEBP2 cDNA has revealed that it encodes a protein containing three Gli-Kruppel (Cys2-His2)-type zinc fingers. Northern blot analysis reveals two transcripts (4.5 and 3.5 kilobases) that are ubiquitously expressed in every mouse tissue examined. AEBP2 repressed transcription from the homologous aP2 promoter containing multiple copies of the AE-1 sequence. Moreover, a chimeric construct encoding a fusion AEBP2 protein with the Gal4 DNA-binding domain is able to repress the transcriptional activity of a heterologous promoter containing the Gal4-binding sequence. The transcriptional repression function of AEBP2 is completely abolished when one of the conserved histidine residues and a flanking serine residue in the middle zinc finger are replaced with an arginine residue. The defective transcriptional repression function of the mutant derivative is due neither to lack of expression nor to a failure to localize to the nucleus. Moreover, both the wild-type and mutant derivative of either the histidine-tagged recombinant AEBP2 proteins or the in vitro translated Gal4-AEBP2 fusion proteins are equally able to bind to the target DNA. These results suggest that a portion of the zinc finger structure may play a direct role in transcriptional repression function, but not in DNA binding (He, 1999).

Recent studies have revealed the intrinsic histone methyltransferase (HMTase) activity of the EED-EZH2 complex and its role in Hox gene silencing, X inactivation, and cancer metastasis. This study focuses on the function of individual components. It was found that the HMTase activity requires a minimum of three components -- EZH2, EED, and SUZ12 -- while AEBP2 (a mammalian Jing homolog) is required for optimal enzymatic activity. Using a stable SUZ12 knockdown cell line, it has been shown that SUZ12 knockdown results in cell growth defects, which correlate with genome-wide alteration on H3-K27 methylation as well as upregulation of a number of Hox genes. Chromatin immunoprecipitation (ChIP) assay identified a 500 bp region located 4 kb upstream of the HoxA9 transcription initiation site as a SUZ12 binding site, which responds to SUZ12 knockdown and might play an important role in regulating HoxA9 expression. Thus, this study establishes a critical role for SUZ12 in H3-lysine 27 methylation and Hox gene silencing (Cao, 2004).

date revised: 10 June 2002

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