The association of Kis-L with most sites of active transcription suggests that it might be required for one or more stages of the transcription cycle. To investigate this possibility, the levels of Pol IIa, Pol IIoser5 and Pol IIoser2 on polytene chromosomes from wild-type and kisk13416 larvae were compared. Although the total level of Pol II in wild-type and kis mutant larvae is similar by Western blotting, the level of Pol IIoser2 associated with the polytene chromosomes of kisk13416 larvae is dramatically reduced relative to wild type. This striking phenotype suggests that Kis-L, like Brm, plays a global role in transcription by Pol II. By contrast, the level and distribution of Pol IIa in kis mutant larvae is unchanged relative to wild type, indicating that Kis-L is not required for the recruitment of Pol II to promoters. The level and distribution of Pol IIoser5 is also unchanged in kisk13416 larvae relative to wild type, suggesting that the initial stages of elongation are not dependent on Kis-L function. Taken together, these data demonstrate that Kis-L facilitates a relatively early step in transcriptional elongation after serine 5 phosphorylation but prior to serine 2 phosphorylation (Srinivasan, 2005).
The absence of Pol IIoser2 on the polytene chromosomes of kis mutant larvae might result from a failure to phosphorylate serine 2, as opposed to a block in an early step of elongation. Inactivation of kinases that phosphorylate serine 2 in yeast (ctk1) (Ahn, 2004) and Drosophila (the Cdk9 subunit of P-TEFb) (Ni, 2004) do not impair transcriptional elongation. However, serine 2 phosphorylation is necessary for the recruitment of factors required for processing the 3' ends of mRNAs (Ahn, 2004; Hirose, 2000; Proudfoot, 2002). Defects in 3' end processing resulting from the loss of serine 2 phosphorylation led to the rapid degradation of mRNAs. The failure to detect Pol IIoser2 in kis mutants could result from either the failure to recruit the serine 2 kinase P-TEFb to promoters, or a defect in transcriptional elongation. To clarify the mechanism of action of Kis-L, the distribution of the elongation factor SPT6 on the polytene chromosomes of kisk13416 larvae was examined. SPT6 was examined for these experiments because the association of this elongation factor with Pol II is not dependent on serine 2 phosphorylation in yeast (Ahn, 2004). The loss of kis function dramatically reduces the level of SPT6 associated with polytene chromosomes without affecting the overall level of this protein in the salivary gland as assayed by Western blotting. It was therefore concluded that the absence of Pol IIoser2 on the polytene chromosomes of kis mutant larvae is probably due to an early defect in elongation (Srinivasan, 2005).
Whether Kis-L is required for the recruitment of other chromatin-remodeling factors to their target genes was examined. The association of Brm with chromosomes is not affected by the loss of Kis-L function. This observation is consistent with previous studies indicating that Brm is required for relatively early stages of transcription, including the recruitment of Pol II to promoters (Armstrong, 2002). By contrast, loss of Kis-L function blocks the association of CHD1 with chromatin without affecting the overall level of CHD1 as assayed by Western blotting. Since CHD1 has been implicated in transcriptional elongation and colocalizes with Pol IIoser2, this result provides additional evidence that an early step in transcriptional elongation is blocked in kisk13416 larvae. The association of Mi-2 with chromatin is not dependent on Kis-L function, suggesting that Mi-2 may act prior to or independently of Kis-L in the transcription cycle. These data suggest that chromatin-remodeling factors act sequentially during the transcription cycle, with Brm acting prior to Kis-L and CHD1 acting during the later stages of elongation (Srinivasan, 2005).
Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L) is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II). Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, this study characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although significant overlap was observed between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L does not bind methylated histone tails in vitro, and loss of TRX or ASH1 function does not alter the association of KIS-L with chromatin. By contrast, loss of kis function leads to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and is accompanied by increased histone H3 lysine 27 methylation - a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. These findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin (Srinivasan, 2008).
The development of the Drosophila wing depends on its subdivision into anterior and posterior compartments, which constitute two independent cell lineages since their origin in the embryonic ectoderm. The anterior-posterior compartment boundary is the place where signaling by the Hedgehog pathway takes place, and this requires pathway activation in anterior cells by ligand expressed exclusively in posterior cells. Several mechanisms ensure the confinement of hedgehog expression to posterior cells, including repression by Cubitus interruptus, the co-repressor Groucho and Master of thick veins. This work identifies Kismet, a chromodomain-containing protein of the SNF2-like family of ATPases, as a novel component of the hedgehog transcriptional repression mechanism in anterior compartment cells. In kismet mutants, hedgehog is ectopically expressed in a domain of anterior cells close to the anterior-posterior compartment boundary, causing inappropriate activation of the pathway and changes in the development of the central region of the wing. The contribution of Kismet to the silencing of hedgehog expression is limited to anterior cells with low levels of the repressor form of Cubitus interruptus. Knockdown of CHD8, the kismet homolog in Xenopus tropicalis, is also associated with ectopic sonic hedgehog expression and up-regulation of one of its target genes in the eye, Pax2, indicating the evolutionary conservation of Kismet/CHD8 function in negatively controlling hedgehog expression (Terriente-Félix, 2011).
This work has used a genetic approach to analyse the role of Kis in the patterning of the Drosophila wing. The main finding is that Kis is required, among other processes, for the repression of hh in anterior cells close to the A/P boundary. This conclusion is based in the phenotype of kis clones in the wing, the changes in the expression of Hh-target genes in these mutant cells, and more directly, in the observation of hh ectopic expression in wing discs of kis loss-of-function alleles. A similar requirement was identified for CHD8, a Kis homolog in X. tropicalis, suggesting conservation in the mechanisms of hh/Shh regulation during evolution. Finally, it was determined that the repression of hh mediated by Kis is not needed when the repressor form of Ci, Ci75, is present in the cell (Terriente-Félix, 2011).
As a way to identify the functional requirements of Kis, the phenotype caused by several kis loss-of-function alleles was studied. For six of these alleles similar results were found , and consequently this study will refer to all of them together. Two main alterations were identified in wings mutant for kis: the formation of ectopic veins and defects in the patterning of the central region of the wing. These phenotypes are diagnostic of failures in the regulation of the level or domain of activity of two signaling pathways, EGFR and Hh, and it is likely that they identify independent requirements for Kis in the modulation of these pathways during wing development. The formation of ectopic veins is observed in all situations when the activity of the EGFR pathway is not correctly restricted to the positions occupied by the normal veins. Thus, over-activation of EGFR and loss-of-function alleles in a variety of the known antagonists of the pathway, such as MKP3 and sprouty, result in the formation of ectopic veins in similar positions to those observed in kis alleles. The implication of Kis in regulating EGFR signaling is also supported by the identification of kis alleles in several genetic screens searching for modifiers of EGFR phenotypes in different developmental stages and tissues. For example, kis was found in a screen of kinase-suppressor of Ras modifiers in the eye and in a screen of EGFR modifiers affecting border cells migration during oogenesis. kis alleles were also identified as modifiers of the Notch phenotype caused by dominant-negative mastermind over-expression in the wing disc. However, in this study the vein phenotype of loss of kis does not appear related to Notch signalling, because Notch-related defects such as thickened veins or loss of wing margin were never pbserved in kis mutant wings (Terriente-Félix, 2011).
The implication of Kis in the modulation of EGFR signalling has never been directly analyzed. However, it is interesting to notice in this context that the two SNF2-familiy chromatin-remodeling complexes containing Brm as the catalytic subunit are also involved in this pathway. In this manner, the BAP (Brahma associated proteins) and PBAP (Polybromo-Brahma associated proteins) complexes are required to modulate positively or negatively, respectively, EGFR signaling in the wing. Furthermore, Brm and Kis share some domain architecture, as they both have an ATPase domain N-terminal to a BRK (Brahma related to Kismet) domain. They also bind to identical sites in polytenic chromosomes, and both are part of the Trithorax group of genes (TrxG). Therefore, it is possible that BAP, PBAP and Kis participate in the transcriptional regulation of EGFR targets using a conserved mechanism involving chromatin modifications in collaboration with other PcG and TrxG proteins. Similarly, the function of CHD7 in the regulation of neural crest cell identities requires the function of PBAF (Polybromo, Brg1-Associated Factors), the homolog of PBAP, suggesting that Kis and its vertebrate homologs can collaborate with other SNF2-helicases (Terriente-Félix, 2011).
This work focussed in the second phenotype observed in kis mutant cells, which consists in duplications of the L3 vein and increase in the distance between the L3 and L4 veins. These two defects are limited to the pattern elements regulated by the Hh pathway, and correspond to an enlargement of the domain of Hh signalling. Hh activity and diffusion in anterior cells are linked to each other by the function of the receptor Ptc, because ptc expression is activated by Ci155 and Hh diffusion is prevented by Ptc. In this manner the phenotype of kis clones might be caused by a reduction in Hh signaling leading to loss of Ptc and consequently to an increase in the range of Hh diffusion. However, it was found that kis clones do not affect the normal domain of ptc expression, but cause ectopic ptc expression in cells localised anterior to this domain. This observation indicates that Hh signaling itself is not affected in kis mutant clones, and points towards changes in hh expression as a likely cause for the kis phenotype. Indeed, ectopic expression of hh is found in kis mutant cells localised in the anterior compartment close to the A/P boundary. Furthermore, ectopic and cell autonomous expression of hh-lacZ reporters is observed in small kis clones (less than 5 cells) localised in this region. These results suggest that Kis is needed during wing imaginal development to maintain hh expression turned-off in anterior cells that, due to Hh signalling, don't have enough levels of the repressor Ci75. This requirement for Kis readily explains both the phenotype of kis alleles in the central region of the wing and the changes in the expression of hh and its target genes observed in kis mutant cells. Interestingly, this activity of Kis in the repression of hh expression appears conserved in its Xenopus homolog CHD8 (Terriente-Félix, 2011).
The regulation of hh expression relies on a combination of several mechanisms acting in different domains of the wing disc. First, Ci75, a form of Ci that is produced by proteolysis from Ci155 when Hh signaling is not active, represses hh in anterior cells. In those anterior cells exposed to Hh the levels of Ci75 are low, and in this domain a second mechanism involving Mtv and Gro represses hh expression. Finally, several genes of the Polycomb group (PcG), such as Polycomb (Pc) and Polyhomeotic (Ph) are also involved in the repression of hh transcription in a tissue-specific manner. In this way, the PcG is involved in maintaining the repressive transcriptional state of hh in anterior cells, while the TrxG maintains the active transcriptional state of hh in posterior cells. This regulation seems direct, because the Pc protein and the TrxG member GAF/Trl bind two regions of the hh gene, and one of them, situated upstream of the hh transcription start site, exhibits cellular memory module (CMM) characteristics. The activity of this CMM is also regulated by PcG and TrxG proteins in experimental situations in which hh is activated by ectopic En in anterior cells, or turndown by loss of En in posterior cells. Interestingly, ectopic expression of En in anterior cells located along the dorso-ventral boundary induces hh in most of the cells of the wing blade except those nearest to the A/P boundary, implying that the activity of this hh CMM in the anterior compartment is excluded or less efficient in the territory where Kis represses hh (Terriente-Félix, 2011).
How Kis regulates hh in anterior cells is not known, but several arguments suggest that Kis is related to the repression mediated by Mtv/Gro. Thus, mtv/gro and kis mutations cause ectopic expression of hh in a similar domain of the wing disc, and in both cases they are not required when the repressor Ci75 is present. In this scenario, it is proposed that the chromatin remodeling activity of Kis could make the hh regulatory region accessible to the Mtv/Gro repressor complex. The putative function of Kis as part of the Mtv/Gro repressor complex would be independent of other functions assigned for the protein in, for example, the control of transcriptional elongation and Histone methylation. Similarly, this function of Kis on hh regulation would be independent of its role as a TrxG protein, because it is only effective in a spatial domain complementary to that in which PcG and TrxG regulate hh expression (Terriente-Félix, 2011).
Interestingly, heterozygous mutations in human CHD7 result in congenital anomalies called CHARGE syndrome, which is caused by the abnormal development of the neural crest. The function of CHD7 in the regulation of neural crest cell identities implies the regulation of several transcription factors expressed in these cells, and requires the function of the PBAF chromatin remodeler. In this manner, Kis and other CHD proteins might form part of different multiprotein complexes regulating different promoters using independent molecular mechanisms. It is remarkable that CHD8 is required for the regulation of Shh, as this implies a strong conservation in the mechanism of hh and Shh transcriptional regulation during evolution. Future experiments should address the mechanisms by which Kis/CHD8 are recruited to the hh/Shh regulatory regions (Terriente-Félix, 2011).
Physical interactions between Kis-L and Brm, Mi-2 or Pol II could account for their similar chromosomal distributions. Indeed, physical interactions between yeast SWI/SNF complexes and Pol II holoenzyme have been reported, but these findings remain somewhat controversial. To investigate this possibility, attempts were made to co-immunoprecipitate Kis-L, other chromatin-remodeling factors and Pol II from embryo extracts using antibodies against Kis-L. Kis-L, but not Kis-S, could be efficiently immunoprecipitated from embryo extracts using affinity-purified antibodies against the N-terminal segment unique to the Kis-L protein. In addition to confirming the specificity of this antibody, this result suggests that Kis-L and Kis-S do not stably interact with each other. It was also not possible to co-immunoprecipitate Kis-L with other chromatin-remodeling factors (including Brm, Mi-2 and ISWI) or Pol II, even when very mild conditions were used. These findings are consistent with gel filtration data, suggesting that Kis-L, Brm and Mi-2 are subunits of distinct protein complexes. Thus, physical interactions between Kis-L, Brm, Mi-2 and Pol II are probably not responsible for their similar chromosomal distributions (Srinivasan, 2005).
To monitor specifically the expression of Kis-L, antibodies were raised against a region unique to this protein (residues 100-300). Affinity-purified rabbit polyclonal antibodies against this segment specifically recognize the 574 kDa Kis-L protein in Drosophila embryo extracts by Western blotting. By contrast, antibodies against the C-terminal segment common to Kis-L and Kis-S detect both the 574 kDa Kis-L and 225 kDa Kis-S proteins. Antibodies against Kis-L were used to monitor its temporal and spatial expression. As previously observed for other trxG proteins and Kis-S (Daubresse, 1999), Kis-L is ubiquitously expressed in nuclei throughout embryogenesis (Srinivasan, 2005).
The majority of Drosophila ATP-dependent chromatin-remodeling factors characterized to date, including Brm, Mi-2, ISWI and Domino, function as subunits of large protein complexes. To determine if Kis-L is also a subunit of a protein complex, its native molecular mass in embryo extracts was determined by gel filtration chromatography. Kis-L elutes from a Superose 6 gel filtration column with an apparent molecular mass of 1 MDa; Kis-S fractionates as a distinct complex with an apparent molecular mass of 650 kDa. The molecular mass of the Kis-L complex is smaller than that of the ~2 MDa Brm complex. The elution profiles of Kis-L and Kis-S are also distinct from those of the chromatin-remodeling factors CHD1 and Mi-2. These findings suggest that Kis-L functions as the ATPase subunit of a novel chromatin-remodeling complex (Srinivasan, 2005).
To directly visualize interactions between Kis-L and chromatin, its distribution on salivary gland polytene chromosomes was examined. Antibodies against Kis-L recognize ~300 sites in euchromatin. The vast majority of these sites reside in interbands: regions of less condensed DNA that stain lightly with DAPI. By contrast, Kis-L is not associated with the heterochromatic chromocenter of polytene chromosomes. These findings suggest that Kis-L plays a relatively general role in transcription or other processes, perhaps by creating open regions of chromatin. The distribution of Kis-S on polytene chromosomes could not be examined because it is not expressed in the salivary gland (Srinivasan, 2005).
To gain insight into the potential role of Kis-L in gene expression, its distribution on polytene chromosomes was compared with that of Pol II. For the initial experiments, Pol II was detected using an antibody against its second largest subunit, Pol IIc. This antibody recognizes the initiating, paused and elongating forms of Pol II. The distributions of Kis-L and Pol II overlap extensively, as observed for the Brm complex. Although the relative levels of Kis-L and Pol II vary from site to site, it is clear that Kis-L is associated with the vast majority (~98%) of transcriptionally active regions in this tissue. This striking observation suggests that Kis-L plays a global role in transcription by Pol II (Srinivasan, 2005).
During the transcriptional cycle, the C-terminal domain (CTD) of the largest subunit of Pol II (which contains multiple repeats of the heptad sequence YSPTSPS) is differentially phosphorylated (Sims, 2004). When recruited to promoters, the CTD is unphosphorylated (Pol IIa) (O'Brien, 1994; Weeks, 1993); after promoter clearance and during early stages of elongation, serine 5 of the CTD is phosphorylated (Pol IIoser5); at later stages of transcriptional elongation, serine 2 is phosphorylated (Pol IIoser2) (Komarnitsky, 2000; Svejstrup, 2004). The distribution of a protein relative to the different forms of Pol II can provide clues to its role in the transcription cycle (Kaplan, 2000; Saunders, 2003) (Srinivasan, 2005 and references therein).
CHD1, which is related to Kis-L, has been implicated in transcriptional elongation in yeast and mammals. CHD1 associates with transcriptional elongation factors in yeast (Krogan, 2003; Simic, 2003) and mammalian cell lines (Kelley, 1999). CHD1 is also associated with interband regions of Drosophila polytene chromosomes (Stokes, 1996) and the body of actively transcribed genes in yeast (Simic, 2003). Consistent with a role in transcriptional elongation, CHD1 and Pol IIoser2 have identical patterns on polytene chromosomes. By contrast, the distribution of Kis-L is not identical to any one form of Pol II. Instead, staining of Kis-L extensively overlaps with that of Pol IIoser2 and Pol IIa and to a lesser extent with Pol IIoser5. These findings suggest that Kis-L is required for an earlier step in transcriptional initiation or elongation than CHD1 (Srinivasan, 2005).
To clarify the functional relationship between Kis-L and other chromatin-remodeling factors, their distributions on polytene chromosomes were compared. The distributions of Kis-L and Brm were compared, since previous studies have suggested that the two proteins have similar functions. For example, brm and kis were both identified in genetic screens for dominant suppressors of Pc (Kennison, 1988) and mutations in the two genes cause similar homeotic transformations (Daubresse, 1999). In addition, Brm plays an extremely general role in transcription by Pol II and, like Kis-L, is associated with almost all transcriptionally active regions of polytene chromosomes. Consistent with a close functional relationship between the two proteins, it was found that the distributions of Brm and Kis-L on polytene chromosomes are virtually identical. In addition, the relative levels of the two proteins do not vary from site to site. The striking similarities between the chromosomal distributions of Brm and Kis-L strongly suggest that the functions of the two trxG proteins are intimately related (Srinivasan, 2005).
Next, the chromosomal distribution of Kis-L was compared with members of the CHD family of ATPases, including CHD1 and Mi-2. CHD1 has been implicated in transcriptional elongation and perfectly co-localizes with the elongating form of Pol II (Pol IIoser2) on polytene chromosomes. By contrast, CHD1 and Kis-L have partially overlapping, but not identical, chromosomal distributions, suggesting that they play distinct roles in the transcription cycle (Srinivasan, 2005).
Based on its association with histone deacetylases and transcriptional repressors, Mi-2 is thought to be involved in transcriptional repression. Genetic studies in Drosophila also suggest that Mi-2 acts in concert with PcG proteins to repress Hox transcription. It was therefore anticipated that the chromosomal distributions of Kis-L and Mi-2 would be very different, if not mutually exclusive. Surprisingly, it was found that the patterns of Kis-L and Mi-2 are actually very similar. Although the relative levels of Kis-L and Mi-2 vary from site to site, only 1 to 2% of the binding sites of the two proteins fail to overlap. These data suggest that Mi-2 plays an unanticipated and relatively general role in transcription by Pol II (Srinivasan, 2005).
The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that is structurally and functionally similar to mammalian glutamatergic synapses. These synapses can, as a result of changes in activity, alter the strength of their connections via processes that require chromatin remodeling and changes in gene expression. The chromodomain helicase DNA binding (CHD) protein, Kismet (Kis), is expressed in both motor neuron nuclei and postsynaptic muscle nuclei of the Drosophila larvae. This study shows that Kis is important for motor neuron synaptic morphology, the localization and clustering of postsynaptic glutamate receptors, larval motor behavior, and synaptic transmission. The data suggest that Kis is part of the machinery that modulates the development and function of the NMJ. Kis is the homolog to human CHD7, which is mutated in CHARGE syndrome. Thus, the data suggest novel avenues of investigation for synaptic defects associated with CHARGE syndrome (Ghosh, 2014: PubMed).
kis is an essential gene and individuals homozygous for extreme kis alleles die prior to the third larval instar (Daubresse, 1999). By contrast, individuals homozygous for a hypomorphic, P element-induced kis allele (kisk13416) survive until late larval or early pupal development. The recessive lethality of kisk13416 is rescued by a duplication spanning the kis gene [Dp(2;Y)L124], but not by a copy of this duplication bearing a kis mutation [Dp(2;Y)L124, kis7] (Kennison, 1988). Thus, the recessive lethality of the kisk13416 chromosome is due to the P-element insertion in the kis gene, as opposed to another mutation. No Kis-L protein could be detected in extracts of salivary glands from kisk13416 homozygotes by Western blotting. Consistent with this observation, Kis-L was absent from the polytene chromosomes of kisk13416 larvae. Although the polytene chromosomes of kisk13416 homozygotes sometimes appear slightly thinner than normal, the loss of Kis-L function does not significantly alter their overall morphology or banding pattern (Srinivasan, 2005).
Whether Kis-L regulates the association of PC with chromatin was examined. PC binds to ~60 sites on polytene chromosomes. Consistent with the functional antagonism between PC and Kis-L, the chromosomal distributions of the two proteins are predominantly non-overlapping. The number and intensity of PC-binding sites is not altered in kisk13416 larvae. Thus, Kis-L does not appear to play a general role in blocking the association of PC with chromatin in vivo. Upon close examination, ~80% of the PC-binding sites lie adjacent to or slightly overlap Kis-L. It remains possible that Kis-L restricts or regulates the activity of PcG proteins in the immediate vicinity of PREs (Srinivasan, 2005).
The Drosophila kismet gene was identified in a screen for dominant suppressors of Polycomb, a repressor of homeotic genes. kismet mutations suppress the Polycomb mutant phenotype by blocking the ectopic transcription of homeotic genes. Loss of zygotic kismet function causes homeotic transformations similar to those associated with loss-of-function mutations in the homeotic genes Sex combs reduced and Abdominal-B. kismet is also required for proper larval body segmentation. Loss of maternal kismet function causes segmentation defects similar to those caused by mutations in the pair-rule gene even-skipped. The kismet gene encodes several large nuclear proteins that are ubiquitously expressed along the anteriorposterior axis. The Kismet proteins contain a domain conserved in the trithorax group protein Brahma and related chromatin-remodeling factors, providing further evidence that alterations in chromatin structure are required to maintain the spatially restricted patterns of homeotic gene transcription (Daubresse, 1999).
The genetic interactions between kis and Pc provided the first clue that kis plays an important role in the determination of body segment identity. kis mutations suppress the adult Pc phenotype by preventing the ectopic transcription of homeotic genes. Thus, kis is a member of the trithorax group of homeotic gene activators. Mosaic analyses reveal that loss of kis function causes homeotic transformations, including the transformation of first leg to second leg and the fifth abdominal segment to a more anterior identity. These phenotypes are identical to those associated with loss-of-function Scr and Abd-B mutations, respectively. Taken together, these findings suggest that kis acts antagonistically to Pc to activate the transcription of both Scr and Abd-B. It is intriguing that kis mutations alter the fate of only the fifth abdominal segment, since the identities of the fifth through ninth abdominal segments are determined by a single homeotic gene, Abd-B (Daubresse, 1999).
Variations in the levels of Abd-B protein result in the differences between these abdominal segments, with Abd-B expression being lowest in the fifth abdominal segment. Parasegment-specific cis-regulatory regions, termed infra-abdominal (iab) regions control Abd-B expression. Each iab region is named for the segment that it affects (iab-5 through iab-9). Mutations in both iab-5 and kis affect the identity of only the fifth abdominal segment, suggesting that the Kis protein may interact specifically with the iab-5 cis-regulatory element of Abd-B (Daubresse, 1999).
kis probably interacts not only with Scr and Abd-B, but with other homeotic genes as well. For example, the isolation of kis mutations as enhancers of loss-of-function Deformed (Dfd) mutations (Gellon, 1997) suggests that kis is probably also required to activate transcription of this ANTC homeotic gene. Furthermore, kis duplications strongly enhance the transformation of wing to haltere in Pc heterozygotes, a phenotype caused by the ectopic transcription of Ubx in the wing imaginal disc. However, kis mutations do not cause haltere-to-wing transformations due to decreased Ubx transcription. A possible explanation for the lack of homeotic transformations in kis clones in segments other than the prothoracic and fifth abdominal segment is that the mutations used in these studies are not null alleles. kis1 is a strong loss-of-function mutation. It has not been characterized at the molecular level, however, and may not completely eliminate kis function. It is also possible that sufficient levels of Kis protein persist in homozygous mutant tissue following mitotic recombination to support normal development. Further genetic studies, including the analysis of conditional kis alleles, will be necessary to distinguish between these possibilities (Daubresse, 1999).
Germline clonal analysis has revealed an unanticipated role for kis in segmentation. Embryos from mosaic kisS females exhibit a deletion or alteration of every other segment, while mutant embryos from mothers bearing germline clones of the stronger kis1 allele usually develop only half of the normal number of segments. This variation in phenotypic severity is closely correlated with the extent to which en expression is disrupted. The phenotypes associated with loss of maternal kis function resemble those caused by mutations in pair-rule segmentation genes that cause the deletion of the odd-numbered parasegments. kis thus appears to be necessary for the expression (or function) of one or more pair-rule genes. Recent genetic studies have suggested that kis may also be involved in the Notch signaling pathway. Thus it appears that kis plays roles in addition to the regulation of homeotic genes (Daubresse, 1999).
What pair-rule genes might require kis for their activity? Based on the kis mutant phenotype, perhaps the best candidates are eve and hairy (h), both of which are required for the formation of odd-numbered parasegments. Unlike eve, h and most other segmentation genes, kis is uniformly expressed in the early embryo. This raises the possibility that Kis functions as an essential cofactor or modifier of Eve or other pair-rule proteins. It is also possible that loss of kis function might result in pair-rule genes being transcribed outside of their normal expression domains. Additional work will be necessary to determine the molecular basis of the segmentation defects resulting from loss of maternal kis function (Daubresse, 1999).
CHARGE syndrome (CS, OMIM #214800) is a rare, autosomal dominant disorder, two-thirds of which are caused by haplo-insufficiency in the Chd7 gene. This study shows that the Drosophila homolog of Chd7, kismet, is required for proper axonal pruning, guidance and extension in the developing fly's central nervous system. In addition to defects in neuroanatomy, flies with reduced kismet expression show defects in memory and motor function, phenotypes consistent with symptoms observed in CS patients. It is suggested that the analysis of this disease model can complement and expand upon the existing studies for this disease, allowing a better understanding of the role of kismet in neural developmental, and Chd7 in CS pathogenesis (Melicharek, 2010).
The data presented in this study describe a requirement for kis gene function in adult Drosophila early climbing behavior, memory, eye development and neural development. These results suggest that the reduced early climbing ability observed in kis mutants can at least partially be attributed to a requirement for kis gene function in muscle cells. This is particularly of interest when it is considered that knockdown of kis function in motor neurons, as well as pan-neural knockdown of kis function has no effect on the early climbing behavior analyzed. Thus, kis may either be regulating the expression of critical post-synaptic target gene(s) in muscle cells required to facilitate synaptic transmission and muscle coordination and/or may be required for the morphology and/or development of the muscle cells themselves. Further, the postural defect observed in adult flies with reduced Kismet protein is also reminiscent of defects associated with muscle cells, and both phenotypes are consistent with hypotonia, impaired motor coordination and muscle-related posture problems observed in CHARGE patients. Based on these similarities, the results may suggest that there is a similar requirement for Chd7 function in muscles of vertebrates and may help to elucidate a possible mechanism for these symptoms often observed in CHARGE patients (Melicharek, 2010).
The data suggest that decreased kis function does not alter the fly's ability to learn, although it does have an effect on immediate recall memory. The data have also shown that kis function is required for the proper development of the Kenyon cells, as kis mutants show defective axonal pruning and axonal migration in these neuronal populations. The Kenyon neurons are associated with learning and memory in multiple experimental paradigms in Drosophila, including the conditioned courtship suppression behavior. Thus, it may be that the defects observed in Kenyon cell development are responsible for the impaired learning behavior observed in adult flies with decreased kis function. kis-mediated transcriptional regulation of genes involved in the pruning and/or migration of axons in this cell population would therefore make attractive targets for further investigation into this memory defect (Melicharek, 2010).
In analyzing the morphology of neuronal populations mutant for kis, defects were consistently observed in axon morphology and positioning. These defects may be due to abnormal axonal pruning observed in some kis mutant neurons or may be due to defective axonal migration or defects in axonal extension and/or retraction. Interestingly, in each of the neuronal populations studied, dendritic development was normal. When taken together, these data suggest that kis may function to regulate the expression of target gene(s) normally required for axon morphology and connectivity, as opposed to dendritic morphology. The specific target genes that Kismet regulates most likely differ between different neuronal populations. However, the defects observed in kis mutant DCNs suggest that kis functions in DCN positioning as well as axonal morphology. Abnormal positioning of larval DC soma is also observed in larval brains mutant for ato gene function. As kis regulates ato transcription in the larval retina, this neuron migration defect may be due to kis mediated regulation of ato transcription in these cells as well (Melicharek, 2010).
Taken together, it is suggested that the analyses presented in this study can complement and expand upon the studies done in cell culture and vertebrate model organisms toward a better understanding of the role of kis in neural developmental and Chd7 in CS pathogenesis (Melicharek, 2010).
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