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).
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).
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).
Reference names in red indicate recommended papers.
Ahn, S. H., Kim, M. and Buratowski, S. (2004). Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing. Mol. Cell 13: 67-76. Medline abstract: 14731395
Armstrong, J. A., Papoulas, O., Daubresse, G., Sperling, A. S., Lis, J. T., Scott, M. P. and Tamkun, J. W. (2002). The Drosophila BRM complex facilitates global transcription by RNA polymerase II. EMBO J. 21: 5245-5254. 12356740
Cho, E. J., Kobor, M. S., Kim, M., Greenblatt, J. and Buratowski, S. (2001). Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain. Genes Dev. 15: 3319-3329. 11751637
Daubresse, G., Deuring, R., Moore, L., Papoulas, O., Zakrajsek, I., Waldrip, W. R., Scott, M. P., Kennison, J. A. and Tamkun, J. W. (1999). The Drosophila kismet gene is related to chromatin-remodeling factors and is required for both segmentation and segment identity. Development 126: 1175-1187. 10021337
Dellino, G. I., Schwartz, Y. B., Farkas, G., McCabe, D., Elgin, S. C. and Pirrotta, V. (2004). Polycomb silencing blocks transcription initiation. Mol. Cell 13: 887-893. 15053881
Gellon, G., Harding, K. W., McGinnis, N., Martin, M. M. and McGinnis, W. (1997). A genetic screen for modifiers of Deformed homeotic function identifies novel genes required for head development. Development 124, 3321-3331. 9310327
Go, M. J. and Artavanis-Tsakonas, S. (1998). A genetic screen for novel components of the Notch signaling pathway during Drosophila bristle development. Genetics 150: 211-220. 9725840
Hirose, Y. and Manley, J. L. (2000). RNA polymerase II and the integration of nuclear events. Genes Dev. 14: 1415-1429. Medline abstract: 10859161
Kaplan, C. D., Morris, J. R., Wu, C. and Winston, F. (2000). Spt5 and Spt6 are associated with active transcription and have characteristicsof general elongation factors in D. melanogaster. Genes Dev. 14: 2623-2634. 11040216
Kelley, D. E., Stokes, D. G. and Perry, R. P. (1999). CHD1 interacts with SSRP1 and depends on both its chromodomain and its ATPase/helicase-like domain for proper association with chromatin. Chromosoma 108: 10-25. 10199952
Kennison, J. A. and Tamkun, J. W. (1988). Dosage-dependent modifiers of Polycomb and Antennapedia mutations in Drosophila. Proc. Natl. Acad. Sci. USA 85: 8136-8140. 3141923
Komarnitsky, P., Cho, E. J. and Buratowski, S. (2000). Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 14: 2452-2460. 11018013
Krogan, N. J., Kim, M., Tong, A., Golshani, A., Cagney, G., Canadien, V., Richards, D. P., Beattie, B. K., Emili, A., Boone, C., et al. (2003). Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 23: 4207-4218. 12773564
Ni, Z., Schwartz, B. E., Werner, J., Suarez, J. R. and Lis, J. T. (2004). Coordination of transcription, RNA processing, and surveillance by P-TEFb kinase on heat shock genes. Mol. Cell 13: 55-65. 14731394
O'Brien, T., Hardin, S., Greenleaf, A. and Lis, J. T. (1994). Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation. Nature 370: 75-77. 8015613
Pokholok, D. K., Hannett, N. M. and Young, R. A. (2002). Exchange of RNA polymerase II initiation and elongation factors during gene expression in vivo. Mol. Cell 9: 799-809. 11983171
Proudfoot, N. J., Furger, A. and Dye, M. J. (2002). Integrating mRNA processing with transcription. Cell 108: 501-512. Medline abstract: 11909521
Saunders, A., Werner, J., Andrulis, E. D., Nakayama, T., Hirose, S., Reinberg, D. and Lis, J. T. (2003). Tracking FACT and the RNA polymerase II elongation complex through chromatin in vivo. Science 301: 1094-1096. 12934007
Schuster, E. F. and Stoger, R. (2002). CHD5 defines a new subfamily of chromodomain-SWI2/SNF2-like helicases. Mamm. Genome 13: 117-119. 11889561
Simic, R., Lindstrom, D. L., Tran, H. G., Roinick, K. L., Costa, P. J., Johnson, A. D., Hartzog, G. A. and Arndt, K. M. (2003). Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes. EMBO J. 22: 1846-1856. 12682017
Sims, R. J., 3rd, Belotserkovskaya, R. and Reinberg, D. (2004). Elongation by RNA polymerase II: the short and long of it. Genes Dev. 18: 2437-2468. 15489290
Smith, S. T., Petruk, S., Sedkov, Y., Cho, E., Tillib, S., Canaani, E. and Mazo, A. (2004). Modulation of heat shock gene expression by the TAC1 chromatin-modifying complex. Nat. Cell Biol. 6: 162-7. 14730313
Srinivasan, S., et al. (2005). The Drosophila trithorax group protein Kismet facilitates an early step in transcriptional elongation by RNA Polymerase II. Development 132: 1623-1635. 15728673
Stokes, D. G., Tartof, K. D. and Perry, R. P. (1996). CHD1 is concentrated in interbands and puffed regions of Drosophila polytene chromosomes. Proc. Natl. Acad. Sci. USA 93: 7137-7142. Medline abstract: 8692958
Svejstrup, J. Q., Li, Y., Fellows, J., Gnatt, A., Bjorklund, S. and Kornberg, R. D. (1997). Evidence for a mediator cycle at the initiation of transcription. Proc. Natl. Acad. Sci. USA 94: 6075-6078. 9177171
Svejstrup, J. Q. (2004). The RNA polymerase II transcription cycle: cycling through chromatin. Biochim. Biophys. Acta 1677: 64-73. 15020047
Therrien, M., Morrison, D. K., Wong, A. M. and Rubin, G. M. (2000). A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in Drosophila. Genetics 156: 1231-1242. 11063697
Verheyen, E. M., Purcell, K. J., Fortini, M. E. and Artavanis-Tsakonas, S. (1996). Analysis of dominant enhancers and suppressors of activated Notch in Drosophila. Genetics 144: 1127-1141. 8913755
Wang, L., Brown, J. L., Cao, R., Zhang, Y., Kassis, J. A. and Jones, R. S. (2004). Hierarchical recruitment of Polycomb group silencing complexes. Mol. Cell 14: 637-646. 15175158
Weeks, J. R., Hardin, S. E., Shen, J., Lee, J. M. and Greenleaf, A. L. (1993). Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing. Genes Dev. 7: 2329-2344. 8253380
date revised: 1 May 2005
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