The Drosophila trithorax group gene kismet (kis) was identified in a screen for extragenic suppressors of Polycomb (Pc) and subsequently shown to play important roles in both segmentation and the determination of body segment identities. One of the two major proteins encoded by kis (Kis-L) is related to members of the SWI2/SNF2 and CHD families of ATP-dependent chromatin-remodeling factors. To clarify the role of Kis-L in gene expression, its distribution on larval salivary gland polytene chromosomes was examined. Kis-L is associated with virtually all sites of transcriptionally active chromatin in a pattern that largely overlaps that of RNA Polymerase II (Pol II). The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of Kis-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These data suggest that Kis-L facilitates an early step in transcriptional elongation by Pol II (Srinivasan, 2005).

The homeotic (Hox) genes of the Antennapedia and bithorax complexes encode homeodomain transcription factors that specify the identities of body segments by regulating the transcription of downstream target genes. The transcription of Hox genes must be regulated precisely, because their inappropriate expression leads to dramatic alterations in segmental identities. In Drosophila, the initial patterns of Hox transcription are established early in embryogenesis by transcription factors encoded by segmentation genes. During subsequent development, these patterns are maintained by two ubiquitously expressed groups of regulatory proteins: the Polycomb group (PcG) of repressors and the trithorax group (trxG) of activators. Counterparts of Drosophila PcG and trxG genes play conserved roles in other metazoans, including humans. Although the molecular mechanisms used to maintain heritable states of Hox transcription remain relatively mysterious, a growing body of evidence suggests that they involve changes in chromatin structure. PcG proteins silence the transcription of their target genes via cis-regulatory elements known as Polycomb-response elements (PREs). Two complexes of Drosophila PcG proteins have been identified: PRC1 (which contains PC and other PcG proteins) and the ESC/E(Z) complex. The ESC/E(Z) complex methylates lysine 27 of histone H3; this modification is required for PcG silencing in vivo and may help recruit PRC1 or stabilize the binding of PRC1 to PREs (Srinivasan, 2005 and references therein).

How does PRC1 silence transcription once targeted to a PRE? One popular model is that PRC1 packages chromatin into a configuration that is inaccessible to transcription factors or the general transcription machinery. However, recent studies have indicated that PRC1 may repress transcription via more selective mechanisms. For example, PRC1 may block transcription via direct interactions with components of the basal transcription machinery, as evidenced by the presence of TFIID subunits in PRC1. Furthermore, DNA-binding activators TBP and Pol II are present at promoters repressed by PRC1, suggesting that PRC1 selectively interferes with events downstream of Pol II recruitment (Srinivasan, 2005 and references therein).

Other potential targets of PRC1 include the members of the trxG of activators. Mutations in many trxG genes suppress Pc mutations and cause homeotic transformations because of the failure to maintain active states of Hox transcription. The majority of trxG proteins characterized to date have been implicated in either chromatin remodeling or the covalent modification of nucleosomal histones. For example, the trxG genes trithorax (trx) and absent, small or homeotic 1 (ash1) encode SET domain proteins with histone methyltransferase activity; these histone-modifying enzymes counteract silencing by PcG proteins in vivo. Another trxG gene, brahma (brm), encodes a member of the SWI2/SNF2 family of ATPases. The Brm ATPase, together with the trxG proteins Moira (Mor) and Osa, are subunits of a 2 MDa chromatin-remodeling complex that is highly related to the yeast SWI/SNF and RSC, and the human BAF and PBAF complexes. By altering the structure or positioning of nucleosomes, these complexes facilitate the binding of transcription factors and other regulatory proteins to chromatin. The BRM complex plays a global role in transcription by Pol II and is therefore an excellent candidate for a target of PRC1 repression. Consistent with this possibility, PRC1 strongly inhibits chromatin remodeling by human SWI/SNF in vitro (Srinivasan, 2005 and references therein).

Like brm, mor and osa, the trxG gene kismet (kis) was identified in a screen for extragenic suppressors of Pc, suggesting that it acts antagonistically to Pc to activate homeotic gene expression. Loss of maternal kis function causes segmentation defects identical to those caused by mutations in the pair-rule gene even-skipped (eve) (Daubresse, 1999). Loss of zygotic kis function causes homeotic transformations, including the transformation of first leg to second leg and the fifth abdominal segment to a more anterior identity (Daubresse, 1999). These phenotypes are identical to those resulting from the decreased transcription of the Hox genes Sex combs reduced (Scr) and Abdominal-B (Abd-B). Thus, kis plays a dual role during development; maternal kis activity is required for embryonic segmentation and zygotic kis activity is required for the control of cell fate. Mutations in kis have also been recovered in screens for genes involved in the Notch and Ras signaling pathways, suggesting that its function is not limited to segmentation and the determination of body segment identities (Go, 1998; Therrien, 2000; Verheyen, 1996, Srinivasan, 2005 and references therein).

Pc mutants are exquisitely sensitive to changes in kis levels; kis mutations and deficiencies strongly suppress homeotic phenotypes resulting from Hox derepression in Pc heterozygotes, while kis duplications enhance them (Daubresse, 1999; Kennison, 1988). This strong, dose-dependent interaction suggests that the functions of kis and Pc are intimately related. A functional antagonism between kis and Pc could occur at several levels. For example, Kis-L may directly counteract the binding of PRC1 or the ESC/E(Z) complex to chromatin by altering the structure or positioning of nucleosomes at PREs. Alternatively, Kis-L might play a more global role in transcription by Pol II, as has been observed for the BRM complex. In this case, PRC1 might silence the transcription of Hox genes by blocking chromatin remodeling by Kis-L (Srinivasan, 2005).

To clarify the mechanism of action of kis, the distribution of Kis-L and other proteins involved in chromatin remodeling and transcription was compared on salivary gland polytene chromosomes. Kis-L, like Brm, is associated with virtually all transcriptionally active regions of the Drosophila genome. The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of Kis-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These findings suggest that Kis-L facilitates an early step in transcriptional elongation by Pol II (Srinivasan, 2005).

Eukaryotic transcription involves a highly coordinated cycle of events, including the assembly of the pre-initiation complex, initiation, promoter clearance, elongation and termination. In theory, any step in the transcription cycle could be subject to developmental regulation by PcG and trxG proteins. Basal transcription factors and Pol II are recruited to the promoter during the initial stages of transcription. After Pol II is recruited to a promoter, the phosphorylation of the C-terminal domain (CTD) of its largest subunit coincides with promoter clearance and the transition from initiation to the elongation phase of transcription (Svejstrup, 2004). A number of CTD kinases have been identified, including CDK7 (a subunit of TFIIH) and CDK9 (a component of P-TEFb), which phosphorylate, respectively, serine 5 and serine 2 residues of the CTD. The phosphorylation of serine 5 is highest near the promoter, whereas the phosphorylation of serine 2 increases as Pol II proceeds toward the 3' end of genes (Cho, 2001; Komarnitsky, 2000). CTD phosphorylation modulates interactions between Pol II and other factors involved in transcription. For example, phosphorylation of the CTD may disrupt interactions between Pol II and mediator, thus facilitating promoter clearance (Pokholok, 2002; Svejstrup, 1997). The phosphorylated CTD can also act as a docking site for factors involved in mRNA processing and termination (Hirose, 2000; Proudfoot, 2002) (Srinivasan, 2005).

The current findings strongly suggest that Kis-L plays a global role in transcription by Pol II. Consistent with this view, Kis-L is associated with virtually all transcriptionally active regions of chromatin in salivary gland nuclei. The initial stages of transcription are normal in kis mutant larvae; Pol II is efficiently recruited to promoters and the phosphorylation of serine 5 of the CTD is not affected. However, the absence of Pol IIo^ser2 and the elongation factors SPT6 and CHD1 on the polytene chromosomes of kis mutant larvae strongly suggests that Kis-L is required for an early step in transcriptional elongation (Srinivasan, 2005).

The discovery that Kis-L plays a global role in transcription by Pol II was unanticipated, since previous genetic studies have suggested that kis plays relatively specialized roles in development (Daubresse, 1999). The limited phenotypes resulting from the loss of zygotic kis function may be due to the high maternal contribution of kis gene products. It is also possible that the mutations used in previous genetic studies of kis are not null alleles, or that other factors can partially compensate for the loss of kis function in tissues other than the salivary gland (Srinivasan, 2005).

How might Kis-L facilitate an early step in transcriptional elongation by Pol II? Based on its similarity to chromatin-remodeling factors, it is likely that Kis-L promotes transcription by altering chromatin structure. Nucleosomes and other components of chromatin can repress transcription at many different levels. For example, nucleosomes can interfere with the assembly of the preinitiation complex by blocking access of gene-specific and general transcription factors to promoter regions. Nucleosomes also present a physical barrier to Pol II during transcriptional elongation. Histone-modifying enzymes, chromatin-remodeling factors and numerous other factors are therefore crucial for transcriptional initiation and elongation in a chromatin environment (Srinivasan, 2005).

Recent studies of the mammalian hsp70 gene have suggested that the remodeling of nucleosomes near promoters is important for early stages of transcriptional elongation. Prior to induction, a paused polymerase is located just downstream of the hsp70 promoter. Following induction, heat shock factor 1 targets mammalian SWI/SNF to the hsp70 promoter resulting in the disruption of this nucleosome, thus allowing elongation to proceed. By analogy, Kis-L may promote elongation by remodeling nucleosomes immediately downstream of promoters (Srinivasan, 2005 and references therein).

The presence of two chromodomains in Kis-L suggests that the methylation of N-terminal histone tails may also be important for its targeting or function. This possibility is intriguing in light of recent studies suggesting that histone methyltransferases modulate distinct stages of transcriptional elongation by Pol II. The phosphorylation of serine 5 of the CTD promotes interactions between Pol II and the SET1 methyltransferase, resulting in the methylation of lysine 4 of histone H3 in the vicinity of promoters. The subsequent phosphorylation of serine 2 of the CTD promotes interactions with the SET2 methyltransferase, resulting in the methylation of lysine 36 of histone H3 in the body of transcribed genes. The pattern of histone methylation resulting from the dynamic interactions between Pol II and histone methyltransferases may facilitate the transition from early to late stages of elongation by regulating interactions between chromatin-remodeling factors and nucleosomes near promoters (Srinivasan, 2005 and references therein).

The yeast SET1 histone methyltransferase is a subunit of a large protein complex known as COMPASS. Functional counterparts of yeast COMPASS have been identified in humans; these complexes contain subunits related to Drosophila TRX (human MLL1 and MLL2) and ASH2 (human ASH2L). Human MLL1 and MLL2 methylate lysine 4 of histone H3, as does Drosophila TRX (Smith, 2004), suggesting that they are functional counterparts of SET1. Another Drosophila trxG protein, ASH1, also methylates lysine 4 of histone H3 both in vitro and in vivo (Srinivasan, 2005).

The above findings suggest a plausible model for how Kis-L interacts with other trxG proteins to activate transcription. Perhaps Kis-L, like SWI/SNF and other chromatin-remodeling factors, is targeted to promoters via interactions with transcriptional activators or components of the general transcription machinery. Once targeted to the vicinity of a promoter, Kis-L may recognize promoter-proximal nucleosomes methylated on lysine 4 of histone H3 (by TRX or ASH1) via its chromodomains, leading to the localized remodeling of nucleosomes that pose a barrier to elongation by Pol II (Srinivasan, 2005).

The findings of this study may help explain the functional antagonism between kis and PcG proteins. PcG proteins do not merely render chromatin inaccessible to the general transcription machinery; transcriptional activators, basal transcription factors (including TFIID and TFIIF) and even Pol II are associated with targets of PcG repression. Thus, PcG proteins may act directly on components of the general transcription machinery assembled at promoters. Consistent with this possibility, Pol II is efficiently recruited to an hsp26 promoter silenced by the bxd PRE, but is unable to melt the promoter and initiate transcription (Dellino, 2004). A separate study of a promoter silenced by PcG proteins in its natural context (the Ubx promoter in wing imaginal discs) has revealed that PcG proteins bind to both PREs and a very narrow region just downstream of the start of transcription (Wang, 2004). Although their precise mechanism of action remains to be determined, the above studies suggest that PcG proteins exert their influence during the later stages of transcriptional initiation or early stages of elongation. It is therefore tempting to speculate that PcG proteins may repress transcription by blocking Kis-L activity. Further analysis of the role of Kis-L in transcription, together with the development of systems for analyzing its function in vitro, will be necessary to test this hypothesis and clarify the role of Kis-L in gene expression and development (Srinivasan, 2005).

GENE STRUCTURE

Genomic length - 30 kb

cDNA clone length - 17424 base pairs

Bases in 5' UTR - 496

Exons - 18 (Kis-A)

Bases in 3' UTR - 959

PROTEIN STRUCTURE

Amino Acids - Kis-PA (5322 aa); Kis-PB (2151 aa)

Structural Domains

kis encodes two major nuclear proteins with molecular weights of 574 kDa (Kis-L) and 225 kDa (Kis-S) (Daubresse, 1999; Therrien, 2000). Kis-L contains an ATPase domain that is highly related to those found in chromatin-remodeling factors, suggesting that Kis-L, like BRM, catalyzes ATP-dependent alterations in chromatin structure. Kis-L also contains two chromodomains and a BRK domain. Chromodomains mediate protein-protein or protein-RNA interactions, and are found in members of the CHD subfamily of ATPases (including Mi-2 and CHD1) and other proteins that interact with chromatin. Some chromodomains are involved in the selective recognition of methylated histone tails. The BRK domain is a 41 amino acid segment of unknown function that is conserved in BRM and its human homologs BRG1 and HBRM (Daubresse, 1999). The Kis-L protein lacks PHD fingers, a domain characteristic of CHD ATPases, and a bromodomain, a domain conserved in SWI2/SNF2 ATPases that mediates interactions with acetylated histone tails. Thus, Kis-L is unusual in that it has characteristics of both the CHD and SWI2/SNF2 subfamilies of ATPases, but is a clear member of neither class. Potential orthologs of kis are present in nematodes, mice and humans, but not yeast, suggesting that it may play a specialized role in transcription or development in higher eukaryotes (Daubresse, 1999; Schuster, 2002; Therrien, 2000; Srinivasan, 2005 and references therein).

kis encodes two major protein isoforms – Kis-S and Kis-L – which share a common 2105 amino acid C-terminal segment. This common segment contains a BRK domain related to those found in Drosophila Brm and its human orthologs. The long N-terminal segment unique to Kis-L contains an ATPase domain and two chromodomains. By contrast, Kis-S lacks these domains and is therefore unlikely to have chromatin-remodeling activity. The absence of an ATPase domain in Kis-S suggests that it may function as a naturally occurring dominant-negative form of the Kis protein (Srinivasan, 2005).

date revised: 1 May 2005

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