The CHD6-9 family of proteins has only recently been identified, and very little is known about the function of these proteins (29
). The first report concerning CHD8, however, was focused not on full-length CHD8 but on an N-terminal fragment of rat CHD8 termed Duplin. This fragment of CHD8, which lacks the catalytic Snf2 helicase domain and part of the second chromodomain, was found to interact directly with β-catenin. Characterization of this fragment led to the conclusion that Duplin is a negative regulator of the Wnt signaling pathway and functions by blocking the binding of β-catenin to TCF (61
). However, further studies also indicated that Duplin could attenuate the Wnt signaling pathway downstream of β-catenin target genes (38
). Bioinformatic analysis suggests that Duplin arose from an improper splicing event, and no evidence for the existence of a human counterpart can be found (data not shown).
This information on Duplin suggested that full-length CHD8 may regulate β-catenin target genes. To investigate this possibility, we first examined the binding of full-length CHD8 to β-catenin both in vivo and in vitro. We found that, like Duplin, full-length CHD8 can interact directly with β-catenin, further suggesting that CHD8 may also play a role in the regulation of β-catenin target genes. To strengthen this hypothesis, we investigated the ability of CHD8 to bind β-catenin target genes in vivo. Using ChIP experiments, we demonstrate that CHD8 is localized to the 5′ ends and not the 3′ ends of several β-catenin-responsive genes. We also show that this binding is specific for the promoter region and possibly sequences immediately downstream of the transcriptional start site but is not localized to the coding region of the gene.
To directly test our hypothesis that CHD8 regulates β-catenin target genes, we chose to utilize shRNA techniques to deplete CHD8 and then to examine the transcription of endogenous targets identified by ChIP analysis. We found that CHD8 does indeed function in the regulation of endogenous β-catenin target genes, since depletion of CHD8 results in an increase in the expression of the β-catenin target genes tested. To verify these results in an independent system, we chose to investigate the Drosophila CHD8 ortholog Kismet. This system also circumvents the complication of the three other CHD8 paralogs possibly functioning at these loci. As discovered with CHD8 in human cells, depletion of kismet by RNAi results in an increase in the transcription of the Wnt target gene nkd.
Kismet was originally identified as an extragenic suppressor of Polycomb and was therefore suggested to be a member of the trxG of activators (15
). Further studies examining the distribution of Kismet on polytene chromosomes suggested a more general role in transcriptional regulation. Kismet staining primarily overlaps that of RNA Pol II. Also, kismet
mutant larvae display reduced levels of elongating polymerase without affecting Pol II recruitment, as determined solely by immunostaining for the phosphorylation status of the carboxy-terminal tail of the large subunit of Pol II and by the loss of immunostaining for SPT6 and CHD1, factors associated with elongating polymerase (69
). These data would suggest that Kismet assists an early step in transcriptional elongation and would therefore be a positive regulator of transcription. By analogy, CHD8 should also act to facilitate transcription. Our results suggest that while Kismet may affect the overall levels of elongating polymerase, as determined by immunostaining, CHD8 and Kismet may act conversely at specific target genes, such as the Wnt-responsive genes, and obstruct optimal elongation.
Given the predicted function for Kismet described above, it is noteworthy that our purification of human CHD8 identifies spliceosome components that overlap with a set of CHD1-interacting proteins (66
). These are components of both the U2 and the U5 snRNPs. Knockdown of CHD1 in HeLa cells reduces the association of spliceosomal components with transcribed regions and also reduces the efficiency of splicing of these genes (66
). This may suggest that, like CHD1, CHD8 may also play a role in regulating the recruitment of spliceosomal components to actively transcribed genes. This function would, however, be upstream of CHD1, since kismet
mutant larvae display a failure to recruit CHD1 to polytene chromosomes (69
). Experiments to address the roles of CHD1 and CHD8, and the interplay between them, are required to advance our understanding of chromatin, transcriptional elongation, and splicing.
A recent study has identified CHD8 as a factor that associates with the zinc finger transcription factor hStaf/ZNF143 (82
). hStaf/ZNF143 is one of two human homologues of Xenopus
Staf, a transcriptional activator of snRNA genes (56
). CHD8 was identified as a factor in HeLa nuclear extracts that can bind to recombinant hStaf and was also shown by ChIP to bind to both Pol II and Pol III snRNA promoters as well as the hStaf-responsive IRF3 promoter (82
). Depletion of CHD8 by small interfering RNA resulted in a modest decrease in transcription at these promoters. These data are possibly consistent with the studies on Kismet as a positive regulator of transcriptional elongation. Again, taken with our results, this suggests that CHD8 can act conversely on different sets of genes and that this specificity may be controlled by the interaction with the underlying transcription factor.
An alternate explanation for the discrepancy between the previous studies on CHD8 and Kismet and our data on CHD8 could lie in the identification of CHD8 as a CTCF binding protein and the finding that CHD8 is required for CTCF-dependent insulator activity (34
). CTCF has many roles in transcriptional regulation beyond insulator activity, including transcriptional repression (19
). While CTCF has not been identified as a regulator of β-catenin-responsive genes, it is possible that CTCF can act in concert with CHD8 as a repressor at β-catenin-responsive genes, or even as an insulator to protect β-catenin-responsive promoters from proximal enhancer activities. Our affinity purification of CHD8 and MS-MS identification of associated proteins, however, did not identify CTCF in association with CHD8, suggesting that the majority of CHD8 may exist outside of a functional CTCF complex. Further studies to address the possible role of CTCF in β-catenin-mediated gene transcription will need to be performed.
As previously mentioned, CHD8 is closely related to CHD6, CHD7, and CHD9. Recent reports on both CHD7 and CHD9 show functional association with various nuclear hormone receptors. CHD9/CReMM/PRIC320 has been shown to interact specifically with peroxisome proliferator-activated receptor α (PPARα), PPARγ, RXR, ERα, CAR, and GR, and CHD9 also functions as a coactivator for PPARα reporter constructs (51
). CHD7 has recently been identified as a component of a corepressor complex that inactivates PPARγ-mediated transcription (73
). It has yet to be determined whether CHD8 also interacts with various nuclear hormone receptors performing a functional role in nuclear hormone signaling, and if so, to what extent CHD8 functions as a coactivator or a corepressor.
The identification of CHD8 in a high-molecular-mass complex of ~900 kDa suggests that CHD8 may reside in a multisubunit complex, like members of the CHD3 family (29
). These additional subunits may prove to be important for the proper function and localization of CHD8. The identification and characterization of these associated factors will play an important role in understanding the physiological role of CHD8. Previously, CHD8 has been identified as a component of a WDR5-containing complex (18
). This complex also contains the histone methyltransferase MLL1, suggesting a possible interplay between the activity of CHD8 and covalent histone modifications. Since this initial purification of CHD8 and the MLL-WDR5 complex was achieved via Flag-WDR5 purification, it has yet to be determined whether CHD8 is a component of this larger complex or whether it represents a new, as yet unidentified WDR5-CHD8 complex. Indeed, our purification could not identify MLL as a component of the CHD8 complex. While future experiments will have to be performed to address the molecular composition of this complex, our current functional characterization of CHD8 at the biochemical and cellular levels provides the required foundation for these studies.
An important question regarding the mechanism for regulation by CHD8 is centered on the prediction that CHD8 is indeed a chromatin remodeling enzyme. While previous reports have demonstrated that members of the CHD6-9 family do indeed possess ATPase activity (48
), here we report the first demonstration of bona fide chromatin remodeling activity for a member of the CHD6-9 family. This would then suggest that CHD8 indeed regulates transcription through the mobilization of chromatin structure. Given the similarity of the Snf2 helicase domains of members of this family (26
), these data suggest that all members of this subfamily should also share this activity. This result thus has important implications for the study of CHD7. Loss of CHD7 function via mutation leads to CHARGE syndrome in humans. CHARGE syndrome is characterized by various developmental defects, including growth retardation, ear malformations, and genital defects, among other symptoms (76
). By characterizing the activities and functions of the CHD6-9 family of enzymes, we can begin to develop a mechanistic link between chromatin structure and human disease.