has a single CHD family member, CHD1. In metazoans, the family has expanded to four CHD proteins in D. melanogaster
and nine in Homo sapiens
). In this study, we have analyzed dCHD3, a novel chromatin remodeler of D. melanogaster
. By analyzing genomes of different Drosophila
species, we have traced the gene duplication event giving rise to dCHD3. We postulate that the dCHD3 gene originated from integration of a truncated, reverse-transcribed dMi-2 mRNA. This hypothesis is supported by the fact that the dCHD3 gene lacks introns.
We have performed a detailed analysis of the nucleosome remodeling activities of dCHD3 in vitro. Like dMi-2, dCHD3 binds DNA and nucleosomes and is a nucleosome-stimulated ATPase. However, dCHD3 and dMi-2 differ in the extent to which their ATPases are activated by DNA. Whereas the activation of dMi-2 by DNA is insignificant, the activation of dCHD3 is more pronounced. Interestingly, DNA stimulation of dMi-2 is increased in a deletion mutant lacking the C-terminal 700 residues (5
). dCHD3 lacks most of this region, and when the remaining C-terminal residues are removed, the stimulation of dCHD3 ATPase activity by DNA is further increased. These results strengthen the hypothesis that the C-terminal sequences of dMi-2 and dCHD3 have a negative regulatory effect on DNA-stimulated ATPase activity (5
dCHD3 mobilizes mononucleosomes in an ATP-dependent manner. Mobilization is effected from both a position close to one end of the DNA fragment to a more central one and a central position to more distal ones. Recombinant dMi-2 displays a strong preference for distal-to-central mobilization when a nucleosome assembled on the mouse 5S rDNA fragment is used (9
). In the current study, mononucleosomes were assembled on DNA fragments containing the high-affinity 601 sequence (28
). Neither dCHD3 nor dMi-2 displayed a significant preference for the direction of nucleosome movement. This demonstrates that direction preference is not an invariant property of the remodeling machine but is influenced by the underlying DNA sequence.
dCHD3 removes a fraction of histone octamers from DNA during the mobilization assay. It is possible that some histone octamers are pushed over the edge during mobilization and become unstable. The kinetics of the mobilization reaction are in agreement with this view: mobilized nucleosomes are produced within seconds, whereas free DNA becomes detectable only after 10 min, when most nucleosomes have already been relocalized.
We have tested the ability of recombinant dCHD3 and dMi-2 to remodel polynucleosomal substrates in vitro. Both factors increased the accessibility of nucleosomal restriction sites. Interestingly, this effect was obtained when the restriction enzyme was present during the remodeling reaction as well as when it was added after remodeling had taken place. This result indicates that both factors can create stably remodeled polynucleosomes in the absence of other proteins.
Removal of histone tails by partial proteolysis does not affect nucleosome stimulation of the dCHD3 ATPase. Likewise, dMi-2 is stimulated by nucleosomes assembled from recombinant histones lacking all N-terminal tails and NuRD complex purified from Xenopus laevis
is activated by trypsinized nucleosomes (8
). These results suggest that histone tail interactions do not play a critical role during remodeling catalyzed by this CHD subfamily, despite the fact that both factors contain chromodomains and PHD fingers. Both of these domains have recently been demonstrated to specifically bind methylated histone tails (10
). For example, the chromodomains of human CHD1 recognize histone H3 methylated at K4 (14
). Moreover, human CHD4 (Mi2β) has been suggested to bind histone H3 methylated at K36 (33
). There is no data suggesting that chromodomains and PHD fingers of dCHD3 and dMi-2 bind methylated histone tails. Indeed, based on sequence differences between chromodomains of CHD3/CHD4 and CHD1, it has been suggested that CHD3/CHD4-type chromodomains do not bind methylated histone tails (36
). Our results indicate that the chromodomains of dCHD3 are critical for nucleosome remodeling by providing an interaction surface for DNA. These findings are in agreement with results that we have previously reported for the chromodomains of dMi-2 (5
). However, we cannot formally rule out the possibility that PHD fingers and chromodomains of dCHD3 carry out redundant functions. Moreover, it remains possible that specific tail modifications positively or negatively influence the interactions of these remodelers with chromatin in vivo.
Most ATP-dependent chromatin remodelers exist in stable complexes with one or more nonenzymatic subunits implicated in targeting and regulation of enzymatic activity. Given the similarity between dCHD3 and dMi-2, it was conceivable that dCHD3 would interact with NuRD subunits. However, our results strongly suggest that dCHD3 exists as a monomer in vivo. In this respect, dCHD3 resembles the CHD1 protein which has been reported both to exist in a complex and as a monomer in S. cerevisiae
and has been shown to be largely monomeric in D. melanogaster
dCHD3 and dMi-2 antibodies stain the same bands on polytene chromosomes. We have ruled out that this is due to antibody cross-reactivity (see Fig. S2 in the supplemental material). The high degree of colocalization of dCHD3 and dMi-2 suggests a common way of targeting. However, the targeting mechanisms that have been proposed for dMi-2 do not seem to be applicable to dCHD3. dMi-2 interacts with DNA bound transcriptional repressors via a C-terminal domain that is missing in dCHD3 (20
). An alternative targeting mechanism of NuRD complexes is provided by MBD-domain-containing subunits which could guide the complex to regions of methylated DNA (31
). However, since dCHD3 exists as a monomer, such a mechanism appears unlikely. Of course, we cannot exclude the possibility that transient interactions might be involved in dCHD3 recruitment. Given that dCHD3 and dMi-2 colocalize with RNA polymerase II at transcriptionally active regions, it is conceivable that targeting involves binding to components of the transcription apparatus. Indeed, such interactions have been demonstrated for yeast CHD1 and SWI2/SNF2 (39
). However, the immunoprecipitation of dCHD3 and dMi-2 from nuclear extracts did not coprecipitate RNA polymerase II and vice versa (data not shown). Like dMi-2 and dCHD3, dChd1, Kismet, and BRM associate with actively transcribed regions on polytene chromosomes, although no physical interactions with RNA polymerase II can be detected (1
). This result suggests that these ATP-dependent chromatin remodelers recognize an as-yet-unidentified component of actively transcribed regions.
It is important to note that dMi-2 and dCHD3 do not colocalize under all circumstances. Our analysis of early embryos suggests different affinities for binding to condensed chromosomes during mitosis. dCHD3 remains associated with condensed chromosomes, whereas dMi-2 is distributed more evenly throughout the nucleus.
Their similar expression patterns, remodeling properties, and colocalization on polytene chromosomes raise the question of whether dMi-2 and dCHD3 carry out redundant functions. dMi-2-deficient flies are generally nonviable (although escapers have been reported) and die during larval stages (20
). Their survival to larval stage has been attributed to maternal contribution of dMi-2. Indeed, our immunofluorescence and Western blot analysis results show a high level of dMi-2 protein in embryos prior to the onset of zygotic transcription. dMi-2-deficient germ cells are not viable (20
). The depletion of dMi-2 by RNAi in KC cells decreases cell growth, supporting the view that dMi-2 is required for viability on a cell autonomous level. By contrast, we have not detected adverse effects of dCHD3 depletion. In addition, no dCHD3 mutant flies have been reported to date. These results suggest that dCHD3 and dMi-2 are not fully redundant and that dCHD3 cannot compensate for the loss of dMi-2. However, we cannot formally exclude the possibility that the differential response of cells to dMi-2 and dCHD3 depletion is due to incomplete knockdown of dCHD3 or a similar dosage effect.
It is remarkable that all four CHD family members colocalize with RNA polymerase II to sites of active transcription on polytene chromosomes (41
; this study). This finding is not easily reconcilable with the proposed role of dMi-2 and dNuRD in transcriptional repression. However, it has recently been demonstrated that HDAC complexes play important roles in silencing cryptic promoters in transcribed genes in S. cerevisiae
). Srinivasan and colleagues have used mutants to demonstrate that Kismet likely acts at an early stage of the transcription process (41
). CHD1 mutants are deficient in the replication-independent incorporation of the histone H3.3 variant both during the restructuring of the male pronucleus and at later stages following the onset of zygotic transcription (21
). Suitable dmi-2
mutants are not yet available to perform similar studies. Whether the four CHD remodelers are all involved at different stages of transcription and what their molecular function is remain to be analyzed.