InteractiveFly: GeneBrief

Chromodomain-helicase-DNA-binding protein: Biological Overview | References

Gene name - Chromodomain-helicase-DNA-binding protein

Synonyms -

Cytological map position- 23C4-23C4

Function - enzyme, chromatin assembly

Keywords - ATP-utilizing chromatin assembly factor

Symbol - Chd1

FlyBase ID: FBgn0250786

Genetic map position - 2L: 2,979,697..2,987,677 [-]

Classification - SNF2 family N-terminal domain

Cellular location - nuclear

NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Kim, S., Bugga, L., Hong, E. S., Zabinsky, R., Edwards, R. G., Deodhar, P. A. and Armstrong, J. A. (2015). An RNAi-based candidate screen for modifiers of the CHD1 chromatin remodeler and assembly factor in Drosophila melanogaster.G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26596648
The conserved chromatin remodeling and assembly factor CHD1 (chromodomains, helicase, DNA-binding domain) is present at active genes where it participates in histone turnover and recycling during transcription. In order to gain a more complete understanding of the mechanism of action of CHD1 during development a novel genetic assay was created in Drosophila melanogaster to evaluate potential functional interactions between CHD1 and other chromatin factors. Over-expression of the CHD1 results in defects in wing development, and this fully penetrant and reliable phenotype was used to conduct a small-scale RNAi-based candidate screen to identify genes that functionally interact with Chd1 in vivo. The results indicate that CHD1 may act in opposition to other remodeling factors, including INO80, and that the recruitment of CHD1 to active genes by RTF1 is conserved in flies.
Berson, A., Sartoris, A., Nativio, R., Van Deerlin, V., Toledo, J. B., Porta, S., Liu, S., Chung, C. Y., Garcia, B. A., Lee, V. M., Trojanowski, J. Q., Johnson, F. B., Berger, S. L. and Bonini, N. M. (2017). TDP-43 promotes neurodegeneration by impairing chromatin remodeling. Curr Biol 27(23):3579-3590.. PubMed ID: 29153328
Regulation of chromatin structure is critical for brain development and function. However, the involvement of chromatin dynamics in neurodegeneration is less well understood. This study found, launching from Drosophila models of amyotrophic lateral sclerosis and frontotemporal dementia, that TDP-43 impairs the induction of multiple key stress genes required to protect from disease by reducing the recruitment of the chromatin remodeler Chd1 to chromatin. Chd1 depletion robustly enhances TDP-43-mediated neurodegeneration and promotes the formation of stress granules. Conversely, upregulation of Chd1 restores nucleosomal dynamics, promotes normal induction of protective stress genes, and rescues stress sensitivity of TDP-43-expressing animals. TDP-43-mediated impairments are conserved in mammalian cells, and, importantly, the human ortholog CHD2 physically interacts with TDP-43 and is strikingly reduced in level in temporal cortex of human patient tissue. These findings indicate that TDP-43-mediated neurodegeneration causes impaired chromatin dynamics that prevents appropriate expression of protective genes through compromised function of the chromatin remodeler Chd1/CHD2. Enhancing chromatin dynamics may be a treatment approach to amyotrophic lateral scleorosis (ALS)/frontotemporal dementia (FTD).


CHD1 is a chromodomain-containing protein in the SNF2-like family of ATPases. This study shows that CHD1 exists predominantly as a monomer and functions as an ATP-utilizing chromatin assembly factor. This reaction involves purified CHD1, NAP1 chaperone, core histones and relaxed DNA. CHD1 catalyzes the ATP-dependent transfer of histones from the NAP1 chaperone to the DNA by a processive mechanism that yields regularly spaced nucleosomes. The comparative analysis of CHD1 and ACF revealed that CHD1 assembles chromatin with a shorter nucleosome repeat length than ACF. In addition, ACF, but not CHD1, can assemble chromatin containing histone H1, which is involved in the formation of higher-order chromatin structure and transcriptional repression. These results suggest a role for CHD1 in the assembly of active chromatin and a function of ACF in the assembly of repressive chromatin (Lusser, 2005).

The packaging of DNA into chromatin is a critical step in the organization and utilization of the genome. In this process, DNA is assembled into arrays of nucleosomes, each of which contains an octamer of the core histone proteins. The nucleosome cores are joined together by linker DNA that is typically ~20-60 base pairs (bp) in length. In metazoans, an additional histone, termed histone H1, interacts with both the linker DNA and the nucleosome core, and promotes the higher-order folding of chromatin (Lusser, 2005).

Chromatin assembly involves the combined action of core histone chaperones and ATP-utilizing motor proteins. Several distinct histone chaperones, such as CAF-1 (chromatin assembly factor-1), Asf1 (anti-silencing function-1), NAP1 (nucleosome assembly protein-1), and HirA (histone regulatory protein A), participate in the deposition of the histones onto the DNA and/or mediate the translocation of core histones from the cytoplasm to the nucleus. The assembly of extended, periodic nucleosome arrays has thus far been observed in vitro with the ATP-utilizing factors ACF (ATP-utilizing chromatin assembly and remodeling factor), CHRAC (chromatin accessibility complex), and RSF (remodeling and spacing factor). ACF and CHRAC are nearly identical and seem to be conserved from yeast to humans. In Drosophila melanogaster, ACF and CHRAC each contain the Acf1 protein and the ISWI (imitation switch) ATPase, but CHRAC additionally comprises the small CHRAC-14 and CHRAC-16 polypeptides. In addition, recombinant ISWI polypeptide alone has ATP-dependent chromatin assembly. RSF has been studied in humans and consists of Rsf1 and hSNF2h, a human homolog of D. melanogaster ISWI. Thus, these ATP-utilizing chromatin assembly factors share an ATPase subunit that is a member of the ISWI/SNF2L subfamily of proteins (Lusser, 2005 and references therein).

In Drosophila, the ISWI ATPase has been found in four protein complexes: ACF, CHRAC, NURF (nucleosome remodeling factor), and TRF2 (TBP-related factor-2) complex. Aside from ISWI, the NURF and TRF2 complexes do not share any subunits with ACF or CHRAC, and a role for the NURF or TRF2 complex in chromatin assembly has not been described. Drosophila melanogaster ISWI is essential for viability, and the loss of ISWI function in vivo results in the misregulation of transcription and global alterations in chromosome structure (Lusser, 2005).

The function of the ACF and CHRAC chromatin assembly factors in vivo in D. melanogaster has been examined by the generation and characterization of acf1 null mutant flies (Fyodorov, 2004). Unlike ISWI, the acf1 gene is not absolutely required for viability. The loss of Acf1 results in a decrease in nucleosome periodicity as well as a shorter nucleosome repeat length in chromatin derived from embryos. Extracts prepared from homozygous null acf1 embryos exhibit reduced yet detectable ATP-dependent chromatin assembly activity. These results support a role for Acf1 in chromatin assembly in vivo, and also suggest that D. melanogaster has at least one additional ATP-dependent chromatin assembly factor that is distinct from ACF or CHRAC (Lusser, 2005).

This work explored the function of ATPases other than ISWI in the ATP-dependent assembly of periodic nucleosome arrays. These studies led to the characterization of the CHD1 (chromo-ATPase/helicase-DNA-binding protein 1) protein. CHD1 was originally found as a chromodomain-helicase-DNA-binding domain-containing protein (Delmas, 1993). It is a member of the CHD1 subfamily of DNA-stimulated ATPases. Both the CHD1 and ISWI/SNF2L subfamilies are members of the SNF2-like family of proteins. One distinctive feature of proteins of the CHD1 subfamily is the presence of two chromodomains (Lusser, 2005).

The analysis of CHD1 has suggested that it functions in the elongation of transcription by RNA polymerase II as well as in chromatin dynamics. For example, CHD1 has been found to bind to DNA (Stokes, 1995), to localize to regions of decondensed chromatin (interbands) and high transcriptional activity (puffs) in D. melanogaster polytene chromosomes (Stokes, 1996), to participate in transcriptional termination (Stokes, 1996), and to interact with the FACT (facilitates chromatin transcription; mammalian SSRP1-Spt16 complex, or yeast Pob3-Spt16/Cdc68 complex; see Drosophila FACT), Rtf1 and Spt5 transcriptional elongation factors as well as with casein kinase II. In addition, studies in S. cerevisiae have revealed synthetic genetic interactions between ISW1 and ISW2 (which encode ISWI-related proteins) and CHD1 (Tsukiyama, 1999) as well as a partial loss of chromatin assembly activity in vitro by crude DEAE fractions derived from strains lacking either Asf1 or Chd1, but not Isw1, Isw2, Snf2, Swr1, NAP1 or CAC-1 (large subunit of CAF-1) (Robinson, 2003). Moreover, purified yeast CHD1 has been shown to remodel mononucleosomes in vitro (Tran, 2000; Lusser, 2005 and references therein).

This study has explored the function of CHD1 in chromatin assembly and found that the purified protein is an ATP-utilizing chromatin assembly factor that is distinct from the ISWI-containing factors. Moreover, comparison of the biochemical functions of CHD1 and ACF revealed differences in their properties that may reflect their participation in distinct biological processes in vivo (Lusser, 2005).

To investigate the biochemical activities of CHD1, Drosophila CHD1 was synthesized with a C-terminal Flag-tag by using a baculovirus expression system and then the protein was purified by Flag immunoaffinity chromatography. To determine whether the purified recombinant CHD1 was enzymatically active, ATPase assays nd were carried out and observed strong stimulation of ATPase activity by DNA or chromatin but not by free histones or RNA was observed, as seen previously with yeast CHD1 protein (Lusser, 2005).

Many proteins in the SNF2 like family are present in multi-subunit complexes. In addition, CHD1 proteins have been found to interact with factors involved in transcriptional elongation and with casein kinase II. Hence, before characterizing the purified recombinant CHD1 protein, whether native D. melanogaster CHD1 protein is a component of a multiprotein complex was investigated by carrying out glycerol gradient sedimentation experiments with crude nuclear extracts derived from Drosophila embryos. Western blot analysis of gradient fractions with an antibody against CHD1 revealed that bulk native CHD1 sediments with an apparent molecular mass of ~200 kDa, which corresponds closely to the calculated molecular mass of the CHD1 polypeptide (212 kDa). In parallel, it was also found that recombinant Flag-tagged CHD1 sediments with an apparent mass similar to that of native CHD1. These results indicate that native and recombinant CHD1 proteins are predominantly present as monomers (Lusser, 2005).

Thus, the majority of native D. melanogaster CHD1 seems to exist as a monomeric protein rather than as a subunit of a stable, multiprotein complex. This conclusion is also consistent with studies of yeast CHD1, which has been found to be a single polypeptide upon purification from a whole-cell extract (Tran, 2000). Hence, it is likely that subsequent analyses of purified recombinant CHD1 reflect the properties of the native form of the protein (Lusser, 2005).

To analyze the functional properties of CHD1, chromatin assembly assays were carried with a completely purified system consisting of recombinant Drosophila CHD1, recombinant D. melanogaster NAP1, native D. melanogaster core histones and relaxed circular plasmid DNA. Analysis of the reaction products by the micrococcal nuclease digestion assay revealed that CHD1 catalyzes the formation of extended arrays of regularly spaced nucleosomes. CHD1-mediated chromatin assembly requires ATP as well as the NAP1 chaperone. Therefore, CHD1 is an ATP-utilizing chromatin assembly factor. Hence, catalysis of the ATP-dependent assembly of periodic nucleosome arrays can be mediated by the CHD1 ATPase as well as by the ISWI ATPase (Lusser, 2005).

The generation of periodic nucleosome arrays by CHD1 could be achieved by different mechanisms. It is possible, for instance, that chromatin assembly by CHD1 occurs via a passive histone deposition mechanism in which the histones are randomly deposited onto the DNA by a chaperone such as NAP1 in an ATP-independent process, and then the resulting nucleosomes are redistributed into periodic arrays by CHD1 acting as an ATP-utilizing nucleosome mobilization factor. Alternatively, chromatin assembly may occur by an active histone deposition mechanism in which CHD1 function is involved in the transfer of histones to DNA as well as the formation of periodic arrays of nucleosomes. Thus, to clarify the mechanism of chromatin assembly by CHD1, its biochemical properties were further analyzed (Lusser, 2005).

Whether CHD1 can rearrange randomly distributed nucleosomes to give periodic arrays was tested, as in the second step of the passive deposition mechanism. To this end, chromatin was reconstituted by using salt dialysis, and the chromatin was purified from free histones and DNA by sucrose gradient sedimentation. In the salt dialysis reconstitution procedure, purified histones are combined with DNA in 2 M NaCl, and then, upon removal of the salt by dialysis, the histones are randomly deposited onto the DNA in an ATP-independent process. When subjected to micrococcal nuclease digestion analysis, the salt dialysis-reconstituted chromatin gave poorly defined bands with a repeat length of ~145 bp. Upon incubation with either CHD1 or ACF in the presence of ATP, the randomly distributed nucleosomes were converted into extended periodic arrays with a repeat length of ~160 bp. Therefore, CHD1, like ISWI-containing remodeling factors, is a nucleosome spacing factor. These results indicate that CHD1 can rearrange existing nucleosomes. This activity could be related to its participation in a passive deposition mechanism. To test this possibility, the function of CHD1 was further analyzed during the assembly of nucleosomes (Lusser, 2005).

Chromatin assembly reactions were carried out to determine whether or not CHD1 catalyzes the ATP-dependent transfer of histones onto the DNA. In an active transfer mechanism, CHD1 would facilitate histone deposition, whereas in a passive transfer mechanism, CHD1 would not affect the transfer of histones from NAP1 chaperone to the DNA. To distinguish between these mechanisms, nucleosome assembly was monitored by using the DNA supercoiling assay, which detects the formation of negative supercoiling in a circular template that results from the induction of approximately one negative supercoil for each nucleosome that is assembled. A series of reactions were carried out with relaxed circular DNA templates in the presence of purified topoisomerase I. These experiments revealed that CHD1 substantially enhances the extent of nucleosome assembly relative to that seen with either NAP1 alone or NAP1 with ATP (Lusser, 2005).

Hence, these results indicate that CHD1 catalyzes the transfer of histones from the NAP1 chaperone onto DNA in an ATP-dependent manner. This property of CHD1 suggests that it functions by an active rather than a passive histone deposition mechanism. In this respect, CHD1 is similar to ACF, which also mediates the transfer of histones onto DNA in an ATP-dependent manner. Moreover, in a related process, the SWR1 complex catalyzes histone H2A.Z-H2B exchange in conjunction with the NAP1 chaperone by an ATP-dependent mechanism. These findings collectively support an active role of ATP-utilizing motor proteins in nucleosome assembly and histone exchange (Lusser, 2005).

The mechanism of CHD1 function was further investigated by testing whether it acts processively in the assembly of chromatin. To this end, chromatin assembly reactions were carried out at a substoichiometric amount of CHD1 relative to DNA templates (approximately one CHD1 molecule per five DNA templates), and then the early reaction products were analyzed by two-dimensional DNA supercoiling analysis. Under these conditions, if CHD1 functions by a processive mechanism, then CHD1 would assemble multiple nucleosomes on a subset of the templates, and two distinct populations of reaction products would be observed: partially assembled chromatin and naked DNA. Alternatively, if CHD1 functions in a nonprocessive manner, then a single normal distribution of supercoiled DNA species would be seen (Lusser, 2005).

In the assembly of chromatin by CHD1, two populations of reaction products were seen: partially assembled chromatin templates with a peak of nine nucleosomes, and naked DNA templates with a peak of zero nucleosomes. Micrococcal nuclease digestion analysis also revealed short arrays of nucleosomes, which indicate local clustering of nucleosomes. These results indicate that CHD1 assembles chromatin processively, as seen with ACF. Moreover, the processivity of CHD1 supports an active histone deposition mechanism (Lusser, 2005).

Activities of CHD1 that may be distinct from those of ACF were examined, and therefore a comparative analysis of the two factors was carried out. A notable difference between CHD1 and ACF was revealed upon examination of the nucleosome repeat lengths of chromatin assembled by each of these factors. Under identical reaction conditions, except for the presence of CHD1 or ACF, CHD1 assembled chromatin with a repeat length of ~162 bp, whereas ACF assembled chromatin with a substantially longer repeat length of ~175 bp. These results indicate that the nucleosome repeat length is dictated by the ATP-driven factor that assembles the chromatin (Lusser, 2005).

In metazoans, bulk native chromatin contains approximately one molecule of the linker histone H1 (and/or H5) per nucleosome. H1 histones have a broad range of effects on chromatin compaction and organization as well as the regulation of gene expression. Whether CHD1 can catalyze the assembly of histone H1-containing chromatin was examined. To this end, chromatin assembly reactions were carried out in the absence or presence of purified histone H1 (at a 1:1 molar ratio of H1/core histone octamers) with either CHD1 or ACF. In the absence of H1, nucleosome repeat lengths of ~162 bp were seen with CHD1 and 172 bp with ACF, consistent with the results described above. Upon addition of H1 to ACF assembly reactions, a distinct micrococcal nuclease digestion pattern was observed that reveals an increase in the nucleosome repeat length from ~172 bp in the absence of H1 to ~200 bp in the presence of histone H1. This alteration in the repeat length is a consequence of the incorporation of histone H1. In contrast, under identical conditions, a dispersed, smeary micrococcal nuclease digestion pattern was observed upon inclusion of H1 in CHD1 assembly reactions, indicating a disruption of the periodicity of the nucleosomes that may be due to random association of the free H1 with nucleosomes. Further attempts were made to assemble H1-containing chromatin with CHD1 under a variety of other reaction conditions, but incorporation of histone H1 was not observed. It therefore seems that histone H1-containing chromatin can be assembled with ACF but not with CHD1 (Lusser, 2005).

The incorporation of histone H1 into chromatin was further characterized by native nucleoprotein gel electrophoresis of mononucleosome species that are generated upon extensive micrococcal nuclease digestion of chromatin. With chromatin assembled by either CHD1 or ACF in the absence of histone H1, core particles as well as some dinucleosomes were detected. Upon inclusion of histone H1 in the assembly reactions, the formation of chromatosomes (mononucleosomes containing histone H1) was observed with ACF but not with CHD1. CHD1 assembly reactions with histone H1 yielded a heterogeneous mixture of mononucleosome species that migrated faster than the chromatosomes. It is possible that nonspecific interactions of free histone H1 with the core particles result in a retardation of their migration through the nondenaturing gel. The addition of excess free competitor DNA after chromatin assembly but before micrococcal nuclease digestion did not affect the efficiency of formation of the chromatosome species (Lusser, 2005).

The DNA fragments present in the mononucleosome preparations were additionally analyzed by deproteinization of the micrococcal nuclease-digested chromatin followed by agarose gel electrophoresis. These experiments revealed that the mononucleosomal DNA fragments derived from ACF-assembled chromatin exhibited a ~25-bp increase in length upon addition of H1 to the chromatin assembly reactions, whereas mononucleosomal DNA fragments derived from CHD1-assembled chromatin were found to be nearly the same length whether or not H1 was present in the assembly reactions. Hence, these results, combined with the observation of chromatosome species with ACF but not with CHD1, provide further evidence for the incorporation of histone H1 into chromatin by ACF but not by CHD1 (Lusser, 2005).

It was also of interest to testing whether the ISWI ATPase subunit of ACF is sufficient for the assembly of histone H1-containing chromatin. Therefore, assembly reactions were carried out with purified ISWI polypeptide, and it was observed that ISWI alone can catalyze the formation of histone H1-containing chromatin. The quality of the H1-containing chromatin generated with ISWI was, however, consistently lower than the quality of that assembled with ACF. Notably, the smearing between the micrococcal nuclease bands, which reflects irregularities in the nucleosome arrays, is more prominent with ISWI-assembled H1-containing chromatin than with ACF-assembled chromatin. Thus, ISWI can catalyze the assembly of histone H1-containing chromatin, but is less effective than ACF in the assembly of periodic arrays of H1-containing nucleosomes. These findings indicate that the difference in the abilities of ACF and CHD1 to assemble H1-containing chromatin is due, at least in part, to the activities of the ISWI versus the CHD1 ATPases (Lusser, 2005).

Thus, chromatin assembly by CHD1 can be carried out with a completely purified system that consists of recombinant CHD1, recombinant NAP1, native core histones and DNA. Therefore, the ATP-dependent assembly of periodic nucleosome arrays can be mediated by CHD1 as well as by ISWI-containing proteins. In contrast, the catalysis of chromatin assembly by other related ATPases such as BRG-1 and Rad54 was not observed. Hence, chromatin assembly is not a general property of members of the SNF2-like family of ATPases (Lusser, 2005).

CHD1 catalyzes the transfer of histones from a chaperone to the DNA template by a processive mechanism that yields periodic nucleosome arrays. CHD1 can also convert randomly distributed nucleosomes into periodic nucleosome arrays. Notably, CHD1 and ACF catalyze the assembly of chromatin with different internucleosomal spacing. In addition, ACF, but not CHD1, can assemble histone H1-containing chromatin. Hence, CHD1 is an ATP-utilizing chromatin assembly and remodeling factor with activities that are distinct from those of ACF (Lusser, 2005).

Transcriptionally active chromatin generally has a shorter nucleosome repeat length than transcriptionally repressed chromatin. Thus, the ability of CHD1 to mediate the reconstitution of chromatin with relatively short internucleosomal spacing might reflect its proposed involvement in active transcription in vivo. The lack of H1 assembly activity by CHD1 supports this model and, in addition, suggests a mechanistic link between these two activities. It is relevant to note that bulk chromatin in Drosophila lacking the Acf1 subunit of ACF and CHRAC has a shorter repeat length than chromatin in wild-type flies. This reduction in the nucleosome repeat length could be due to the loss of ACF (and CHRAC), which catalyzes the assembly of chromatin with a longer repeat length than CHD1-assembled chromatin. Moreover, the shorter repeat length of chromatin in the Acf1-deficient flies could also be due to a defect in the incorporation of histone H1 into the chromatin upon loss of ACF (and CHRAC) (Lusser, 2005).

From a broader perspective, the findings from this study contribute to the current understanding of the biological roles of CHD1 and ACF. ACF seems to promote the assembly of repressive chromatin. For instance, Drosophila Acf1 contributes to heterochromatic repression, such as that seen in position-effect variegation. In addition, Drosophila ISWI is mostly associated with nontranscribed regions of polytene chromosomes. Consistent with these observations, ACF can assemble transcriptionally repressive histone H1-containing chromatin. In contrast, CHD1 associates with factors that promote transcriptional elongation, such as FACT, Rtf1 and Spt5, and localizes to transcriptionally active regions of the genome. The function of CHD1 as a chromatin assembly factor fits well with its proposed role in the reassembly of nucleosomes subsequent to their disruption during transcription (Robinson, 2003; Belotserkovskaya, 2003). Moreover, the ability of CHD1 to assemble H1-deficient chromatin, but not H1-containing chromatin, is consistent with a function in the assembly of transcriptionally active DNA into chromatin. It was also observed that the majority of native Drosophila CHD1 seems to exist as a monomer. These results further suggest that CHD1 and the elongation factors interact transiently rather than as components of a stable complex, or that only a small fraction of CHD1 is present in a multiprotein complex. Future studies may address whether there are other ATP-utilizing chromatin assembly factors and examine their shared and unique functions (Lusser, 2005).

CHD1 is required for deposition of histone variant H3.3 into chromatin in vivo

The organization of chromatin affects all aspects of nuclear DNA metabolism in eukaryotes. H3.3 is an evolutionarily conserved histone variant and a key substrate for replication-independent chromatin assembly. Elimination of chromatin remodeling factor CHD1 in Drosophila embryos abolishes incorporation of H3.3 into the male pronucleus, renders the paternal genome unable to participate in zygotic mitoses, and leads to the development of haploid embryos. Furthermore, CHD1, but not ISWI, interacts with HIRA in cytoplasmic extracts. These findings establish CHD1 as a major factor in replacement histone metabolism in the nucleus and reveal a critical role for CHD1 in the earliest developmental instances of genome-scale, replication-independent nucleosome assembly. Furthermore, these results point to the general requirement of adenosine triphosphate (ATP)-utilizing motor proteins for histone deposition in vivo (Konev, 2007).

Histone-DNA interactions constantly change during various processes of DNA metabolism. Recent studies have highlighted the importance of histone variants, such as H3.3, CENP-A (centromere protein A), or H2A.Z, in chromatin dynamics. Incorporation of replacement histones into chromatin occurs throughout the cell cycle, whereas nucleosomes containing canonical histones are assembled exclusively during DNA replication. A thorough understanding of the replication-independent mechanisms of chromatin assembly, however, is lacking (Konev, 2007).

In vitro, chromatin assembly requires the action of histone chaperones and adenosine triphosphate (ATP)-utilizing factors. Histone chaperones may specialize for certain histone variants. For example, H3.3 associates with a complex containing HIRA, whereas canonical H3 is in a complex with CAF-1 (chromatin assembly factor 1) (Tagami, 2004). The molecular motors known to assemble nucleosomes are ACF (ATP-utilizing chromatin assembly and remodeling factor), CHRAC (chromatin accessibility complex), and RSF (nucleosome-remodeling and spacing factor), which contain the Snf2 family member ISWI as the catalytic subunit, and CHD1, which belongs to the CHD subfamily of Snf2-like adenosine triphosphatases (ATPases). These factors have not been shown to mediate deposition of histones in vivo. It has been demonstrated that CHD1, together with the chaperone NAP-1, assembles nucleosome arrays from DNA and histones in vitro (Lusser, 2005). This study investigated the role of CHD1 in chromatin assembly in vivo in Drosophila (Konev, 2007).

Chd1 alleles were generated by P element-mediated mutagenesis. Two excisions, Df(2L)Chd1[1] and Df(2L)Chd1[2], deleted fragments of the Chd1 gene and fragments of unrelated adjacent genes. Heterozygous combinations, however, of Chd1[1] or Chd1[2] with Df(2L)Exel7014 affect both copies of the Chd1 gene only. Also a single point mutation was identified that results in premature translation termination of Chd1 (Q1394*) in a previously described lethal allele, l(2)23Cd[A7-4]. Hence, l(2)23Cd[A7-4] was renamed Chd1[3] (Konev, 2007).

Analysis of Western blots of embryos from heterozygous Chd1[3] fruit flies revealed the presence of a truncated polypeptide besides full-length CHD1. No truncated polypeptides were detected in heterozygous Chd1[1] or Chd1[2] embryos. Therefore, the corresponding deficiencies result in null mutations of Chd1. Crosses of heterozygous Chd1 mutant alleles with Df(2L)JS17/CyO or Df(2L)Exel7014/CyO produced subviable adult homozygous mutant progeny. Both males and females were sterile. Homozygous null females mated to wildtype males laid fertilized eggs that died before hatching. Therefore, maternal CHD1 is essential for embryonic development (Konev, 2007).

When the chromosome structure of 0- to 4-hour-old embryos laid by Chd1-null females were examined, it was observed that, during syncytial mitoses (cycles 3 to 13), the nuclei appeared to be abnormally small. The observed numbers of anaphase chromosomes suggested that they were haploid. To confirm this observation, wild-type or Chd1-null females were mated with males that carried a green fluorescent protein (GFP) transgene. Embryonic DNA was amplified with primers detecting male-specific GFP and a reference gene, Asf1. In wild-type embryos, both primer pairs produced polymerase chain reaction (PCR)-amplified products, whereas only the Asf1 fragment was amplified in the mutants. Thus, Chd1 embryos develop with haploid, maternally derived chromosome content (Konev, 2007).

To investigate the causes of haploidy in mutant animals, distributions of various developmental stages were compared in samples of wildtype and Chd1-null embryos. The lack of maternal CHD1 dramatically changed this distribution. Most notably, at 0 to 4 hours after egg deposition, the majority of Chd1 embryos (56%) remained at a very early stage of development in contrast to the wild type (24%) (Konev, 2007).

In Drosophila eggs, meiosis gives rise to four haploid nuclei. When the egg is fertilized, one of them is selected as a female pronucleus; the remaining three form the polar body. After breakdown of the sperm nuclear envelope, the compacted sperm chromatin is decondensed, and sperm-specific protamines are replaced with maternal histones. The male and female pronuclei juxtapose in the middle of the embryo and undergo one round of separate haploid mitoses. The resulting products fuse with their counterparts to give two diploid nuclei. In the majority of Chd1 embryos, partial decondensation of the sperm chromatin and normal apposition of parental pronuclei were observed. Then, however, one pronucleus underwent mitosis; the other one did not. Considering the subsequent loss of paternal DNA, it is concluded that mitotic progression of the male pronucleus is hindered in Chd1 embryos (Konev, 2007).

Because CHD1 can assemble nucleosomes in vitro, it was asked whether the absence of CHD1 affects histone incorporation into the male pronucleus. Embryos from wild-type or Chd1-null females were stained with an antibody against histone H3. In wild-type embryos, uniform staining was observed in both parental pronuclei. In contrast, in Chd1-null embryos only the female chromatin was brightly stained. The male pronucleus contained considerably less histone H3. These observations indicate that CHD1 is necessary for nucleosome assembly during sperm decondensation (Konev, 2007).

Sperm DNA does not replicate during decondensation, and histones are deposited by replication-independent assembly mechanisms, which involve the variant histone H3.3 but not canonical H3. It has been shown in Drosophila and mice that H3.3 is specifically present in the male pronucleus (Loppin, 2005; Torres-Padilla, 2006). The distribution of H3.3 was analyzed in embryos derived from Chd1-null females that carry a FLAG-tagged H3.3 transgene. In wild-type embryos, colocalization of the H3.3-FLAG signal was observed with male pronuclear DNA during migration and apposition. No H3.3-FLAG was detectable in the maternal pronucleus. In Chd1-null embryos, the male pronucleus showed altered H3.3-FLAG staining. The signal did not co-localize with the DNA but remained constrained to the nuclear periphery in a sacshaped pattern (Konev, 2007).

These findings suggest that in the earliest phases of Drosophila development CHD1 is essential for the incorporation of H3.3 and normal assembly of paternal chromatin. In contrast, CHD1 does not appear to affect the organization of maternal chromatin. It is concluded that CHD1 is required for replication-independent nucleosome assembly in the decondensing male pronucleus, but is dispensable for replication-coupled incorporation of H3 (Konev, 2007).

It has recently been shown that the sèsame (ssm) mutation of Drosophila histone chaperone HIRA causes the development of haploid embryos and abolished H3.3 deposition into the male pronucleus. Chd1 and ssm mutants, however, differ profoundly in the manifestation of this phenotype. In ssm embryos, H3.3 is absent from the male pronucleus. In contrast, in Chd1-null embryos, H3.3 delivery to the male pronucleus appears to be unaffected. Thus, these observations allow the roles of CHD1 and HIRA to be mechanistically discerned. Whereas HIRA is essential for histone delivery to the sites of nucleosome assembly, CHD1 directly facilitates histone deposition. These findings are consistent with observations in vitro that histone chaperones either do not assemble nucleosomes or assemble them at a greatly reduced rate in the absence of ATP-utilizing factors. These data provide evidence that histone deposition in vivo also transpires through an ATP-dependent mechanism (Konev, 2007).

CHD1 has been implicated in transcription elongation-related chromatin remodeling (Sims, 2004). This study demonstrates that CHD1 functions in nucleosome assembly in the early Drosophila embryo, which is transcriptionally silent. The biological role of CHD1, therefore, is not confined to transcription-related processes. The Schizosaccharomyces pombe homolog of CHD1, Hrp1, has been shown to function in loading of the centromere-specific H3 variant CENP-A (Walfridsson, 2005). Similarly to H3.3, incorporation of CENP-A into chromatin is not restricted to S phase. Therefore, CHD1 may have a general role in replication-independent nucleosome assembly (Konev, 2007).

Sperm decondensation involves not only histone incorporation, but also eviction of protamines. To discern whether CHD1 has a role in this process, the fate of protamine B (Mst35Bb) was analyzed in Chd1-null embryos. Although GFP-tagged Mst35Bb was detected in the sperm head immediately upon fertilization, no Mst35Bb-GFP signal was detected in the male pronucleus. Thus, like HIRA, CHD1 is dispensable for protamine removal. This study has shown that the male pronucleus in Chd1-null embryos contains very low amounts of histones, yet the DNA is not packaged with protamines. It remains an open question whether other DNA-protein complexes exist in the male pronucleus (Konev, 2007).

Drosophila eggs contain stores of both known chromatin assembly factors CHD1 and ISWI. Nevertheless, ISWI is unable to substitute for CHD1 in the deposition of H3.3. To examine whether CHD1 and ISWI differ in their ability to interact with the H3.3 chaperone HIRA, coimmunoprecipitation experiments were performed with extracts from embryos expressing FLAG-HIRA. CHD1 signal was readily detected in FLAG-specific immunoprecipitates, whereas ISWI did not coimmunoprecipitate with HIRA. Thus, a fraction of CHD1, but not ISWI, physically associates with HIRA. This property of CHD1 may account for its unique function in the H3.3 deposition process (Konev, 2007).

A subpopulation of Chd1 mutant haploid embryos survives beyond apposition stage. Therefore, it was asked whether H3.3 deposition is altered in Chd1 mutant embryos during later developmental stages. In wild-type nuclei, the H3.3-FLAG signal originating from the male pronucleus becomes undetectable after 2 to 3 divisions. Most maternal H3.3 remains distributed diffusely throughout the syncytium. After cycle 11 (roughly correlating with the onset of zygotic transcription) H3.3-FLAG is redistributed into the nuclei, where it colocalizes with the DNA. In contrast, incorporation of H3.3 into Chd1 mutant nuclei was impaired. H3.3-FLAG produced a speckled staining with numerous bright dots that poorly overlapped with the maxima of DNA staining. It is important to note that, in the ssm (HIRA) mutant, H3.3 incorporation defects in tissues or developmental stages other than the apposition stage were not observed. This result is consistent with the idea that misincorporation of H3.3 in Chd1 embryos is a direct effect of CHD1 deletion rather than a consequence of haploid development. It is also concludes that CHD1 functions in H3.3 deposition during later stages of embryonic development, possibly in a HIRA-independent fashion (Konev, 2007).

This study provides evidence that ATP-dependent mechanisms are used for histone deposition during chromatin assembly in vivo. Thus, molecular motor proteins, such as CHD1, function not only in remodeling of existing nucleosomes but also in de novo nucleosome assembly from DNA and histones. Finally, this work identifies CHD1 as a specific factor in the assembly of nucleosomes that contain variant histone H3.3 (Konev, 2007).

Investigations of CHD1 function in transcription and development of Drosophila melanogaster

Examination of chd1 mutant alleles reveals that the CHD1 chromatin-remodeling factor is important for wing development and fertility. While CHD1 colocalizes with elongating RNA polymerase II (Pol II) on polytene chromosomes, elongating Pol II can persist on chromatin in the absence of CHD1. These results clarify the roles of chromatin remodelers in transcription and provide novel insights into CHD1 function (McDaniel, 2008).

To investigate the function of CHD1, two deletion alleles (chd14 and chd15) were generated by imprecise excision of an EP element inserted into position -2 of the chd1 promoter. No differences were observed in the behavior of the two alleles. chd14 and chd15 homozygous and chd14/chd15 heteroallelic individuals display phenotypes that are less severe than those seen in hemizygous mutants. These genetic data would suggest that chd14 and chd15 are hypomorphic alleles. The possibility was investigated that the two chd1 alleles could generate proteins with N-terminal truncations. Given the location of the earliest in-frame start codon, it was predicted that both chd14 and chd15 alleles would generate a 166-kDa protein. However, Western blot analysis of heterozygous embryo extracts failed to detect an N-terminal truncated protein. While it is formally possible that chd14 and chd15 express an unstable protein that is not detectable by Western blot, it is proposed that the alleles are protein nulls, and it is concluded that chd1 is not an essential gene. chd14 and chd15 homozygous, heteroallelic, and hemizygous mutant individuals are viable, although they display a 1- to 2-day developmental delay, and some marker combinations reduce viability of chd1 mutants. Given these genetic data, it is proposed that 1 of the other 18 genes uncovered by Df(2L)Exel7014 may dominantly enhance chd1 mutant phenotypes. For example, okra (the RAD54 homolog, a SNF2-like helicase) is located 20 kb away from chd1. okra mutant phenotypes include female sterility, one of the chd1 phenotypes that may be dominantly enhanced by the deficiency (McDaniel, 2008).

The distribution of chd1 mRNA was examined by in situ hybridization, and it was found that chd1 is broadly expressed throughout embryogenesis and in imaginal discs. This broad expression pattern is similar to that of brm and kis and suggests that, like BRM and KIS, CHD1 could function globally to regulate transcription (McDaniel, 2008).

While chd1 is broadly expressed, specific phenotypes were observed in mutant animals, suggesting that CHD1 may function as a tissue-specific chromatin-remodeling factor. Wing margins in chd14 and chd15 homozygous, heteroallelic, and hemizygous mutant individuals displayed notching, with 3.8%-36% of heteroallelic individuals and 75%-94% of hemizygous individuals showing notched wing margins. The variability of the wing-notching phenotype was not correlated with developmental delay or viability. Individuals homozygous for the precise excision did not show cut-in wing margins, indicating that the phenotype is due to lack of CHD1. This specific wing phenotype is not observed in animals lacking BRM or KIS and suggests that genes critical for wing-margin formation are especially sensitive to loss of CHD1 (McDaniel, 2008).

chd14 and chd15 homozygous, heteroallelic, and hemizygous males are sterile; CHD1 is therefore required for male fertility. chd1 mutant males displayed normal mating behaviors, and there were no obvious defects in the general morphology of testes of chd14/chd15 mutant males. While mutant males produced zero progeny, control males produced an average of 101 progeny per single male under the same conditions. CHD1 is also important for female fertility; chd1 mutant females produced few offspring (by comparison, a single wild-type female produced 108 progeny under the same conditions). It is proposed that the majority of the fertilized eggs cannot develop due to an inability to repackage the sperm pronuclear DNA into H3.3-containing chromatin. Examination of egg chambers from hemizygous mutant females [chd14/Df(2L)Exel7014] reveals that, while eggs are occasionally formed, oogenesis often fails at stage 8, the start of yolk production. An 8.5-kb genomic chd1 transgene (-456 to + 8019 relative to the chd1 start site) fully rescued all mutant phenotypes: male sterility, reduced female fertility, and notching of wing margins (McDaniel, 2008).

CHD1 protein persisted into early third instar larval stages in chd1 mutant larvae (likely a consequence of maternal perdurance). However, in contrast to CHD1 levels in control chromosomes, CHD1 levels were greatly reduced on chromosomes derived from chd1 mutant individuals in mid-to-late third instar larval development, providing an excellent system in which to dissect the role of CHD1 on chromosomes. Unlike the chromatin-remodeling factor ISWI, CHD1 is not essential for global chromosome structure (McDaniel, 2008).

Given the observation that chromosomes lacking KIS are not bound by CHD1, it is proposed that CHD1 is functionally downstream of Kismet. Alternatively, Kismet and CHD1 could be mutually dependent upon each other for chromosome binding. To distinguish between these two possibilities, it was asked whether KIS was found on chromosomes lacking CHD1. KIS was present at wild-type levels in chd1 mutant individuals, indicating that, while CHD1 localization depends upon KIS, KIS localization is not dependent upon CHD1 (McDaniel, 2008).

To test the hypothesis that CHD1 is required for the continued elongation of Pol II, the levels were examined of Pol IIoser2 on chromosomes derived from mutant chd1 individuals. Chromosomes were observed that lacked CHD1 protein but still possessed normal levels of Pol IIoser2. An antibody recognizing all the forms of Pol II similarly showed normal levels and distribution of total Pol II on chd1 mutant chromosomes. Given that chromosomes were observed lacking observable CHD1 protein that retain elongating Pol II, it is concluded that while CHD1 colocalizes with elongating Pol II, it is not absolutely required for the association of Pol II with chromatin. It is noted that lower levels of CHD1 protein on polytenes from chd1 mutant larvae can correlate with reduced levels of Pol IIoser2. Reduced levels of transcription may be a secondary affect of healthy larvae; alternatively, loss of CHD1 may affect subsequent rounds of transcription, leading to a reduction of Pol IIoser2 levels over time (McDaniel, 2008).

In conclusion, two chd1 null alleles were generated that have revealed roles for CHD1 in male fertility, oogenesis, and wing development. While it is formally possible that transcription is affected in a subtle way, these experiments lead to the conclusion that CHD1 is not absolutely required for the association of elongating Pol II on chromosomes. However, since Pol IIoser2 levels can occasionally be reduced on chromosomes derived from chd1 mutant larvae, CHD1 activity may indirectly impact transcriptional elongation. Yeast Chd1 is implicated in Pol II elongation, termination, and the response to transcriptional stress. Drosophila CHD1 participates in nucleosome assembly in vitro and has been found to repackage the sperm pronuclear DNA into H3.3-containing chromatin in vivo. Whether CHD1 functions to facilitate chromatin disassembly or reassembly during transcription remains to be determined (McDaniel, 2008).

Cdk9 is an essential kinase in Drosophila that is required for heat shock gene expression, histone methylation and elongation factor recruitment: Chromo domain-methylated histone interaction plays a dominant role in targeting CHD1 to active chromatin

Phosphorylation of the large RNA Polymerase II subunit C-terminal domain (CTD) is believed to be important in promoter clearance and for recruiting protein factors that function in messenger RNA synthesis and processing. P-TEFb is a protein kinase that targets the (CTD). The goal of this study was to identify chromatin modifications and associations that require P-TEFb activity in vivo. The catalytic subunit of P-TEFb, Cdk9, was knocked down in Drosophila using RNA interference. Cdk9 knockdown flies die during metamorphosis. Phosphorylation at serine 2 and serine 5 of the CTD heptad repeat were both dramatically reduced in knockdown larvae. Hsp 70 mRNA induction by heat shock was attenuated in Cdk9 knockdown larvae. Both mono- and trimethylation of histone H3 at lysine 4 were dramatically reduced, suggesting a link between CTD phosphorylation and histone methylation in transcribed chromatin in vivo. Levels of the chromo helicase protein CHD1 were reduced in Cdk9 knockdown chromosomes, suggesting that CHD1 is targeted to chromosomes through P-TEFb-dependent histone methylation. Dimethylation of histone H3 at lysine 36 was significantly reduced in knockdown larvae, implicating CTD phosphorylation in the regulation of this chromatin modification. Binding of the RNA Polymerase II elongation factor ELL was reduced in knockdown chromosomes, suggesting that ELL is recruited to active polymerase via CTD phosphorylation (Eissenberg, 2006).

Cdk9, the catalytic subunit of P-TEFb, is highly conserved among eukaryotes. The yeast kinases Ctk1 and Bur1 are both homologs of Cdk9, and both are CTD kinases in Drosophila, although loss of Bur1 has no effect on CTD phosphorylation yeast. Bur1 is essential but Ctk1 is not (Eissenberg, 2006).

RNAi knockdown of Cdk9 in transgenic flies results in lethality at the pupal stage. This is considerably later than the embryonic lethality reported for C. elegans RNAi knockdown of Cdk9. While this difference could reflect differences in the requirements for Cdk9 in these organisms, it is more likely that differences in timing or efficiency of RNAi, Cdk9 protein turnover and/or maternal Cdk9 loading accounts for the much later lethality in knockdown flies. Nevertheless, these results confirm and extend the finding that P-TEFb is essential in metazoan development (Eissenberg, 2006).

In contrast, Cdk9 homologs in fission yeast and Neurospora are not essential. Since CTD phosphorylation has been linked to promoter clearance, pre-mRNA processing and chromatin modification, it is not possible to say what aspect of P-TEFb activity is essential in metazoa. RNAi knockdown of the Drosophila Cdk9 in cultured cells causes arrest of the cell cycle at the G1-S transition, implicating this kinase in cell cycle control. It is unlikely that cell cycle arrest is causing the lethality in the knockdown flies, since cell cycle mutations in Drosophila generally are associated with reduced or missing imaginal discs, and the discs in Cdk9 knockdown larvae appear overtly normal. The finding that Hsp70 transcripts are reduced in Cdk9 knockdown larvae is consistent with the reduced Hsp70 transcription previously reported in Cdk9 RNAi cultured cells. Hsp 70 is not essential in Drosophila, but the effects on Hsp70 suggest that defects in gene expression could underlie the essential requirement for Cdk9 in Drosophila development (Eissenberg, 2006).

Cdk9 knock-down flies show dramatic reductions in both serine 2 and serine 5 phosphorylation. In contrast, flavopiridol treatment of cultured cells has been found to selectively reduce serine 2 phosphorylation. The significance of this difference is unclear, but could reflect differences in experimental protocol. For example, flavopiridol treatments were limited to 15-20 min, while RNAi knockdown third instar larvae are subject to knockdown conditions for several days before assay. Longer periods of Cdk9 inactivation may be required for reduction in serine 5 phosphorylation. Alternatively, it is possible that knockdown of Cdk9 protein levels results in inhibition of TFIIH, the other known CTD kinase. Regardless of the mechanism, the RNAi knockdown clearly results in reduced phosphorylation of the CTD, enabling a test of the consequences of loss of CTD phosphorylation on chromatin modification and recruitment of RNA Polymerase II-associated factors (Eissenberg, 2006).

Loss of CTD phosphorylation in Cdk9 knockdown larvae is associated with reduced binding of the RNA Polymerase II elongation factor ELL genome-wide. ELL is broadly co-localized with phosphorylated RNA Polymerase II on polytene chromosomes, and is rapidly recruited to heat shock loci after a brief heat shock. These results suggest that the efficient recruitment of ELL to transcribed loci requires CTD phosphorylation. Whether this reflects a direct interaction of ELL with the CTD is unknown (Eissenberg, 2006).

Despite the fact that Elongin A affects the same kinetic parameter in RNA Polymerase II catalysis as ELL, Elongin A binding is not reduced by loss of CTD phosphorylation. As with ELL, the nature of Elongin A binding to RNA Polymerase II is unknown, but these observations suggest their binding can be distinguished by sensitivity to the phosphorylation state of the CTD. Since no increase of Elongin A was observed under conditions of reduced ELL binding, it seems unlikely that ELL and Elongin A compete for RNA Polymerase II binding (Eissenberg, 2006).

Spt4 and Spt5 are subunits of DSIF, which is implicated in the regulation of RNA Polymerase II elongation. Previous work suggested that reduced serine 2 phosphorylation of the RNA Polymerase II CTD has no effect on Spt5 recruitment to a heat shock gene in cultured cells (Ni, 2004). In Cdk9 knockdown flies, in which both serine 2 and 5 phosphorylation are reduced, the chromosomal distribution of Spt5 is unchanged genome-wide. This is consistent with previous reports that Spt5 interacts with both phosphorylated and unphosphorylated RNA Polymerase II (Wen, 1999; Lindstrom, 2001; Lindstrom, 2003; Eissenberg, 2006 and references therein).

The chromo domain motif is a binding site for methylated histone tails. The role of the CHD1 chromo domain in methylated histone binding is controversial. However, recent structural data determined that the double chromo domain of mammalian CHD1 binds methylated H3K4 in vitro (Flanagan, 2005). This study shows that Cdk9 knockdown leads to a loss of chromosomal CHD1. This observation is most easily interpreted as the result of loss of H3K4 methylation that also occurs in Cdk9 knockdown chromosomes. Thus, the finding reported in this study lends support to the in vitro binding data and strongly suggests that the chromo domain-methylated histone interaction plays a dominant role in targeting CHD1 to active chromatin in vivo (Eissenberg, 2006).

The observation that both H3K4 and H3K36 methylation are significantly reduced in Cdk9 knockdown chromosomes suggests a linkage between phosphorylation of the CTD and histone methylation at transcribed genes. In this respect, Cdk9 subsumes activities found in yeast Bur1/Bur2 and yeast Ctk1. Since no significant difference was observed in ASH1 protein levels on Cdk9 knockdown chromosomes, a model is favored in which Cdk9-dependent RNA Polymerase II elongation plays a mechanistic role in H3 tail methylation. In this model, RNA Polymerase II passage destabilizes histone-DNA contacts, making the histones better substrates for efficient methylation. Reduced CTD phosphorylation would lead to reduced rates of RNA Polymerase II transcription genome-wide, resulting in reduced efficiency of histone tail methylation. While the mechanism connecting CTD phosphorylation to RNA Polymerase II elongation rate is likely to be complex in vivo, the observation that reduced CTD phosphorylation is associated with reduced dELL binding suggests that loss of dELL association could be a contributing factor (Eissenberg, 2006).

Mutation in Ash1 in Drosophila results in loss of all detectable H3K4 methylation, but has no effect on H3K36 methylation. This is consistent with independent mechanisms for these two chromatin modifications. A Polymerase II passage model provides a simple mechanism to account for similar effects on both modifications based on substrate availability (Eissenberg, 2006).

The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are reduced on polytene chromosomes from kis mutants

The Drosophila trithorax group gene kismet (kis) was identified in a screen for extragenic suppressors of Polycomb 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).

Double chromodomains cooperate to recognize the methylated histone H3 tail

Chromodomains are modules implicated in the recognition of lysine-methylated histone tails and nucleic acids. CHD (for chromo-ATPase/helicase-DNA-binding) proteins regulate ATP-dependent nucleosome assembly and mobilization through their conserved double chromodomains and SWI2/SNF2 helicase/ATPase domain. The Drosophila CHD1 localizes to the interbands and puffs of the polytene chromosomes, which are classic sites of transcriptional activity (Stokes, 1996). Other CHD isoforms (CHD3/4 or Mi-2) are important for nucleosome remodelling in histone deacetylase complexes. Deletion of chromodomains impairs nucleosome binding and remodelling by CHD proteins. This study describes the structure of the tandem arrangement of the human CHD1 chromodomains, and its interactions with histone tails. Unlike HP1 and Polycomb proteins that use single chromodomains to bind to their respective methylated histone H3 tails, the two chromodomains of CHD1 cooperate to interact with one methylated H3 tail. The human CHD1 double chromodomains target the lysine 4-methylated histone H3 tail (H3K4me), a hallmark of active chromatin. Methylammonium recognition involves two aromatic residues, not the three-residue aromatic cage used by chromodomains of HP1 and Polycomb proteins. Furthermore, unique inserts within chromodomain 1 of CHD1 block the expected site of H3 tail binding seen in HP1 and Polycomb, instead directing H3 binding to a groove at the inter-chromodomain junction (Flanagan, 2005).

Double chromodomains occur in CHD proteins, which are ATP-dependent chromatin remodeling factors implicated in RNA polymerase II transcription regulation. Biochemical studies suggest important differences in the histone H3 tail binding of different CHD chromodomains. In human and Drosophila, CHD1 double chromodomains bind lysine 4-methylated histone H3 tail, which is a hallmark of transcriptionally active chromatin in all eukaryotes. This study presents the crystal structure of the yeast CHD1 double chromodomains, and pinpoints their differences with that of the human CHD1 double chromodomains. The most conserved residues in these double chromodomains are the two chromoboxes that orient adjacently. Only a subset of CHD chromoboxes can form an aromatic cage for methyllysine binding, and methyllysine binding requires correctly oriented inserts. These factors preclude yeast CHD1 double chromodomains from interacting with the histone H3 tail. Despite great sequence similarity between the human CHD1 and CHD2 chromodomains, variation within an insert likely prevents CHD2 double chromodomains from binding lysine 4-methylated histone H3 tail as efficiently as in CHD1. By using the available structural and biochemical data this study highlights the evolutionary specialization of CHD double chromodomains, and provide insights about their targeting capacities (Flanagan, 2007).

CHD1 Contributes to intestinal resistance against infection by P. aeruginosa in Drosophila melanogaster

Drosophila SNF2-type ATPase CHD1 catalyzes the assembly and remodeling of nucleosomal arrays in vitro and is involved in H3.3 incorporation in vivo during early embryo development. Evidence for a role as transcriptional regulator comes from its colocalization with elongating RNA polymerase II as well as from studies of fly Hsp70 transcription. This study used microarray analysis to identify target genes of CHD1. A fraction of genes that were misregulated in Chd1 mutants were functionally linked to Drosophila immune and stress response. Infection experiments using different microbial species revealed defects in host defense in Chd1-deficient adults upon oral infection with P. aeruginosa but not upon septic injury, suggesting a so far unrecognized role for CHD1 in intestinal immunity. Further molecular analysis showed that gut-specific transcription of antimicrobial peptide genes was overactivated in the absence of infection in Chd1 mutant flies. Moreover, microbial colonization of the intestine was elevated in Chd1 mutants and oral infection resulted in strong enrichment of bacteria in the body cavity indicating increased microbial passage across intestinal epithelia. However, no enhanced epithelial damage or alterations of the intestinal stem cell population were detected. Collectively, these data provide evidence that intestinal resistance against infection by P. aeruginosa in Drosophila is linked to maintaining proper balance of gut-microbe interactions and that the chromatin remodeler CHD1 is involved in regulating this aspect (Sebald, 2012).

Different CHD chromatin remodelers are required for expression of distinct gene sets and specific stages during development of Dictyostelium discoideum

Control of chromatin structure is crucial for multicellular development and regulation of cell differentiation. The CHD (chromodomain-helicase-DNA binding) protein family is one of the major ATP-dependent, chromatin remodeling factors that regulate nucleosome positioning and access of transcription factors and RNA polymerase to the eukaryotic genome. There are three mammalian CHD subfamilies and their impaired functions are associated with several human diseases. This study identified three CHD orthologs (ChdA, ChdB and ChdC) in Dictyostelium discoideum. These CHDs are expressed throughout development, but with unique patterns. Null mutants lacking each CHD have distinct phenotypes that reflect their expression patterns and suggest functional specificity. Accordingly, using genome-wide (RNA-seq) transcriptome profiling for each null strain, it was shown that the different CHDs regulate distinct gene sets during both growth and development. ChdC is an apparent ortholog of the mammalian Class III CHD group that is associated with the human CHARGE syndrome, and GO analyses of aberrant gene expression in chdC nulls suggest defects in both cell-autonomous and non-autonomous signaling, which have been confirmed through analyses of chdC nulls developed in pure populations or with low levels of wild-type cells. This study provides novel insight into the broad function of CHDs in the regulation development and disease, through chromatin-mediated changes in directed gene expression (Platt, 2013).


Search PubMed for articles about Drosophila Chd1

Belotserkovskaya, R., et al. (2003). FACT facilitates transcription-dependent nucleosome alteration. Science 301: 1090-1093. PubMed ID: 12934006

Delmas, V., Stokes, D. G. and Perry, R. P. (1993). A mammalian DNA-binding protein that contains a chromodomain and an SNF2/SWI2-like helicase domain. Proc. Natl. Acad. Sci. 90: 2414-2418. PubMed ID: 8460153

Eissenberg, J. C., Shilatifard, A., Dorokhov, N., Michener, D. E.. (2006). Cdk9 is an essential kinase in Drosophila that is required for heat shock gene expression, histone methylation and elongation factor recruitment. Mol. Genet. Genomics. 277(2): 101-14. PubMed ID: 17001490

Flanagan, J. F., Mi, L.-Z., Chruszcz, M., Cymborowski, M., Clines, K. L., Kim, Y., Minor, W., Rastinejad, F. and Khorasanizadeh, S. (2005). Double chromodomains cooperate to recognize the methylated histone H3 tail. Nature 438: 1181-1185. PubMed ID: 16372014

Flanagan, J. F., et al. (2007). Molecular implications of evolutionary differences in CHD double chromodomains. J. Mol. Biol. 369(2): 334-42. PubMed ID: 17433364

Fyodorov, D. V., Blower, M. D., Karpen, G. H. and Kadonaga, J. T. (2004). Acf1 confers unique activities to ACF/CHRAC and promotes the formation rather than disruption of chromatin in vivo. Genes Dev. 18: 170-183. PubMed ID: 14752009

Konev, A. Y., et al. (2007). CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo. Science 317(5841): 1087-90. PubMed ID: 17717186

Ni, Z., Schwartz, B. E., Werner, J., Suarez, R.-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. PubMed ID: 147313946

Lindstrom, D. L. and Hartzog, G. A. (2001). Genetic interactions of Spt4-Spt5 and TFIIS with the RNA polymerase II CTD and CTD modifying enzymes in Saccharomyces cerevisiae. Genetics 159: 487-497. PubMed ID: 11606527

Lindstrom, D. L., et al. (2003). Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins. Mol. Cell Biol. 23: 1368-1378. PubMed ID: 12556496

Loppin, B., Bonnefoy, E., Anselme, C., Laurencon, A., Karr, T. L. and Couble, P. (2005). The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus. Nature 437(7063): 1386-90. PubMed ID: 16251970

Lusser, A., Urwin, D. L. and Kadonaga, J. T. (2005). Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly. Nat. Struct. Mol. Biol. 12(2): 160-6. PubMed ID: 15643425

McDaniel, I. E., Lee, J. M., Berger, M. S., Hanagami, C. K. and Armstrong, J. A. (2008). Investigations of CHD1 function in transcription and development of Drosophila melanogaster. Genetics 178(1): 583-7. PubMed ID: 18202396

Platt, J. L., Rogers, B. J., Rogers, K. C., Harwood, A. J. and Kimmel, A. R. (2013). Different CHD chromatin remodelers are required for expression of distinct gene sets and specific stages during development of Dictyostelium discoideum. Development 140: 4926-4936. PubMed ID: 24301467

Robinson, K. M. and Schultz, M. C. (2003). Replication-independent assembly of nucleosome arrays in a novel yeast chromatin reconstitution system involves antisilencing factor Asf1p and chromodomain protein Chd1p. Mol. Cell. Biol. 23: 7937-7946. PubMed ID: 14585955

Sebald, J., et al. (2012). CHD1 Contributes to intestinal resistance against infection by P. aeruginosa in Drosophila melanogaster. PLoS One. 7(8):e43144. PubMed ID: 22912810

Sims, R. J., Belotserkovskaya, R. and Reinberg, D. (2004). Elongation by RNA polymerase II: the short and long of it. Genes Dev. 18: 2437-68. PubMed ID: 15489290

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. PubMed ID: 15728673

Stokes, D. G. and Perry, R. P. (1995). DNA-binding and chromatin localization properties of CHD1. Mol. Cell. Biol. 15: 2745-2753. PubMed ID: 7739555

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. 93(14): 7137-42. PubMed ID: 8692958

Tagami, H., Ray-Gallet, E., Almouzni, G. and Nakatani, Y. (2004). Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 116: 51-61. PubMed ID: 14718166

Torres-Padilla, M. E., et al. (2006). Dynamic distribution of the replacement histone variant H3.3 in the mouse oocyte and preimplantation embryos. Int. J. Dev. Biol. 50: 455-61. PubMed ID: 16586346

Tran, H. G., Steger, D. J., Iyer, V. R. and Johnson, A. D. (2000). The chromo domain protein chd1p from budding yeast is an ATP-dependent chromatin-modifying factor. EMBO J. 19: 2323-2331. PubMed ID: 10811623

Tsukiyama, T., Palmer, J., Landel, C. C., Shiloach, J. and Wu, C. (1999). Characterization of the imitation switch subfamily of ATP-dependent chromatin-remodeling factors in Saccharomyces cerevisiae. Genes Dev. 13: 686-697. PubMed ID: 10090725

Walfridsson, J., et al. (2005). The CHD remodeling factor Hrp1 stimulates CENP-A loading to centromeres. Nucleic Acids Res. 33: 2868-79. PubMed ID: 15908586

Wen, Y. and Shatkin, A. J. (1999). Transcription elongation factor hSPT5 stimulates mRNA capping. Genes Dev 13: 1774-1779. PubMed ID: 10421630

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date revised: 10 February 2014

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