The chromodomains of CHD1 are critical for enzymatic activity but less important for chromatin localization

Abstract

The molecular motor protein CHD1 has been implicated in the regulation of transcription and in the transcription-independent genome-wide incorporation of H3.3 into paternal chromatin in Drosophila melanogaster. A key feature of CHD1 is the presence of two chromodomains, which can bind to histone H3 methylated at lysine 4 and thus might serve to recruit and/or maintain CHD1 at the chromatin. Here, we describe genetic and biochemical approaches to the study of the Drosophila CHD1 chromodomains. We found that overall localization of CHD1 on polytene chromosomes does not appreciably change in chromodomain-mutant flies. In contrast, the chromodomains are important for transcription-independent activities of CHD1 during early embryonic development as well as for transcriptional regulation of several heat shock genes. However, neither CHD1 nor its chromodomains are needed for RNA polymerase II localization and H3K4 methylation but loss of CHD1 decreases transcription-induced histone eviction at the Hsp70 gene in vivo. Chromodomain mutations negatively affect the chromatin assembly activities of CHD1 in vitro, and they appear to be involved in linking the ATP-dependent motor to the chromatin assembly function of CHD1.

Figure 1.

Chromatin localization of CHD1 appears similar in wild-type and chromodomain-mutant larvae. Polytene chromosomes were isolated from wild-type (A and D), Chd1^CD (B and E) and Chd1^WT larvae (C and F) and stained with antibodies against CHD1 (green) and the elongating form of RNA polymerase II (Pol II_ser2; red). Merge (A–C) and split (D–F) images indicate extensive colocalization between CHD1 and Pol II_ser2 in both wild-type and chromodomain mutant chromosomes.

Figure 2.

The Chd1^CD transgene partially rescues viability and embryonic development of Chd1-deficient flies. (A) Transgenes were recombined onto the chromosomes bearing either the Chd1¹ or the Chd1² mutant allele or the deficiency Exel7014, respectively. Flies were mated and heterozygous combinations of either mutant allele with the deficiency chromosome were counted and normalized against the expected number of progeny. Column color indicates the genetic background. (B) Embryos (n = 100) laid by flies expressing the indicated transgene in a Chd1-deficient genetic background (indicated by column color) were transferred to apple juice agar plates and hatching was monitored after 24 h incubation. (C) Embryos laid by Chd1-deficient females (Chd1²/Exel7014) bearing the indicated transgenes were collected at 0–2 h after egg deposition and aged at 25°C for another 2 h (Chd1^WT, n = 410; Chd1^CD, n = 306; Chd1 ⁻, n = 545). Developmental distribution of the embryos was monitored by DAPI staining.

Figure 3.

HS-induced transcription of Hsp70, Hsp83 and Hsp22 genes is decreased in Chd1-deficient and Chd1^CD-rescued larvae. Larvae bearing the indicated transgenes in a Chd1²/Exel7014 genetic background and wild-type larvae were subjected to the indicated periods of HS before RNA was prepared and expression of the HS genes was analyzed by real-time PCR. All values were normalized against the non-HS (0) sample of the respective fly strain. Error bars indicate standard deviations of three independent experiments. (A) Hsp70, (B) Hsp83 and (C) Hsp22.

Figure 4.

Pol II _ser2 levels at HS puffs 87A/C and overall H3K4 methylation appear to be unaltered in Chd1 mutant larvae. (A) Polytene chromosomes were isolated from wild-type, Chd1^WT, Chd1-deficient and Chd1^CD-rescued larvae after 20 min of heat stress and stained with antibodies against CHD1 (green) and Pol II_ser2 (red). DNA is visualized in blue. Pol II_ser2 is readily redistributed to the Hsp70 puffs in the Chd1-deficient and Chd1^CD-rescued larvae. Also, there are no apparent differences between the recruitment of chromodomain-mutated CHD1 and wild-type CHD1. (B) H3K4 methylation is not affected in Chd1-mutant larvae. Polytene chromosomes were isolated from Chd1^WT-rescued (left), Chd1-deficient (middle) and Chd1^CD-rescued (right) larvae and stained with antibodies against H3K4me3 (green). DNA is visualized in blue. (C) Western blot analysis of total protein extracts from Chd1^WT, Chd1^CD and Chd1-deficient third instar larvae reveals similar levels of H3K4me3 in all three strains. Actin was probed to control for equal loading.

Figure 5.

Mutations in the chromodomains affect nucleosome assembly activities of CHD1. (A) Diagram of CHD1 mutant proteins. ATPase domain (boxes), DNA binding domain (ovals), chromodomains (circles). The positions of tryptophan to alanine mutations are indicated. (B) Mutant proteins were synthesized in Sf9 cells via the baculovirus expression system. Flag-tagged proteins were purified by Flag-affinity chromatography, equal amounts were separated on a 6% SDS–PAGE and visualized by Coommassie staining. (C) DNA supercoiling analysis of nucleosome assembly products. Chromatin assembly reactions were carried out with the indicated mutant proteins or without CHD1 in the absence or presence of ATP. Reactions were terminated after 90 min and reaction products were analyzed by agarose gelelectrophoresis of deproteinized samples. N, nicked circular DNA; R, relaxed circular DNA; SC, supercoiled DNA. (D) Quantification of nucleosome assembly activity. The percentage of DNA supercoiling was determined as described in ‘Materials and Methods’ section. The amount of signal obtained in the absence of CHD1 (none in C) was subtracted as background. The resulting data were normalized against those obtained with wild-type CHD1. Error bars denote standard deviations of three independent experiments.

Figure 6.

Effects of Chd1 mutation on chromatin structure. (A) Chd1 mutations do not perturb overall chromatin architecture. Chromatin was prepared from Chd1 deficient larvae and larvae expressing either the Chd1^WT or the Chd1^CD transgene and subjected to digestion with micrococcal nuclease. Deproteinized reaction products were separated on a 1% agarose gel and visualized by staining with ethidium bromide. M, 123 bp ladder (Invitrogen). (B) Chd1 deletion causes decreased histone H3 loss at the activated Hsp70 locus. ChIP analysis with antibodies against H3 was performed on chromatin isolated from Chd1^WT, Chd1^CD and Chd1⁻ larvae that were either not heat shocked (0) or exposed to 30 min of heat treatment. Immunoprecipitated material was amplified by RT–PCR and expressed relative to the input material. Black columns represent an amplicon between bp +334 and +423 of the Hsp70 gene, white columns correspond to an intergenic region. Histone H3 occupancy differences upon HS at the Hsp70 locus are highly significant between the Chd1^WT and Chd1⁻ lines (P = 0.003), but statistically not significant between the Chd1^WT and Chd1^CD lines (P = 0.197).