HP1a recruitment to promoters is independent of H3K9 methylation in Drosophila melanogaster

Abstract

Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila melanogaster three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1, and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, we have examined correlations between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. We show here that Su(var)3-9 controls H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. Our results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.

Figure 1. HP1a profiles in HKMT mutants.

(A) HP1a binding profiles for the entire chromosome arm 2L and the 4^th chromosome in salivary gland tissue from wild type and null Su(var)3-9, Setdb1 and G9a mutants. p2L and 2L:31 indicate the centromere proximal heterochromatin in chromosome 2L and the HP1a-enriched cytogenetic region in the middle of the chromosome arm, respectively. Numbers along the x-axis denote chromosomal positions along the chromosomes in Mb. The y-axis shows the ChIP enrichment in log₂ ratios. (B) Immunostaining of polytene chromosomes from wild type and Su(var)3-9, Setdb1 and G9a mutants with DAPI (blue) and HP1a (yellow). The fourth chromosomes are indicated by boxes. Note that HP1a is decreased on the 4^th chromosome in Setdb1 mutants and in the pericentromeric heterochromatin in Su(var)3-9 mutants. (C) Western blot showing total HP1a and tubulin from dissected salivary glands from wild type and Su(var)3-9, Setdb1 and G9a mutants.

Figure 2. H3K9 methylation profiles in HKMT mutants.

H3K9me3 (gray) and H3K9me2 (green) profiles for the entire chromosome arm 2L and the 4^th chromosome in salivary gland tissue from wild type and null Su(var)3-9, Setdb1 and G9a mutants. p2L and 2L:31 indicate the centromere proximal heterochromatin in chromosome 2L and the HP1a-enriched cytogenetic region in the middle of the chromosome arm, respectively. Numbers along the x-axis denote chromosomal positions along the chromosomes in Mb. The y-axis shows the ChIP enrichment in log₂ ratios. Note that both H3K9me2 and H3K9me3 are strongly reduced in region 2L:31 and chromosome 4 in Setdb1 mutants, and strongly reduced in p2L in Su(var)3-9 mutants.

Figure 3. HP1a binds independently of H3K9me to promoters and spreads H3K9me-dependently into gene bodies.

(A) HP1a and H3K9me profiles in three illustrative regions of the 4^th chromosome in wild type and Setdb1 mutant backgrounds. Numbers along the x-axis denote chromosomal positions along the chromosomes in Mb. The y-axis shows the ChIP enrichment in log₂ ratios. Genes expressed from left to right and vice versa are shown above and below the horizontal lines, respectively. The HP1a methylation-independent promoter peaks are indicated by yellow boxes (B) Average metagene profiles of HP1a, H3K9me2 and H3K9me3 on the 4^th chromosome, region 2L:31,the pericentromeric regions and the euchromatic regions, based on eight enrichment values for each active gene in the respective regions (x-axis). The y-axis shows the ChIP enrichment in log₂ ratios. The first points (IG) of the curves show average values for the intergenic regions upstream of the designated promoters of the genes. The promoter (P) is defined as the 500 bp region upstream of the TSS. The gene bodies are divided into five bins (E1–E5) and the average enrichment in introns (IN) is indicated by the last point of each curve. The average profiles for wild type (solid black), Su(var)3-9 mutants (solid gray) and Setdb1 mutants (dashed gray) are shown.

Figure 4. HP1a-enriched promoters are A/T rich, bound by HP2 and DNase sensitive.

(A) Sequence motif over-represented in HP1a-enriched promoters. (B) Motif scores in promoters from chromosome 4 (red), 2L:31 (green), pericentromeric region (blue) and, as a control, chromosome 2L (pink). (C) A/T content in promoters. In (B) and (C) significant differences are indicated by * (Mann-Whitney U test, p<0.01). (D) Binary heat-map showing significant enrichment of indicated chromatin factors at promoters on chromosomes 2L and 4. Presence of a protein at a promoter is indicated by a gray line. Note the almost perfect correlation between HP1a and HP2. (E) Average metagene profiles showing the ChIP enrichments of HP1a in wild type (black), HP1a in Setdb1 mutants (dashed gray) and HP2 in wild type (gray) on the 4^th chromosome (log₂ ratios). (F) HP2 enrichment (log₂ ratios) plotted around HP1a peak centers of 34 methylation-independent HP1a peaks. (G) Average metagene profiles of DNase sensitivity for the 4^th chromosome (red) and chromosome 2L (blue). The y-axis shows the density of mapped DNaseI cleavages in a 150 bp sliding window .

Figure 5. HP1a binds independently of POF and Setdb1 to promoters.

(A) HP1a profiles in five illustrative regions of the 4^th chromosome in wild type, Pof and Setdb1 backgrounds. Numbers along the x-axis denote chromosomal positions along the chromosomes in Mb. The y-axis shows the ChIP enrichment in log₂ ratios. Genes expressed from left to right and vice versa are shown above and below the horizontal lines, respectively. The HP1a methylation-independent promoter peaks are indicated by yellow boxes. (B) Average metagene profiles of HP1a enrichments on the 4^th chromosome in wild type (solid black), Pof (solid gray) and Setdb1 (dashed gray) backgrounds.

Figure 6. HP1a binds two unexpressed genes in the middle of the 4^th chromosome independently of Setdb1 and POF.

HP1a enrichment profiles at a 90 kb region in the middle of the 4^th chromosome in wild type, Pof and Setdb1 backgrounds. Numbers along the x-axis denote chromosomal positions along the chromosomes in Mb. The y-axis shows the ChIP enrichment in log₂ ratios. Genes expressed from left to right and vice versa are shown above and below the horizontal lines, respectively. The peak within the yellow box corresponds to the CG1970 Ephrin promoter peak. The two genes CG1909 and onecut within the gray box are unexpressed genes that bind HP1a independently of POF and Setdb1.