HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle

Abstract

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the ‘used’ cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

Figure 1. HDAC8 is an SMC3 deacetylase

a, HeLa cells were transfected with HDAC8 siRNA (Lanes 1-4) or incubated with 25 μM PCI, the HDAC8-specific inhibitor (Lanes 5-9) for the indicated times. Total cell lysates were prepared and analyzed by immunoblotting using anti-SMC3-ac, SMC3 and HDAC8 antibodies. Numbers beneath SMC3-ac bands indicate quantification of SMC3-ac levels normalized to SMC3 levels and the 0 hour time point. b, Acetylated SMC3 was prepared by co-immunoprecipitation with SMC1A from HeLa cell chromatin extracts. Immunoprecipitates were incubated with recombinant purified HDAC8 or mutant HDAC8 protein at 30°C for 1 h and analyzed by immunoblotting as in (a). c, Unsynchronized HeLa cells transfected with HDAC8 siRNA for 48 h were fractionated into soluble and chromatin fractions and immunoblotted as in (a). d, HeLa cells were synchronized by double thymidine arrest and released into the presence or absence of 25 μM PCI for the indicated time and verified by FACS analysis for cell cycle progression. e, Cell extracts of the PCI-treated cells in (d) were fractionated into soluble and chromatin fractions and were analyzed by immunoblotting as in (a). Histone H3 Serine 10 phosphorylation (H3S10P) is a marker of prophase and the onset of mitosis.

Figure 2. Cohesin and SMC3-ac localization sites in control- and HDAC8 RNAi-treated HeLa cells

a, Example of ChIP-Seq data (Ensemble Gene position 1.68–2.10 Mb of human chromosome 12). ChIP-Seq data are shown in reads per million. Regions in which signals were significantly enriched (see Methods) are in red. Binding profiles for RAD21 and acetylated SMC3 in control and HDAC8 RNAi-treated HeLa cells are shown. b, Venn diagrams showing numbers and overlap of localization sites. c, Example of region-specific localization showing RAD21 and SMC3-ac binding sites in the MYC gene locus. Ensemble Gene position 128.728-128.773 Mb of human chromosome 8 is shown. d, Classification of RAD21 and SMC3-ac localization sites as a log ratio to background reads. Significance was calculated using a 2-sample test for equality of proportions; *, **, *** and -, indicate p<0.05, p<0.001, p<0.0001 and p>0.05, respectively. “Upstream” and “downstream” are defined as within 5kb from gene boundaries, respectively. Acetylated SMC3 preferentially localizes to the downstream end of genes.

Figure 3. HDAC8 mutations in CdLS

a, Facial features of individuals with HDAC8 mutations labeled with corresponding mutation and sex; ♂=male, ♀=female. b, Western blotting of protein from lymphoblastoid cell lines (LCL) and fibroblasts (Fib). Sex is indicated under Controls (1,2,3) or mutation designation. Primary antibody is indicated at the left. c, HDAC8 mutations disrupt deacetylase activity. Bar graphs demonstrate the effect of HDAC8 mutations on deacetylase specific activity (nmol substrate. μmol enzyme⁻¹. min⁻¹). All assays were performed in triplicate. Error bars indicate standard deviation. Enzymatic activity for all mutations is significantly less than wild type (p<0.05). d, Localization of HDAC8 mutations on the crystal structure (PDB accession code 3F06). Mutated residues are in red. e, Expression profile of HDAC8-mutant LCL is consistent with that seen in those with NIPBL mutations. The summed standard deviations of gene expression compared to NIPBL-mutant from control LCLs for 32 genes was determined for each sample. Box graphs demonstrate 25th, median and 75th percentiles of each group. Whiskers indicate minimum and maximum values. Note that for the HDAC8 male (p.G320R) the values are for biological replicate samples for a single cell line (denoted by *). Unpaired two-tailed t test significances are indicated.

Figure 4. Retention of RAD21-N and Sororin on cohesin in the absence of HDAC8

a, RAD21 separase cleavage sites. Vertical lines indicate cleavage sites. Gray region indicates the RAD21-N antibody epitope. b, SMC3, STAG1 and STAG2 were immunoprecipitated from soluble extracts from normal (C) and HDAC8-mutant (H) cell lines. Full-length RAD21 (FL), the cleaved N-terminal fragment of RAD21 (N), acetylated SMC3 (SMC3-ac), and STAG1/2 were analyzed by SDS-PAGE and immunoblotting. c, Experimental schema of panel (d). HeLa cells were synchronized by double thymidine arrest (DTA) and released (R) into the presence or absence of PCI. Seven hours after release, cells were treated with RO-3306 (RO) to arrest at the G2/M boundary. After removing RO-3306, cells were cultured in the presence or absence of nocodazole (Noc) or MG-132 (MG) to arrest in metaphase. Cells were harvested at indicated time points after release from RO-3306 arrest. d, Cell extracts were fractionated into soluble and chromatin-bound fractions. Full length RAD21 (FL), cleaved C-terminus fragment of RAD21 (C), cleaved N-terminus fragment of RAD21 (N), acetylated SMC3 (SMC3-ac), SMC3, phosphorylated Histone H3 at Serine 10 (H3S10-P), and α-Tubulin were analyzed by SDS-PAGE and immunoblotting. H3S10-P is used as a metaphase marker. e, Experimental schema of panel F and G. HeLa cells were synchronized and released and treated with RO-3306 (RO) as in D. After removing RO-3306, cells were cultured in the presence or absence of PCI and harvested at the indicated times. f, FACS analysis of HeLa cells in (g). g, Cell extracts were fractionated into soluble and chromatin-bound fractions. Levels of SMC3-ac, RAD21-N, full length RAD21 (FL), Sororin, WAPAL, and Cyclin B (a G2 marker) and SMC3 were analyzed by SDS-PAGE and immunoblotting.