Mutations in pericentrin cause Seckel syndrome with defective ATR-dependent DNA damage signaling

Abstract

Large brain size is one of the defining characteristics of modern humans. Seckel syndrome (MIM 210600), a disorder of markedly reduced brain and body size, is associated with defective ATR-dependent DNA damage signaling. Only a single hypomorphic mutation of ATR has been identified in this genetically heterogeneous condition. We now report that mutations in the gene encoding pericentrin (PCNT)--resulting in the loss of pericentrin from the centrosome, where it has key functions anchoring both structural and regulatory proteins--also cause Seckel syndrome. Furthermore, we find that cells of individuals with Seckel syndrome due to mutations in PCNT (PCNT-Seckel) have defects in ATR-dependent checkpoint signaling, providing the first evidence linking a structural centrosomal protein with DNA damage signaling. These findings also suggest that other known microcephaly genes implicated in either DNA repair responses or centrosomal function may act in common developmental pathways determining human brain and body size.

Figure 1. Schematic of the Sckl4 critical region and the PCNT gene depicting location of identified mutations

(a) Genetic map of chromosome 21q22.3 with the refined Sckl4 locus, defined by overlapping homozygous chromosomal segments in two consanguineous families, and extending over a 2.9 Mbp interval between SNP markers rs1598206 and rs2330591 (43,883,204 – 46,751,852, UCSC Browser, March 2006 Assembly). Genetic distances, Decode genetic map. (b) PCNT spans 122kb of genomic sequence in 47 exons and encodes a 3336 amino acid protein. (c) Sequence electropherograms of PCNT mutations. (d) Schematic of the pericentrin protein indicating position of identified mutations and protein structure. Pericentrin contains internal repeats (red), coiled-coil regions (green), and a PACT domain (blue). Protein structural regions as predicted by SMART. Sites of interaction with PKA, PKC βII and calmodulin indicated by asterisks.

Figure 2. Pericentrin localisation and function is disrupted in PCNT-Seckel cell lines

(a) Pericentrin is not localised to the pericentiolar material in PCNT-Seckel cells. Deconvolved immunofluorescent images from PCNT-Seckel and control lymphoblastoid cell lines. Pericentrin (ab448-100, green) and centrin (red). Scale bar 5μm. Control, heterozygote relative, PCNT^220X/+. (b) Immunoblot of LCL cell lysates with Pericentrin ab448-100 antibody, that detects both Pericentrin A and B isoforms. Two pericentrin (arrowheads) isoforms are absent from PCNT-Seckel cells, but present in control lymphoblastoid cells from heterozygous relatives. A smaller protein product of ∼170kD is detected in the PCNT^220X cell line,that might represent an aberrant truncated PCNT protein product. Loading control, alpha-tubulin. (c,d) γ–tubulin localisation is frequently reduced or absent during mitosis in PCNT-Seckel cells. (c) Quantification of γ-tubulin signal in PCNT-Seckel mitotic cells at prometaphase and metaphase, relative to wild-type. n=20. error bar, s.d. γ-tubulin signal intensity is significantly reduced relative to wild type cells (p<0.05, S629fs; p<0.001, E220X) (d) In PCNT-Seckel mitotic cells (astericks), γ–tubulin centrosomal staining is reduced (arrows) or absent, relative to centrosomal staining in adjacent interphase cells, or in control cells from a heterozygous relative.

Figure 3. PCNT is required for ATR dependent DNA damage signalling

(a) G2-M checkpoint arrest was observed 2 h post UV treatment in control but not in ATR- or PCNT-Seckel LCLs. The mitotic index was examined 2 hours after treatment with 5 J m⁻² UV. Average of 3 experiments, error bars, s.d. Controls:- heterozygote relatives PCNT^E220X/+ and PCNT^S629fs/+ ; and an unrelated wild type (WT) control. UNT, untreated; UV, UV-C treated. Mitotic index is significantly increased in PCNT-Seckel cells compared with Wild-Type cells after UV treatment (p<0.001, E220X; p<0.01, S629fs; p<0.05, C1190fs) (b) ATR- and PCNT-Seckel LCL cells have increased hydroxyurea-induced nuclear fragmentation. Nuclear fragmentation was examined 24 h after treatment with 5mM hydroxyurea (HU). Percentage of cells displaying nuclear fragmentation (NF) is shown (p<0.001 WT versus PCNT-Seckel E220X) (c) ATR- and PCNT-Seckel LCL cells exhibit elevated levels of supernumerary centrosomes in mitosis. Percentage of mitotic (phosphohistoneH3 (Ser10) positive cells) with > 2 γ-tubulin stained foci (centrosomes) determined following 24 h incubation with nocodazole (p<0.05 WT versus E220X) (d) γH2AX foci formation is normal in PCNT-Seckel LCLs. Phosphorylation of the histone H2AX (termed γH2AX) by the PI3K-kinases is one of the earliest detectable responses to DNA damage, and is ATR-signalling specific after hydroxyurea treatment. Percentage of γH2AX foci was determined 2 hours after treatment with 5mM hydroxyurea. (e) ATR- and PCNT-Seckel cells show significantly reduced 53BP1 foci formation after treatment with 5mM hydroxyurea for 2 hrs, reflecting impairment in ATR/Chk1 dependent signalling. Immunofluorescent staining with anti-BrdU confirmed that all LCLs had equivalent S-phase populations (range: 20-23%) prior to exposure to HU (p<0.005 E220X; and p<0.001, S629fs versus WT after HU). (f) G2-M checkpoint arrest after Ionizing Radiation (IR) is normal in PCNT-Seckel and ATR-Seckel LCLs but not in Ataxia Telangiectasia mutated (ATM) LCLs.

Figure 4. Model of Pericentrin's role in ATR-dependent G2/M checkpoint arrest

Low-dose UV and Hydroxyurea activate ATR kinase which then phosphorylates downstream targets including Chk1 kinase. Activating phosphorylation is required for accumulation of Chk1 at the centrosome after DNA damage. Direct or indirect binding of Chk1 by pericentrin could mediate Chk1's localisation to the centrosome. Chk1 localisation at the centrosome inhibits Cdc25, preventing Cyclin B/Cdk1 activation and thus the transition from G2 in to mitosis.