Fluorescence in situ hybridization.
Region-specific BAC clones for FISH mapping were selected from the NCBI and UCSC genome browsers. FISH analysis was performed using metaphase chromosomes prepared from the patient’s cell suspension and lymphoblastoid cell lines. Genomic BAC DNA was fluorescently labeled with Cy3-dCTP (Amersham Biosciences) by standard nick translation with the DIG-Nick-Translation Kit (Roche). The probes were blocked with Cot-1 DNA. Metaphase spreads were hybridized at 37°C overnight with the Cy3-labeled BAC probes and a specific FluoroX-labeled (Amersham Biosciences) subtelomeric control probe. After posthybridization washes, chromosomes were counterstained with DAPI (Serva). Images were captured on a Zeiss Axioplan 2 microscope with a CCD camera and processed with the Isis Software (Metasystems).
Long-template PCR and mini-FISH analyses.
All long-template PCRs were performed with the Expand Long Template PCR-System (Roche Diagnostics) according to the manufacturer instructions, with primer pairs (480K16-F1-F ACCATTAGGTGTCCATTTTTAAGAAACA, 480K16-F1-R CAGCTTACGGAGAATGTAGGAGACTTAG, 88E10-F5-F TGCTTTAGCTAGAATAGGGACAGAAAGA, 88E10-F5-R TGCACTACAGTCACAGCACAATAAAATA) chosen from the genomic sequence of the breakpoint-spanning BAC clones. Purified long-template PCR-products (QIAquick PCR Purification Kit, Qiagen) were labeled with Cy3-dCTPs by standard nick translation as described above, and FISH was performed on metaphase spreads.
Primer pairs for breakpoint-spanning PCRs were chosen from the genomic sequence of the breakpoint regions on chromosomes 10 and 13. Different combinations of forward and reverse primers were used to amplify a breakpoint-spanning PCR product with the Expand 20kbPlus PCR-System, dNTPack (Roche Diagnostics), according to the manufacturer’s instructions.
Cell culture and transfection of HeLa and HEK293 cells.
HeLa and HEK293 cells were maintained in DMEM/HAM’s F12 (BIOCHROM AG), supplemented with 10% heat-inactivated fetal bovine serum (BIOCHROM AG) and 1% penicillin/streptomycin (10,000 U/10,000 μg/ml) (BIOCHROM AG) at 37°C, 5% CO2, 91% relative air moisture. Three independent RNAi siRNAs targeting MYST4 mRNA (MYST4 HSS118880, HSS177468 and HSS177469; Invitrogen) were validated by real-time RT-PCR. Cells were then transfected with 1 out of 3 different Stealth RNAi siRNA targeting MYST4 mRNA (HSS118879, HSS118880, HSS118881; Invitrogen) or Scrambled Negative Control Stealth RNA (Invitrogen) according to the manufacturer’s instructions. Medium was changed to growth medium without antibiotics 12 hours after transfection. Cells were harvested 51.75 hours after transfection. After evaluation of siRNA efficiency by quantitative RT-PCR and histone acetylation assay, HSS118880 was used for further studies.
Global histone H3/H4 acetylation assay.
Histone extraction and detection of global histone H3/H4 acetylation were performed using EpiQuik Global Histone H3/H4 Acetylation Assay Kits, according to the manufacturer’s instructions (Epigentek), with an input of 1 μg of histone proteins extracted from lymphoblastoid cell lines of the patient and 3 unrelated healthy controls and HEK293 and HeLa cells. Data analysis was performed according to the manufacturer’s instruction. Acetylation levels were calculated for the patient with reference to the 3 healthy controls and for the HEK293 and HeLa MYST4 siRNA cell lines with reference to Scrambled Negative Control Stealth RNA transfected cell lines.
HAT activity assay.
Histone extraction and detection of HAT activity were performed using the EpiQuik HAT Activity/Inhibition Assay Kit, according to the manufacturer’s instructions (Epigentek). Briefly, histone substrate was stably spotted on wells where active HATs bind and acetylate histone substrate. The acetylated substrate was colorimetrically quantified after binding of high-affinity antiacetylated histone antibody.
RNA isolation from human cells.
Harvested cell pellets were washed using PBS. RNA was isolated using the QIAcube instrument in combination with the RNeasy Mini Kit, QIAshredder, and RNase-Free DNase Set, according to the manufacturer’s instructions (QIAGEN).
cDNA was synthesized using the SuperScript II Reverse Transcriptase Kit with random hexamer primers (Invitrogen) from RNA extracted from fresh blood samples collected in PAXgene tubes (Becton Dickinson) or from cell cultures.
Relative mRNA expression levels of all MYST4/MORF isoforms in fetal and adult human tissues.
For relative expression analysis of MYST4/MORF isoforms, real-time PCR primers and mgb probes were designed according to the manufacturer’s instructions (Applied Biosystems) to obtain primer/probe sets specific for each isoform: MORF forward (F), TGTCTGTAACCAGTGATGAAGGA; MORF probe (P), 6FAM-TCACCTGATACTGAAATAA; MORFα F, AAAAAGGTCTCTCAGAAACAGTCATG; MORFα P, 6FAM-TGTTGGCTACAGATACTGAA; MORFβ F, GAGCTTGACAGACGGAAGGATT; MORFβ P, 6FAM-CAGGATGATGATACTGAAATA; and all isoforms reverse, CATCTGCACTTTCTTGTTTGATGTT.
The tissue expression pattern was determined using 4 commercial available cDNA panels (Human Fetal MTCTM Panel and Human MTCTM Panel I [both from Clonetech]; C8244525 and C8234504 [both from BioChain Institute]). All real-time RT-PCRs were performed in quadruplicates in 384-well plates, with a final volume of 20 μl each on an ABI 7900HT, using the TaqMan Gene Expression Mastermix, according to the manufacturer’s instructions (Applied Biosystems). The expression levels of the individual isoforms were calculated using the ΔΔCt method with 2 endogenous controls (B2m, phosphoglyceratekinase 1 [PGK1]). Levels of significance were determined by a 2-sample equal-variance t test applied to the log-expression values.
All exon and flanking intron sequences of MYST4
gene, except the 3ι UTR, were amplified from patient DNA by PCR using intronic primers designed with the Primer3 software. The PCR products were purified with the AMPure Kit (Agencourt) on a Biomek NX instrument (Beckman Coulter). Sequencing reactions were performed on both strands using the BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems) according to the manufacturer’s instructions. After purification using the CleanSEQ Kit (Agencourt), the products were analyzed on an ABI Genetic Analyzer 3730 (Applied Biosystems), and the traces were evaluated using the SeqPilot 220.127.116.11 software (JSI Medical Systems). Variants were explored against the SNP database (NM_012330.2;
Extraction of protein lysates from human cells.
Harvested cell pellets were washed using PBS and frozen at –70°C for a minimum of 1 hour. After thawing on ice, pellets were resuspended in lysis buffer (0.01 M Tris; 0.15 M NaCl; 1% Triton-X, pH 7.5) and shook at 4°C for 20 minutes. Shearing of cells was performed using a 14-gauge needle. Lysates were stored at –70°C.
Western blot analysis using protein lysates extracted from lymphoblastoid cell lines of the patient, HeLa and HEK293 cells, respectively, transfected with either Stealth RNA siRNA targeting MYST4 mRNA (MYST4 HSS118880, Invitrogen) or Scrambled Negative Control Stealth RNA (Invitrogen) was performed. Electrophoresis was performed on a 3%–8% Tris-Acetate gel using the NuPage Novex system from Invitrogen. The KAT6B/MORF antibody (ab58823, Abcam) and the β-actin antibody (ab8227, Abcam) were used as loading controls. As secondary antibody, we used the HRP-conjugated goat anti-rabbit IgG antibody (170-6515, Bio-Rad).
Mice heterozygous for a mutation in the Querkopf
(also known as Qkf
, and Kat6b
) gene, Qkfgt/+
), were used to generate E15.5 and newborn Qkfgt/gt
control pups. Skeletal preparations were conducted as described previously (51
), with modifications. In brief, bodies were eviscerated; fixed in ethanol and acetone for 4 days each; rinsed in H2
O; stained in a solution of 0.015% Alcian Bue 8GX (Sigma-Aldrich), 0.005% Alizarin Red S (Sigma-Aldrich), and 0.05% acetic acid in H2
O for 10 days; and then cleared in a solution of 1% potassium hydroxide and 20% glycerol in H2
O, followed by further clearing through 40%, 60%, and 80% glycerol in H2
β-Galactosidase staining, in situ hybridization, and histology of mice.
β-Galactosidase staining was conducted as described previously (52
), and in situ hybridization was carried out as described previously (17
). Embryos and pups were fixed in 4% paraformaldehyde and Bouin’s fixative, respectively, and processed for serial paraffin sections as described previously (53
). Each block of 4 consecutive sections of the hind limb was stained with (a) hematoxylin and eosin, (b) toluidine blue and fast green, (c) safranin O and fast green, and (d) Masson’s trichrome to visualize specific cell types in developing bone and cartilage, cartilage matrix, ossifying cartilage, and bone structures.
Expression array and analysis of data.
The RNA quality was controlled by an Agilent Bioanalyzer (Agilent Technologies). Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix) were used for HEK293 and HeLa gene expression analyses, and Affymetrix GeneChip Mouse Exon 1.0 ST arrays were used for Qkfgt/gt
and control mouse gene expression analysis. Probe synthesis, hybridization, and initial expression analysis of the Affymetrix GeneChip microarrays were performed according to the protocol recommended by Affymetrix. MYST4
siRNA silencing in HEK293 and HeLa cell lines was performed in 3 individual experiments and hybridized on separate arrays (i.e., 6 arrays were run for HEK293 and 6 arrays were run for HeLa cell lines). All data are MIAME compliant and were uploaded to Gene expression omnibus (GEO;
). The 12 arrays were analyzed together. Data processing and normalization were performed using the Partek Genomic Suite Software Package (Partek Incorporated). For each gene, a 2-way ANOVA was calculated, and an F-test was conducted for an MYST4 effect, adjusting for differences between the 2 cell lines. Genes were determined as regulated by MYST4
if the false discovery rates were less than 0.05 and the fold change was greater than 1.5. Experiments for the Qkfgt/gt
and control mouse expression were performed likewise. Qkfgt/gt
versus wild-type adult dorsal cortex and Qkfgt/gt
versus wild-type E12.5 dorsal telencephalon were analyzed in triplicates with 3 animals per genotype, 1 array per animal and tissue. The Qkfgt/gt
arrays were analyzed using Bioconductor software (
). Data from all tissues were analyzed together. Expression values were normalized using the gcRMA algorithm. Gene-wise linear models were fitted using the limma software package. Empirical Bayes-moderated t-statistics were computed for each tissue to test for differences between the Qkfgt/gt
mutants and normal controls (54
). The t-statistics were adjusted for age differences and for correlation between sibs (55
values were adjusted to control the false discovery rate using the method of Benjamini and Hochberg. Genes were determined as regulated by MYST4
if the unadjusted P
value was less than 0.05 and the fold change was greater than 1.5.
HeLa and HEK293 cells were processed with the LowCell ChIP Kit (Diagenode) shearing the DNA with a Bioruptor (Diagenode) according to the manufacturer’s instructions, including positive and negative controls (10,000 cells/IP). Two micrograms of MYST4 (KAT6B/MORF) antibody (ab58823, Abcam) were used in parallel to 2 μg of negative control IgG from rabbit (Diagenode). The immunoprecipitates were amplified using the GenomePlex Complete Whole Genome Amplification (WGA2) Kit (Sigma-Aldrich) as described in the Farnham laboratory WGA protocol for ChIP amplicons (
Tiling array and analysis of data.
ChIP and control input DNA samples were hybridized in triplicates on separate Affymetrix human promoter 1.0 arrays, as described in the Affymetrix Analysis Technical Manual. All data are MIAME compliant and were uploaded to ArrayExpress (ArrayExpress accession, E-MEXP-2591;
). Enrichment values (ChIP/control input DNA) were calculated using the MAT algorithm implemented in the Partek Genomic Suite Software. RefSeq genes were assigned to the MAT regions with MAT score P
values of 0.1, 0.05, 0.01, and 0.0001.
Quantitative real-time PCR validation of whole-genome expression array and ChIP-CHIP results.
All real-time RT-PCRs were performed in quadruplicates in 384-well plates with a final volume of 20 μl each on an ABI 7900HT using the TaqMan Gene Expression Mastermix according to the manufacturer’s instructions (Applied Biosystems). The expression levels of the individual isoforms were calculated using the ΔΔCt method with 4 endogenous controls (B2m, PGK1, β-actin, and TATA-box binding protein). Levels of significance were determined by a 2-sample equal-variance t test applied to the log-expression values. Nine significantly differentially expressed genes of the MAPK signaling pathway were validated using the following gene expression assays (Applied Biosystems): SMAD4 (Hs00929647_m1), MAX (Hs00231142_m1), PRKX (Hs00746337_s1), AKT2 (Hs01086102_m1), MRAS (Hs00171926_m1), ELK1 (Hs00428286_g1), SRF (Hs00182371_m1), PPP3CB (Hs00184176_m1), and MAP2K3 (Hs00177127_m1) and, 2 unaltered control genes, NTRK1 (Hs01021011_m1) and DUSP2 (Hs00358879_m1).
Gene ontology and pathway analysis.
Gene ontology and pathway analysis (KEGG pathways; ref. 26
) was carried out using the DAVID functional annotation tool (
) and Pathway-Express as part of Onto-Tools (
) scoring the pathways, with a significant P
value/corrected gamma P
value of less than 0.05 in both methods. This was performed individually for the HEK293/HeLa MYST4
siRNA expression data, the mouse Qkfgt/gt
adult dorsal cortex versus control expression data, the mouse Qkfgt/gt
E12.5 dorsal telencephalon versus control expression data, and the HEK293/HeLa MYST4
siRNA ChIP-CHIP data.
MEK, ERK, and AKT activation assays.
The patient cell line was transiently transfected with a human MYST4 wild-type construct (SC308942, Origene) using Lipofectamine 2000 (Invitrogen). Normalization of expression levels were measured by quantitative real-time PCR as mentioned above. Amounts of MEK1/2, ERK1/2, AKT, phospho-MEK1/2, phospho-ERK1/2, and phospho-AKT were determined by Western blotting of the cell lysates from patient and control lymphoblastoid cell lines as well as from the MYST4-transfected patient cell line. Cells were lysed (lysis buffer containing 50 mM Tris/HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1% Igepal CA-630, 10% glycerol, 20 mM β-glycerolphosphate, 1 mM orthovanadate, EDTA-free inhibitor cocktail-Roche), and the lysates were cleared by centrifugation. For similar loading the protein amount was normalized by Bradford assay (Bio-Rad), and the proteins were separated by SDS-PAGE (10% polyacrylamide) and detected by Western blotting using antibodies against MEK1/2 (Cell Signaling Technology), ERK1/2 (Cell Signaling Technology), AKT (Cell Signaling Technology), phospho-MEK1/2 (Ser 217/221, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling Technology), and phospho-AKT (Ser473, Cell Signaling Technology).
Quantitative real-time PCR statistical analysis was performed according to the ΔΔCt method. All experiments were performed in triplicate, and error bars represent SD unless otherwise stated. All statistical analysis of the expression and ChIP-CHIP results were generated with Partek Genomic Suite 6.5. These analyses were performed in triplicate, and the ANOVA calculation was applied to identify the fold changes of differentially expressed genes and the MAT score of ChIP-CHIP binding sites. Unless otherwise stated, P values of less than 0.05 were considered significant.
This study was approved by the Ethical Review Board of the Medical Faculty of the Friedrich-Alexander University Erlangen-Nuremberg. After written informed consent, peripheral blood samples for DNA and RNA extraction and cell culture were obtained from the family members.