Knockdown of ALR (MLL2) Reveals ALR Target Genes and Leads to Alterations in Cell Adhesion and Growth

Confirmation of ALR complex composition. (A) Size fractionation of the enriched ALR complex. The ALR-enriched K562 nuclear extracts, obtained from subsequent purification steps on P11 and Q-Sepharose columns, were applied to a preparative Superose 6 column. One percent of each fraction collected from Superose 6 was analyzed by immunoblotting using Abs directed to various ALR complex components. fr#, fraction number. The positions of molecular mass markers determined in a parallel run are indicated at the top. (B) Coprecipitation of ALR with other ALR complex components from A549 cells. Crude nuclear extracts obtained from A549 cells were utilized in reciprocal coimmunoprecipitations. Proteins coprecipitated (IP) with anti-ALR, anti-PTIP, anti-UTX, and anti-RBQ3 Abs, as well as with control IgG, were separated on SDS-PAGE and immunoblotted (IB) with the indicated Abs. Since different amounts of Abs were used in some IPs, the analysis is qualitative rather than quantitative.

Histone methyltransferase activities of the ALR complex and of the ALR SET domain. (A) The ALR complex methylates histone H3. Anti-ALR, anti-PTIP, anti-UTX, or control immunoprecipitate (IP) was incubated with core histones and the methyl donor H³-SAM. Samples were resolved on 15% SDS-PAGE, stained with Coomassie blue (right), amplified, dried, and fluorographed (left). Histone subunits are indicated on the right. (B) ALR SET domain methylates H3K4. Two ALR SET domain-containing fragments, encompassing the ALR amino acids 5009 to 5261 and 5089 to 5261 (designated SET a and SET b, respectively), were fused to GST and overexpressed in 293T cells. Cells overexpressing GST only were used as a control. The overexpressed polypeptides were immunoprecipitated from cell lysates using anti-GST Ab and incubated with N-terminal H3 peptides containing unmodified K4 and K9, dimethylated K4, dimethylated K9, or trimethylated K9. Samples were resolved on 20% SDS-PAGE, stained with Coomassie blue (bottom), and fluorographed (top). (C) The recombinant ALR SET domain does not interact with the ASH2, RBQ3, and WDR5 proteins. GST-SET fusions or control GST was precipitated from 293T cell lysates with anti-GST Ab and analyzed by immunoblotting using anti-GST, -ASH2, -RBQ3, or -WDR5 Abs. Five percent of each GST-SET- or control GST-overexpressing lysate was loaded as an input control. (D) The ALR complex methylates H3 peptides containing either unmodified or dimethylated K4. Anti-ALR, anti-PTIP, anti-UTX, or control immunoprecipitate was incubated with unmodified or appropriately methylated H3 peptides. The reaction was analyzed as described above.

(A) Effects of ALR shRNAs on the level of ALR. Four stable cell lines generated by infection of HeLa cells with retroviruses carrying four different ALR shRNA constructs (lanes shALR SP1 to shALR A10) and two control HeLa cell lines, infected with the retroviral vector or with noncompetent ALR shRNA D, were examined by immunoblotting for expression of the ALR protein and some ALR complex components. (B) Confirmation of the microarray results by RT-PCR analysis. RNAs isolated from the above-described cell lines were used for semiquantitative RT-PCR analysis to monitor the expression of selected genes. PCR cycle numbers were individually optimized for each gene so that each reaction fell into the linear range of product amplification. The genes analyzed included 11 downregulated genes (CSPG4, CRIP1, CRIP2, DKK1, FABP3, ENO3, LOXL1, NT5E, GPR56, LAMB3, and MEOX1), 1 upregulated gene (DPT), and 2 control genes (β-actin and HoxA9).

ChIP analysis of CRIP2 (A), ENO3 (B), CSPG4 (C), DKK1 (D), and HoxA9 (E) loci in vector-infected and SP3 cells. The presence of the ALR, PTIP, and UTX proteins, as well as of histone H3K4 trimethylation, on several regions of CRIP2, ENO3, CSPG4, and DKK1 loci and on the 5′ end of the HoxA9 locus was examined. Chromatin was prepared from the control vector-infected cells and from the ALR-deprived SP3 cell line. The analyzed portions of the genes are depicted to the right of panels A to D and at the bottom of panel E. The boxes shown on the schemes indicate the first exons, and the dark areas correspond to the coding regions. The arrows point to the transcription initiation sites; a to e correspond to the sequences amplified by PCR following immunoprecipitation with each of the six Abs (two anti-ALR Abs, anti-PTIP, anti-UTX, control IgG, and anti-trimethylated H3K4). For input, 0.1% or 0.5% of the amount of sonicated chromatin to be subsequently processed for ChIP was removed and PCR amplified.

Changes in the morphology of ALR knockdown cells during spreading. (A and B) Differences in dynamics and modes of cell spreading. (A) Control (Vector) and ALR-deficient (SP3) cells were plated on fibronectin-coated dishes and analyzed by time-lapse photography during the subsequent 4 h. The numbers represent the time after plating in min. (B) Two control (Vector and D) and two ALR-underexpressing (SP3 and A10) cell lines were seeded on fibronectin-coated coverslips and fixed with 3.7% paraformaldehyde at different time points during the subsequent 15 h. Typical photographs of the cells at 6 and 15 h after being plated are shown. (C and D) Decreased numbers of FA and alterations in actin cytoskeleton organization in spreading ALR-deficient cells. (C) Control (Vector) and ALR knockdown (SP3) cells were processed as described for panel B, and FA were double stained with anti-vinculin and anti-paxillin Abs. The numbers of FA formed by each cell type at the indicated time points were calculated. The data represent the average FA number of 30 cells plus standard deviation. (D) The cells described above, fixed at 3 h after being plated, were stained for filamentous actin with tetramethyl rhodamine isothiocyanate-phalloidin (red) and for FA with anti-paxillin Ab (green). Bars (A, B, and D), 50 μm.

ALR knockdown affects cell growth, apoptosis, and migration. (A) Cell growth assay. The growth rates of three ALR-deprived HeLa cell lines (SP3, SP4, and A10) and of the control cells (Vector and D) were determined by plating 10⁵ cells in triplicate on six-well plates and counting the cells every 24 h during the subsequent 4 days. The error bars represent the standard deviations of three independent experiments. (B) Increased apoptosis in the ALR-deprived cells. Control (Vector and D) and ALR-deprived (SP1, SP3, SP4, and A10) HeLa cells were stained with the TUNEL assay kit. Percentages of TUNEL-positive (apoptotic) populations are shown. The error bars represent the standard deviations of three independent experiments. (C) ALR knockdown inhibits soft-agar growth of HeLa cells. Five hundred and 1,000 cells/3-cm dish were seeded on 0.3% agar in triplicate. Three weeks later, the dishes were photographed and the colonies were counted. The experiment was repeated twice. Typical photographs of plates seeded with control (Vector) and ALR-deficient (SP3) cells are shown on the left. The histogram shows the numbers of colonies formed in each cell line at both plating concentrations as a percentage of the control vector-expressing cells. Absolute colony numbers are shown above the bars. (D) Effect of ALR knockdown on cell migration. A scratch injury was introduced with a micropipette tip into confluent cultures of the control (Vector) and ALR-depleted (SP3) A549 cells. Phase-contrast images of cultures were taken either immediately after scratching (0 h) or 16 h later.