MBD4 is induced in activated B cells and interacts with MLH1 and PMS2.

<p><b>A)</b> A schematic of the murine MBD4 protein, including the methyl-binding domain (MBD) (white oval; aa 49–147), the glycosylase domain (gray oval; aa 411–554) spanning exons II–III and V–VIII, respectively, and predicted NLS and NES. <b>B,C</b>) Resting splenic B cells were activated with LPS+IL4 for (<b>B</b>) 72 hours or (<b>C</b>) the indicated times. <b>B</b>) Total protein was determined by Bradford assay and either cytoplasmic (100 µg), or nuclear (25 µg) extracts were examined in a Western blot with Abs against MBD4, β-ACTIN, and LAMIN B1. <b>C)</b> Nuclear (50 µg) extracts were examined in Western analysis using anti-MBD4 and anti-β-ACTIN Abs. Extracts were prepared from two independent mice. One representative experiment is shown. <b>D</b>) Nuclear extracts from CH12.F3 cells induced with CIT for 24 hours were immunoprecipitated with α-MLH1, α-MBD4, or α-GFP Abs then analyzed by Western blot (WB) with the indicated Abs. IPs were performed in duplicate in two independent experiments. One representative experiment is shown.</p

Complex Relationship between Mismatch Repair Proteins and MBD4 during Immunoglobulin Class Switch Recombination

<div><p>Mismatch repair (MMR) safeguards against genomic instability and is required for efficient Ig class switch recombination (CSR). Methyl CpG binding domain protein 4 (MBD4) binds to MutL homologue 1 (MLH1) and controls the post-transcriptional level of several MMR proteins, including MutS homologue 2 (MSH2). We show that in WT B cells activated for CSR, MBD4 is induced and interacts with MMR proteins, thereby implying a role for MBD4 in CSR. However, CSR is in the normal range in Mbd4 deficient mice deleted for exons 2–5 despite concomitant reduction of MSH2. We show by comparison in Msh2^+/− B cells that a two-fold reduction of MSH2 and MBD4 proteins is correlated with impaired CSR. It is therefore surprising that CSR occurs at normal frequencies in the Mbd4 deficient B cells where MSH2 is reduced. We find that a variant Mbd4 transcript spanning exons 1,6–8 is expressed in Mbd4 deficient B cells. This transcript can be ectopically expressed and produces a truncated MBD4 peptide. Thus, the 3′ end of the Mbd4 locus is not silent in Mbd4 deficient B cells and may contribute to CSR. Our findings highlight a complex relationship between MBD4 and MMR proteins in B cells and a potential reconsideration of their role in CSR.</p></div

Mbd4 deficient B cells undergo CSR despite reduced MSH2 and MLH1.

<p>Splenic B cells from WT, Mbd4^Δ2−5/+, or Mbd4^{Δ2−5/Δ2−5} mice were activated with LPS+IL4 for 48 hours and nuclear extracts (<b>A</b>) or cDNA (<b>B</b>) were prepared in two independent experiments. <b>A</b>) Immunoblot analyses of nuclear extracts were developed with Abs against MBD4 (69 kD), MSH2 (107 kD), MLH1 (95 kD), and β-ACTIN (43 kD). One representative experiment is shown. <b>B</b>) Mlh1 or Msh2 transcripts were analyzed by qRT-PCR and normalized to 18S rRNA. The averages from 2 to 3 independent experiments are shown with standard error of mean (SEM). <b>C-E</b>) Splenic B cells from WT or Mbd4^{Δ2−5/Δ2−5} from two independent mice were activated with LPS or LPS+IL4 to induce IgG3 or IgG1 CSR, respectively, for 4 days (<b>C,D</b>), or 48 hours (<b>E</b>). <b>C</b>) A representative FACS analysis of anti-B220 (APC) versus anti-IgG1 or anti-IgG3 (FITC) is shown. <b>D</b>) Cumulative CSR frequencies are shown with SEM. WT was set to 1. <b>E</b>) GLT γ3 and γ1 expression were analyzed by qRT-PCR and normalized to 18S rRNA and SEM are shown.</p

Msh2 ^+/⁻ B cells are happloinsufficient for CSR.

<p>Splenic B cells from WT and Mlh1^+/− (<b>A</b>) or WT and Msh2^+/− (<b>B</b>) littermates were stimulated with LPS or LPS+IL4 to induce IgG3 or IgG1 switching, respectively. P values are from Student’s two tailed <i>t</i> test; p <0.05 (*), p<0.01 (**), p<0.001 (***). <b>A, B</b>) Two-three independent mice from each genotype were analyzed. Representative FACS analyses of anti-B220 (APC) versus anti-IgG1 or anti-IgG3 (FITC) are shown for WT, Mlh1 (<b>A</b>) and Msh2 (<b>B</b>) deficient B cells. <b>C</b>) CSR frequency averages from FACS experiments are shown with SEM. WT was set to 1. <b>D, E</b>) Splenic B cells from two independent WT and Msh2^+/− mice were activated with LPS+IL4 for 72 hours (<b>D</b>), or 48 hours (<b>E</b>). <b>D</b>) Nuclear extracts were analyzed by immunoblot for MMR protein expression, as indicated. Proteins were quantitated by densitometry and normalized to the βACTIN loading control. WT is set to 1. Triangles represent serial two fold dilutions of nuclear extracts. <b>E</b>) Averages of Q-RT-PCR of Msh2, Mbd4, and Mlh1 transcripts are normalized to 18S rRNA and shown with SEM.</p

Exon 6 Kozak sequence directs expression of a truncated MBD4 peptide.

<p><b>A</b>) Diagram of the Mbd4 locus, (chr6: 115,840,698-115,853,371 (mm8)) with a partial representation of the antisense CN781668 and BY200253 lncRNAs. The RNA splicing pattern for the full length Mbd4 transcript (solid line) is shown with exons 1–8 depicted as black blocks. The splicing patterns for CN781668 exons 1–4 (long dashed lines) and BY200253 (short dashed lines) are shown as white blocks. PCR primers (arrowheads) are indicated. <b>B,C</b>) WT or Mbd4^{Δ2−5/Δ2−5} B cells were activated with LPS+IL4 for 48 hours (<b>B</b>), or as indicated (<b>C</b>) and cDNA prepared. <b>B</b>) RT-PCR was carried out for Mbd4 (primers F1/R1), lncRNA CN781668 (primers F3/R3), or AID. Mbd4 1–8 (circle), Mbd4 1/4–8 (asterisk), Mbd4 1/6–8 (square) are indicated. In the lower panel a schematic is shown of Mbd4 and CN781668 transcripts with exons indicated by alternating black and white blocks followed by poly-A tails. <b>C</b>) QRT-PCR using F5/R1 primers detect WT Mbd4 1-8 and Mbd4^{Δ2−5/Δ2−5} 1/6–8 transcripts. Results are averaged from 2 independent experiments and shown with SEM. <b>D</b>) CH12.F3 cells were transduced in three independent experiments with empty (E) or Mbd4 exons 6–8 (M) expression constructs. Transductants were stimulated with CIT for 24 hours, nuclear extracts prepared and then analyzed by Western with anti-MBD4 or anti-βACTIN and a representative blot is shown.</p

Inhibitor of DNA Binding 4 (ID4) Is Highly Expressed in Human Melanoma Tissues and May Function to Restrict Normal Differentiation of Melanoma Cells

<div><p>Melanoma tissues and cell lines are heterogeneous, and include cells with invasive, proliferative, stem cell-like, and differentiated properties. Such heterogeneity likely contributes to the aggressiveness of the disease and resistance to therapy. One model suggests that heterogeneity arises from rare cancer stem cells (CSCs) that produce distinct cancer cell lineages. Another model suggests that heterogeneity arises through reversible cellular plasticity, or phenotype-switching. Recent work indicates that phenotype-switching may include the ability of cancer cells to dedifferentiate to a stem cell-like state. We set out to investigate the phenotype-switching capabilities of melanoma cells, and used unbiased methods to identify genes that may control such switching. We developed a system to reversibly synchronize melanoma cells between 2D-monolayer and 3D-stem cell-like growth states. Melanoma cells maintained in the stem cell-like state showed a striking upregulation of a gene set related to development and neural stem cell biology, which included SRY-box 2 (SOX2) and Inhibitor of DNA Binding 4 (ID4). A gene set related to cancer cell motility and invasiveness was concomitantly downregulated. Intense and pervasive ID4 protein expression was detected in human melanoma tissue samples, suggesting disease relevance for this protein. SiRNA knockdown of ID4 inhibited switching from monolayer to 3D-stem cell-like growth, and instead promoted switching to a highly differentiated, neuronal-like morphology. We suggest that ID4 is upregulated in melanoma as part of a stem cell-like program that facilitates further adaptive plasticity. ID4 may contribute to disease by preventing stem cell-like melanoma cells from progressing to a normal differentiated state. This interpretation is guided by the known role of ID4 as a differentiation inhibitor during normal development. The melanoma stem cell-like state may be protected by factors such as ID4, thereby potentially identifying a new therapeutic vulnerability to drive differentiation to the normal cell phenotype.</p></div

Staining of human melanoma tissue microarray samples with anti-ID4.

<p>Staining of human melanoma tissue microarray samples with anti-ID4.</p

Knockdown of ID4 or SOX2 inhibit MB formation.

<p><b>(A)</b> Diagram of experimental design. (<b>B)</b> Effectiveness of siRNAs in knockdown of ID4, SOX2 and NOTCH1 in hESC media. Western blots were carried out using LIN28 siRNA as a negative control. (<b>C)</b> The 1205Lu cells were treated with the indicated siRNAs, followed by challenge with hESC medium. MB formation was monitored. (<b>D)</b> The1205Lu cells were treated with individual ID4 siRNA, followed by challenge with hESC medium. Cell morphology was monitored after 7 days of culture in hESC media. (<b>E)</b> Western blots were carried out to analyze the effectiveness of individual ID4 siRNA. Lanes: mix, a mix of three siRNAs targeting ID4; con, untreated; 1,2,3, independent siRNAs targeting ID4.</p

Expression microarray analysis of monolayer versus 3D-MB cultures.

<p>(<b>A)</b> Diagram of experimental design for comparison of transcriptomes of monolayer and 3D-MB cells. OM, original monolayer; MB, melanoma bodies; NM, new monolayer. <b>(B)</b> Raw microarray data for six housekeeping genes in OM, MB and NM RNA samples: ACTB, Beta-Actin; ACTG1, Gamma 1 Actin; GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase; LDH1, Lactate Dehydrogenase; UBC, Ubiquitin C; VIM, Vimentin. An average value was calculated from 10 spots, for each gene. (<b>C)</b> Sampling of genes upregulated 5-fold or greater in the MB sample compared to the OM and NM samples (MB>OM, MB>NM). Genes are ordered according to the fold-upregulation in the MB sample versus OM sample (MB>OM). Samples were normalized versus an average of three housekeeping genes, GAPDH, ACTB and HSP90 (see Panel B), and error bars represent the standard deviation. Grey filled circles indicate genes categorized with the GO term “developmental process.” (<b>D)</b> Sampling of genes downregulated 5-fold or greater in the MB sample compared to the OM and NM samples (MBE) Venn diagram showing the number of upregulated and downregulated (unshared and shared) genes when OM, MB and NM are compared. (<b>F)</b> Heat maps of genes upregulated and downregulated in MB samples compared to OM and NM samples.</p

Analysis of defects in MB formation.

<p><b>(A)</b> Diagram of experimental design. Nuclear DsRed-labeled 1205Lu cells were treated with siRNAs, mixed with untreated GFP-labeled 1205Lu cells, and plated under hESC conditions. (<b>B)</b> Imaging of MB formation. Merged image shows readout of red-green mixed (yellow dashed circle) versus green-only (green dashed circle) MBs. In the phase contrast image, the MBs corresponding to the merged images are indicated (white dashed circle). Right, FACS analysis was used to independently monitor cell growth or survival of green and red cells in hESC medium.</p