Question

Please explain figures 6C, 6D, 6F, and 6G in simple terms! I am having difficulty understanding the figures. I have copy and pasted a section in the article. Thank you!!

Figure 6. MLL5 Suppresses Differentiation of GBM Self-Renewing Cells

(A) Western blot to assess the effects of MLL5 knockdown in the GBM primary culture G432NS.
(B) Western blots on cell lysates from two GBM cultures differentiated over a 21-day period.

(C) Heatmap and unsupervised hierarchical clustering of differentially regulated genes (p < 0.05, < 1.2-fold or >1.2-fold) between MLL5 overexpressing cells and EGFP-expressing control cells (n = 3 biological replicates for each treatment).

(D) Gene ontology analysis of genes downregulated in MLL5 overexpressing cells.

(E) Gene set enrichment analysis of genes differentially regulated in MLL5 overexpressing cells. NES, normalized enrichment score. FDR, false discovery rate.

(F) Expression levels of NEUROD4 based on gene expression arrays.
(G) Expression levels of MIRLET72A based on gene expression arrays.

MLL5 Suppresses Differentiation of GBM Self-Renewing Cells
We next investigated whether the changes in epigenetic marks and chromatin structure mediated by MLL5 functionally resulted in alterations of the self-renewal and tumorigenic potential of GBM primary cultures. We observed rapid and increased expression of the astrocytic marker glial fibrillary acidic protein (GFAP) upon knockdown of MLL5 (Figure 6A), supporting the notion that MLL5 may contribute to the neoplastic phenotype by suppressing differentiation. In a complementary experiment, directed differentiation by growth factor withdrawal in two patient-derived GBM primary cultures, G411NS and G432NS, was associated with a significant decrease of MLL5 levels coin- cident with upregulation of GFAP and/or the neuronal marker bIII-tubulin (TUBB3) (Figure 6B). Importantly, as MLL5 levels decreased during induced differentiation, H3K4me3 levels increased, consistent with our studies above (Figures 3B and 6A). Interestingly, we observed increased H3.3 levels with GBM differentiation, reminiscent of what is observed with H3.3 changes during normal brain development (Pin ̃a and Suau, 1987) and neural differentiation (Hake et al., 2006), supporting the notion that appropriate levels of H3.3 protein incorporation into chromatin are required for cellular differentiation.

To gain insight into the suppression of neural differentiation mediated by MLL5, we overexpressed MLL5 in G514NS and performed gene expression array analysis (Figure S5A). EGFP- expressing G514NS cells were used as controls and 252 genes were differentially expressed between MLL5 overexpressing and control cells (p < 0.05, >1.2-fold change), with more than two- thirds of them being repressed (Figure 6C). Gene ontology analysis of the genes downregulated in MLL5-overexpressing cells showed significant enrichment for genes involved in mmune response (Figures 6D and S5B). Gene set enrichment analysis specifically showed suppression of interferon signaling-related genes (Figure 6E). Interestingly, interferon signaling was previously shown to be a potent inducer of neural differentiation in neural stem and progenitor cells (Kim et al., 2007; Wong et al., 2004) and to induce differentiation and sup- press the malignant phenotype of neuroblastoma cells (Cinatl et al., 2002). Furthermore, MLL5 overexpression caused signifi- cant downregulation of NEUROD4 (Figure 6F), a master regu- lator of neurogenesis (Lee and Pfaff, 2003), and upregulation of a miRNA belonging to the LET-7 family (Figure 6G), which is known to suppress neurogenesis (Cimadamore et al., 2013).

А NT shRNA ShMLL5 #3 shMLL5 #4 G411NS G432NS Time (days) 0 7 1416 18 21 0 7 1416 18 21 MLL5E АСТВ 1.0 0.3 0.7 MLL5 H3K4me3 АСТВ GFAP АСТВ H3K4me3 H3.3 Total H3 TUBB3 GFAPE ACTBL MLL5 OE GENES DOWN IN MLL5 OE CELLS Gene count 0 10 20 Immune response Antigen processing/ presentation MHC class I protein complex MHC class I receptor activity 10-1110-9 10-710-5 Benjamini EGFP Normalized log2 values -1.761 +1.76 m INTERFERON SIGNALLING Enrichment score (ES) NEUROD4 expression MIRLET7A2 expression> -0.7 NES -3.665 p=0 FDR = 0 EGFP MLL5 OE EGFP MLL5 OE

Answer 1

6C)

This is a heatmap that shows the genes that are upregulated or downregulated when MLL5 is Overexpressed compared to cells expressing EGFP. The data shown here is from 3 replicates, and the clustering of genes (which gene is placed next to which gene is unsupervised). MLL5 Overexpression suppresses the expression of a large fraction of genes by 2-3 fold while increasing the expression of some genes 2-3 fold.

6D)

This figure shows gene pathways/gene groups that are downregulated in MLL5 overexpression. The red dots labeled Benjamini is a p-value correction, that adjusts data to account for the False Discovery Rate (False +ve error correction). The various gene classes shown on the y-axis are downregulated when MLL5 is overexpressed.

6F-6G)

Both these figures show a comparative analysis of the expression level of one gene in the Wild-type and MLL5 overexpression genotype. The data shown is in triplicates, such that gene expression levels for NEUROD4 (6F) and MIRLET7A2 (6G) were assayed three times in wild type cells and three times in MLL5 overexpression cells.

NEUROD4 expression is suppressed in MLL5 overexpression. NEUROD4 is a regulator of Neurogenesis and the downregulation of NEUROD4 indicates that MLL5 overexpression is inhibiting the differentiation of cells into Neuronal cells.

On the other hand, MIRLET7A2 is an miRNA species that represses neurognesis. This miRNA is a repressor of Neurogenesis and the overepression of this RNA species again allows MLL5 Overexpression cells to escape differentiation.