Fig 1: Under Proliferation conditions, Rest transcription is activated by MLL4, HAT and BRD proteins and negatively regulated minimally by PRC2 and demethylase, KDM7A, where we hypothesize it helps in chromatin recruitment of the repressive complexes. In contrast, during differentiation, the repressive complexes G9A and PRC2 play a major role in controlling Rest transcription. At the same time, a lower level of Rest expression is maintained by activating marks and the demethylases KDM6A and KDM7A. The level of action of the proteins are denoted by the boldness of the font, brighter denotes stronger activity and lighter font denotes lower activity.
Fig 2: KDM7A up- or down-regulation is associated with significantly poor survival in patient with REST-elevated SHH MBs. Hierarchical clustering analysis identified six distinct clusters based on expression profiles of neuronal differentiation markers in SHH MB patient samples (www.ncbi.nlm.nih.gov/geo; dataset GSE85217, Sci Signal. 2019). Vertical column corresponds to one individual with subtype (alpha, beta, gamma, and delta). Six-colored boxes represent the six-clusters. The bar graphs show the gene expression values (Z score) of each patient. (A) Differential expression of REST, MLL4, KDM6A, EZH2, G9A, GLP1, SUV39H1, KDM7A and BRD4 in the different clusters are shown. (B) KDM7A mRNA expression profile in SHH MB patient samples. Hierarchical clustering based on expression of neuronal differentiation markers divided the SHH MB patient samples into six distinct clusters (Cluster 1; n = 39, Cluster 2; n = 31, Cluster 3; n = 32, Cluster 4; n = 61, Cluster 5; n = 39, Cluster 6; n = 21). Each dot corresponds to an individual patient. Data show individual variability and means ± SD. P-values were obtained using the unpaired t-test with Welch’s correction. (C) Four patient groups based on REST and KDM7A expression levels. Overall survival of four patient groups based on REST and KDM7A in patients with SHH MB (P value; log-rank Mantel-Cox test).
Fig 3: MLL4 activity at the REST locus is diminished in differentiating CGNPs. (A) H3K4me1, 2 and 3 levels were measured by ChIP-qPCR and plotted as fold enrichment over IgG in differentiating progenitors. (B) ChIP-qPCR showing enrichment of MLL4 at the REST promoter represented as fold change over IgG. (C) H3K27Ac was measured by ChIP-qPCR in differentiating progenitors and plotted as fold enrichment over IgG (n = 4 for differentiating progenitors). (D) Western blot analysis of BRD4 protein in proliferating and differentiating progenitors and ACTIN as loading control. Blot shown is a representative from n=2. (E) ChIP-qPCR showing enrichment of BRD4 at the REST promoter represented as fold change over IgG. (F) Effect of BRD4 inhibition with JQ1 on REST mRNA was shown by qRT-PCR in DMSO and JQ1 treated progenitors in differentiating conditions (n = 2).
Fig 4: MLL4 activity is associated with transcriptional activation of REST in proliferating CGNPs. (A) Schematic of Rest locus in mouse showing H3K4me3 and H3K27me3 in the cerebellum and representing primer design strategy to include CDS and upstream promoter at -3kb, -2kb and -1kb. (B) H3K4me1,2 and 3 levels were measured by ChIP-qPCR and plotted as fold enrichment over IgG in proliferating progenitors. (C) ChIP-qPCR showing enrichment of MLL4 at the Rest promoter represented as fold change over IgG. (D) Western blot analysis of MLL4 protein and Actin from proliferating and differentiating progenitors. Shown is a representative blot from n = 2. (E) H3K27Ac was measured by ChIP-qPCR in proliferating progenitors and plotted as fold enrichment over IgG (n = 4 for proliferating progenitors). (F) Effect of BRD4 inhibition with JQ1 on Rest mRNA was shown by qRT-PCR in DMSO and JQ1 treated progenitors in proliferating and differentiating conditions (n = 2).
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