Numerous pieces of evidence support the complex, 3D spatial organization of the genome dictates gene expression. CTCF is essential to define topologically associated domain boundaries and to facilitate the formation of insulated chromatin loop structures. To understand CTCF’s direct role in global transcriptional regulation, we integrated the miniAID-mClover3 cassette to the endogenous CTCF locus in a human pediatric B-ALL cell line, SEM, and an immortal erythroid precursor cell line, HUDEP-2, to allow for acute depletion of CTCF protein by the auxin-inducible degron system. In SEM cells, CTCF loss notably disrupted intra-TAD loops and TAD integrity in concurrence with a reduction in CTCF-binding affinity, while showing no perturbation to nuclear compartment integrity. Strikingly, the overall effect of CTCF’s loss on transcription was minimal. Whole transcriptome analysis showed hundreds of genes differentially expressed in CTCF-depleted cells, among which MYC and a number of MYC target genes were specifically downregulated. Mechanically, acute depletion of CTCF disrupted the direct interaction between the MYC promoter and its distal enhancer cluster residing ∼1.8 Mb downstream. Notably, MYC expression was not profoundly affected upon CTCF loss in HUDEP-2 cells suggesting that CTCF could play a B-ALL cell line specific role in maintaining MYC expression.
We report our progress on the development of new synthetic anti-cancer lead compounds that modulate the splicing of mRNA. We also report the synthesis evaluation of new biologically active ester and carbamate analogs. Further, we describe initial animal studies demonstrating the antitumor efficacy of compound 5 in vivo. Additionally, we report the enantioselective and diastereospecific synthesis of a new 1,3-dioxane series of active analogs. We confirm that compound 5 inhibits the splicing of mRNA in both cell-free nuclear extracts and in a cell-based dual-reporter mRNA splicing assay. In summary, we have developed totally synthetic novel spliceosome modulators as therapeutic lead compounds for a number of highly aggressive cancers. Future efforts will be directed toward the more complete optimization of these compounds as potential human therapeutics.
IMP dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo synthesis of GTP. Yeast with mutations in the transcription elongation machinery are sensitive to inhibitors of this enzyme such as 6-azauracil and mycophenolic acid, at least partly because of their inability to transcriptionally induce IMPDH. To understand the molecular basis of this drug-sensitive phenotype, we have dissected the expression and function of a four-gene family in yeast called IMD1 through IMD4. We show here that these family members are distinct, despite a high degree of amino acid identity between the proteins they encode. Extrachromosomal copies of IMD1, IMD3, or IMD4 could not rescue the drug-sensitive phenotype of IMD2 deletants. When overexpressed, IMD3 or IMD4 weakly compensated for deletion of IMD2. IMD1 is transcriptionally silent and bears critical amino acid substitutions compared with IMD2 that destroy its function, offering strong evidence that it is a pseudogene. The simultaneous deletion of all four IMD genes was lethal unless growth media were supplemented with guanine. This suggests that there are no other essential functions of the IMPDH homologs aside from IMP dehydrogenase activity. Although neither IMD3 nor IMD4 could confer drug resistance to cells lacking IMD2, either alone was sufficient to confer guanine prototrophy. The special function of IMD2 was provided by its ability to be transcriptionally induced and the probable intrinsic drug resistance of its enzymatic activity.
Summary The most aggressive of four medulloblastoma (MB) subgroups are cMyc-driven Group 3 (G3) tumors, some of which overexpress EZH2, the histone H3K27 mono-, di- and trimethylase of polycomb repressive complex 2. Ezh2 has a context dependent role in different cancers as an oncogene or tumor suppressor and retards tumor progression in a mouse model of G3 MB. Engineered deletions of Ezh2 in G3 MBs by gene editing nucleases accelerated tumorigenesis, whereas Ezh2 re-expression reversed attendant histone modifications and slowed tumor progression. Candidate oncogenic drivers suppressed by Ezh2 included Gfi1, a proto-oncogene frequently activated in human G3 MBs. Gfi1 disruption antagonized the tumor promoting effects of Ezh2 loss; conversely, Gfi1 overexpression collaborated with Myc to bypass effects of Trp53 inactivation in driving MB progression in primary cerebellar neuronal progenitors. Although negative regulation of Gfi1 by Ezh2 may restrain MB development, Gfi1 activation can bypass these effects.
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