SUMMARY Recent studies have demonstrated that MyoD initiates a feed-forward regulation of skeletal muscle gene expression, predicting that MyoD binds directly to many genes expressed during differentiation. We have used chromatin immunoprecipitation and high throughput sequencing to identify genome-wide binding of MyoD in several skeletal muscle cell types. As anticipated, MyoD preferentially binds to a VCASCTG sequence that resembles the in vitro selected site for a MyoD:E-protein heterodimer, and MyoD binding increases during differentiation at many of the regulatory regions of genes expressed in skeletal muscle. Unanticipated findings were that MyoD was constitutively bound to thousands of additional sites in both myoblasts and myotubes, and that the genome-wide binding of MyoD was associated with regional histone acetylation. Therefore, in addition to regulating muscle gene expression, MyoD binds genome-wide and has the ability to broadly alter the epigenome in myoblasts and myotubes.

The joining of different genomes in allotetraploids played a major role in plant evolution, but the molecular implications of this event are poorly understood. In synthetic allotetraploids of Arabidopsis and Cardaminopsis arenosa, we previously demonstrated the occurrence of frequent gene silencing. To explore the involvement of epigenetic phenomena, we investigated the occurrence and effects of DNA methylation changes. Changes in DNA methylation patterns were more frequent in synthetic allotetraploids than in the parents. Treatment with 5-aza-2Ј-deoxycytidine, an inhibitor of DNA methyltransferase, resulted in the development of altered morphologies in the synthetic allotetraploids, but not in the parents. We profiled mRNAs in control and 5-aza-2Ј-deoxycytidine-treated parents and allotetraploids by amplified fragment length polymorphism-cDNA. We show that DNA demethylation induced and repressed two different transcriptomes. Our results are consistent with the hypothesis that synthetic allotetraploids have compromised mechanisms of epigenetic gene regulation.Allotetraploids are formed by hybridization between two species and inherit a complete diploid set of chromosomes from each parental species. Although many established wild and cultivated allopolyploids are fertile, well adapted, and genetically stable, allopolyploids of more recent origin commonly display genomic and phenotypic instability (Soltis and Soltis, 1995;Pikaard, 1999; Comai, 2000).As a consequence of the union of two genomes, abnormal phenotypes have been reported (Comai, 2000;Schranz and Osborn, 2000). The causes of these phenotypes are largely unknown. McClintock (1984) described similar phenomena as "genomic shock," which she defined as a preprogrammed response to an unusual challenge resulting in extensive restructuring of the genome. This "unusual challenge" may involve epigenetic gene silencing, which results from homologous DNA-DNA or DNA-RNA interactions. The hybridization of redundant and diverged homeologous sets of genes in allopolyploids might trigger widespread gene silencing and changes in chromatin structure and DNA methylation patterns.Recent molecular data are consistent with the gene silencing hypothesis. Previously, we have reported about 1% changes in gene expression by comparing synthetic allotetraploids derived by hybridizing 4x Arabidopsis and 4x Cardaminopsis arenosa (also known as Arabidopsis arenosa; Comai et al., 2000). These changes can involve both normal genes and genes related to transposons. The corresponding natural allotetraploid, Arabidopsis suecica, was examined by Lee and Chen (2001), who demonstrated similar silencing levels. Furthermore, they found that silencing was related to methylation and could be reversed by treatment with the DNA demethylating agent 5-aza-2Ј-deoxycytidine (azadC). Instability can also be manifested by genomic rearrangements. Synthetic hybrids of wheat (Triticum aestivum) displayed rapid and widespread loss of DNA sequences and changes in DNA methylation Shaked et al., 2001). These...

SUMMARY To identify therapeutic targets for Glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 "knockout" (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit Cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, likely as a result of oncogenic signaling, causing GBM-specific lethality.

Differential genomic targeting of the transcription factor TAL1 in alternate haematopoietic lineagesExpression of the basic helix-loop-helix transcription factor TAL1/SCL is required for erythrocyte differentiation; aberrant expression in lymphoid cells leads to oncogenic transformation. Here, global analysis of TAL1 binding in erythroid and malignant T cells identifies cell type specific functional interaction with the transcription factors RUNX and ETS1.

To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancerspecific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 39 splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies.