Alon Kahana

The ends of chromosomes in Saccharomyces cerevisiae initiate a repressive chromatin structure that spreads internally and inhibits the transcription of nearby genes, a phenomenon termed telomeric silencing. To investigate the molecular basis of this process, we carried out a genetic screen to identify genes whose overexpression disrupts telomeric silencing. We thus isolated 10 DOT genes (disruptor of telomeric silencing). Among these were genes encoding chromatin component Sir4p, DNA helicase Dna2p, ribosomal protein L32, and two proteins of unknown function, Asf1p and Ifh1p. The collection also included genes that had not previously been identified: DOT1, DOT4, DOT5, DOT6, and TLC1, which encodes the RNA template component of telomerase. With the exception of TLC1, all these genes, particularly DOT1 and DOT4, also reduced silencing at other repressed loci (HM loci and rDNA) when overexpressed. Moreover, deletion of the latter two genes weakened silencing as well, suggesting that DOT1 and DOT4 normally play important roles in gene repression. DOT1 deletion also affected telomere tract length. The function of Dot1p is not known. The sequence of Dot4p suggests that it is a ubiquitin-processing protease. Taken together, the DOT genes include both components and regulators of silent chromatin.

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.

Intra-tumoral heterogeneity is a hallmark of glioblastoma that challenges treatment efficacy. However, the mechanisms that set up tumor heterogeneity and tumor cell migration remain poorly understood. Herein, we present a comprehensive spatiotemporal study that aligns distinctive intra-tumoral histopathological structures, oncostreams, with dynamic properties and a specific, actionable, spatial transcriptomic signature. Oncostreams are dynamic multicellular fascicles of spindle-like and aligned cells with mesenchymal properties, detected using ex vivo explants and in vivo intravital imaging. Their density correlates with tumor aggressiveness in genetically engineered mouse glioma models, and high grade human gliomas. Oncostreams facilitate the intra-tumoral distribution of tumoral and non-tumoral cells, and potentially the collective invasion of the normal brain. These fascicles are defined by a specific molecular signature that regulates their organization and function. Oncostreams structure and function depend on overexpression of COL1A1. Col1a1 is a central gene in the dynamic organization of glioma mesenchymal transformation, and a powerful regulator of glioma malignant behavior. Inhibition of Col1a1 eliminates oncostreams, reprograms the malignant histopathological phenotype, reduces expression of the mesenchymal associated genes, induces changes in the tumor microenvironment and prolongs animal survival. Oncostreams represent a pathological marker of potential value for diagnosis, prognosis, and treatment.

In the anterior vertebrate head, a population of neural crest cells (NCCs) migrates to the periocular mesenchyme and makes critical contributions to the developing eye and orbit. Improper migration and differentiation of these NCCs have been implicated in human diseases such as congenital glaucoma and anterior segment dysgenesis syndromes. The mechanisms by which these cells migrate to their target tissues within and around the eye are not well understood. We present a fate map of zebrafish diencephalic and mesencephalic NCC contributions to the eye and orbit. The fate map closely resembles that in chick and mice, demonstrating evolutionary conservation. To gain insight into the mechanisms of anterior NCC guidance, we used the eyeless mutant chokh/rx3. We show that, in chokh mutants, dorsal anterior NCC migration is severely disorganized. Time-lapse analysis shows that NCCs have significantly reduced migration rates and directionality in chokh mutants.