Transcription-associated DNA DSBs activate p53 during hiPSC-based neurogenesis.

Neurons are overproduced during cerebral cortical development. Neural progenitor cells (NPCs) divide rapidly and incur frequent DNA double-strand breaks (DSBs) throughout cortical neurogenesis. Although half of the neurons born during neurodevelopment die, many neurons with inaccurate DNA repair survive leading to brain somatic mosaicism. Recurrent DNA DSBs during neurodevelopment are associated with both gene expression level and gene length. We used imaging flow cytometry and a genome-wide DNA DSB capture approach to quantify and map DNA DSBs during human induced pluripotent stem cell (hiPSC)-based neurogenesis. Reduced p53 signaling was brought about by knockdown (p53KD); p53KD led to elevated DNA DSB burden in neurons that was associated with gene expression level but not gene length in neural progenitor cells (NPCs). Furthermore, DNA DSBs incurred from transcriptional, but not replicative, stress lead to p53 activation in neurotypical NPCs. In p53KD NPCs, DNA DSBs accumulate at transcription start sites of genes that are associated with neurological and psychiatric disorders. These findings add to a growing understanding of how neuronal genome dynamics are engaged by high transcriptional or replicative burden during neurodevelopment.

Topoisomerase II contributes to DNA secondary structure-mediated double-stranded breaks.

DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability.

Pausing sites of RNA polymerase II on actively transcribed genes are enriched in DNA double-stranded breaks.

DNA double-stranded breaks (DSBs) are strongly associated with active transcription, and promoter-proximal pausing of RNA polymerase II (Pol II) is a critical step in transcriptional regulation. Mapping the distribution of DSBs along actively expressed genes and identifying the location of DSBs relative to pausing sites can provide mechanistic insights into transcriptional regulation. Using genome-wide DNA break mapping/sequencing techniques at single-nucleotide resolution in human cells, we found that DSBs are preferentially located around transcription start sites of highly transcribed and paused genes and that Pol II promoter-proximal pausing sites are enriched in DSBs. We observed that DSB frequency at pausing sites increases as the strength of pausing increases, regardless of whether the pausing sites are near or far from annotated transcription start sites. Inhibition of topoisomerase I and II by camptothecin and etoposide treatment, respectively, increased DSBs at the pausing sites as the concentrations of drugs increased, demonstrating the involvement of topoisomerases in DSB generation at the pausing sites. DNA breaks generated by topoisomerases are short-lived because of the religation activity of these enzymes, which these drugs inhibit; therefore, the observation of increased DSBs with increasing drug doses at pausing sites indicated active recruitment of topoisomerases to these sites. Furthermore, the enrichment and locations of DSBs at pausing sites were shared among different cell types, suggesting that Pol II promoter-proximal pausing is a common regulatory mechanism. Our findings support a model in which topoisomerases participate in Pol II promoter-proximal pausing and indicated that DSBs at pausing sites contribute to transcriptional activation.

Myt1 and Myt1l transcription factors limit proliferation in GBM cells by repressing YAP1 expression.

Myelin transcription factor 1 (Myt1) and Myt1l (Myt1-like) are zinc finger transcription factors that regulate neuronal differentiation. Reduced Myt1l expression has been implicated in glioblastoma (GBM), and the related St18 was originally identified as a potential tumor suppressor for breast cancer. We previously analyzed changes in gene expression in a human GBM cell line with re-expression of either Myt1 or Myt1l. This revealed largely overlapping gene expression changes, suggesting similar function in these cells. Here we show that re-expression of Myt1 or Myt1l reduces proliferation in two different GBM cell lines, activates gene expression programs associated with neuronal differentiation, and limits expression of proliferative and epithelial to mesenchymal transition gene-sets. Consistent with this, expression of both MYT1 and MYT1L is lower in more aggressive glioma sub-types. Examination of the gene expression changes in cells expressing Myt1 or Myt1l suggests that both repress expression of the YAP1 transcriptional coactivator, which functions primarily in the Hippo signaling pathway. Expression of YAP1 and its target genes is reduced in Myt-expressing cells, and there is an inverse correlation between YAP1 and MYT1/MYT1L expression in human brain cancer datasets. Proliferation of GBM cell lines is reduced by lowering YAP1 expression and increased with YAP1 over-expression, which overcomes the anti-proliferative effect of Myt1/Myt1l expression. Finally we show that reducing YAP1 expression in a GBM cell line slows the growth of orthotopic tumor xenografts. Together, our data suggest that Myt1 and Myt1l directly repress expression of YAP1, a protein which promotes proliferation and GBM growth.

P190RhoGAP prevents mitotic spindle fragmentation and is required to activate Aurora A kinase at acentriolar poles.

Assembly of the mitotic spindle is essential for proper chromosome segregation during mitosis. Maintenance of spindle poles requires precise regulation of kinesin- and dynein-generated forces, and improper regulation of these forces disrupts pole integrity leading to pole fragmentation. The formation and function of the mitotic spindle are regulated by many proteins, including Aurora A kinase and the motor proteins Kif2a and Eg5. Here, we characterize a surprising role for the RhoA GTPase-activating protein, p190RhoGAP, in regulating the mitotic spindle. We show that cells depleted of p190RhoGAP arrest for long periods in mitosis during which cells go through multiple transitions between having bipolar and multipolar spindles. Most of the p190RhoGAP-depleted cells finally achieve a stable bipolar attachment and proceed through anaphase. The multipolar spindle phenotype can be rescued by low doses of an Eg5 inhibitor. Moreover, we show that p190RhoGAP-depleted multipolar cells localize Aurora A to all the poles, but the kinase is only activated at the two centriolar poles. Overall, our data identify an unappreciated connection between p190RhoGAP and the proteins that control spindle poles including Aurora A kinase and Eg5 that is required to prevent or correct spindle pole fragmentation.

Analysis of transcriptional activity by the Myt1 and Myt1l transcription factors.

Myt1 and Myt1l (Myelin transcription factor 1, and Myt1-like) are members of a small family of closely related zinc finger transcription factors, characterized by two clusters of C2HC zinc fingers. Both are widely expressed during early embryogenesis, but are largely restricted to expression within the brain in the adult. Myt1l, as part of a three transcription factor mix, can reprogram fibroblasts to neurons and plays a role in maintaining neuronal identity. Previous analyses have indicated roles in both transcriptional activation and repression and suggested that Myt1 and Myt1l may have opposing functions in gene expression. We show that when targeted to DNA via multiple copies of the consensus Myt1/Myt1l binding site Myt1 represses transcription, whereas Myt1l activates. By targeting via a heterologous DNA binding domain we mapped an activation function in Myt1l to an amino-terminal region that is poorly conserved in Myt1. However, genome wide analyses of the effects of Myt1 and Myt1l expression in a glioblastoma cell line suggest that the two proteins have largely similar effects on endogenous gene expression. Transcriptional repression is likely mediated by binding to DNA via the known consensus site, whereas this site is not associated with the transcriptional start sites of genes with higher expression in the presence of Myt1 or Myt1l. This work suggests that these two proteins function similarly, despite differences observed in analyses based on synthetic reporter constructs.

Genetic and Molecular Analyses indicate independent effects of TGIFs on Nodal and Gli3 in neural tube patterning.

Holoprosencephaly (HPE) is a prevalent craniofacial developmental disorder that has both genetic and environmental causes. The gene encoding TG-interacting factor 1 (TGIF1) is among those that are routinely screened in HPE patients. However, the mechanisms by which TGIF1 variants cause HPE are not fully understood. TGIF1 is a transcriptional repressor that limits the output of the Transforming Growth Factor ß (TGFß)/Nodal signaling pathway, and HPE in patients with TGIF1 variants has been suggested to be due to increased Nodal signaling. Mice lacking both Tgif1 and its paralog, Tgif2, have HPE, and embryos lacking Tgif function do not survive past mid-gestation. Here, we show that in the presence of a Nodal heterozygous mutation, proliferation defects are rescued and a proportion of embryos lacking all Tgif function survive to late gestation. However, these embryos have a classic HPE phenotype, suggesting that this is a Nodal-independent effect of Tgif loss of function. Further, we show that the Gli3 gene is a direct target for repression by Tgifs, independent of TGFß/Nodal signaling, consistent with Tgif mutations causing HPE via Nodal-independent effects on the Sonic Hedgehog (Shh) pathway. Based on this work, we propose a model for distinct functions of Tgifs in the Nodal and Shh/Gli3 pathways during forebrain development.

Synchronization of Yeast.

The budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe are amongst the simplest and most powerful model systems for studying the genetics of cell cycle control. Because yeast grows very rapidly in a simple and economical media, large numbers of cells can easily be obtained for genetic, molecular, and biochemical studies of the cell cycle. The use of synchronized cultures greatly aids in the ease and interpretation of cell cycle studies. In principle, there are two general methods for obtaining synchronized yeast populations. Block-and-release methods can be used to induce cell cycle synchrony. Alternatively, centrifugal elutriation can be used to select synchronous populations. Because each method has innate advantages and disadvantages, the use of multiple approaches helps in generalizing results. An overview of the most commonly used methods to generate synchronized yeast cultures is presented along with working Notes: a section that includes practical comments, experimental considerations and observations, and hints regarding the pros and cons innate to each approach.

A complex of p190RhoGAP-A and anillin modulates RhoA-GTP and the cytokinetic furrow in human cells.

The cytokinetic furrow is organized by the RhoA GTPase, which recruits actin and myosin II to the furrow and drives contractility. Here, we show that the RhoA GTPase-activting protein (GAP) p190RhoGAP-A (also known as ARHGAP35) has a role in cytokinesis and is involved in regulating levels of RhoA-GTP and contractility. Cells depleted of p190RhoGAP-A accumulate high levels of RhoA-GTP and markers of high RhoA activity in the furrow, resulting in failure of the cytokinetic furrow to progress to abscission. The loss of p190RhoGAP-A can be rescued by a low dose of the myosin II inhibitor blebbistatin, suggesting that cells fail cytokinesis because they have too much myosin activity. p190RhoGAP-A binds the cytokinetic organizer anillin, and mutants of p190RhoGAP-A that are unable to bind anillin or unable to inactivate RhoA fail to rescue cytokinesis defects in p190RhoGAP-A-depleted cells. Taken together, these data demonstrate that a complex of p190RhoGAP-A and anillin modulates RhoA-GTP levels in the cytokinetic furrow to ensure progression of cytokinesis.

FASTA barcodes: a simple method for the identification of yeast ORF deletions.

A consortium of yeast geneticists have created -6000 individual ORF deletions, representing > 96% of the currently verified or predicted ORFs in S. cerevisiae. Importantly, molecular barcodes (each a unique 20 bp sequence termed either Uptag or Downtag) were used as identifiers for every ORF deletion. Microarray analyses of pooled yeast deletions has been used to identify thousands of genes involved in general fitness, haploinsufficiency, drug resistance and DNA damage repair. However, application of this powerful technology requires considerable expense, expertise and specialized equipment. While standard PCR techniques and specifically designed PCR primers can be used to confirm that a given ORF is in fact deleted, this procedure cannot be used to identify unknown deletions. In theory, every ORF deletion could be determined by barcode sequencing. However, neither a consolidated barcode database nor a reliable search engine is currently available for this purpose. To address this need, we have adapted a FASTA sequence program that utilizes the unique barcode database to allow users to identify individual ORF deletions, based upon simple sequencing reactions of PCR amplifications of either Uptag or Downtag barcodes. In silico and practical testing of this application reveals that it is an inexpensive, reliable and reproducible method for rapidly identifying unknown deletions. This approach allows laboratories to conduct small- or large-scale genetic screens with pooled yeast deletion strains and identify or verify any ORF deletion without the need for microarray technology.

Synchronization of yeast.

The budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe are amongst the simplest and most powerful model systems for studying the genetics of cell cycle control. Because yeast grows very rapidly in simple and economical media, large numbers of cells can easily be obtained for genetic, molecular, and biochemical studies of the cell cycle. The use of synchronized cultures greatly aids in the ease and interpretation of cell cycle studies. In principle, there are two general methods for obtaining synchronized yeast populations. Block and release methods can be used to induce cell cycle synchrony. Alternatively, centrifugal elutriation can be used to select synchronous populations. Because each method has innate advantages and disadvantages, the use of multiple approaches helps in generalizing results. An overview of the most commonly used methods to generate synchronized yeast cultures is presented along with working Notes, a section that includes practical comments, experimental considerations and observations, and hints regarding the pros and cons innate to each approach.

Cell size and growth rate are major determinants of replicative lifespan.

Yeast cells, like mammalian cells, enlarge steadily as they age. Unabated cell growth can promote cellular senescence; however, the significance of the relationship between size and cellular lifespan is not well understood. Herein, we report a genetic link between cell size, growth rate and lifespan. Mutations that increase cell size concomitantly increase growth rate and decrease lifespan. As a result, large cells grow, divide and age dramatically faster than small cells. Conversely, small cell mutants age slowly and are long-lived. Investigation of the mechanisms involved suggests that attainment of a maximal size modulates lifespan. Indeed, cumulative results revealed that life expectancy is size-dependent, and that the rate at which cells age is determined in large part by the amount of cell growth per generation.

Gene regulation: global transcription rates scale with size.

Is bigger better? Scientists have long puzzled over the potential relationship between cell size and the rate of mRNA production. A recent report builds a strong case that global transcription rates scale with size.

Ccr4 alters cell size in yeast by modulating the timing of CLN1 and CLN2 expression.

Large, multisubunit Ccr4-Not complexes are evolutionarily conserved global regulators of gene expression. Deletion of CCR4 or several components of Ccr4-Not complexes results in abnormally large cells. Since yeast must attain a critical cell size at Start to commit to division, the large size of ccr4 delta cells implies that they may have a size-specific proliferation defect. Overexpression of CLN1, CLN2, CLN3, and SWI4 reduces the size of ccr4 delta cells, suggesting that ccr4 delta cells have a G(1)-phase cyclin deficiency. In support of this, we find that CLN1 and CLN2 expression and budding are delayed in ccr4 delta cells. Moreover, overexpression of CCR4 advances the timing of CLN1 expression, promotes premature budding, and reduces cell size. Genetic analyses suggest that Ccr4 functions independently of Cln3 and downstream of Bck2. Thus, like cln3 delta bck2 delta double deletions, cln3 delta ccr4 delta cells are also inviable. However, deletion of Whi5, a transcriptional repressor of CLN1 and CLN2, restores viability. We find that Ccr4 negatively regulates the half-life of WHI5 mRNAs, and we conclude that, by modulating the stability of WHI5 mRNAs, Ccr4 influences the size-dependent timing of G1-phase cyclin transcription.