Genomic Insights into the Different Layers of Gene Regulation in Yeast

The model organism Saccharomyces cerevisiae has allowed the development of new functional genomics techniques devoted to the study of transcription in all its stages. With these techniques, it has been possible to find interesting new mechanisms to control gene expression that act at different levels and for different gene sets apart from the known cis-trans regulation in the transcription initiation step. Here we discuss a method developed in our laboratory, Genomic Run-On, and other new methods that have recently appeared with distinct technical features. A comparison between the datasets generated by them provides interesting genomic insights into the different layers of gene regulation in eukaryotes

Defects in the NC2 repressor affect both canonical and non-coding RNA polymerase II transcription initiation in yeast.

BACKGROUND: The formation of the pre-initiation complex in eukaryotic genes is a key step in transcription initiation. The TATA-binding protein (TBP) is a universal component of all pre-initiation complexes for all kinds of RNA polymerase II (RNA pol II) genes, including those with a TATA or a TATA-like element, both those that encode proteins and those that transcribe non-coding RNAs. Mot1 and the negative cofactor 2 (NC2) complex are regulators of TBP, and it has been shown that depletion of these factors in yeast leads to defects in the control of transcription initiation that alter cryptic transcription levels in selected yeast loci. RESULTS: In order to cast light on the molecular functions of NC2, we performed genome-wide studies in conditional mutants in yeast NC2 essential subunits Ydr1 and Bur6. Our analyses show a generally increased level of cryptic transcription in all kinds of genes upon depletion of NC2 subunits, and that each kind of gene (canonical or ncRNAs, TATA or TATA-like) shows some differences in the cryptic transcription pattern for each NC2 mutant. CONCLUSIONS: We conclude that NC2 plays a general role in transcription initiation in RNA polymerase II genes that is related with its known TBP interchange function from free to promoter bound states. Therefore, loss of the NC2 function provokes increases in cryptic transcription throughout the yeast genome. Our results also suggest functional differences between NC2 subunits Ydr1 and Bur6

Aberrations of Genomic Imprinting in Glioblastoma Formation

In human glioblastoma (GBM), the presence of a small population of cells with stem cell characteristics, the glioma stem cells (GSCs), has been described. These cells have GBM potential and are responsible for the origin of the tumors. However, whether GSCs originate from normal neural stem cells (NSCs) as a consequence of genetic and epigenetic changes and/or dedifferentiation from somatic cells remains to be investigated. Genomic imprinting is an epigenetic marking process that causes genes to be expressed depending on their parental origin. The dysregulation of the imprinting pattern or the loss of genomic imprinting (LOI) have been described in different tumors including GBM, being one of the earliest and most common events that occurs in human cancers. Here we have gathered the current knowledge of the role of imprinted genes in normal NSCs function and how the imprinting process is altered in human GBM. We also review the changes at particular imprinted loci that might be involved in the development of the tumor. Understanding the mechanistic similarities in the regulation of genomic imprinting between normal NSCs and GBM cells will be helpful to identify molecular players that might be involved in the development of human GBM

The mRNA degradation factor Xrn1 regulates transcription elongation in parallel to Ccr4

Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4–Not complex conditions its posttranscriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 action in gene transcription. This double strategy brought us to conclude that both Xrn1-decaysome and Ccr4–Not regulate RNAPII elongation, and that they do it in parallel. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII. We found that xrn1Δ and ccr4Δ exhibited very different patterns of TFIIS versus RNAPII occupancy, which confirmed their distinct role in controlling transcription elongation. We also found that the relative influence of Xrn1 and Ccr4 is different in the genes encoding ribosomal proteins as compared to the rest of the genome

Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning.

BACKGROUND: TFIIS stimulates RNA cleavage by RNA polymerase II and promotes the resolution of backtracking events. TFIIS acts in the chromatin context, but its contribution to the chromatin landscape has not yet been investigated. Co-transcriptional chromatin alterations include subtle changes in nucleosome positioning, like those expected to be elicited by TFIIS, which are elusive to detect. The most popular method to map nucleosomes involves intensive chromatin digestion by micrococcal nuclease (MNase). Maps based on these exhaustively digested samples miss any MNase-sensitive nucleosomes caused by transcription. In contrast, partial digestion approaches preserve such nucleosomes, but introduce noise due to MNase sequence preferences. A systematic way of correcting this bias for massively parallel sequencing experiments is still missing. RESULTS: To investigate the contribution of TFIIS to the chromatin landscape, we developed a refined nucleosome-mapping method in Saccharomyces cerevisiae. Based on partial MNase digestion and a sequence-bias correction derived from naked DNA cleavage, the refined method efficiently mapped nucleosomes in promoter regions rich in MNase-sensitive structures. The naked DNA correction was also important for mapping gene body nucleosomes, particularly in those genes whose core promoters contain a canonical TATA element. With this improved method, we analyzed the global nucleosomal changes caused by lack of TFIIS. We detected a general increase in nucleosomal fuzziness and more restricted changes in nucleosome occupancy, which concentrated in some gene categories. The TATA-containing genes were preferentially associated with decreased occupancy in gene bodies, whereas the TATA-like genes did so with increased fuzziness. The detected chromatin alterations correlated with functional defects in nascent transcription, as revealed by genomic run-on experiments. CONCLUSIONS: The combination of partial MNase digestion and naked DNA correction of the sequence bias is a precise nucleosomal mapping method that does not exclude MNase-sensitive nucleosomes. This method is useful for detecting subtle alterations in nucleosome positioning produced by lack of TFIIS. Their analysis revealed that TFIIS generally contributed to nucleosome positioning in both gene promoters and bodies. The independent effect of lack of TFIIS on nucleosome occupancy and fuzziness supports the existence of alternative chromatin dynamics during transcription elongation

Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5ʹ exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3ʹ was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.Ministerio de Economía y Competitividad BFU2016-77728- C3-1-P, BFU2016-77728-C3-3-P, BFU2016- 77728-C3-2-P, RED2018-102467-TJunta de Andalucía BIO271, US-1256285, BIO258, UJA 1260360Generalitat Valenciana AICO/2019/08

The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manne

Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5ʹ exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3ʹ was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.This work has been supported by grants from the Ministerio de Economía, Industria y Competitividad – Agencia Estatal de Investigación, and European Union funds (FEDER) [BFU2016-77728-C3-1-P to S. C.],[BFU2016-77728-C3-3-P to J.E.P-O & P.A], [BFU2016-77728-C3-2-P to F.N.] and RED2018‐102467‐T to J.E.P-O, F.N. and S.C.; by FPI grants from the Spanish Government to V.B and A.C-B; by the Regional Andalusian Government [BIO271 and US-1256285 to S. C.], [BIO258 and FEDER-UJA 1260360 to F.N.] and from the Regional Valencian Government [AICO/2019/088 to J.E.P-O]. Funding for open access charge: [BFU2016-77728-C3-1-P]