Nuclear functions of prefoldin

Prefoldin is a cochaperone, present in all eukaryotes, that cooperates with the chaperonin CCT. It is known mainly for its functional relevance in the cytoplasmic folding of actin and tubulin monomers during cytoskeleton assembly. However, both canonical and prefoldin-like subunits of this heterohexameric complex have also been found in the nucleus, and are functionally connected with nuclear processes in yeast and metazoa. Plant prefoldin has also been detected in the nucleus and physically associated with a gene regulator. In this review, we summarize the information available on the involvement of prefoldin in nuclear phenomena, place special emphasis on gene transcription, and discuss the possibility of a global coordination between gene regulation and cytoplasmic dynamics mediated by prefoldin.Ministerio de Economía y Competitividad, BFU-2010- 21975-C03-03Junta de Andalucía 08-CVI-03508Andalucía, Junta de Andalucía P12-BIO-1938

Use of Arctium lappa Extract Against Acetaminophen-Induced Hepatotoxicity in Rats

AbstractBackgroundSevere destructive hepatic injuries can be induced by acetaminophen overdose and may lead to acute hepatic failure.ObjectiveTo investigate the ameliorative effects of Arctium lappa root extract on acetaminophen-induced hepatotoxicity.MethodsRats were divided into 4 groups: normal control group, Arctium lappa extract group, acetaminophen-injected group, and acetaminophen treated with Arctium lappa extract group.ResultsThe treatment with Arctium lappa extract reduced serum alanine transaminase, aspartate aminotransferase, and alkaline phosphatase in the acetaminophen group when compared with the control group. DNA fragments in the acetaminophen-injected group were also significantly increased (P < 0.05). The comet assay revealed increased detaching tail length and DNA concentration during the hepatic toxicity in the acetaminophen group. The malondialdehyde content was inhibited by Arctium lappa treatment (12.97±0.89 nmol/mg) when compared with the acetaminophen-treated-only group (12.97±0.89 nmol/mg). Histopathologic examination revealed that acetaminophen administration produced hepatic cell necrosis, infiltrate of lymphocytes, and vacuolation that were associated with the acetaminophen-treated animal group, but the degree of acetaminophen-induced hepatotoxicity was mediated by treatment with Arctium lappa extract.ConclusionsArctium lappa can prevent most of the hepatic tissue damage caused by acetaminophen overdose in rats

The ribosome assembly gene network is controlled by the feedback regulation of transcription elongation

Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the upregulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesismutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.Ministerio de Economía y Competitividad BFU2013-48643-C3-1-P, BFU2016-77728-C3-1-P, BFU2013-48643-C3- 3-P, BFU2013-42958-PJunta de Andalucía P12-BIO1938MO, P08-CVI-03508Comunidad Valenciana 2015/00

RNA Binding by Histone Methyltransferases Set1 and Set2.

Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional posttranscriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity

One step back before moving forward: regulation of transcription elongation by arrest and backtracking

RNA polymerase II backtracking is a well-known phenomenon, but its involvement in gene regulation is yet to be addressed. Structural studies into the backtracked complex, new reactivation mechanisms and genome-wide approaches are shedding some light on this interesting aspect of gene transcription. In this review, we briefly summarise these new findings, comment about some results recently obtained in our laboratory, and propose a new model for the influence of the chromatin context on RNA polymerase II backtracking.Ministerio de Economía y Competitividad de España. BFU2007-67575-C03-02 y BFU-2010-21975-C03-03Junta de Andalucía. P07-CVI-02623 y P08-CVI-0350

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

Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control.

N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia

Chromatin Reassembly Factors Are Involved in Transcriptional Interference Promoting HIV Latency▿ †

The establishment of a stable reservoir of latently infected cells allows HIV to persist in the host. Usually, HIV infection of T cells results in integration of the viral genome, with a preference for regions in the human genome containing active genes, viral expression, and production of new viruses. However, in rare cases T cells become latently infected, and this is presumed to be due to a combination of two factors: integrated viruses are not efficiently transcribed and infected T cells revert to a resting memory state. HIV latency has been associated with provirus integration in regions of constitutive heterochromatin, gene deserts, or very highly expressed genes. We have investigated the transcriptional consequences of latent HIV integration into cellular genes and the involvement of chromatin reassembly factors (CRFs) in the transcriptional interference that a host gene exerts on the integrated cryptic HIV promoter. Chimeric transcripts containing sequences from the host gene and HIV can be detected, having been initiated at promoters of either the cell or the virus. Reactivation of HIV downregulates host gene expression. Cryptic promoters might remain inactive due to the repressive chromatin configuration established by CRFs during transcription elongation. Depletion of CRFs such as Spt6, Chd1, and FACT, or the histone chaperones ASF1a and HIRA, promoted HIV reactivation, concomitantly with chromatin relaxation and a decrease in general RNA polymerase activity. Overall, our results indicate that CRFs play a role in maintaining HIV latency by transcriptional interference when the provirus is integrated into an intron of a highly active gene

The Prefolding Complex Regulates Chromatin Dynamics during Transcriptional Elongation

Trabajo presentado en la 26th International Conference on Yeast Genetics and Molecular Biology, celebrada en Frankfurt (Alemania) del 29 de agosto al 3 de septiembre de 2013Peer Reviewe

Topoisomerase IIα represses transcription by enforcing promoter-proximal pausing

Accumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) and needs to be resolved by DNA topoisomerases to sustain productive transcriptional elongation. Topoisomerases are therefore considered positive facilitators of transcription. Here, we show that, in contrast to this general assumption, human topoisomerase IIα (TOP2A) activity at promoters represses transcription of immediate early genes such as c-FOS, maintaining them under basal repressed conditions. Thus, TOP2A inhibition creates a particular topological context that results in rapid release from promoter-proximal pausing and transcriptional upregulation, which mimics the typical bursting behavior of these genes in response to physiological stimulus. We therefore describe the control of promoter-proximal pausing by TOP2A as a layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription, possibly by regulating the accumulation of DNA supercoiling at promoter regions.This work was funded with grants from the Spanish and Andalusian governments (SAF2017-89619-R, CVI-7948, and European Regional Development Fund) and the European Research Council (ERC-CoG-2014-647359), and with individual fellowships for A.H.-R. (Contratos para la Formación de Doctores, BES-2015-071672, and Ministerio de Economía y Competitividad); S.J.-G. (Ramó n y Cajal, RYC-2015-17246, and Ministerio de Economía y Competitividad); J.T.-B. (Formación Profesorado Universitario, FPU15/03656, and Ministerio de Educación, Cultura y Deporte); and G.M.-Z. (AECC Postdoctoral Fellowships). CABIMER is supported by the Andalusian Government.Ye