Analysis by RNA-seq of transcriptomic changes elicited by heat shock in Leishmania major.

Besides their medical relevance, Leishmania is an adequate model for studying post-transcriptional mechanisms of gene expression. In this microorganism, mRNA degradation/stabilization mechanisms together with translational control and post-translational modifications of proteins are the major drivers of gene expression. Leishmania parasites develop as promastigotes in sandflies and as amastigotes in mammalians, and during host transmission, the parasite experiences a sudden temperature increase. Here, changes in the transcriptome of Leishmania major promastigotes after a moderate heat shock were analysed by RNA-seq. Several of the up-regulated transcripts code for heat shock proteins, other for proteins previously reported to be amastigote-specific and many for hypothetical proteins. Many of the transcripts experiencing a decrease in their steady-state levels code for transporters, proteins involved in RNA metabolism or translational factors. In addition, putative long noncoding RNAs were identified among the differentially expressed transcripts. Finally, temperature-dependent changes in the selection of the spliced leader addition sites were inferred from the RNA-seq data, and particular cases were further validated by RT-PCR and Northern blotting. This study provides new insights into the post-transcriptional mechanisms by which Leishmania modulate gene expression.

Chromatin conformation regulates the coordination between DNA replication and transcription.

Chromatin is the template for the basic processes of replication and transcription, making the maintenance of chromosomal integrity critical for cell viability. To elucidate how dividing cells respond to alterations in chromatin structure, here we analyse the replication programme of primary cells with altered chromatin configuration caused by the genetic ablation of the HMGB1 gene, or three histone H1 genes. We find that loss of chromatin compaction in H1-depleted cells triggers the accumulation of stalled forks and DNA damage as a consequence of transcription-replication conflicts. In contrast, reductions in nucleosome occupancy due to the lack of HMGB1 cause faster fork progression without impacting the initiation landscape or fork stability. Thus, perturbations in chromatin integrity elicit a range of responses in the dynamics of DNA replication and transcription, with different consequences on replicative stress. These findings have broad implications for our understanding of how defects in chromatin structure contribute to genomic instability.

Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major.

Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance.

Strand-specific, high-resolution mapping of modified RNA polymerase II.

Reversible modification of the RNAPII C-terminal domain links transcription with RNA processing and surveillance activities. To better understand this, we mapped the location of RNAPII carrying the five types of CTD phosphorylation on the RNA transcript, providing strand-specific, nucleotide-resolution information, and we used a machine learning-based approach to define RNAPII states. This revealed enrichment of Ser5P, and depletion of Tyr1P, Ser2P, Thr4P, and Ser7P in the transcription start site (TSS) proximal ~150 nt of most genes, with depletion of all modifications close to the poly(A) site. The TSS region also showed elevated RNAPII relative to regions further 3', with high recruitment of RNA surveillance and termination factors, and correlated with the previously mapped 3' ends of short, unstable ncRNA transcripts. A hidden Markov model identified distinct modification states associated with initiating, early elongating and later elongating RNAPII. The initiation state was enriched near the TSS of protein-coding genes and persisted throughout exon 1 of intron-containing genes. Notably, unstable ncRNAs apparently failed to transition into the elongation states seen on protein-coding genes.

R-loops and initiation of DNA replication in human cells: a missing link?

The unanticipated widespread occurrence of stable hybrid DNA/RNA structures (R-loops) in human cells and the increasing evidence of their involvement in several human malignancies have invigorated the research on R-loop biology in recent years. Here we propose that physiological R-loop formation at CpG island promoters can contribute to DNA replication origin specification at these regions, the most efficient replication initiation sites in mammalian cells. Quite likely, this occurs by the strand-displacement reaction activating the formation of G-quadruplex structures that target the origin recognition complex (ORC) in the single-stranded conformation. In agreement with this, we found that R-loops co-localize with the ORC within the same CpG island region in a significant fraction of these efficient replication origins, precisely at the position displaying the highest density of G4 motifs. This scenario builds on the connection between transcription and replication in human cells and suggests that R-loop dysregulation at CpG island promoter-origins might contribute to the phenotype of DNA replication abnormalities and loss of genome integrity detected in cancer cells.

High-resolution analysis of DNA synthesis start sites and nucleosome architecture at efficient mammalian replication origins.

DNA replication origins are poorly characterized genomic regions that are essential to recruit and position the initiation complex to start DNA synthesis. Despite the lack of specific replicator sequences, initiation of replication does not occur at random sites in the mammalian genome. This has lead to the view that DNA accessibility could be a major determinant of mammalian origins. Here, we performed a high-resolution analysis of nucleosome architecture and initiation sites along several origins of different genomic location and firing efficiencies. We found that mammalian origins are highly variable in nucleosome conformation and initiation patterns. Strikingly, initiation sites at efficient CpG island-associated origins always occur at positions of high-nucleosome occupancy. Origin recognition complex (ORC) binding sites, however, occur at adjacent but distinct positions marked by labile nucleosomes. We also found that initiation profiles mirror nucleosome architecture, both at endogenous origins and at a transgene in a heterologous system. Our studies provide a unique insight into the relationship between chromatin structure and initiation sites in the mammalian genome that has direct implications for how the replication programme can be accommodated to diverse epigenetic scenarios.