Experimental and Bioinformatic upgrade for genome-wide mapping of nucleosomes in Trypanosoma cruzi

In Trypanosoma cruzi, as in every eukaryotic cell, DNA is packaged into chromatin by octamers of histone proteins that constitute nucleosomes. Besides compacting DNA, nucleosomes control DNA dependent processes by modulating the access of DNA binding proteins to regulatory elements on the DNA; or by providing the platform for additional layers of regulation given by histone variants and histone post-translational modifications. In trypanosomes, protein coding genes are constitutively transcribed as polycistronic units. Therefore, gene expression is controlled mainly post transcriptionally. However, chromatin organization and the histone code influence transcription, cell cycle progression, replication and DNA repair. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities of these processes in trypanosomes. Digestion of chromatin with micrococcal nuclease followed by deep sequencing has been widely applied for genome-wide mapping of nucleosomes in several organisms. Nonetheless, this parasite presents numerous singularities. On one hand, special growth conditions and cell manipulation are required. On the other hand, chromatin organization shows some uniqueness that demands a specially designed analytical approach. An additional entanglement is given by the nature of its genome harboring a large content of repetitive sequences and the poor quality of the genome assembly and annotation of many strains. Here, we adapted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we developed an exhaustive and thorough computational workflow for data analysis, highlighting the relevance of using its whole genome as a reference instead of the commonly used Esmeraldo-like haplotype. Moreover, the performance of two aligners, Bowtie2 and HISAT2 was tested to find the most appropriate tool to map any genomic read to reference genomes bearing this complexity.

The MuvB Complex Binds and Stabilizes Nucleosomes Downstream of the Transcription Start Site of Cell-Cycle Dependent Genes

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene-expression.

The chromatin remodeler Ino80 mediates alternative RNAPII pausing site determination

Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step in early transcription elongation for the precise regulation of gene expression. Here, we provide evidence of promoter-proximal pausing-like distributions of RNAPII in S. cerevisiae. We found that genes bearing an alternative pausing site utilize Ino80p to properly localize RNAPII pausing at the first pausing site and to suppress the accumulation of RNAPII at the second pausing site, which is tightly associated with the +1 nucleosome. This alternative pausing site determination was dependent on the remodeling activity of Ino80p to modulate the +1 nucleosome position and might be controlled synergistically with Spt4p. Furthermore, we observed similar Ino80-dependent RNAPII pausing in mouse embryonic stem cells (mESCs). Based on our collective results, we hypothesize that the chromatin remodeler Ino80 plays a highly conserved role in regulating early RNAPII elongation to establish intact pausing.

Nucleosome positioning on episomal human papillomavirus DNA in cultured cells

Several human papillomaviruses (HPV) are associated with the development of cervical carcinoma. HPV DNA synthesis is increased during the differentiation of infected host keratinocytes as they migrate from the basal layer of the epithelium to the spinous layer, but the molecular mechanism is unclear. Nucleosome positioning affects various cellular processes such as DNA replication and repair by permitting the access of transcription factors to promoters to initiate transcription. In this study, nucleosome positioning on virus chromatin was investigated in normal immortalized keratinocytes (NIKS) stably transfected with HPV16 or HPV18 genomes to determine if there is an association with the viral life cycle. Micrococcal nuclease-treated DNA analyzed by Southern blotting using probes against HPV16 and HPV18 and quantified by nucleosome scanning analysis using real-time PCR revealed mononucleosomal-sized fragments of 140–200 base pairs that varied in their location within the viral genome according to whether the cells were undergoing proliferation or differentiation. Notably, changes in the regions around nucleotide 110 in proliferating and differentiating host cells were common to HPV16 and HPV18. Our findings suggest that change in nucleosome position on viral DNA during host cell differentiation is an important regulatory event in the viral life cycle.

Decoding nucleosome positions with ATAC-seq data at single-cell level

As the basal bricks, the dynamics and arrangement of nucleosomes orchestrate the higher architecture of chromatin in a fundamental way, thereby affecting almost all nuclear biology processes. Thanks to its rather simple protocol, ATAC-seq has been rapidly adopted as a major tool for chromatin-accessible profiling at both bulk and single-cell level. However, to picture the arrangement of nucleosomes per se remains a challenge with ATAC-seq. In the present work, we introduce a novel ATAC-seq analysis toolkit, named deNOPA, to predict nucleosome positions. Assessments showed that deNOPA not only outperformed state-of-the-art tools, but it is the only tool able to predict nucleosome position precisely with ultrasparse ATAC-seq data. The remarkable performance of deNOPA was fueled by the reads from short fragments, which compose nearly half of sequenced reads and are normally discarded from nucleosome position detection. However, we found that the short fragment reads enrich information on nucleosome positions and that the linker regions were predicted by reads from both short and long fragments using Gaussian smoothing. We applied deNOPA to a single-cell ATAC-seq dataset and deciphered the intrapopulation heterogeneity of the human erythroleukemic cell line (K562). Last, using deNOPA, we showed that the dynamics of nucleosome organization may not directly couple with chromatin accessibility in the cis-regulatory regions when human cells respond to heat shock stimulation. Our deNOPA provides a powerful tool with which to analyze the dynamics of chromatin at nucleosome position level in the single-cell ATAC-seq age.

MNase Profiling of Promoter Chromatin in Salmonella typhimurium-Stimulated GM12878 Cells Reveals Dynamic and Response-Specific Nucleosome Architecture

The nucleosome is the primary unit of chromatin structure and commonly imputed as a regulator of nuclear events, although the exact mechanisms remain unclear. Recent studies have shown that certain nucleosomes can have different sensitivities to micrococcal nuclease (MNase) digestion, resulting in the release of populations of nucleosomes dependent on the concentration of MNase. Mapping MNase sensitivity of nucleosomes at transcription start sites genome-wide reveals an important functional nucleosome organization that correlates with gene expression levels and transcription factor binding. In order to understand nucleosome distribution and sensitivity dynamics during a robust genome response, we mapped nucleosome position and sensitivity using multiple concentrations of MNase. We used the innate immune response as a model system to understand chromatin-mediated regulation. Herein we demonstrate that stimulation of a human lymphoblastoid cell line (GM12878) with heat-killed Salmonella typhimurium (HKST) results in changes in nucleosome sensitivity to MNase. We show that the HKST response alters the sensitivity of -1 nucleosomes at highly expressed promoters. Finally, we correlate the increased sensitivity with response-specific transcription factor binding. These results indicate that nucleosome sensitivity dynamics reflect the cellular response to HKST and pave the way for further studies that will deepen our understanding of the specificity of genome response.

The SWI/SNF subunits BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA

BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes. The function of the SWI/SNF complexes in transcriptional initiation has been well studied, while a function in alternative splicing has only been studied for a few cases for BRM-containing SWI/SNF complexes. Here, we have expressed BRG1 in C33A cells, a BRG1 and BRM-deficient cell line, and we have analysed the effects on the transcriptome by RNA sequencing. We have shown that BRG1 expression affects the splicing of a subset of genes. For some, BRG1 expression favours exon inclusion and for others, exon skipping. Some of the changes in alternative splicing induced by BRG1 expression do not require the ATPase activity of BRG1. Among the exons regulated through an ATPase-independent mechanism, the included exons had signatures of high GC-content and lacked a positioned nucleosome at the exon. By investigating three genes in which the expression of either wild-type BRG1 or a BRG1-ATPase-deficient variant favoured exon inclusion, we showed that expression of the ATPases promotes the local recruitment of RNA binding factors to chromatin and RNA in a differential manner. The hnRNPL, hnRNPU and SAM68 proteins associated to chromatin in C33A cells expressing BRG1 or BRM, but their association with RNA varied. We propose that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and altering their binding to the nascent pre-mRNA, which changes RNP structure.Author summarySplicing, in particular alternative splicing, is a combinatorial process which involves splicing factor complexes and many RNA binding splicing regulatory proteins in different constellations. Most splicing events occur during transcription, which also makes the DNA sequence, the chromatin state and the transcription rate at the exons important components that influence the splicing outcome. We show here that the ATP-dependent chromatin remodelling complex SWI/SNF influences the interactions of splicing regulatory factors with RNA during transcription on certain exons that have a high GC-content. The splicing on this type of exon rely on the ATPase BRG1 and favour inclusion of alternative exons in an ATP-independent manner. SWI/SNF complexes are known to alter the chromatin structure at promoters in transcription initiation, and have been previously shown to alter the transcription rate or nucleosome position in splicing. Our results suggests a further mechanism for chromatin remodelling proteins in splicing: to change the interaction patterns of RNA binding splicing regulatory factors at alternative exons to alter the splicing outcome.

Integrated Genomic Analysis Reveals Key Features of Long Undecoded Transcript Isoform (LUTI)-based Gene Repression

SUMMARYLong Undecoded Transcript Isoforms (LUTIs) represent a class of non-canonical mRNAs that downregulate gene expression through the combined act of transcriptional and translational repression. While single gene studies revealed some important aspects of LUTI-based repression, how these features impact gene regulation at a global scale is unknown. By using transcript leader and direct RNA sequencing, here we identify 74 LUTI candidates that are expressed specifically during meiotic prophase. Translational repression of these candidates is ubiquitous and dependent on upstream open reading frames. However, LUTI-based transcriptional repression is highly variable. In only 50% of the cases, LUTI transcription causes downregulation of the protein-coding transcript isoform. Higher LUTI expression, enrichment of histone 3 lysine 36 trimethylation, and changes in nucleosome position are the strongest predictors of LUTI-based transcriptional repression. We conclude that LUTIs downregulate gene expression in a manner that integrates translational repression, chromatin state changes, and the magnitude of LUTI expression.