Poly(dA:dT) Tracts Differentially Modulate Nucleosome Remodeling Activity of RSC and ISW1a Complexes, Exerting Tract Orientation-Dependent and -Independent Effects.

The establishment and maintenance of nucleosome-free regions (NFRs) are prominent processes within chromatin dynamics. Transcription factors, ATP-dependent chromatin remodeling complexes (CRCs) and DNA sequences are the main factors involved. In Saccharomyces cerevisiae, CRCs such as RSC contribute to chromatin opening at NFRs, while other complexes, including ISW1a, contribute to NFR shrinking. Regarding DNA sequences, growing evidence points to poly(dA:dT) tracts as playing a direct role in active processes involved in nucleosome positioning dynamics. Intriguingly, poly(dA:dT)-tract-containing NFRs span asymmetrically relative to the location of the tract by a currently unknown mechanism. In order to obtain insight into the role of poly(dA:dT) tracts in nucleosome remodeling, we performed a systematic analysis of their influence on the activity of ISW1a and RSC complexes. Our results show that poly(dA:dT) tracts differentially affect the activity of these CRCs. Moreover, we found differences between the effects exerted by the two alternative tract orientations. Remarkably, tract-containing linker DNA is taken as exit DNA for nucleosome sliding catalyzed by RSC. Our findings show that defined DNA sequences, when present in linker DNA, can dictate in which direction a remodeling complex has to slide nucleosomes and shed light into the mechanisms underlying asymmetrical chromatin opening around poly(dA:dT) tracts.

The linker histone Hho1 modulates the activity of ATP-dependent chromatin remodeling complexes.

Diverse factors play roles in chromatin dynamics, including linker proteins. Among them are high mobility group (HMG) box family proteins and linker histones. In the yeast Saccharomyces cerevisiae, Hmo1 has been identified as an HMG-box protein. This protein displays properties that are in agreement with this allocation. However, a number of studies have postulated that Hmo1 functions as a linker histone in yeast. On the other hand, when discovered, the Hho1 protein was identified as a linker histone. While multiple studies support this classification, some findings point to characteristics of Hho1 that are dissimilar to those commonly assigned to linker histones. In order to better understand the roles played by Hmo1 and Hho1 in chromatin dynamics and transcriptional regulation, we performed several analyses directly comparing these two proteins. Our analyses of genome-wide binding profiles support the belonging of Hmo1 to the HMGB family and Hho1 to the linker histones family. Interestingly, by performing protein-protein interaction analyses we found that both Hmo1 and Hho1 display physical interaction with the ATP-dependent chromatin remodeling complexes RSC, ISW1a and SWI/SNF. Moreover, by carrying out nucleosome remodeling assays, we found that both proteins stimulate the activity of the ISW1a complex. However, in the case of RSC, Hmo1 and Hho1 displayed differential properties, with Hho1 mainly showing an inhibitory effect. Our results are in agreement with the opposite roles played by RSC and ISW1a in chromatin dynamics and transcriptional regulation, and expand the view for the roles played by Hho1 and linker histones.

Search for potential biomarkers for saxitoxin detection.

The Neuro-2a cell assay has been a promising in vitro alternative for the detection of saxitoxin (STX)-like toxins. However, its application is problematic in samples with complex matrices containing different toxins, whose mechanisms of action could be antagonistic. In the search of alternative procedures that reduce or avoid this interference, we evaluated the transcriptional modulation produced by a 24-h exposure to STX in Neuro-2a cells under three conditions: exposure to STX (33 nM), a mussel meat matrix (12.5 mg meat/mL) and a fortified sample (STX-fortified matrix). Differential gene expression was evaluated by RNA-seq after Illumina high-throughput sequencing, and data were analyzed to identify genes differentially expressed regardless of the matrix. From the 9487 identified genes, 213 were differentially expressed of these, 10 genes were identified as candidate markers for STX detection due to their regulation by STX regardless of the matrix interference. Expression dynamics of 7 of these candidate genes (Fgf-1, Adgrb2, Tfpt, Zfr2, Nup 35, Fam195a, and Dusp7) was further evaluated by qRT-PCR analysis of cells exposed to different concentrations of STX for up to 24 h. A downregulation of some markers expression was observed, namely Nup35 (involved in nucleo-cytoplasmic transporter activity) and Zfr-2 (involved in nucleic acids binding), whereas Fgf-1 (apoptosis signaling) was significantly upregulated. Markers' expression was not influenced by the matrix, suggesting that gene expression variations are directly related to STX response. These results support the potential of these genes as biomarkers for the development of an alternative STX-like toxins screening method.