Combinatorial Control of Light Induced Chromatin Remodeling and Gene Activation in Neurospora

Light is an important environmental cue that affects physiology and development of Neurospora crassa. The light-sensing transcription factor (TF) WCC, which consists of the GATAfamily TFs WC1 and WC2, is required for light-dependent transcription. SUB1, another GATA-family TF, is not a photoreceptor but has also been implicated in light-inducible gene expression. To assess regulation and organization of the network of light-inducible genes, we analyzed the roles of WCC and SUB1 in light-induced transcription and nucleosome remodeling. We show that SUB1 co-regulates a fraction of light-inducible genes together with the WCC. WCC induces nucleosome eviction at its binding sites. Chromatin remodeling is facilitated by SUB1 but SUB1 cannot activate light-inducible genes in the absence of WCC. We identified FF7, a TF with a putative O-acetyl transferase domain, as an interaction partner of SUB1 and show their cooperation in regulation of a fraction of light-inducible and a much larger number of non light-inducible genes. Our data suggest that WCC acts as a general switch for light-induced chromatin remodeling and gene expression. SUB1 and FF7 synergistically determine the extent of light-induction of target genes in common with WCC but have in addition a role in transcription regulation beyond light-induced gene expression

Prevalencia del trastorno de estrés postraumático en veteranos de guerra inscritos en UCSF San Luis mayo-junio 2018

Esta investigación fue realizada con el objetivo de establecer la prevalencia del Trastorno de Estrés Post Traumático en veteranos de guerra inscritos en la UCSF San Luis, así como factores asociados a la presentación de esta morbilidad.La investigación es de tipo cualitativa, alcance descriptivo, corte transversal y no experimental; el periodo de estudio fue de mayo a junio de 2018. Con una muestra de 81 veteranos de guerra 41 femeninos y 40 masculinos, elegidos/as a conveniencia de los investigadores que consultaron a la UCSF San Luis, por inscripción, control de adulto mayor, morbilidad o visita domiciliar; durante el periodo de estudio antes citado. Los resultados obtenidos muestran que el 52% de la población en estudio cumplen los criterios diagnósticos de Trastorno de Estrés postraumático del DSM 5; dentro de los cuales 57% de estos corresponden al sexo masculino y el restante al femenino. Entre los resultados del estudio se encontró,que el rango de edad en el que se presenta mayormente el TEPT es de 40-59 años con un 62%, que la mayor parte de la población eran de origen demográfico rural, nivel socio económico bajo y nivel académico básico; que de la población con diagnóstico de TEPT el 71% presentaban secuelas físicas y un 17%síntomas disociativos

Thermoregulation of Capsule Production by Streptococcus pyogenes

The capsule of Streptococcus pyogenes serves as an adhesin as well as an anti-phagocytic factor by binding to CD44 on keratinocytes of the pharyngeal mucosa and the skin, the main entry sites of the pathogen. We discovered that S. pyogenes HSC5 and MGAS315 strains are further thermoregulated for capsule production at a post-transcriptional level in addition to the transcriptional regulation by the CovRS two-component regulatory system. When the transcription of the hasABC capsular biosynthetic locus was de-repressed through mutation of the covRS system, the two strains, which have been used for pathogenesis studies in the laboratory, exhibited markedly increased capsule production at sub-body temperature. Employing transposon mutagenesis, we found that CvfA, a previously identified membrane-associated endoribonuclease, is required for the thermoregulation of capsule synthesis. The mutation of the cvfA gene conferred increased capsule production regardless of temperature. However, the amount of the capsule transcript was not changed by the mutation, indicating that a post-transcriptional regulator mediates between CvfA and thermoregulated capsule production. When we tested naturally occurring invasive mucoid strains, a high percentage (11/53, 21%) of the strains exhibited thermoregulated capsule production. As expected, the mucoid phenotype of these strains at sub-body temperature was due to mutations within the chromosomal covRS genes. Capsule thermoregulation that exhibits high capsule production at lower temperatures that occur on the skin or mucosal surface potentially confers better capability of adhesion and invasion when S. pyogenes penetrates the epithelial surface

Experimental validation for computationally predicted small RNAs of Streptococcus pyogenes

The human pathogen Streptococcus pyogenes (Group A Streptococcus or GAS) are a versatile Gram-positive cocci that havw shown complex modes of regulation of its different virulence factors. Discoveries of a few small non-coding RNAs (sRNAs) in S. pyogenes and their influence on the expression of virulence factors revealed an important role of sRNAs on S. pyogenes virulence. The genome-wide analysis of bacterial genomes for the discovery of sRNAs through computational methods has become an effective way to discover new sRNAs. In this study we provided a computational scheme where three different algorithms (RNAz, eQRNA, and sRNAPredict) were combined to increase the probabilities of predicting putative sRNAs within S. pyogenes\u27 intergenic regions (IGR). A total of 46 candidates were chosen based on our criteria, and through Northern blot we analyzed each candidate. We obtained hybridization signals from twelve newly discovered sRNAs in S. pyogenes. Subsequently, we analyzed their sequence and their location within the IGR to find a putative -10 promoter region and possible Rho-independent terminator site, and their possible targets through computational methods. We further expanded our analysis of the new sRNAs by using Real-Time RT-PCR to determine the expression of sRNAs during different phases of growth. Our results showed that our computational scheme and experimental method was effective in predicting sRNAs previously undiscovered in S. pyogenes, and that more sRNAs are yet to be discovered and characterized, helping to further understand the regulation of virulence factors in S. pyogene

Novel regulatory small RNAs in Streptococcus pyogenes

Streptococcus pyogenes (Group A Streptococcus or GAS) is a Gram-positive bacterial pathogen that has shown complex modes of regulation of its virulence factors to cause diverse diseases. Bacterial small RNAs are regarded as novel widespread regulators of gene expression in response to environmental signals. Recent studies have revealed that several small RNAs (sRNAs) have an important role in S. pyogenes physiology and pathogenesis by regulating gene expression at the translational level. To search for new sRNAs in S. pyogenes, we performed a genomewide analysis through computational prediction followed by experimental verification. To overcome the limitation of low accuracy in computational prediction, we employed a combination of three different computational algorithms (sRNAPredict, eQRNA and RNAz). A total of 45 candidates were chosen based on the computational analysis, and their transcription was analyzed by reverse-transcriptase PCR and Northern blot. Through this process, we discovered 7 putative novel trans-acting sRNAs. Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals. Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis. This study provides a significant resource for future study of small RNAs and their roles in physiology and pathogenesis of S. pyogenes

Combinatorial Control of Light Induced Chromatin Remodeling and Gene Activation in <i>Neurospora</i>

<div><p>Light is an important environmental cue that affects physiology and development of <i>Neurospora crassa</i>. The light-sensing transcription factor (TF) WCC, which consists of the GATA-family TFs WC1 and WC2, is required for light-dependent transcription. SUB1, another GATA-family TF, is not a photoreceptor but has also been implicated in light-inducible gene expression. To assess regulation and organization of the network of light-inducible genes, we analyzed the roles of WCC and SUB1 in light-induced transcription and nucleosome remodeling. We show that SUB1 co-regulates a fraction of light-inducible genes together with the WCC. WCC induces nucleosome eviction at its binding sites. Chromatin remodeling is facilitated by SUB1 but SUB1 cannot activate light-inducible genes in the absence of WCC. We identified FF7, a TF with a putative O-acetyl transferase domain, as an interaction partner of SUB1 and show their cooperation in regulation of a fraction of light-inducible and a much larger number of non light-inducible genes. Our data suggest that WCC acts as a general switch for light-induced chromatin remodeling and gene expression. SUB1 and FF7 synergistically determine the extent of light-induction of target genes in common with WCC but have in addition a role in transcription regulation beyond light-induced gene expression.</p></div

Thermoregulation of Capsule Production by Streptococcus pyogenes

The capsule of Streptococcus pyogenes serves as an adhesin as well as an anti-phagocytic factor by binding to CD44 on keratinocytes of the pharyngeal mucosa and the skin, the main entry sites of the pathogen. We discovered that S. pyogenes HSC5 and MGAS315 strains are further thermoregulated for capsule production at a post-transcriptional level in addition to the transcriptional regulation by the CovRS two-component regulatory system. When the transcription of the hasABC capsular biosynthetic locus was de-repressed through mutation of the covRS system, the two strains, which have been used for pathogenesis studies in the laboratory, exhibited markedly increased capsule production at sub-body temperature. Employing transposon mutagenesis, we found that CvfA, a previously identified membrane-associated endoribonuclease, is required for the thermoregulation of capsule synthesis. The mutation of the cvfA gene conferred increased capsule production regardless of temperature. However, the amount of the capsule transcript was not changed by the mutation, indicating that a post-transcriptional regulator mediates between CvfA and thermoregulated capsule production. When we tested naturally occurring invasive mucoid strains, a high percentage (11/53, 21%) of the strains exhibited thermoregulated capsule production. As expected, the mucoid phenotype of these strains at sub-body temperature was due to mutations within the chromosomal covRS genes. Capsule thermoregulation that exhibits high capsule production at lower temperatures that occur on the skin or mucosal surface potentially confers better capability of adhesion and invasion when S. pyogenes penetrates the epithelial surface

A high-throughput newborn screening approach for SCID, SMA, and SCD combining multiplex qPCR and tandem mass spectrometry.

Early diagnosis of severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD) improves health outcomes by providing a specific treatment before the onset of symptoms. A high-throughput nucleic acid-based method in newborn screening (NBS) has been shown to be fast and cost-effective in the early detection of these diseases. Screening for SCD has been included in Germany's NBS Program since Fall 2021 and typically requires high-throughput NBS laboratories to adopt analytical platforms that are demanding in terms of instrumentation and personnel. Thus, we developed a combined approach applying a multiplexed quantitative real-time PCR (qPCR) assay for simultaneous SCID, SMA, and 1st-tier SCD screening, followed by a tandem mass spectrometry (MS/MS) assay for 2nd-tier SCD screening. DNA is extracted from a 3.2-mm dried blood spot from which we simultaneously quantify T-cell receptor excision circles for SCID screening, identify the homozygous SMN1 exon 7 deletion for SMA screening, and determine the integrity of the DNA extraction through the quantification of a housekeeping gene. In our two-tier SCD screening strategy, our multiplex qPCR identifies samples carrying the HBB: c.20A>T allele that is coding for sickle cell hemoglobin (HbS). Subsequently, the 2nd tier MS/MS assay is used to distinguish heterozygous HbS/A carriers from samples of patients with homozygous or compound heterozygous SCD. Between July 2021 and March 2022, 96,015 samples were screened by applying the newly implemented assay. The screening revealed two positive SCID cases, while 14 newborns with SMA were detected. Concurrently, the qPCR assay registered HbS in 431 samples which were submitted to 2nd-tier SCD screening, resulting in 17 HbS/S, five HbS/C, and two HbS/β thalassemia patients. The results of our quadruplex qPCR assay demonstrate a cost-effective and fast approach for a combined screening of three diseases that benefit from nucleic-acid based methods in high-throughput NBS laboratories

Cistrome analysis of WCC and SUB1.

<p><b>A</b>. Heat-map showing the light-induced WCC occupancy at 92 binding sites identified by both, MNase-WC2 ChIP-seq and TAP-WC2 ChIP-seq. 5 kb region covering the binding sites are shown. Left panel: WCC binding in the dark. Right panel: WCC binding 30 min after light-exposure. <b>B</b>. Occurrence of tandem GATC motifs with the indicated spacing at WCC binding sites. 300 bp DNA regions covering the peaks of 92 highly confident WCC binding sites were analyzed. The dashed line corresponds to the occurrence of tandem GATC motifs in a set of randomly chosen 300 bp regions. <b>C</b>. Potential light response elements (LREs) at WCC binding sites contain multiple GATC motifs. GATC motifs in WCC binding sites of the indicated genes are shown. <i>frq</i>_<i>as</i>: <i>frq</i> antisense [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005105#pgen.1005105.ref019" target="_blank">19</a>]. <i>vvd</i>_<i>prox</i> and <i>vvd</i>_<i>dis</i>: proximal and distal WCC binding sites in <i>vvd</i> promoter (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005105#pgen.1005105.s008" target="_blank">S2 Table</a>). <b>D</b>. Distribution of tandem GATC motifs with < 30 bp spacing at WCC binding sites. The grey area represents the sequence coverage of the WCC ChIP (MNase-WC2 ChIP, 30 min) at the highly confident 92 WCC binding sites. The red line shows the occurrence of tandem GATC motifs. <b>E</b>. Heat-map showing the SUB1 occupancy at binding sites. Left panel: SUB1 binding in the dark. Right panel: SUB1 binding 30 min after light-exposure. <b>F</b>. SUB1 binding motifs identified by MEME are shown. The major sequence motif shown in the upper panel is found in 171 sites. The GTA-rich motifs shown in the lower left and right panels are present in 82 and 63 sites, respectively. <b>G</b>. Distribution of the major SUB1 binding motif (a/cGAT-x6-a/cTGc/t) at SUB1 binding sites. The grey area represents the sequence coverage of the SUB1 ChIP (SUB1 30 min) at 617 SUB1 binding sites. The red line shows the occurrence of the SUB1 binding motif.</p

FF7 interacts weakly with SUB1 and co-regulates light-inducible and non light-inducible genes.

<p><b>A-B</b>. Western blots showing co-immunoprecipitation (co-IP) of <b>(A)</b> SUB1 with FF7_FLAG-HIS and <b>(B)</b> FF7_FLAG-HIS with SUB1. FLAG antibody was used for FF7_FLAG-HIS IP and α-SUB1 antibody was used for SUB1 IP. The asterisks (*) indicate cross-reactions of the FLAG antibody. <b>C</b>. FF7 binding motifs identified by MEME. The top 200 binding sites identified by FF7 ChIP-seq were used for the motif analysis. The upper motif is found in 117 / 200 binding sites whereas the lower motif is found in 36 / 200 binding sites. <b>D</b>. Occurrence of the major FF7 motif at FF7 binding sites. The grey area shows the occupancy of FF7 binding sites determined by ChIP-seq. The red line shows the occurrence of the FF7 binding motif “t/c AAGCG c/a”. <b>E</b>. Wig file showing MNase-WC2, SUB1 and FF7 ChIP-seq signals at the <i>rds1</i> promoter. Numbers on the ChIP-seq panels correspond the maximum coverage shown in the wig file. <b>F</b>. Venn-diagram showing the overlap between SUB1, WC2 and FF7 ChIP-seq signals. <b>G</b>. Heat-map showing light-inducible genes with significantly lower RNA levels in Δ<i>sub1</i> and in Δ<i>ff7</i> strains in comparison to <i>wt</i>. <b>H</b>. Wig file (left panel) showing the nucleosome position and occupancy at the <i>rds1</i> promoter in <i>wt</i> and Δ<i>ff7</i> strains in the dark and after light-exposure. The MNase-WC2 ChIP-seq (blue) is shown below the nucleosome signals. Numbers on the ChIP-seq panels show the maximum coverage shown in the wig file. ChIP-PCR analysis (right panel) of H2A occupancy at the binding sites of WCC and SUB1 at <i>rds1</i> promoter in the dark and 20 min after light-exposure (± SEM, n = 4). a<i>ctin</i> DNA was used for normalization. w<i>t</i> dark level was set to 1. <b>I</b>. Nucleosome occupancy at binding sites of WCC (n = 92) and in Δ<i>ff7</i> in dark (dotted lines) and 20 min after light-exposure (solid lines).</p