Biology subjects

Lorenzi, H.

Publications and source records attributed to Lorenzi, H..

3 recordsLinked to original sources

bioRxiv2025

Kar4 acts as a Ste12 regulator in Saccharomyces cerevisiae, promoting Ste12 binding to a specific DNA motif genome-wide

Kar4 is a putative transcription factor required for efficient mating in the budding yeast Saccharomyces cerevisiae. Kar4 functions with Ste12, the master transcriptional regulator of the yeast mating pheromone response, to promote the transcription of a subset of Ste12 targets required for mating. However, the mechanism by which Kar4 modulates Ste12 activity has remained uncertain. Here, we examined Kar4s function at the levels of transcription (RNA-seq) and Ste12 DNA binding (ChIP-exo). We show that Kar4 promotes Ste12 binding to nearly all Ste12 DNA-binding sites associated with transcriptionally-upregulated genes, even if their upregulation is not dependent on Kar4. We further found that the majority of Ste12-binding sites have two pheromone response elements (PREs) separated by four nucleotides in a head-to-tail orientation (H-T 4 motif). Sites associated with Kar4-dependent transcription have PREs with more mismatches and substantially lower Ste12 occupancy in kar4{Delta} cells than sites linked to Kar4-independent transcription. During the pheromone response in kar4{Delta} cells, Ste12 exhibits increased binding to non-H-T 4 motifs, particularly T-T 3 motifs, resulting in the abnormal upregulation of many genes not transcribed during the wild-type pheromone response. Therefore, we propose that Kar4 functions by increasing the DNA-binding specificity of Ste12 globally, promoting its binding primarily to H-T 4 motifs. Our model is consistent with previously-observed slower induction kinetics of Kar4-dependent genes via a feed-forward mechanism. Lastly, we uncovered several novel aspects of the pheromone response, including a broad role for the Crz1 transcription factor, induction of stress responses, and the identification of pheromone-responsive intergenic transcripts.

genetics↗

bioRxiv2024

A highly conserved sRNA downregulates multiple genes, including a sigma54 transcriptional activator, in the virulence mode of Bordetella pertussis

Bacterial sRNAs together with the RNA chaperone Hfq post-transcriptionally regulate gene expression by affecting ribosome binding or mRNA stability. In the human pathogen Bordetella pertussis, the causative agent of whooping cough, hundreds of sRNAs have been identified, but their roles in B. pertussis biology are mostly unknown. Here we characterize a Hfq-dependent sRNA (S17), whose level is dramatically higher in the virulence (Bvg+) mode. We show that transcription from a {sigma}A-dependent promoter yields a long form of 190 nucleotides (nts) that is processed by RNase E to generate a shorter, more stable form (S17S) of 67 nts. Using RNA-seq and RT-qPCR, we identify 92 genes whose expression significantly increases in the absence of S17. Of these genes, 70 contain sequences at/near their ribosome binding sites (RBSs) that are complementary to single-stranded (ss) regions (Sites 1 or 2) of S17S. The identified genes include those encoding multiple transporters and 3 transcriptional regulators. Using a lacZ translational reporter system, we demonstrate that S17S directly represses one of these genes, BP2158, a {sigma}54- dependent transcriptional regulator, suggesting the repression of a {sigma}54 regulon in the Bvg+ mode. We find that the S17S region containing Sites 1 and 2 is 100% conserved throughout various Betaproteobacteria species, and the S17S target sites are often conserved in the homologs of the B. pertussis target genes. We speculate that S17S regulation represents a highly conserved process that fine-tunes gene expression in the Bvg+ mode of B. pertussis and perhaps under other conditions in related bacteria. IMPORTANCERegulation of gene expression involves controlling transcription, translation, and transcript degradation. sRNAs with short sequences complementary to an mRNA sequence are involved in post-transcriptional regulation by aiding or interfering with either ribosome binding or nuclease attack. In the human pathogen Bordetella pertussis, the causative agent of whooping cough, hundreds of sRNA have been identified, but their functions remain largely unknown. We have characterized a sRNA that is abundant in the virulence mode of B. pertussis and serves to down-regulate multiple genes including transcriptional regulators and various transporters. We demonstrate that this sRNA directly represses a transcriptional factor, suggesting that it influences the regulation of specific B. pertussis regulons. The high conservation of this sRNA and its targets within Betaproteobacteria suggests a conserved pathway for gene regulation.

microbiology↗

bioRxiv2024

Phylogenomic reconstruction of Cryptosporidium spp. captured directly from clinical samples reveals extensive genetic diversity

Cryptosporidium is a leading cause of severe diarrhea and mortality in young children and infants in Africa and southern Asia. More than twenty Cryptosporidium species infect humans, of which C. parvum and C. hominis are the major agents causing moderate to severe diarrhea. Relatively few genetic markers are typically applied to genotype and/or diagnose Cryptosporidium. Most infections produce limited oocysts making it difficult to perform whole genome sequencing (WGS) directly from stool samples. Hence, there is an immediate need to apply WGS strategies to 1) develop high-resolution genetic markers to genotype these parasites more precisely, 2) to investigate endemic regions and detect the prevalence of different genotypes, and the role of mixed infections in generating genetic diversity, and 3) to investigate zoonotic transmission and evolution. To understand Cryptosporidium global population genetic structure, we applied Capture Enrichment Sequencing (CES-Seq) using 74,973 RNA-based 120 nucleotide baits that cover [~]92% of the genome of C. parvum. CES-Seq is sensitive and successfully sequenced Cryptosporidium genomic DNA diluted up to 0.005% in human stool DNA. It also resolved mixed strain infections and captured new species of Cryptosporidium directly from clinical/field samples to promote genome-wide phylogenomic analyses and prospective GWAS studies.

genomics↗