Transcription Start Regions in PTU-intergenic regions drive cell cycle-dependent transcriptional activation events in Leishmania donovani

Evolution of a genome-architecture-encoded gene regulation system in trypanosomatids

Transcriptional regulation of protein-coding genes is a hallmark of eukaryotic gene expression. Yet, a group of parasitic protists, trypanosomatids, appear to lack this capability. Here, we analyzed genomic, nascent transcriptomic, RNA polymerase occupancy and gene organization data to reconstruct the evolutionary origin and biological consequences of their unusual regulatory strategy. Across 59 Discoba protists, we show stepwise evolutionary erosion of conventional transcription regulation components in trypanosomatida lineage, including gene consolidation into polycistronic transcription units (PTUs), shortening of intra-PTU non-coding regions, and depletion of transcription factors and their enriched DNA-binding motifs. This transition was associated with near-constitutive expression of most genes, indicating broad loss of conditional gene expression. However, trypanosomatids retain some differential regulation at the PTU level, with >70% PTUs featuring significantly different nascent transcription than their neighbors or resident chromosomes. Moreover, gene expression is not uniform within PTUs: nascent transcription, translation efficiency, and protein abundance progressively decline with distance from the transcription start site. Consistent with this architecture-encoded regulatory logic, co-complex subunits and co-pathway enzymes preferentially occupy adjacent positions within PTUs despite each PTU’s overall functional heterogeneity. These findings reveal an evolutionary shift from gene-specific transcriptional regulation toward a regime where genome architecture becomes a regulator of gene expression.

Activation of mTOR pathway by human cytomegalovirus promoting host ribosomal protein expression by coordinated transcriptional and translational controls

ABSTRACT Human cytomegalovirus (HCMV) profoundly reprograms host transcription and RNA metabolism, yet its impact on transcription start site (TSS) regulation of host genes remains poorly understood. Here, we employed NanoCap Analysis of Gene Expression sequencing (NanoCAGE-seq) to investigate HCMV-driven changes in alternative TSS usage across the host transcriptome. We identified widespread TSS switching, with ribosomal protein genes (RPGs) emerging as a highly enriched category. Alternative TSS usage produced isoforms with distinct 5’untranslated regions (UTRs), thereby altering cis-regulatory elements that shape translational efficiency. Integrative transcriptomic and proteomic analyses revealed a paradoxical accumulation of RPG proteins despite transcriptional downregulation during infection. Using 5’ Rapid Amplification of cDNA Ends (5’RACE), we characterized four RPGs of RPL4, RPS11, RPS23, and RPS24 that generated 5’UTR variants through alternative TSS usage. Notably, isoforms containing a 5’terminal oligopyrimidine (5’TOP) motif were significantly enriched, correlating with mTOR activation induced by HCMV. Functional assays with bicistronic reporter constructs in HEK293 cells and infection models in human embryonic lung fibroblasts demonstrated that the RPL4 5’TOP isoform exhibited enhanced mTORC1-driven translation compared with non-5’TOP counterparts. Importantly, RPL4 upregulation facilitated viral protein synthesis and boosted production of infectious virions. Together, our findings reveal that dynamic TSS switching of RPGs provides a simple, yet effective, mechanism for fine-tuning mTORC1-responsive translation. By co-opting host transcriptional and translational programs, HCMV enhances ribosome function to optimize the cellular environment for productive viral replication.

TALE-independent transcriptional activation of the rice executor gene Xa23 is regulated via histone acetylation during zygote development

Transcription activator-like effectors (TALEs) from Xanthomonas activate transcription of “executor” (E) genes in host plants, leading to cell death and thereby restricting proliferation of biotrophic pathogens. Because E gene transcripts had only been detected upon activation by cognate Xanthomonas TALEs, E genes were thought to function exclusively in plant immunity. Here, we detect TALE-independent transcription of the rice E gene Xa23 in zygotes 4-6 hours after gamete fusion. Histone deacetylase inhibition induces Xa23 transcription in unfertilized egg cells, implicating histone acetylation in Xa23 regulation. We identified potential cis-regulatory elements and transcription start sites associated with native Xa23 transcription during zygote development. Together, our findings suggest that Xa23 is a developmentally regulated gene with a native role during early zygote development. This supports a previously proposed model in which E genes have native functions in development, while fortuitous upstream polymorphisms can create TALE-binding sites that convert them into immune executors.

The molecular basis of transcription initiation by RNA polymerase II.

Cis- and trans-regulatory factors contributing to divergent activity of the TDH3 promoter in Saccharomyces yeast

Abstract Changes in regulatory sequences controlling the timing and activity of gene products underlie much of natural phenotypic variation. Yet, identifying which divergent sites matter and how they impact gene expression remains challenging. Here, we investigate how transcriptional activity and homeostatic responsiveness of orthologous promoters of the metabolic gene TDH3 evolved among Saccharomyces yeast. We found that promoter expression level increased specifically in the Saccharomyces cerevisiae lineage and that a substantial part of this increase was caused by genetic variants located between the well-characterized, conserved binding sites for two direct transcriptional regulators. These nucleotide changes altered the promoters’ expression levels while leaving the expression dynamics conserved. Further, the effects of these nucleotide changes were only seen in the presence of a third transcription factor, TYE7p, which is known to be recruited by the other transcription factors through protein–protein interactions. These results suggest that the cis-regulatory changes act through their influence on the collective assembly/activation of a transcription factor complex and that changes acting through such a mechanism can allow distinct parts of gene expression, such as expression level and dynamics, to separately diverge.

Mechanisms of transcription termination across the coding and noncoding loci of the genome.