Faculty Bibliography

Cerebellar granule cells (GCs) are critical for motor and cognitive functions. Lineage tracing studies have identified a hierarchical developmental progression of GC neurogenesis, transitioning from Sox2⁺ stem-like cells to Atoh1⁺ rapidly proliferating granule cell precursors (GCPs), and ultimately to NeuN⁺ mature GCs. However, the molecular mechanisms governing these transitions remain poorly understood. In this study, we identified a transient, slow-cycling progenitor population defined by co-expression of Sox2 and Atoh1. We show that GC maturation depends critically on the repressive function of the Sin3A/Hdac1 complex, which sequentially silences Sox2 and then Atoh1 to ensure orderly progression through developmental stages. Loss of these repressions prolongs progenitor states, compromises survival, and markedly reduces GC output. We also identify NeuroD1 as a co-repressor that collaborates with Sin3A/Hdac1 to inhibit Atoh1 transcription. Our findings highlight the central role of the Sin3A complex in orchestrating distinct stages of cerebellar GC lineage development and may provide insights into Sin3A-related cerebellar disorders and medulloblastoma in human.

Stress-induced dinucleoside tetraphosphates (Np₄Ns, where N is adenosine, guanosine, cytosine or uridine) are ubiquitous in living organisms, yet their function has been largely elusive for over 50 years. Recent studies have revealed that RNA polymerase can influence the cellular lifetime of transcripts by incorporating these alarmones into RNA as 5'-terminal caps. Here we present structural and biochemical data that reveal the molecular basis of noncanonical transcription initiation from Np₄As by Escherichia coli and Thermus thermophilus RNA polymerases. Our results show the influence of the first two nucleotide incorporation steps on capping efficiency and the different interactions of Np₄As with transcription initiation complexes. These data provide critical insights into the substrate selectivity that dictates levels of Np₄ capping in bacterial cells.

Unravelling how genomes are spatially organized and how their three-dimensional (3D) architecture drives cellular functions remains a major challenge in biology^1,2. In bacteria, genomic DNA is compacted into a highly ordered, condensed state called nucleoid^3-5. Despite progress in characterizing bacterial 3D genome architecture over recent decades^6-8, the fine structure and functional organization of the nucleoid remain elusive due to low-resolution contact maps from methods such as Hi-C^9-11. Here we developed an enhanced Micro-C chromosome conformation capture, achieving 10-base pair (bp) resolution. This ultra-high-resolution analysis reveals elemental spatial structures in the Escherichia coli nucleoid, including chromosomal hairpins (CHINs) and chromosomal hairpin domains (CHIDs). These structures, organized by histone-like proteins H-NS and StpA, have key roles in repressing horizontally transferred genes. Disruption of H-NS causes drastic reorganization of the 3D genome, decreasing CHINs and CHIDs, whereas removing both H-NS and StpA results in their complete disassembly, increased transcription of horizontally transferred genes and delayed growth. Similar effects are observed with netropsin, which competes with H-NS and StpA for AT-rich DNA binding. Interactions between CHINs further organize the genome into isolated loops, potentially insulating active operons. Our Micro-C analysis reveals that all actively transcribed genes form distinct operon-sized chromosomal interaction domains (OPCIDs) in a transcription-dependent manner. These structures appear as square patterns on Micro-C maps, reflecting continuous contacts throughout transcribed regions. This work unveils the fundamental structural elements of the E. coli nucleoid, highlighting their connection to nucleoid-associated proteins and transcription machinery.

Enigmatic dinucleoside tetraphosphates, known as 'alarmones' (Np₄Ns), have recently been shown to function in bacteria as precursors to Np₄ caps on transcripts, likely influencing RNA longevity and cellular adaptation to stress. In proteobacteria, ApaH is the predominant enzyme that hydrolyzes Np₄Ns and decaps Np₄-capped RNAs to initiate their 5'-end-dependent degradation. Here we conducted a biochemical and structural study to uncover the catalytic mechanism of Escherichia coli ApaH, a prototypic symmetric Np₄N hydrolase, on various Np₄Ns and Np₄-capped RNAs. We found that the enzyme uses a unique combination of nonspecific and semispecific substrate recognition, enabling substrates to bind in two orientations with a slight orientational preference. Despite such exceptional recognition properties, ApaH efficiently decaps various Np₄-capped mRNAs and sRNAs, thereby impacting their lifetimes. Our findings highlight the need to determine substrate orientation preferences before designing substrate-mimicking drugs, as enzymes may escape activity modulation with one of the alternative substrate orientations.

Exposure to Bacillus Calmette-Guérin (BCG) or Canarypox ALVAC/Alum vaccine elicits pro- or antiinflammatory innate responses, respectively. We tested whether prior exposure of macaques to these immunogens protected against SARS-CoV-2 replication in lungs and found more efficient replication control after the pro-inflammatory immunity elicited by BCG. The decreased virus level in lungs was linked to early infiltrates of classical monocytes producing IL-8 with systemic neutrophils, Th2 cells, and Ki67+CD95+CD4+ T cells producing CCR7. At the time of SARS-CoV-2 exposure, BCG-treated animals had higher frequencies of lung infiltrating neutrophils and higher CD14+ cells expressing efferocytosis marker MERTK, responses correlating with decreased SARS-CoV-2 replication in lung. At the same time point, plasma IL-18, TNF-α, TNFSF-10, and VEGFA levels were also higher in the BCG group and correlated with decreased virus replication. Finally, after SARS-CoV-2 exposure, decreased virus replication correlated with neutrophils producing IL-10 and CCR7 preferentially recruited to the lungs of BCG-vaccinated animals. These data point to the importance of the spatiotemporal distribution of functional monocytes and neutrophils in controlling SARS-CoV-2 levels and suggest a central role of monocyte efferocytosis in curbing replication.

The primary objective of life is to ensure the faithful transmission of genetic material across generations, despite the constant threat posed by DNA-damaging factors. To counter these challenges, life has evolved intricate mechanisms to detect, signal, and repair DNA damage, thereby preventing mutations that can cause developmental abnormalities or diseases. DNA repair is especially vital during development - a period of rapid cell proliferation and differentiation. Failure to repair DNA damage in somatic cells can result in tissue dysfunction, while during embryonic development, it is often fatal. Transcription machinery plays a key role in the mechanisms of DNA repair. This review highlights current insights into DNA repair pathways that are driven or facilitated by transcription and their essential contribution to preserving genome stability.

Establishment and maintenance of cellular sex identity is essential for reproduction. Sex identity of somatic and germline cells must correspond for sperm or oocytes to be produced, with mismatched identity causing infertility in all organisms from flies to humans. In adult Drosophila testes, Chronologically inappropriate morphogenesis (Chinmo) is required for maintenance of male somatic identity. Loss of chinmo leads to feminization of the male soma, including adoption of female-specific cell morphologies and gene expression. However, the degree to which feminized somatic cells engage female-specific cellular behaviors or influence the associated XY germline is unknown. Using extended live imaging, we find that chinmo-depleted somatic cells acquire cell behaviors characteristic of ovarian follicle cells, including incomplete cytokinesis and rotational migration. Importantly, migration in both contexts require the basement membrane protein Perlecan and adhesion protein E-cadherin. Finally, we find that sex- converted soma non-autonomously induce expression of an early oocyte specification protein in XY germ cells. Taken together, our work reveals a dramatic transformation of somatic cell behavior during sex conversion and provides a powerful model to study soma-derived induction of oocyte identity.

In Escherichia coli, ribose-5-phosphate (R5P) biosynthesis occurs via two distinct pathways: an oxidative branch of the pentose phosphate pathway (PPP) originating from glucose-6-phosphate, and a reversed non-oxidative branch originating from fructose-6-phosphate, which relies on transaldolases TalA and TalB. Remarkably, we found that disrupting the oxidative PPP branch by deleting the zwf gene significantly increased bacterial susceptibility to killing by a variety of antibiotics. Surprisingly, additional mutations in the talA and talB genes further enhanced bacterial sensitivity to oxidative stress and antibiotic-mediated killing though they had little impact on the minimal inhibitory concentrations (MICs). The hypersensitivity observed in the zwf talAB mutant could be fully reversed by the processes that either utilize R5P or limited its accumulation. Specifically, activating the purine biosynthetic regulon or inhibiting nucleoside catabolism via deoB gene inactivation, which blocks the conversion of ribose-1-phosphate to R5P, restored bacterial tolerance. Furthermore, enhancing the biosynthesis of cell wall component ADP-heptose from sedoheptulose-7-phosphate suppressed antibiotic killing of the zwf talAB mutant. Biochemical analysis confirmed a direct link between elevated intracellular R5P levels and increased bacterial susceptibility to antibiotics-induced killing. These findings suggest that targeting the PPP could be a promising strategy for developing new therapeutic approaches aimed at potentiating clinically relevant antibiotics.IMPORTANCERecent studies have revealed the crucial role of bacterial cell's metabolic status in its susceptibility to the lethal action of antibacterial drugs. However, there is still no clear understanding of which key metabolic nodes are optimal targets to improve the effectiveness of bacterial infection treatment. Our study establishes that the disruption of the canonical pentose phosphate pathway induces one-way anabolic synthesis of pentose phosphates (aPPP) in E. coli cells, increasing the killing efficiency of various antibiotics. It is also demonstrated that the activation of ribose-5-phosphate utilization processes restores bacterial tolerance to antibiotics. We consider the synthesis of ribose-5-phosphate to be one of the determining factors of bacterial cell stress resistance. Understanding bacterial metabolic pathways, particularly the aPPP's role in antibiotic sensitivity, offers insights for developing novel adjuvant therapeutic strategies to enhance antibiotic potency.

INTRODUCTION/BACKGROUND:Presenilin (PS) gene mutations cause memory impairment in early-onset familial Alzheimer's disease (FAD), but the underlying mechanisms remain unclear. METHODS:activity and CREB phosphorylation in the primary motor cortex. RESULTS:levels are altered in a cortical layer and neuron type-specific manner in PS1 mutant mice as compared to WT control mice. Notably, while running caused a significant increase of CREB phosphorylation in WT mice, it led to a significant decrease of CREB phosphorylation in layer 5 neurons of mutant mice. DISCUSSION/CONCLUSIONS:activity and CREB phosphorylation in deep cortical layers are early events leading to memory impairment in the PS1 mutation-related familial form of AD.