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Interferons (IFNs) play a crucial role in the regulation and evolution of host-virus interactions. Here, we conducted a genome-wide arrayed CRISPR knockout screen in the presence and absence of IFN to identify human genes that influence Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. We then performed an integrated analysis of genes interacting with SARS-CoV-2, drawing from a selection of 67 large-scale studies, including our own. We identified 28 genes of high relevance in both human genetic studies of Coronavirus Disease 2019 (COVID-19) patients and functional genetic screens in cell culture, with many related to the IFN pathway. Among these was the IFN-stimulated gene PLSCR1. PLSCR1 did not require IFN induction to restrict SARS-CoV-2 and did not contribute to IFN signaling. Instead, PLSCR1 specifically restricted spike-mediated SARS-CoV-2 entry. The PLSCR1-mediated restriction was alleviated by TMPRSS2 overexpression, suggesting that PLSCR1 primarily restricts the endocytic entry route. In addition, recent SARS-CoV-2 variants have adapted to circumvent the PLSCR1 barrier via currently undetermined mechanisms. Finally, we investigate the functional effects of PLSCR1 variants present in humans and discuss an association between PLSCR1 and severe COVID-19 reported recently., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Le Pen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Biallelic pathogenic variants in the essential DNA repair gene BRCA2 causes Fanconi anemia, complementation group FA-D1. Patients in this group are highly prone to develop embryonal tumors, most commonly medulloblastoma arising from the cerebellar granule cell progenitors (GCPs). GCPs undergo high proliferation in the postnatal cerebellum under SHH activation, but the type of DNA lesions that require the function of the BRCA2 to prevent tumorigenesis remains unknown. To identify such lesions, we assessed both GCP neurodevelopment and tumor formation using a mouse model with deletion of exons three and four of Brca2 in the central nervous system, coupled with global Trp53 loss. Brca2 Δex3-4 ;Trp53 -/- animals developed SHH subgroup medulloblastomas with complete penetrance. Whole-genome sequencing of the tumors identified structural variants with breakpoints enriched in areas overlapping G-quadruplexes (G4s). Brca2 -deficient GCPs exhibited decreased replication speed in the presence of the G4-stabilizer pyridostatin. Pif1 helicase, which resolves G4s during replication, was highly upregulated in tumors, and Pif1 knockout in primary MB tumor cells resulted in increased genome instability upon pyridostatin treatment. These data suggest that G4s may represent sites prone to replication stalling in highly proliferative GCPs and without BRCA2, G4s become a source of genome instability. Tumor cells upregulate G4-resolving helicases to facilitate rapid proliferation through G4s highlighting PIF1 helicase as a potential therapeutic target for treatment of BRCA2 -deficient medulloblastomas., Competing Interests: Competing Interest Statement A.S. is an advisor to Rocket Pharmaceuticals. Other authors have declared that no conflict of interest exists.

Alterations in the tumor suppressor ATRX are recurrently observed in mesenchymal neoplasms. ATRX has multiple epigenetic functions including heterochromatin formation and maintenance and regulation of transcription through modulation of chromatin accessibility. Here, we show in murine mesenchymal progenitor cells (MPCs) that Atrx deficiency aberrantly activated mesenchymal differentiation programs. This includes adipogenic pathways where ATRX loss induced expression of adipogenic transcription factors and enhanced adipogenic differentiation in response to differentiation stimuli. These changes are linked to loss of heterochromatin near mesenchymal lineage genes together with increased chromatin accessibility and gains of active chromatin marks. We additionally observed depletion of H3K9me3 at transposable elements, which are derepressed including near mesenchymal genes where they could serve as regulatory elements. Finally, we demonstrated that loss of ATRX in a mesenchymal malignancy, undifferentiated pleomorphic sarcoma, results in similar epigenetic disruption and de-repression of transposable elements. Together, our results reveal a role for ATRX in maintaining epigenetic states and transcriptional repression in mesenchymal progenitors and tumor cells and in preventing aberrant differentiation in the progenitor context., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Post-translational modifications (PTMs) on histones are a key source of regulation on chromatin through impacting cellular processes, including gene expression 1 . These PTMs often arise from metabolites and are thus impacted by metabolism and environmental cues 2-7 . One class of metabolically regulated PTMs are histone acylations, which include histone acetylation, butyrylation, crotonylation and propionylation 3,8 . As these PTMs can be derived from short-chain fatty acids, which are generated by the commensal microbiota in the intestinal lumen 9-11 , we aimed to define how microbes impact the host intestinal chromatin landscape, mainly in female mice. Here we show that in addition to acetylation, intestinal epithelial cells from the caecum and distal mouse intestine also harbour high levels of butyrylation and propionylation on lysines 9 and 27 of histone H3. We demonstrate that these acylations are regulated by the microbiota and that histone butyrylation is additionally regulated by the metabolite tributyrin. Tributyrin-regulated gene programmes are correlated with histone butyrylation, which is associated with active gene-regulatory elements and levels of gene expression. Together, our study uncovers a regulatory layer of how the microbiota and metabolites influence the intestinal epithelium through chromatin, demonstrating a physiological setting in which histone acylations are dynamically regulated and associated with gene regulation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The properties of the cell types that are selectively vulnerable in Huntington's disease (HD) cortex, the nature of somatic CAG expansions of mHTT in these cells, and their importance in CNS circuitry have not been delineated. Here, we employed serial fluorescence-activated nuclear sorting (sFANS), deep molecular profiling, and single-nucleus RNA sequencing (snRNA-seq) of motor-cortex samples from thirteen predominantly early stage, clinically diagnosed HD donors and selected samples from cingulate, visual, insular, and prefrontal cortices to demonstrate loss of layer 5a pyramidal neurons in HD. Extensive mHTT CAG expansions occur in vulnerable layer 5a pyramidal cells, and in Betz cells, layers 6a and 6b neurons that are resilient in HD. Retrograde tracing experiments in macaque brains identify layer 5a neurons as corticostriatal pyramidal cells. We propose that enhanced somatic mHTT CAG expansion and altered synaptic function act together to cause corticostriatal disconnection and selective neuronal vulnerability in HD cerebral cortex., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships among CAG expansions, death of specific cell types and molecular events associated with these processes are not established. Here, we used fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise at mHTT in striatal medium spiny neurons (MSNs), cholinergic interneurons and cerebellar Purkinje neurons, and at mutant ATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSβ), which can inhibit nucleolytic excision of CAG slip-outs by FAN1. Our data support a model in which CAG expansions are necessary but may not be sufficient for cell death and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity., (© 2024. The Author(s).)

Insects and mammals have independently evolved odorant receptor genes that are arranged in large genomic tandem arrays. In mammals, each olfactory sensory neuron chooses to express a single receptor in a stochastic process that includes substantial chromatin rearrangements. Here, we show that ants, which have the largest odorant receptor repertoires among insects, employ a different mechanism to regulate gene expression from tandem arrays. Using single-nucleus RNA sequencing, we found that ant olfactory sensory neurons choose different transcription start sites along an array but then produce mRNA from many downstream genes. This can result in transcripts from dozens of receptors being present in a single nucleus. Such rampant receptor co-expression at first seems difficult to reconcile with the narrow tuning of the ant olfactory system. However, RNA fluorescence in situ hybridization showed that only mRNA from the most upstream transcribed odorant receptor seems to reach the cytoplasm where it can be translated into protein, whereas mRNA from downstream receptors gets sequestered in the nucleus. This implies that, despite the extensive co-expression of odorant receptor genes, each olfactory sensory neuron ultimately only produces one or very few functional receptors. Evolution has thus found different molecular solutions in insects and mammals to the convergent challenge of selecting small subsets of receptors from large odorant receptor repertoires., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although lung disease is the primary clinical outcome in COVID-19 patients, how SARS-CoV-2 induces lung pathology remains elusive. Here we describe a high-throughput platform to generate self-organizing and commensurate human lung buds derived from hESCs cultured on micropatterned substrates. Lung buds resemble human fetal lungs and display proximodistal patterning of alveolar and airway tissue directed by KGF. These lung buds are susceptible to infection by SARS-CoV-2 and endemic coronaviruses and can be used to track cell type-specific cytopathic effects in hundreds of lung buds in parallel. Transcriptomic comparisons of infected lung buds and postmortem tissue of COVID-19 patients identified an induction of BMP signaling pathway. BMP activity renders lung cells more susceptible to SARS-CoV-2 infection and its pharmacological inhibition impairs infection by this virus. These data highlight the rapid and scalable access to disease-relevant tissue using lung buds that recapitulate key features of human lung morphogenesis and viral infection biology., Competing Interests: Conflict of interests A.H.B. is a co-founder of startup companies RUMI Viro Inc., RUMI Scientific Inc., and OvaNova Laboratories, LLC, and serves on their scientific advisory boards. Both A.H.B. and E.A.R. are shareholders of RUMI Viro Inc. and RUMI Scientific Inc. C.M.R. is a founder of Apath LLC; a Scientific Advisory Board member of Imvaq Therapeutics, Vir Biotechnology, and Arbutus Biopharma; and an advisor for Regulus Therapeutics and Pfizer., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Periodontal disease is more common in individuals with rheumatoid arthritis (RA) who have detectable anti-citrullinated protein antibodies (ACPAs), implicating oral mucosal inflammation in RA pathogenesis. Here, we performed paired analysis of human and bacterial transcriptomics in longitudinal blood samples from RA patients. We found that patients with RA and periodontal disease experienced repeated oral bacteremias associated with transcriptional signatures of ISG15 + HLADR hi and CD48 high S100A2 pos monocytes, recently identified in inflamed RA synovia and blood of those with RA flares. The oral bacteria observed transiently in blood were broadly citrullinated in the mouth, and their in situ citrullinated epitopes were targeted by extensively somatically hypermutated ACPAs encoded by RA blood plasmablasts. Together, these results suggest that (i) periodontal disease results in repeated breaches of the oral mucosa that release citrullinated oral bacteria into circulation, which (ii) activate inflammatory monocyte subsets that are observed in inflamed RA synovia and blood of RA patients with flares and (iii) activate ACPA B cells, thereby promoting affinity maturation and epitope spreading to citrullinated human antigens.

Background: Sanitary sewage overflows (SSOs) release raw sewage, which may contaminate the drinking water supply. Boil water advisories (BWAs) are issued during low or negative pressure events, alerting customers to potential contamination in the drinking water distribution system., Objective: We evaluated the associations between SSOs and BWAs and diagnoses of gastrointestinal (GI) illness in Columbia, South Carolina, and neighboring communities, 2013-2017., Methods: A symmetric bi-directional case-crossover study design was used to assess the role of SSOs and BWAs on Emergency Room and Urgent Care visits with a primary diagnosis of GI illness. Cases were considered exposed if an SSO or BWA occurred 0-4 days, 5-9 days, or 10-14 days prior to the diagnosis, within the same residential zip code. Effect modification was explored via stratification on participant-level factors (e.g., sex, race, age) and season (January-March versus April-December)., Results: There were 830 SSOs, 423 BWAs, and 25,969 cases of GI illness. Highest numbers of SSOs, BWAs and GI cases were observed in a zip code where >80% of residents identified as Black or African-American. SSOs were associated with a 13% increase in the odds of a diagnosis for GI illness during the 0-4 day hazard period, compared to control periods (Odds Ratio: 1.13, 95% Confidence Interval: 1.09, 1.18), while no associations were observed during the other hazard periods. BWAs were not associated with increased or decreased odds of GI illness during all three hazard periods. However, in stratified analyses BWAs issued between January-March were associated with higher odds of GI illness, compared to advisories issued between April-December, in all three hazard periods., Significance: SSOs (all months) and BWAs (January-March) were associated with increased odds of a diagnosis of GI illness. Future research should examine sewage contamination of the drinking water distribution system, and mechanisms of sewage intrusion from SSOs., Impact: Sewage contains pathogens, which cause gastrointestinal (GI) illness. In Columbia, South Carolina, USA, between 2013-2017, there were 830 sanitary sewage overflows (SSOs). There were also 423 boil water advisories, which were issued during negative pressure events. Using case-crossover design, SSOs (all months) and boil water advisories (January-March) were associated with increased odds of Emergency Room and Urgent Care diagnoses of GI illness, potentially due to contamination of the drinking water distribution system. Lastly, we identified a community where >80% of residents identified as Black or African-American, which experienced a disproportionate burden of sewage exposure, compared to the rest of Columbia., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage 1-3 . The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes 4-7 . Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas 8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus 9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Insect societies are tightly integrated, complex biological systems in which group-level properties arise from the interactions between individuals 1-4 . However, these interactions have not been studied systematically and therefore remain incompletely known. Here, using a reverse engineering approach, we reveal that unlike solitary insects, ant pupae extrude a secretion derived from the moulting fluid that is rich in nutrients, hormones and neuroactive substances. This secretion elicits parental care behaviour and is rapidly removed and consumed by the adults. This behaviour is crucial for pupal survival; if the secretion is not removed, pupae develop fungal infections and die. Analogous to mammalian milk, the secretion is also an important source of early larval nutrition, and young larvae exhibit stunted growth and decreased survival without access to the fluid. We show that this derived social function of the moulting fluid generalizes across the ants. This secretion thus forms the basis of a central and hitherto overlooked interaction network in ant societies, and constitutes a rare example of how a conserved developmental process can be co-opted to provide the mechanistic basis of social interactions. These results implicate moulting fluids in having a major role in the evolution of ant eusociality., (© 2022. The Author(s).)

While dysregulation of adipocyte endocrine function plays a central role in obesity and its complications, the vast majority of adipokines remain uncharacterized. We employed bio-orthogonal non-canonical amino acid tagging (BONCAT) and mass spectrometry to comprehensively characterize the secretome of murine visceral and subcutaneous white and interscapular brown adip ocytes. Over 600 proteins were identified, the majority of which showed cell type-specific enrichment. We here describe a metabolic role for leucine-rich α-2 glycoprotein 1 (LRG1) as an obesity-regulated adipokine secreted by mature adipocytes. LRG1 overexpression significantly improved glucose homeostasis in diet-induced and genetically obese mice. This was associated with markedly reduced white adipose tissue macrophage accumulation and systemic inflammation. Mechanistically, we found LRG1 binds cytochrome c in circulation to dampen its pro-inflammatory effect. These data support a new role for LRG1 as an insulin sensitizer with therapeutic potential given its immunomodulatory function at the nexus of obesity, inflammation, and associated pathology., Competing Interests: CC, WB, SZ, CM, AP, MK, ZL, SS, FM, AC, TC, HM, PC No competing interests declared, (© 2022, Choi et al.)

Food intake and body weight are tightly regulated by neurons within specific brain regions, including the brainstem, where acute activation of dorsal raphe nucleus (DRN) glutamatergic neurons expressing the glutamate transporter Vglut3 (DRN Vglut3 ) drive a robust suppression of food intake and enhance locomotion. Activating Vglut3 neurons in DRN suppresses food intake and increases locomotion, suggesting that modulating the activity of these neurons might alter body weight. Here, we show that DRN Vglut3 neurons project to the lateral hypothalamus (LHA), a canonical feeding center that also reduces food intake. Moreover, chronic DRN Vglut3 activation reduces weight in both leptin-deficient (ob/ob) and leptin-resistant diet-induced obese (DIO) male mice. Molecular profiling revealed that the orexin 1 receptor (Hcrtr1) is highly enriched in DRN Vglut3 neurons, with limited expression elsewhere in the brain. Finally, an orally bioavailable, highly selective Hcrtr1 antagonist (CVN45502) significantly reduces feeding and body weight in DIO. Hcrtr1 is also co-expressed with Vglut3 in the human DRN, suggesting that there might be a similar effect in human. These results identify a potential therapy for obesity by targeting DRN Vglut3 neurons while also establishing a general strategy for developing drugs for central nervous system disorders., (© 2022. The Author(s).)

The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals. In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.

Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely, the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule-organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells, hence exposing a vulnerability in cancer that could be exploited therapeutically., Competing Interests: BH, NG, AG, VF, AS, MN, DB, TC, HP No competing interests declared, EF Reviewing editor, eLife, (© 2022, Hurwitz, Guzzi et al.)

Double-strand break (DSB) repair relies on DNA damage response (DDR) factors including BRCA1, BRCA2, and RAD51, which promote homology-directed repair (HDR); 53BP1, which affects single-stranded DNA formation; and proteins that mediate end-joining. Here we show that the CRL4/DDB1/WDR70 complex (CRL4 WDR70 ) controls the expression of DDR factors. Auxin-mediated degradation of WDR70 led to reduced expression of BRCA1, BRCA2, RAD51, and other HDR factors; 53BP1 and its downstream effectors; and other DDR factors. In contrast, cNHEJ factors were generally unaffected. WDR70 loss abrogated the localization of HDR factors to DSBs and elicited hallmarks of genomic instability, although 53BP1/RIF1 foci still formed. Mutation of the DDB1-binding WD40 motif, disruption of DDB1, or inhibition of cullins phenocopied WDR70 loss, consistent with CRL4, DDB1, and WDR70 functioning as a complex. RNA-sequencing revealed that WDR70 degradation affects the mRNA levels of DDR and many other factors. The data indicate that CRL4 WDR70 is critical for expression of myriad genes including BRCA1, BRCA2, and RAD51., Competing Interests: Declaration of Competing Interest For the manuscript: “Expression of BRCA1, BRCA2, RAD51, and other DSB repair factors is regulated by CRL4(WDR70)″: TdL is a member of the Scientific Advisory Board of Calico Life Sciences. The remaining authors declare no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cohesin and CTCF are major drivers of 3D genome organization, but their role in neurons is still emerging. Here, we show a prominent role for cohesin in the expression of genes that facilitate neuronal maturation and homeostasis. Unexpectedly, we observed two major classes of activity-regulated genes with distinct reliance on cohesin in mouse primary cortical neurons. Immediate early genes (IEGs) remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the IEGs Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterized by long-range chromatin contacts. Cohesin-dependence of constitutive neuronal genes with key functions in synaptic transmission and neurotransmitter signaling also scaled with chromatin loop length. Our data demonstrate that key genes required for the maturation and activation of primary cortical neurons depend on cohesin for their full expression, and that the degree to which these genes rely on cohesin scales with the genomic distance traversed by their chromatin contacts., Competing Interests: LC, FW, JB, MO, RG, YW, TC, GD, WG, KT, Vd, CW, MU, AF, JP, MM No competing interests declared, (© 2022, Calderon et al.)

The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAP's potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and nonneuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntington's Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAP's transcriptional activity can partially rescue the HD phenotype., Competing Interests: FP, TH, QT, TL, RD, AB, TC, JL No competing interests declared, NK, KG is part to an application for patent protection of derivatives of the Lats inhibitor TRULI used in this study, FE is a shareholder of RUMI Scientific, AH Is part to to an application for patent protection of derivatives of the Lats inhibitor TRULI used in this study, AB is a co-founder and shareholder of RUMI Scientific, (© 2022, Piccolo et al.)

Neurons rely on translation of synaptic mRNAs in order to generate activity-dependent changes in plasticity. Here, we develop a strategy combining compartment-specific crosslinking immunoprecipitation (CLIP) and translating ribosome affinity purification (TRAP) in conditionally tagged mice to precisely define the ribosome-bound dendritic transcriptome of CA1 pyramidal neurons. We identify CA1 dendritic transcripts with differentially localized mRNA isoforms generated by alternative polyadenylation and alternative splicing, including many that have altered protein-coding capacity. Among dendritic mRNAs, FMRP targets were found to be overrepresented. Cell-type-specific FMRP-CLIP and TRAP in microdissected CA1 neuropil revealed 383 dendritic FMRP targets and suggests that FMRP differentially regulates functionally distinct modules in CA1 dendrites and cell bodies. FMRP regulates ~15-20% of mRNAs encoding synaptic functions and 10% of chromatin modulators, in the dendrite and cell body, respectively. In the absence of FMRP, dendritic FMRP targets had increased ribosome association, consistent with a function for FMRP in synaptic translational repression. Conversely, downregulation of FMRP targets involved in chromatin regulation in cell bodies suggests a role for FMRP in stabilizing mRNAs containing stalled ribosomes in this compartment. Together, the data support a model in which FMRP regulates the translation and expression of synaptic and nuclear proteins within different compartments of a single neuronal cell type., Competing Interests: CH, KS, KM, JF, JK, PC, KC, TC, RD No competing interests declared, (© 2021, Hale et al.)

Brain development is regulated by conserved transcriptional programs across species, but little is known about the divergent mechanisms that create species-specific characteristics. Among brain regions, human cerebellar histogenesis differs in complexity compared with nonhuman primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. We report a rapid protocol for the derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSCs). Upon transplantation into juvenile mice, hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification-seq, we identified an unexpected temporal shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, in the human outer external granule layer. This molecular divergence may enable the protracted development of the human cerebellum compared to mice., Competing Interests: HB, AK, DB, TC No competing interests declared, MH Reviewing editor, eLife, (© 2021, Behesti et al.)

BACKGROUNDChildhood cancer survivors who received abdominal radiotherapy (RT) or total body irradiation (TBI) are at increased risk for cardiometabolic disease, but the underlying mechanisms are unknown. We hypothesize that RT-induced adipose tissue dysfunction contributes to the development of cardiometabolic disease in the expanding population of childhood cancer survivors.METHODSWe performed clinical metabolic profiling of adult childhood cancer survivors previously exposed to TBI, abdominal RT, or chemotherapy alone, alongside a group of healthy controls. Study participants underwent abdominal s.c. adipose biopsies to obtain tissue for bulk RNA sequencing. Transcriptional signatures were analyzed using pathway and network analyses and cellular deconvolution.RESULTSIrradiated adipose tissue is characterized by a gene expression signature indicative of a complex macrophage expansion. This signature includes activation of the TREM2-TYROBP network, a pathway described in diseases of chronic tissue injury. Radiation exposure of adipose is further associated with dysregulated adipokine secretion, specifically a decrease in insulin-sensitizing adiponectin and an increase in insulin resistance-promoting plasminogen activator inhibitor-1. Accordingly, survivors exhibiting these changes have early signs of clinical metabolic derangement, such as increased fasting glucose and hemoglobin A1c.CONCLUSIONChildhood cancer survivors exposed to abdominal RT or TBI during treatment exhibit signs of chronic s.c. adipose tissue dysfunction, manifested as dysregulated adipokine secretion that may negatively impact their systemic metabolic health.FUNDINGThis study was supported by Rockefeller University Hospital; National Institute of General Medical Sciences (T32GM007739); National Center for Advancing Translational Sciences (UL1 TR001866); National Cancer Institute (P30CA008748); American Cancer Society (133831-CSDG-19-117-01-CPHPS); American Diabetes Association (1-17-ACE-17); and an anonymous donor (MSKCC).

Known for nearly a century but through mechanisms that remain elusive, cells retain a memory of inflammation that equips them to react quickly and broadly to diverse secondary stimuli. Using murine epidermal stem cells as a model, we elucidate how cells establish, maintain, and recall inflammatory memory. Specifically, we landscape and functionally interrogate temporal, dynamic changes to chromatin accessibility, histone modifications, and transcription factor binding that occur during inflammation, post-resolution, and in memory recall following injury. We unearth an essential, unifying role for the general stress-responsive transcription factor FOS, which partners with JUN and cooperates with stimulus-specific STAT3 to establish memory; JUN then remains with other homeostatic factors on memory domains, facilitating rapid FOS re-recruitment and gene re-activation upon diverse secondary challenges. Extending our findings, we offer a comprehensive, potentially universal mechanism behind inflammatory memory and less discriminate recall phenomena with profound implications for tissue fitness in health and disease., Competing Interests: Declaration of interests The authors declare no competing financial interests. E.F. serves as an advisory board member for Cell Stem Cell and serves on the scientific advisory boards of L’Oreal and Arsenal Biosciences., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The antitumor efficacy of cancer immunotherapy can correlate with the presence of certain bacterial species within the gut microbiome. However, many of the molecular mechanisms that influence host response to immunotherapy remain elusive. In this study, we show that members of the bacterial genus Enterococcus improve checkpoint inhibitor immunotherapy in mouse tumor models. Active enterococci express and secrete orthologs of the NlpC/p60 peptidoglycan hydrolase SagA that generate immune-active muropeptides. Expression of SagA in nonprotective E. faecalis was sufficient to promote immunotherapy response, and its activity required the peptidoglycan sensor NOD2. Notably, SagA-engineered probiotics or synthetic muropeptides also augmented anti-PD-L1 antitumor efficacy. Taken together, our data suggest that microbiota species with specialized peptidoglycan remodeling activity and muropeptide-based therapeutics may enhance cancer immunotherapy and could be leveraged as next-generation adjuvants., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Obesity is a major risk factor for adverse outcomes in breast cancer; however, the underlying molecular mechanisms have not been elucidated. To investigate the role of crosstalk between mammary adipocytes and neoplastic cells in the tumor microenvironment (TME), we performed transcriptomic analysis of cancer cells and adjacent adipose tissue in a murine model of obesity-accelerated breast cancer and identified glycine amidinotransferase (Gatm) in adipocytes and Acsbg1 in cancer cells as required for obesity-driven tumor progression. Gatm is the rate-limiting enzyme in creatine biosynthesis, and deletion in adipocytes attenuated obesity-driven tumor growth. Similarly, genetic inhibition of creatine import into cancer cells reduced tumor growth in obesity. In parallel, breast cancer cells in obese animals upregulated the fatty acyl-CoA synthetase Acsbg1 to promote creatine-dependent tumor progression. These findings reveal key nodes in the crosstalk between adipocytes and cancer cells in the TME necessary for obesity-driven breast cancer progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Blood-feeding mosquitoes survive by feeding on nectar for metabolic energy but require a blood meal to develop eggs. Aedes aegypti females must accurately discriminate blood and nectar because each meal promotes mutually exclusive feeding programs with distinct sensory appendages, meal sizes, digestive tract targets, and metabolic fates. We investigated the syringe-like blood-feeding appendage, the stylet, and discovered that sexually dimorphic stylet neurons taste blood. Using pan-neuronal calcium imaging, we found that blood is detected by four functionally distinct stylet neuron classes, each tuned to specific blood components associated with diverse taste qualities. Stylet neurons are insensitive to nectar-specific sugars and respond to glucose only in the presence of additional blood components. The distinction between blood and nectar is therefore encoded in specialized neurons at the very first level of sensory detection in mosquitoes. This innate ability to recognize blood is the basis of vector-borne disease transmission to millions of people worldwide., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Through recurrent bouts synchronous with the hair cycle, quiescent melanocyte stem cells (McSCs) become activated to generate proliferative progeny that differentiate into pigment-producing melanocytes. The signaling factors orchestrating these events remain incompletely understood. Here, we use single-cell RNA sequencing with comparative gene expression analysis to elucidate the transcriptional dynamics of McSCs through quiescence, activation, and melanocyte maturation. Unearthing converging signs of increased WNT and BMP signaling along this progression, we endeavored to understand how these pathways are integrated. Employing conditional lineage-specific genetic ablation studies in mice, we found that loss of BMP signaling in the lineage leads to hair graying due to a block in melanocyte maturation. We show that interestingly, BMP signaling functions downstream from activated McSCs and maintains WNT effector, transcription factor LEF1. Employing pseudotime analysis, genetics, and chromatin landscaping, we show that following WNT-mediated activation of McSCs, BMP and WNT pathways collaborate to trigger the commitment of proliferative progeny by fueling LEF1- and MITF-dependent differentiation. Our findings shed light upon the signaling interplay and timing of cues that orchestrate melanocyte lineage progression in the hair follicle and underscore a key role for BMP signaling in driving complete differentiation., (© 2020 Infarinato et al.; Published by Cold Spring Harbor Laboratory Press.)

Zbtb11 is a conserved transcription factor mutated in families with hereditary intellectual disability. Its precise molecular and cellular functions are currently unknown, precluding our understanding of the aetiology of this disease. Using a combination of functional genomics, genetic and biochemical approaches, here we show that Zbtb11 plays essential roles in maintaining the homeostasis of mitochondrial function. Mechanistically, we find Zbtb11 facilitates the recruitment of nuclear respiratory factor 2 (NRF-2) to its target promoters, activating a subset of nuclear genes with roles in the biogenesis of respiratory complex I and the mitoribosome. Genetic inactivation of Zbtb11 resulted in a severe complex I assembly defect, impaired mitochondrial respiration, mitochondrial depolarisation, and ultimately proliferation arrest and cell death. Experimental modelling of the pathogenic human mutations showed these have a destabilising effect on the protein, resulting in reduced Zbtb11 dosage, downregulation of its target genes, and impaired complex I biogenesis. Our study establishes Zbtb11 as an essential mitochondrial regulator, improves our understanding of the transcriptional mechanisms of nuclear control over mitochondria, and may help to understand the aetiology of Zbtb11-associated intellectual disability.

Bladder cancer prognosis is closely linked to the underlying differentiation state of the tumor, ranging from the less aggressive and most-differentiated luminal tumors to the more aggressive and least-differentiated basal tumors. Sequencing of bladder cancer has revealed that loss-of-function mutations in chromatin regulators and mutations that activate receptor tyrosine kinase (RTK) signaling frequently occur in bladder cancer. However, little is known as to whether and how these two types of mutations functionally interact or cooperate to regulate tumor growth and differentiation state. Here, we focus on loss of the histone demethylase UTX (also known as KDM6A) and activation of the RTK FGFR3, two events that commonly cooccur in muscle invasive bladder tumors. We show that UTX loss and FGFR3 activation cooperate to disrupt the balance of luminal and basal gene expression in bladder cells. UTX localized to enhancers surrounding many genes that are important for luminal cell fate, and supported the transcription of these genes in a catalytic-independent manner. In contrast to UTX, FGFR3 activation was associated with lower expression of luminal genes in tumors and FGFR inhibition increased transcription of these same genes in cell culture models. This suggests an antagonistic relationship between UTX and FGFR3. In support of this model, UTX loss-of-function potentiated FGFR3-dependent transcriptional effects and the presence of UTX blocked an FGFR3-mediated increase in the colony formation of bladder cells. Taken together, our study reveals how mutations in UTX and FGFR3 converge to disrupt bladder differentiation programs that could serve as a therapeutic target., Competing Interests: The authors declare no competing interest.

Comparative transcriptomics between differentiating human pluripotent stem cells (hPSCs) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate human pluripotent stem cell-derived Purkinje cells (hPSC-PCs) that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2 - L10a-Egfp TRAP hPSC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides a direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders., Competing Interests: The authors declare no competing interest.

Modifications of histone proteins have essential roles in normal development and human disease. Recognition of modified histones by 'reader' proteins is a key mechanism that mediates the function of histone modifications, but how the dysregulation of these readers might contribute to disease remains poorly understood. We previously identified the ENL protein as a reader of histone acetylation via its YEATS domain, linking it to the expression of cancer-driving genes in acute leukaemia 1 . Recurrent hotspot mutations have been found in the ENL YEATS domain in Wilms tumour 2,3 , the most common type of paediatric kidney cancer. Here we show, using human and mouse cells, that these mutations impair cell-fate regulation by conferring gain-of-function in chromatin recruitment and transcriptional control. ENL mutants induce gene-expression changes that favour a premalignant cell fate, and, in an assay for nephrogenesis using murine cells, result in undifferentiated structures resembling those observed in human Wilms tumour. Mechanistically, although bound to largely similar genomic loci as the wild-type protein, ENL mutants exhibit increased occupancy at a subset of targets, leading to a marked increase in the recruitment and activity of transcription elongation machinery that enforces active transcription from target loci. Furthermore, ectopically expressed ENL mutants exhibit greater self-association and form discrete and dynamic nuclear puncta that are characteristic of biomolecular hubs consisting of local high concentrations of regulatory factors. Such mutation-driven ENL self-association is functionally linked to enhanced chromatin occupancy and gene activation. Collectively, our findings show that hotspot mutations in a chromatin-reader domain drive self-reinforced recruitment, derailing normal cell-fate control during development and leading to an oncogenic outcome.

Early life experiences program brain structure and function and contribute to behavioral endophenotypes in adulthood. Epigenetic control of gene expression by those experiences affect discrete brain regions involved in mood, cognitive function and regulation of hypothalamic-pituitary-adrenal (HPA) axis. In rodents, acute restraint stress increases the expression of the repressive histone H3 lysine 9 tri-methylation (H3K9me3) in hippocampal fields, including the CA3 pyramidal neurons. These CA3 neurons are crucially involved in cognitive function and mood regulation as well as activation of glucocorticoid (CORT) secretion. CA3 neurons also exhibit structural and functional changes after early-life stress (ELS) as well as after chronic stress in adulthood. Using a protocol of chronic ELS induced by limited bedding and nesting material followed by acute-swim stress (AS) in adulthood, we show that mice with a history of ELS display a blunted CORT response to AS, despite exhibiting activation of immediate early genes after stress similar to that found in control mice. We find that ELS induced persistently increased expression of the repressive H3K9me3 histone mark in the CA3 subfield at baseline that was subsequently decreased following AS. In contrast, AS induced a transient increase of this mark in control mice. Using translating ribosome affinity purification (TRAP) method to isolate CA3 translating mRNAs, we found that expression of genes of the epigenetic gene family, GABA/glutamate family, and glucocorticoid receptors binding genes were decreased transiently in control mice by AS and showed a persistent reduction in ELS mice. In most cases, AS in ELS mice did not induce gene expression changes. A stringent filtering of genes affected by AS in control and ELS mice revealed a noteworthy decrease in gene expression change in ELS mice compared to control. Only 18 genes were selectively regulated by AS in ELS mice and encompassed pathways such as circadian rhythm, inflammatory response, opioid receptors, and more genes included in the glucocorticoid receptor binding family. Thus, ELS programs a restricted translational response to stress in stress-sensitive CA3 neurons leading to persistent changes in gene expression, some of which mimic the transient effects of AS in control mice, while leaving in operation the immediate early gene response to AS.

Developmental health risks of chronical exposure to low doses of foodborne persistent organic pollutants (POP) are recognized but still largely uncharacterized. Juvenile female BALB/c mice exposed to either HBCD, CB-153 or TCDD at doses relevant to human dietary exposures (49.5 μg, 1.35 μg and 0.90 ng kg -1 bw -1 day -1 , respectively) for 28 days displayed histopathological changes in liver (HBCD, CB-153, TCDD), thymus (HBCD, CB-153) and uterus (HBCD), reduced serum oestradiol 17β (E2) levels (HBCD), increased serum testosterone (T) levels (CB-153) and an increased T/E2 ratio (HBCD). Proteomics analysis of brain provided molecular support for the HBCD-induced reduction in E2. Neural gene expression analysis, confirmed effects on 18 out of 30 genes previously found to be affected after exposure to higher doses to the same pollutants. Our findings indicate that exposure to POP at low doses is associated with subtle, but toxicological relevant effects on post-natal development in female mice., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR) that accumulates in humans and affects the nervous system. To elucidate the mechanisms of HBCD neurotoxicity, we used transcriptomic profiling in brains of female mice exposed through their diet to HBCD (199 mg/kg body weight per day) for 28 days and compared with those of neuronal N2A and NSC-19 cell lines exposed to 1 or 2 µM HBCD. Similar pathways and functions were affected both in vivo and in vitro, including Ca 2+ and Zn 2+ signalling, glutamatergic neuron activity, apoptosis, and oxidative stress. Release of cytosolic free Zn 2+ by HBCD was confirmed in N2A cells. This Zn 2+ release was partially quenched by the antioxidant N-acetyl cysteine indicating that, in accordance with transcriptomic analysis, free radical formation is involved in HBCD toxicity. To investigate the effects of HBCD in excitable cells, we isolated mouse hippocampal neurons and monitored Ca 2+ signalling triggered by extracellular glutamate or zinc, which are co-released pre-synaptically to trigger postsynaptic signalling. In control cells application of zinc or glutamate triggered a rapid rise of intracellular [Ca 2+ ]. Treatment of the cultures with 1 µM of HBCD was sufficient to reduce the glutamate-dependent Ca 2+ signal by 50%. The effect of HBCD on zinc-dependent Ca 2+ signalling was even more pronounced, resulting in the reduction of the Ca 2+ signal with 86% inhibition at 1 µM HBCD. Our results show that low concentrations of HBCD affect neural signalling in mouse brain acting through dysregulation of Ca 2+ and Zn 2+ homeostasis.

The present study assessed if eating a diet of fish, spiked with persistent organic pollutants (POPs), affects gene and protein expression in the maturing mouse brain. Juvenile female Balb/c mice (22 days of age) were exposed for 28 days to fish-based diets spiked with the dioxin 2,3,7,8-tetrachlorodibenzodioxin (TCDD) or the non dioxin-like (NDL) chemicals hexabromocyclodocecane (HBCD), 2,2'4,4'-tetrabromodiphenylether (BDE-47) or 2,2'4,4',5,5'-hexachlorobiphenyl (CB-153) at doses approximating their respective lowest observed adverse effect levels (LOAEL). It was found that all POPs elicited changes in neural gene and protein expression profiles. Bioinformatic analysis of gene expression data highlighted the importance of the aryl hydrocarbon receptor (AHR) in dioxin toxicity and revealed that zinc regulation in the brain is targeted by TCDD through the AHR. Calcium homeostasis was affected by both TCDD and the NDL chemicals. In contrast to the transcriptomic analysis, the proteomics data did not allow for a clear distinction between DL and NDL responses in the juvenile brain but indicated that proteins associated with excitotoxicity were affected in all exposure groups. Integrated interpretation of data led to the conclusion that the dietary contaminants investigated in the present study breach the blood brain barrier (BBB) and accumulate in the juvenile brain where they may induce excitotoxic insults by dysregulation of the otherwise tightly controlled homeostasis of calcium and zinc. Overall, the findings of the present study highlight the need for further assessment of the risks associated with early life exposure to foodborne POPs., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

With the advent of ChIP-seq multiplexing technologies and the subsequent increase in ChIP-seq throughput, the development of working standards for the quality assessment of ChIP-seq studies has received significant attention. The ENCODE consortium's large scale analysis of transcription factor binding and epigenetic marks as well as concordant work on ChIP-seq by other laboratories has established a new generation of ChIP-seq quality control measures. The use of these metrics alongside common processing steps has however not been evaluated. In this study, we investigate the effects of blacklisting and removal of duplicated reads on established metrics of ChIP-seq quality and show that the interpretation of these metrics is highly dependent on the ChIP-seq preprocessing steps applied. Further to this we perform the first investigation of the use of these metrics for ChIP-exo data and make recommendations for the adaptation of the NSC statistic to allow for the assessment of ChIP-exo efficiency.

Fish represents source of nutrients and major dietary vehicle of lipophilic persistent contaminants. The study compared the effects of two legacy and two emerging fish pollutants (Hexabromocyclododecane HBCD; 2,2',4,4'-Tetrabromodiphenyl ether BDE-47; 2,2',4,4',5,5'-Hexachlorobiphenyl PCB-153; 2,3,7,8-Tetrachlorodibenzo-p-doxin TCDD) in juvenile female mice exposed through a salmon based rodent diet for 28 days (dietary doses: HBCD 199 mg/kg bw/day; BDE-47 450 μg/kg bw/day; PCB-153 195 μg/kg bw/day; TCDD 90 ng/kg bw/day). Dose levels were comparable to previously reported developmental Lowest Observed Adverse Effect Levels. None of the treatments elicited signs of overt toxicity, but HBCD increased relative liver weight. All compounds caused changes in liver, thymus and thyroid; spleen was affected by BDE-47 and PCB-153; no effects were seen in uterus and adrenals. Strongest effects in thyroid follicles were elicited by PCB-153, in thymus and liver by BDE-47. HBCD and BDE-47 induced liver fatty changes, but appeared to be less potent in the other tissues. HBCD, BDE-47 and TCDD increased serum testosterone levels and the testosterone/estradiol ratio, suggesting a potential involvement of pathways related to sex steroid biosynthesis and/or metabolism. The results support the role of toxicological studies on juvenile rodents in the hazard characterization of chemicals, due to endocrine and/or immune effects., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Transcription corepressors are general regulators controlling the expression of genes involved in multiple signaling pathways and developmental programs. Repression is mediated through mechanisms including the stabilization of a repressive chromatin structure over control regions and regulation of Mediator function inhibiting RNA polymerase II activity. Using whole-genome arrays we show that the Arabidopsis thaliana corepressor LEUNIG, a member of the GroTLE transcription corepressor family, regulates the expression of multiple targets in vivo. LEUNIG has a role in the regulation of genes involved in a number of different physiological processes including disease resistance, DNA damage response, and cell signaling. We demonstrate that repression of in vivo LEUNIG targets is achieved through histone deacetylase (HDAC)-dependent and -independent mechanisms. HDAC-dependent mechanisms involve direct interaction with HDA19, a class 1 HDAC, whereas an HDAC-independent repression activity involves interactions with the putative Arabidopsis Mediator components AtMED14/SWP and AtCDK8/HEN3. We suggest that changes in chromatin structure coupled with regulation of Mediator function are likely to be utilized by LEUNIG in the repression of gene transcription.