Identifying novel mechanisms of per- and polyfluoroalkyl substance-induced hepatotoxicity using FRG humanized mice.

Per- and polyfluoroalkyl substances (PFAS) such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) and perfluoro-2-methyl-3-oxahexanoic acid (GenX), the new replacement PFAS, are major environmental contaminants. In rodents, these PFAS induce several adverse effects on the liver, including increased proliferation, hepatomegaly, steatosis, hypercholesterolemia, nonalcoholic fatty liver disease and liver cancers. Activation of peroxisome proliferator receptor alpha by PFAS is considered the primary mechanism of action in rodent hepatocyte-induced proliferation. However, the human relevance of this mechanism is uncertain. We investigated human-relevant mechanisms of PFAS-induced adverse hepatic effects using FRG liver-chimeric humanized mice with livers repopulated with functional human hepatocytes. Male FRG humanized mice were treated with 0.067 mg/L of PFOA, 0.145 mg/L of PFOS, or 1 mg/L of GenX in drinking water for 28 days. PFOS caused a significant decrease in total serum cholesterol and LDL/VLDL, whereas GenX caused a significant elevation in LDL/VLDL with no change in total cholesterol and HDL. All three PFAS induced significant hepatocyte proliferation. RNA-sequencing with alignment to the human genome showed a total of 240, 162, and 619 differentially expressed genes after PFOA, PFOS, and GenX exposure, respectively. Upstream regulator analysis revealed that all three PFAS induced activation of p53 and inhibition of androgen receptor and NR1D1, a transcriptional repressor important in circadian rhythm. Further biochemical studies confirmed NR1D1 inhibition and in silico modeling indicated potential interaction of all three PFAS with the DNA-binding domain of NR1D1. In conclusion, our studies using FRG humanized mice have revealed new human-relevant molecular mechanisms of PFAS including their previously unknown effect on circadian rhythm.

Selective targeting of IRAK1 attenuates low molecular weight hyaluronic acid-induced stemness and non-canonical STAT3 activation in epithelial ovarian cancer.

Advanced epithelial ovarian cancer (EOC) survival rates are dishearteningly low, with ~25% surviving beyond 5 years. Evidence suggests that cancer stem cells contribute to acquired chemoresistance and tumor recurrence. Here, we show that IRAK1 is upregulated in EOC tissues, and enhanced expression correlates with poorer overall survival. Moreover, low molecular weight hyaluronic acid, which is abundant in malignant ascites from patients with advanced EOC, induced IRAK1 phosphorylation leading to STAT3 activation and enhanced spheroid formation. Knockdown of IRAK1 impaired tumor growth in peritoneal disease models, and impaired HA-induced spheroid growth and STAT3 phosphorylation. Finally, we determined that TCS2210, a known inducer of neuronal differentiation in mesenchymal stem cells, is a selective inhibitor of IRAK1. TCS2210 significantly inhibited EOC growth in vitro and in vivo both as monotherapy, and in combination with cisplatin. Collectively, these data demonstrate IRAK1 as a druggable target for EOC.

Manzamine A reduces androgen receptor transcription and synthesis by blocking E2F8-DNA interactions and effectively inhibits prostate tumor growth in mice.

The androgen receptor (AR) is the main driver in the development of castration-resistant prostate cancer, where the emergence of AR splice variants leads to treatment-resistant disease. Through detailed molecular studies of the marine alkaloid manzamine A (MA), we identified transcription factor E2F8 as a previously unknown regulator of AR transcription that prevents AR synthesis in prostate cancer cells. MA significantly inhibited the growth of various prostate cancer cell lines and was highly effective in inhibiting xenograft tumor growth in mice without any pathophysiological perturbations in major organs. MA suppressed the full-length AR (AR-FL), its spliced variant AR-V7, and the AR-regulated prostate-specific antigen (PSA; also known as KLK3) and human kallikrein 2 (hK2; also known as KLK2) genes. RNA sequencing (RNA-seq) analysis and protein modeling studies revealed E2F8 interactions with DNA as a potential novel target of MA, suppressing AR transcription and its synthesis. This novel mechanism of blocking AR biogenesis via E2F8 may provide an opportunity to control therapy-resistant prostate cancer over the currently used AR antagonists designed to target different parts of the AR gene.

Kupffer cell diversity maintains liver function in alcohol-associated liver disease.

BACKGROUND AIMS: Liver macrophages are heterogeneous and play an important role in alcohol-associated liver disease (ALD) but there is limited understanding of the functions of specific macrophage subsets in the disease. We used a Western Diet Alcohol (WDA) mouse model of ALD to examine the hepatic myeloid cell compartment by scRNA seq and targeted Kupffer cell (KC) ablation to understand the diversity and function of liver macrophages in ALD. APPROACH AND RESULTS: In the WDA liver, KCs and infiltrating monocytes/macrophages (IMs) each represented about 50% of the myeloid pool. Five major KC clusters all expressed genes associated with receptor mediated endocytosis and lipid metabolism, but most were predicted to be non-inflammatory and antifibrotic with one minor KC cluster having a pro-inflammatory and extracellular matrix degradation gene signature. IM clusters, in contrast, were predicted to be pro-inflammatory and pro-fibrotic. In vivo diphtheria toxin based selective KC ablation during alcohol exposure resulted in a liver failure phenotype with increases in PT/INR and bilirubin, loss of differentiated hepatocyte gene expression, and an increase in expression of hepatocyte progenitor markers such as EpCAM, CK-7 and Igf2bp3. Gene set enrichment analysis of whole liver RNAseq from the KC ablated WDA mice showed a similar pattern as seen in human alcoholic hepatitis. CONCLUSIONS: In this ALD model, KCs are anti-inflammatory and are critical for maintenance of hepatocyte differentiation. IMs are largely pro-inflammatory and contribute more to liver fibrosis. Future targeting of specific macrophage subsets may provide new approaches to treatment of liver failure and fibrosis in ALD.

LH/hCG Regulation of Circular RNA in Mural Granulosa Cells during the Periovulatory Period in Mice.

Ovarian follicles undergo a series of dynamic changes following the ovulatory surge of luteinizing hormone including cumulus expansion, oocyte maturation, ovulation, and luteinization. Post-transcriptional gene regulatory events are critical for mediating LH follicular responses, and among all RNA isoforms, circular RNA (circRNA) is one of the most abundant forms present in cells, yet they remain the least studied. Functionally, circRNA can act as miRNA sponges, protein sponges/decoys, and regulators of transcription and translation. In the context of ovarian follicular development, the identity and roles of circRNA are relatively unknown. In the present study, high throughput RNA sequencing of granulosa cells immediately prior to and 4-h after the LH/hCG surge identified 42,381 circRNA originating from 7712 genes. A total of 54 circRNA were identified as differentially expressed between 0-h and 4-h time points (Fold Change ± 1.5, FDR ≤ 0.1), among them 42 circRNA were upregulated and 12 circRNA were downregulated. All differentially expressed circRNA between the 0-h and 4-h groups were subjected to circinteractome analysis and identified networks of circRNA-protein and circRNA-miRNA were further subjected to "micro-RNA target filter analysis" in Ingenuity Pathway Analyses, which resulted in the identification of miRNA targeted mRNAs. A comparison of these circRNA target mRNAs with LH-induced mRNAs identified Runx2, Egfr, Areg, Sult1el, Cyp19a1, Cyp11a1, and Hsd17b1 as targets of circKif2, circVcan, circMast4, and circMIIt10. These newly identified LH/hCG-induced circRNA, their target miRNA and protein networks provide new insights into the complex interactions associated with periovulatory follicular development.

Notch3 deletion regulates HIV-1 gene expression and systemic inflammation to ameliorate chronic kidney disease.

Antiretroviral therapy (ART) has profoundly decreased HIV-1 associated morbidity. However, despite ART, immune cells remain latently infected and slowly release viral proteins, leading to chronic inflammation and HIV associated comorbidities. Thus, new strategies are needed to reduce the inflammatory effects of HIV-1. In previous studies we found that gamma secretase inhibitor (GSIXX) ameliorated renal lesions of HIV-Tg26 mice carrying replication defective HIV-1 PNL4-3 by inhibiting Notch activation. Since gamma secretase inhibition is not a safe strategy in humans, here we examined the specific role of the Notch3 pathway in the pathogenesis of the renal lesions and outcome of HIV-Tg26 mice. We found that Notch3 is activated in podocytes and other renal cells in HIV-Tg26 mice and human biopsies with HIV-1 associated Nephropathy (HIVAN). Knockdown of Notch3 in HIV-Tg26 mice revealed a marked reduction in the mortality rate, improvement in renal injury and function. RNA sequencing and immunolabeling data revealed that Notch3 deletion drastically reduced infiltrating renal macrophages in HIV-Tg-N3KO mice in association with renal reduction of HIV-nef mRNA expression levels. In fact, bone marrow derived macrophages from HIV-Tg26 mice showed a significant activation of Notch3 signaling. Further, systemic levels of TNF-alpha and MCP-1 and other inflammatory chemokines and cytokines were reduced in Tg-N3KO mice as compared to HIV-Tg26 mice and this translated to a marked reduction of HIV-induced skin lesions. Taken together, these studies strongly point to a dual inhibitory/therapeutic effect of Notch3 inhibition on HIV-induced systemic, skin and renal lesions independently of ART.

Transcriptional metabolic reprogramming implements meiotic fate decision in mouse testicular germ cells.

Nutrient starvation drives yeast meiosis, whereas retinoic acid (RA) is required for mammalian meiosis through its germline target Stra8. Here, by using single-cell transcriptomic analysis of wild-type and Stra8-deficient juvenile mouse germ cells, our data show that the expression of nutrient transporter genes, including Slc7a5, Slc38a2, and Slc2a1, is downregulated in germ cells during meiotic initiation, and this process requires Stra8, which binds to these genes and induces their H3K27 deacetylation. Consequently, Stra8-deficient germ cells sustain glutamine and glucose uptake in response to RA and exhibit hyperactive mTORC1/protein kinase A (PKA) activities. Importantly, expression of Slc38a2, a glutamine importer, is negatively correlated with meiotic genes in the GTEx dataset, and Slc38a2 knockdown downregulates mTORC1/PKA activities and induces meiotic gene expression. Thus, our study indicates that RA via Stra8, a chordate morphogen pathway, induces meiosis partially by generating a conserved nutrient restriction signal in mammalian germ cells by downregulating their nutrient transporter expression.

Landscape of Double-Stranded DNA Breaks in Postmortem Brains from Alzheimer's Disease and Non-Demented Individuals.

BACKGROUND: Alzheimer's disease (AD) brains accumulate DNA double-strand breaks (DSBs), which could contribute to neurodegeneration and dysfunction. The genomic distribution of AD brain DSBs is unclear. OBJECTIVE: To map genome-wide DSB distributions in AD and age-matched control brains. METHODS: We obtained autopsy brain tissue from 3 AD and 3 age-matched control individuals. The donors were men between the ages of 78 to 91. Nuclei extracted from frontal cortex tissue were subjected to Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assay with an antibody against γH2AX, a marker of DSB formation. γH2AX-enriched chromatins were purified and analyzed via high-throughput genomic sequencing. RESULTS: The AD brains contained 18 times more DSBs than the control brains and the pattern of AD DSBs differed from the control brain pattern. In conjunction with published genome, epigenome, and transcriptome analyses, our data revealed aberrant DSB formation correlates with AD-associated single-nucleotide polymorphisms, increased chromatin accessibility, and upregulated gene expression. CONCLUSION: Our data suggest in AD, an accumulation of DSBs at ectopic genomic loci could contribute to an aberrant upregulation of gene expression.

Identification of follicle-stimulating hormone-responsive genes in Sertoli cells during early postnatal mouse testis development.

BACKGROUND: In the mouse testis, Sertoli cells rapidly divide during a narrow window of time pre-pubertally and differentiate thereafter. The number of Sertoli cells determines the testis size and germ cell-carrying capacity. Follicle-stimulating hormone (FSH) binds its cognate FSH-receptors expressed on Sertoli cells and acts as a mitogen to regulate their proliferation. Fshb-/- mutant adult male mice have reduced Sertoli cell number and testis size and reduced sperm number and motility. However, FSH-responsive genes in early postnatal mouse Sertoli cells are unknown. OBJECTIVES: To identify FSH-responsive genes in early postnatal mouse Sertoli cells. MATERIALS AND METHODS: A fluorescence-activated cell sorting method was developed to rapidly purify Sertoli cells from control and Fshb-/- mice carrying a Sox9 GfpKI allele. These pure Sertoli cells were used for large-scale gene expression analyses. RESULTS: We show that mouse Sertoli cells rarely divide beyond postnatal day 7. Our in vivo BrdU labeling studies indicate loss of FSH results in a 30% reduction in Sertoli cell proliferation in mice at 5 days of age. Flowsorted GFP+ Sertoli cells with maximal Fshr expression were 97%-98% pure and mostly devoid of Leydig and germ cells as assessed by Taqman qPCR quantification of gene expression and immunolabeling of the corresponding cell-specific markers. Large-scale gene expression analysis identified several differentially regulated genes in flow-sorted GFP+ Sertoli cells obtained from testis of control and Fshb-/- mice at 5 days of age. The top 25 networks identified by pathway analysis include those related to the cell cycle, cell survival and most importantly, carbohydrate and lipid metabolism and molecular transport. DISCUSSION: Several of the FSH-responsive genes identified in this study could serve as useful markers for Sertoli cell proliferation in normal physiology, toxicant-induced Sertoli cell/testis injury, and other pathological conditions. CONCLUSION: Our studies reveal that FSH-regulates macromolecular metabolism and molecular transport networks of genes in early postnatal Sertoli cells most likely in preparation for establishment of functional associations with germ cells to successfully coordinate spermatogenesis.

Sera of Individuals Chronically Infected with Hepatitis B Virus (HBV) Contain Diverse RNA Types Produced by HBV Replication or Derived from Integrated HBV DNA.

This study aimed to better characterize the repertoire of serum hepatitis B virus (HBV) RNAs during chronic HBV infection in humans, which remains understudied. Using reverse transcription-PCR (RT-PCR), real-time quantitative PCR (RT-qPCR), RNA-sequencing, and immunoprecipitation, we found that (i) >50% of serum samples bore different amounts of HBV replication-derived RNAs (rd-RNAs); (ii) a few samples contained RNAs transcribed from integrated HBV DNA, including 5'-HBV-human-3' RNAs (integrant-derived RNAs [id-RNAs]) and 5'-human-HBV-3' transcripts, as a minority of serum HBV RNAs; (iii) spliced HBV RNAs were abundant in <50% of analyzed samples; (iv) most serum rd-RNAs were polyadenylated via conventional HBV polyadenylation signal; (v) pregenomic RNA (pgRNA) was the major component of the pool of serum RNAs; (vi) the area of HBV positions 1531 to 1739 had very high RNA read coverage and thus should be used as a target for detecting serum HBV RNAs; (vii) the vast majority of rd-RNAs and pgRNA were associated with HBV virions but not with unenveloped capsids, exosomes, classic microvesicles, or apoptotic vesicles and bodies; (viii) considerable rd-RNAs presence in the circulating immune complexes was found in a few samples; and (ix) serum relaxed circular DNA (rcDNA) and rd-RNAs should be quantified simultaneously to evaluate HBV replication status and efficacy of anti-HBV therapy with nucleos(t)ide analogs. In summary, sera contain various HBV RNA types of different origin, which are likely secreted via different mechanisms. In addition, since we previously showed that id-RNAs were abundant or predominant HBV RNAs in many of liver and hepatocellular carcinoma tissues as compared to rd-RNAs, there is likely a mechanism favoring the egress of replication-derived RNAs. IMPORTANCE The presence of integrant-derived RNAs (id-RNAs) and 5'-human-HBV-3' transcripts derived from integrated hepatitis B virus (HBV) DNA in sera was demonstrated for the first time. Thus, sera of individuals chronically infected with HBV contained both replication-derived and integrant-transcribed HBV RNAs. The majority of serum HBV RNAs were the transcripts produced by HBV genome replication, which were associated with HBV virions and not with other types of extracellular vesicles. These and other above-mentioned findings advanced our understanding of the HBV life cycle. In addition, the study suggested a promising target area on the HBV genome to increase sensitivity of the detection of serum HBV RNAs and supported the idea that simultaneous detection of replication-derived RNAs (rd-RNAs) and relaxed circular DNA (rcDNA) in serum provides more adequate evaluation of (i) the HBV genome replication status and (ii) the durability and efficiency of the therapy with anti-HBV nucleos(t)ide analogs, which could be useful for improvement of the diagnostics and treatment of HBV-infected individuals.

Exploring Whether Iron Sequestration within the CNS of Patients with Alzheimer's Disease Causes a Functional Iron Deficiency That Advances Neurodegeneration.

The involvement of iron in the pathogenesis of Alzheimer's disease (AD) may be multifaceted. Besides potentially inducing oxidative damage, the bioavailability of iron may be limited within the central nervous system, creating a functionally iron-deficient state. By comparing staining results from baseline and modified iron histochemical protocols, iron was found to be more tightly bound within cortical sections from patients with high levels of AD pathology compared to subjects with a diagnosis of something other than AD. To begin examining whether the bound iron could cause a functional iron deficiency, a protein-coding gene expression dataset of initial, middle, and advanced stages of AD from olfactory bulb tissue was analyzed for iron-related processes with an emphasis on anemia-related changes in initial AD to capture early pathogenic events. Indeed, anemia-related processes had statistically significant alterations, and the significance of these changes exceeded those for AD-related processes. Other changes in patients with initial AD included the expressions of transcripts with iron-responsive elements and for genes encoding proteins for iron transport and mitochondrial-related processes. In the latter category, there was a decreased expression for the gene encoding pitrilysin metallopeptidase 1 (PITRM1). Other studies have shown that PITRM1 has an altered activity in patients with AD and is associated with pathological changes in this disease. Analysis of a gene expression dataset from PITRM1-deficient or sufficient organoids also revealed statistically significant changes in anemia-like processes. These findings, together with supporting evidence from the literature, raise the possibility that a pathogenic mechanism of AD could be a functional deficiency of iron contributing to neurodegeneration.

Identifying Human Specific Adverse Outcome Pathways of Per- and Polyfluoroalkyl Substances Using Liver-Chimeric Humanized Mice.

Background: Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants with myriad adverse effects. While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are the most common contaminants, levels of replacement PFAS, such as perfluoro-2-methyl-3-oxahexanoic acid (GenX), are increasing. In rodents, PFOA, PFOS, and GenX have several adverse effects on the liver, including nonalcoholic fatty liver disease. Objective: We aimed to determine human-relevant mechanisms of PFAS induced adverse hepatic effects using FRG liver-chimeric humanized mice with livers repopulated with functional human hepatocytes. Methods: Male humanized mice were treated with 0.067 mg/L of PFOA, 0.145 mg/L of PFOS, or 1 mg/L of GenX in drinking water for 28 days. Liver and serum were collected for pathology and clinical chemistry, respectively. RNA-sequencing coupled with pathway analysis was used to determine molecular mechanisms. Results: PFOS caused a significant decrease in total serum cholesterol and LDL/VLDL, whereas GenX caused a significant elevation in LDL/VLDL with no change in total cholesterol and HDL. PFOA had no significant changes in serum LDL/VLDL and total cholesterol. All three PFAS induced significant hepatocyte proliferation. RNA-sequencing with alignment to the human genome showed a total of 240, 162, and 619 differentially expressed genes after PFOA, PFOS, and GenX exposure, respectively. Upstream regulator analysis revealed inhibition of NR1D1, a transcriptional repressor important in circadian rhythm, as the major common molecular change in all PFAS treatments. PFAS treated mice had significant nuclear localization of NR1D1. In silico modeling showed PFOA, PFOS, and GenX potentially interact with the DNA-binding domain of NR1D1. Discussion: These data implicate PFAS in circadian rhythm disruption via inhibition of NR1D1. These studies show that FRG humanized mice are a useful tool for studying the adverse outcome pathways of environmental pollutants on human hepatocytes in situ.

Doublecortin-like kinase 1 is a therapeutic target in squamous cell carcinoma.

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC (HNSCC) and anal SCC (ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virus negative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone) binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.

Loss of the repressor REST affects progesterone receptor function and promotes uterine leiomyoma pathogenesis.

Uterine leiomyomas (UL) are benign tumors that arise in the myometrial layer of the uterus. The standard treatment option for UL is hysterectomy, although hormonal therapies, such as selective progesterone receptor modulators, are often used as temporary treatment options to reduce symptoms or to slow the growth of tumors. However, since the pathogenesis of UL is poorly understood and most hormonal therapies are not based on UL-specific, divergent hormone signaling pathways, hallmarks that predict long-term efficacy and safety of pharmacotherapies remain largely undefined. In a previous study, we reported that aberrant expression of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes activate UL growth due to the near ubiquitous loss of REST. Here, we show that ablation of the Rest gene in mouse uterus leads to UL phenotype and gene-expression patterns analogous to UL, including altered estrogen and progesterone signaling pathways. We demonstrate that many of the genes dysregulated in UL harbor cis-regulatory elements bound by REST and progesterone receptor (PGR) adjacent to each other. Crucially, we identify an interaction between REST and PGR in healthy myometrium and present a putative mechanism for the dysregulation of progesterone-responsive genes in UL ensuing in the loss of REST. Using three Rest conditional knockout mouse lines, we provide a comprehensive picture of the impact loss of REST has in UL pathogenesis and in altering the response of UL to steroid hormones.

TFEB regulates sulfur amino acid and coenzyme A metabolism to support hepatic metabolic adaptation and redox homeostasis.

Fatty liver is a highly heterogenous condition driven by various pathogenic factors in addition to the severity of steatosis. Protein insufficiency has been causally linked to fatty liver with incompletely defined mechanisms. Here we report that fatty liver is a sulfur amino acid insufficient state that promotes metabolic inflexibility via limiting coenzyme A availability. We demonstrate that the nutrient-sensing transcriptional factor EB synergistically stimulates lysosome proteolysis and methionine adenosyltransferase to increase cysteine pool that drives the production of coenzyme A and glutathione, which support metabolic adaptation and antioxidant defense during increased lipid influx. Intriguingly, mice consuming an isocaloric protein-deficient Western diet exhibit selective hepatic cysteine, coenzyme A and glutathione deficiency and acylcarnitine accumulation, which are reversed by cystine supplementation without normalizing dietary protein intake. These findings support a pathogenic link of dysregulated sulfur amino acid metabolism to metabolic inflexibility that underlies both overnutrition and protein malnutrition-associated fatty liver development.

The START domain mediates Arabidopsis GLABRA2 dimerization and turnover independently of homeodomain DNA binding.

Class IV homeodomain leucine-zipper transcription factors (HD-Zip IV TFs) are key regulators of epidermal differentiation that are characterized by a DNA-binding HD in conjunction with a lipid-binding domain termed steroidogenic acute regulatory-related lipid transfer (START). Previous work established that the START domain of GLABRA2 (GL2), a HD-Zip IV member from Arabidopsis (Arabidopsis thaliana), is required for TF activity. Here, we addressed the functions and possible interactions of START and the HD in DNA binding, dimerization, and protein turnover. Deletion analysis of the HD and missense mutations of a conserved lysine (K146) resulted in phenotypic defects in leaf trichomes, root hairs, and seed mucilage, similar to those observed for START domain mutants, despite nuclear localization of the respective proteins. In vitro and in vivo experiments demonstrated that while HD mutations impair binding to target DNA, the START domain is dispensable for DNA binding. Vice versa, protein interaction assays revealed impaired GL2 dimerization for multiple alleles of START mutants, but not HD mutants. Using in vivo cycloheximide chase experiments, we provided evidence for the role of START, but not HD, in maintaining protein stability. This work advances our mechanistic understanding of HD-Zip TFs as multidomain regulators of epidermal development in plants.

Hippo signaling cofactor, WWTR1, at the crossroads of human trophoblast progenitor self-renewal and differentiation.

Healthy progression of human pregnancy relies on cytotrophoblast (CTB) progenitor self-renewal and its differentiation toward multinucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, the underlying molecular mechanisms that fine-tune CTB self-renewal or direct its differentiation toward STBs or EVTs during human placentation are poorly defined. Here, we show that Hippo signaling cofactor WW domain containing transcription regulator 1 (WWTR1) is a master regulator of trophoblast fate choice during human placentation. Using human trophoblast stem cells (human TSCs), primary CTBs, and human placental explants, we demonstrate that WWTR1 promotes self-renewal in human CTBs and is essential for their differentiation to EVTs. In contrast, WWTR1 prevents induction of the STB fate in undifferentiated CTBs. Our single-cell RNA sequencing analyses in first-trimester human placenta, along with mechanistic analyses in human TSCs revealed that WWTR1 fine-tunes trophoblast fate by directly regulating WNT signaling components. Importantly, our analyses of placentae from pathological pregnancies show that extreme preterm births (gestational time ≤28 wk) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish the critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal versus differentiation. It plays positive instructive roles in promoting CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from attaining the STB fate.

GenX induces fibroinflammatory gene expression in primary human hepatocytes.

The toxicity induced by the persistent organic pollutants per- and polyfluoroalkyl substances (PFAS) is dependent on the length of their polyfluorinated tail. Long-chain PFASs have significantly longer half-lives and profound toxic effects compared to their short-chain counterparts. Recently, production of a short-chain PFAS substitute called ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propanoate, also known as GenX, has significantly increased. However, the adverse health effects of GenX are not completely known. In this study, we investigated the dose-dependent effects of GenX on primary human hepatocytes (PHH). Freshly isolated PHH were treated with either 0.1, 10, or 100 µM of GenX for 48 and 96 h; then, global transcriptomic changes were determined using Human Clariom™ D arrays. GenX-induced transcriptional changes were similar at 0.1 and 10 µM doses but were significantly different at the 100 µM dose. Genes involved in lipid, monocarboxylic acid, and ketone metabolism were significantly altered following exposure of PHH at all doses. However, at the 100 µM dose, GenX caused changes in genes involved in cell proliferation, inflammation and fibrosis. A correlation analysis of concentration and differential gene expression revealed that 576 genes positively (R > 0.99) and 375 genes negatively (R < -0.99) correlated with GenX concentration. The upstream regulator analysis indicated HIF1α was inhibited at the lower doses but were activated at the higher dose. Additionally, VEGF, PPARα, STAT3, and SMAD4 signaling was induced at the 100 µM dose. These data indicate that at lower doses GenX can interfere with metabolic pathways and at higher doses can induce fibroinflammatory changes in human hepatocytes.

Heat-induced increases in body temperature in lactating dairy cows: impact on the cumulus and granulosa cell transcriptome of the periovulatory follicle.

Cows acutely heat stressed after a pharmacologically induced luteinizing hormone (LH) surge had periovulatory changes in the follicular fluid proteome that may potentiate ovulation and impact oocyte developmental competence. Because the cellular origins of differentially abundant proteins were not known, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows exhibiting varying levels of hyperthermia when occurring after the LH surge. After pharmacological induction of a dominant follicle, lactating dairy cows were administered gonadotropin releasing hormone (GnRH) and maintained in thermoneutral conditions (~67 temperature-humidity index [THI]) or heat stress conditions where THI was steadily increased for ~12 h (71 to 86 THI) and was sufficient to steadily elevate rectal temperatures. Cumulus-oocyte complexes and mural granulosa cells were recovered by transvaginal aspiration of dominant follicle content ~16 h after GnRH. Rectal temperature was used as a continuous, independent variable to identify differentially expressed genes (DEGs) increased or decreased per each 1 °C change in temperature. Cumulus (n = 9 samples) and granulosa (n = 8 samples) cells differentially expressed (false discovery rate [FDR] < 0.05) 25 and 87 genes, respectively. The majority of DEGs were upregulated by hyperthermia. Steady increases in THI are more like the "turning of a dial" than the "flipping of a switch." The moderate but impactful increases in rectal temperature induced modest fold changes in gene expression (<2-fold per 1 °C change in rectal temperature). Identification of cumulus DEGs involved in cell junctions, plasma membrane rafts, and cell-cycle regulation are consistent with marked changes in the interconnectedness and function of cumulus after the LH surge. Depending on the extent to which impacts may be occurring at the junctional level, cumulus changes may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. Two granulosa cell DEGs have been reported by others to promote ovulation. Based on what is known, several other DEGs are suggestive of impacts on collagen formation or angiogenesis. Collectively these and other findings provide important insight regarding the extent to which the transcriptomes of the components of the periovulatory follicle (cumulus and mural granulosa cells) are affected by varying degrees of hyperthermia.

Approximately 70% of the world's cattle population reside under ambient conditions experiencing some level of heat stress. Heat-stressed cows chronically exposed to elevated ambient temperatures have difficulty getting pregnant. Although the underlying basis for poor fertility during bouts of chronic heat stress remains unclear and is likely because of many different factors, when ambient conditions are sufficient to increase cow body temperature, different ovulatory follicle components are affected (i.e., mural granulosa cells that line the ovulatory follicle, the intrafollicular fluid and or the cumulus-oocyte complex while it matures in preparation for fertilization while resident within). To test this hypothesis, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows. Using steady increases in THI to induce varying levels of elevated body temperature after the luteinizing hormone surge we discovered certain genes in the cumulus cells that may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. We also noted changes in gene expression in granulosa cells that may impact ovulation and corpus luteum formation.

Alcohol-associated fibrosis in females is mediated by female-specific activation of lysine demethylases KDM5B and KDM5C.

Alcohol-associated liver disease is a major cause of alcohol-related mortality. However, the mechanisms underlying disease progression are not fully understood. Recently we found that liver molecular pathways are altered by alcohol consumption differently in males and females. We were able to associate these sex-specific pathways with two upstream regulators: H3K4-specific demethylase enzymes KDM5B and KDM5C. Mice were fed the Lieber-DeCarli alcohol liquid diet for 3 weeks or a combination of a high-fat diet with alcohol in water for 16 weeks (western diet alcohol model [WDA] model). To assess the role of histone demethylases, mice were treated with AAV-shControl, AAV-shKdm5b, and/or AAV-shKdm5c and/or AAV-shAhR vectors. Gene expression and epigenetic changes after Kdm5b/5c knockdown were assessed by RNA-sequencing and H3K4me3 chromatin immunoprecipitation analysis. We found that less than 5% of genes affected by Kdm5b/Kdm5c knockdown were common between males and females. In females, Kdm5b/Kdm5c knockdown prevented fibrosis development in mice fed the WDA alcohol diet for 16 weeks and decreased fibrosis-associated gene expression in mice fed the Lieber-DeCarli alcohol liquid diet. In contrast, fibrosis was not affected by Kdm5b/Kdm5c knockdown in males. We found that KDM5B and KDM5C promote fibrosis in females through down-regulation of the aryl hydrocarbon receptor (AhR) pathway components in hepatic stellate cells. Kdm5b/Kdm5c knockdown resulted in an up-regulation of Ahr, Arnt, and Aip in female but not in male mice, thus preventing fibrosis development. Ahr knockdown in combination with Kdm5b/Kdm5c knockdown restored profibrotic gene expression. Conclusion: KDM5 demethylases contribute to differences between males and females in the alcohol response in the liver. The KDM5/AhR axis is a female-specific mechanism of fibrosis development in alcohol-fed mice.