Biology subjects

Gearhart, M. D.

Publications and source records attributed to Gearhart, M. D..

13 recordsLinked to original sources

bioRxiv2026

Hepatocyte nuclear factor-1β recruits the nucleosome remodeling and deacetylase complex to activate gene transcription

Mutations affecting the transcription factor hepatocyte nuclear factor-1{beta} (HNF-1{beta}) are the most common monogenic cause of congenital anomalies of the kidney and urinary tract. The mechanism whereby HNF-1{beta} regulates gene transcription remains unclear. Here, we performed an unbiased proteomic screen and found that endogenous HNF-1{beta} interacts with core subunits of the nucleosome remodeling and deacetylase complex (NuRD) in renal epithelial cells. Genome-wide ChIP-seq studies demonstrated colocalization of endogenous HNF-1{beta} and the core NuRD subunits GATAD2A, CHD4, and MBD3 at 2,776 primarily intergenic and intronic sites in native chromatin from wild-type renal epithelial cells. NuRD co-occupancy at 628 of the sites (22.6%) was reduced in the absence of HNF-1{beta}. Of the 628 HNF-1{beta}-dependent NuRD-occupied sites, 113 sites (18.2%) showed higher ATAC-seq peaks, indicative of open chromatin, and enrichment of histone H3 lysine-27 (H3K27) acetylation, indicative of active enhancers, in wild-type cells compared to HNF-1{beta}-deficient cells. Correlation with transcriptomic profiles in wild-type and HNF-1{beta}-deficient cells revealed that most of the sites were located near genes that are activated by HNF-1{beta}. Knockdown of CHD4 with shRNA resulted in decreased expression of 67 HNF-1{beta}-regulated genes that contained nearby NuRD and HNF-1{beta} co-occupied sites. Collectively, these results demonstrate that HNF-1{beta} recruits the NuRD complex resulting in H3K27 acetylation and chromatin opening and leading to gene activation. Our findings reveal a new mechanism of HNF-1{beta}-dependent gene expression and expand the role of the NuRD complex in gene activation at distal enhancers.

biochemistry↗

bioRxiv2026

Translation of a small upstream open reading frame functions as a rheostat for the regulation of lin-41 by the Let-7 microRNA in Caenorhabditis elegans

MicroRNAs have been likened to the "dark matter" of eukaryotic genomes, reflecting their pervasive regulatory influence. MicroRNAs were first identified through genetic studies of developmental timing in the nematode Caenorhabditis elegans. Let-7 was the first microRNA recognized to be broadly conserved. The principal target of Let-7 in the developmental timing pathway is the TRIM-NHL RNA-binding protein LIN-41. During the L4 larval stage, Let-7 represses lin-41 translation by binding to two Let-7 complementary sites in the lin-41 3'UTR. Despite the importance of microRNA-based translational regulation, the underlying molecular mechanisms are incompletely understood. Through genetic analysis, we discovered an unrecognized feature of the mechanism by which Let-7 controls lin-41 translation. This mechanism requires a 5'-regulatory exon containing a seven-amino acid upstream open reading frame (uORF) and conserved sequence elements. Genome editing indicates that the specific uORF amino acid sequence itself is not important. Our data suggest that uORF translation and 5'UTR structure limit initiation at the downstream lin-41 start codon, enabling tight control by Let-7. Without this mechanism, the Let-7 microRNA is unable to properly regulate lin-41 to enable proper development.

developmental biology↗

bioRxiv2026

Pichinde virus models intrauterine infection by hemorrhagic fever-causing arenaviruses

Arenavirus infection during pregnancy can cause severe maternal disease, congenital infection, and fetal demise. Many arenaviruses are endemic in economically disadvantaged areas and must be studied in high containment laboratories, which has limited our understanding of arenavirus pathogenesis in the placenta. Pichinde virus (PICV) is a nonpathogenic arenavirus that recapitulates many aspects of viral hemorrhagic fever in guinea pigs. Using a combination of experiments in human placental cells and guinea pigs, we characterized the tropism and impact of PICV infection during pregnancy. PICV replicated in human trophoblast stem cells (TSCs), TSC-derived trophoblasts, and explanted term placenta. When guinea pigs were infected at mid-gestation, PICV caused fetal demise. High infectious titers were recovered from placenta and decidua, but PICV was infrequently detected in fetal tissues or amniotic fluid. In situ hybridization confirmed that the placenta, decidua, and fetal membranes were all infected by the virus. Transcriptional profiling of PICV-infected human trophoblasts and guinea pig tissues revealed that infection upregulated canonical antiviral responses, which could contribute to placental dysfunction and pregnancy loss. Thus, PICV is safe and tractable model of zoonotic arenavirus infection during pregnancy with utility for preclinical therapeutic and vaccine development.

microbiology↗

bioRxiv2025

The SPN-4 Rbfox RNA-binding protein selects maternal mRNAs for CCR4-NOT-dependent clearance in early Caenorhabditis elegans embryos

The elimination of maternal mRNAs is an essential feature of the maternal-to-zygotic transition. We report an essential pathway that clears many maternal transcripts from early C. elegans embryos using the Rbfox-related SPN-4 RNA-binding protein as a specificity factor and the CCR4-NOT deadenylase complex as an effector. We biochemically identified SPN-4-associated mRNAs from late-stage oocytes and found that the set of SPN-4-associated transcripts is enriched for maternal mRNAs that undergo early decay. Single-molecule fluorescence in situ hybridization experiments established that many SPN-4-associated mRNAs fail to be eliminated in the absence of SPN-4. In the 3UTRs of two target mRNAs, we identified Rbfox motifs that are required for SPN-4-dependent clearance in vivo and bind SPN-4 in vitro. In a genetic screen to identify factors that work with SPN-4, we isolated mutant alleles of CCR4-NOT components. Auxin-induced degradation of the LET-711/NOT1 scaffold and the CCF-1 deadenylase disrupted clearance of two SPN-4-associated transcripts. Our results support a model in which SPN-4 initiates expression in late-stage oocytes, associates with maternal mRNA targets through RNA sequences in their 3UTRs and promotes CCR4-NOT-mediated decay during early embryogenesis.

developmental biology↗

bioRxiv2025

Spatial Transcriptomics Reveals that the Local Immune Response to Placental Guinea Pig

Cytomegalovirus infection can disrupt placental development and function either by directly infecting placental cells or by eliciting a pathogenic immune response. The relative contributions of these two mechanisms to adverse pregnancy outcomes remains poorly understood. In this study, we used spatial transcriptomics to quantify host and viral gene expression at the maternal-fetal interface at near single-cell resolution. Guinea pig cytomegalovirus (GPCMV) infection after mid-gestation causes focal infections at the base of the main placenta. Samples for spatial transcriptomics were collected from guinea pigs infected with GPCMV at 35 days gestation. Viral loads and the location of infected cells in placentas were assessed using virus-specific droplet digital PCR and in situ hybridization at 21 days post-infection. Representative placentas were sectioned onto Visium Spatial Gene Expression Slides and sequencing libraries were prepared from six infected and six uninfected tissue sections. Spatial transcriptomes from 33,687 55-{micro}m spots were generated and used in subsequent analyses. To assess how infection affected gene expression at the maternal-fetal interface, a combination of graph-based clustering and manual classification was used to assign spatial transcriptomes to clusters representative of different anatomic regions. Infection dysregulated more transcripts in the decidua and junctional zone than in the labyrinth or non-capillarized syncytium. Notably, infection downregulated transcripts involved in lipid metabolism and upregulated transcripts involved in antiviral defense and chemokine signaling. A second analysis compared the spatial transcriptomes of GPCMV-infected cells and their immediate microenvironment with similar regions in uninfected placentas. A transcriptional signature indicative of immune activation was clear in this comparison, and the local placental response to cytomegalovirus infection was driven by upregulated chemokine signaling. Thus, spatial transcriptomics revealed regional patterns of gene expression in the guinea pig placenta and illuminated how the host response to GPCMV may compromise placental function. Author SummaryThe placenta supports fetal development while also acting as an immune barrier against bloodborne pathogens. Cytomegalovirus (CMV), the most common viral cause of congenital infections and preventable neurologic disability in children, evades host defenses to infect the placenta. How CMV affects the placenta and fetal health remains poorly understood. Using a guinea pig model of CMV infection during pregnancy and spatial transcriptomics, a recently-developed method that enables gene expression to be studied at near-cellular resolution, this study compared normal and infected placentas. The effects of infection on directly infected cells and their immediate environment and indirect effects that occur at more distant sites were revealed. This information may inform the development of therapies to improve placental function after CMV infection.

immunology↗

bioRxiv2025

A sperm-oocyte protein partnership required for egg activation in Caenorhabditis elegans

ABSTRACTFertilization triggers the completion of female meiosis and launches the oocyte-to-embryo transition. C. elegans spe-11 is one of the few known paternal-effect embryonic lethal genes. We report that the sperm protein, SPE-11, forms a complex with an oocyte protein, OOPS-1 (Oocyte Partner of SPE-11), and that the protein complex is required for the completion of meiosis, the block to polyspermy, and eggshell formation. Consistent with the molecular interaction of their encoded proteins, oops-1 and spe-11 exhibit identical null phenotypes, displaying defects in meiotic progression and cytokinesis. We show that the complex binds F-actin in the absence of other proteins and inhibits the nucleation of actin filaments in vitro. Thus, the OOPS-1-SPE-11 complex may function to promote F-actin-mediated meiotic cytokinesis. Both OOPS-1 and SPE-11 are intrinsically disordered proteins that are highly phosphorylated. Biochemical and genetic experiments define interactions with the protein phosphatase 1 homologs GSP-3/4, which appear to promote OOPS-1-SPE-11 function. Genetic results support a model in which the OOPS-1-SPE-11 complex interacts with the cortical EGG complex to promote meiotic cytokinesis and to activate synthesis of the eggshell.

developmental biology↗

bioRxiv2024

Polycomb Repressive Complex 1.1 Component, BCOR, Promotes Syncytiotrophoblast Differentiation in Mice and Humans

Early defects in placenta development are thought to underlie a range of adverse pregnancy conditions including miscarriage, fetal growth abnormalities, preeclampsia, and stillbirth. Differentiating trophoblast stem cells undergo a choreographed allocation of syncytiotrophoblast and extravillous trophoblast cells in response to signaling cues from the developing fetus and the uterine environment. The expression and activity of transcription factors and chromatin modifying enzymes change during differentiation to appropriately reshape the chromatin landscape in each cell type. We have previously found in mice that extraembryonic loss of BCOR, a conserved component of the epigenetic silencing complex Polycomb Repressive Complex 1.1 (PRC1.1), leads to a reduced labyrinth and expanded trophoblast giant cell population in the placenta. Molecular analysis of wild-type and BCOR loss-of-function male and female placentas by RNA-seq identified gene expression changes as early as E6.5. We found that BCOR is required to down regulate stem cell genes and repress factors that promote alternate lineages which leads to reduced levels of syncytiotrophoblasts. ChIP-seq experiments identified a number of directly bound functional targets including Pdgfa and Wnt7b. In humans, BCOR is mutated in X-linked syndromes involving fetal growth restriction and females with a heterozygous null mutation in BCOR can experience recurrent miscarriages. To establish a direct role for BCOR in human placental development, we used CRISPR/Cas9 to knockout BCOR in male (CT29) and female (CT30) human trophoblast stem cells. Mutant cell lines retained capacity for induced differentiation into syncytiotrophoblast and extravillous trophoblasts and exhibited minimal changes in gene expression. However, in 3D cell culture using trophoblast organoid media, BCOR knockout lines had significantly altered gene expression including homologs of stem cell genes upregulated in Bcor knockout mice. CUT&RUN experiments in self-renewing and 3D cell culture identified genes directly bound by BCOR. Single cell profiling of wild type, knockout, and a P85L pathogenic knock-in BCOR mutation showed a reduced capacity to differentiate into syncytiotrophoblasts after four days of differentiation. Together, these results suggest that BCOR is a conserved regulator of trophoblast development that represses stem cell genes during differentiation and maintains lineage fidelity by repressing genes that promote alternate cell fates.

developmental biology↗

bioRxiv2023

Human Trophoblast Stem Cells Restrict Human Cytomegalovirus Replication

Placental infection plays a central role in the pathogenesis of congenital human cytomegalovirus (HCMV) infections and is a cause of fetal growth restriction and pregnancy loss. HCMV can replicate in some trophoblast cell types, but it remains unclear how the virus evades antiviral immunity in the placenta and how infection compromises placental development and function. Human trophoblast stem cells (TSCs) can be differentiated into extravillous trophoblasts (EVTs), syncytiotrophoblasts (STBs), and organoids, and this study assessed the utility of TSCs as a model of HCMV infection in the first trimester placenta. HCMV was found to non-productively infect TSCs, EVTs, and STBs. Immunofluorescence assays and flow cytometry experiments further revealed that infected TSCs frequently only express immediate early viral gene products. Similarly, RNA-sequencing found that viral gene expression in TSCs does not follow the kinetic patterns observed during lytic infection in fibroblasts. Canonical antiviral responses were largely not observed in HCMV-infected TSCs and TSC-derived trophoblasts. Rather, infection dysregulated factors involved in cell identity, differentiation, and WNT signaling. Thus, while HCMV does not replicate in TSCs, infection may perturb trophoblast differentiation in ways that could interfere with placental function. ImportancePlacental infection plays a central role in HCMV pathogenesis during pregnancy, but the species-specificity of HCMV and the limited availability and lifespan of primary trophoblasts have been persistent barriers to understanding how infection impacts this vital organ. Human TSCs represent a new approach to modeling viral infection early in placental development. This study reveals that TSCs, like other stem cell types, restrict HCMV replication. However, infection perturbs the expression of genes involved in differentiation and cell fate determination, pointing to a mechanism by which HCMV could cause placental injury.

microbiology↗

bioRxiv2023

Metformin impairs trophoblast metabolism and differentiation in dose dependent manner

Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing risks of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformins impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 M (therapeutic range) and 2000 M (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200 M metformin treated cells, 2000 M metformin impaired oxidative metabolism and increased abundance of lactate and TCA cycle intermediates, -ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 M, but not 200 M metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impairs trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.

cell biology↗

bioRxiv2023

Mitochondrial citrate metabolism and efflux regulates trophoblast differentiation

Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established cell culture model of trophoblast differentiation. Trophoblast differentiation was associated with increased abundance of the TCA cycle intermediates citrate and -ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.

developmental biology↗

bioRxiv2021

BCOR and BCORL1 mutations disrupt PRC1.1 repressive function in leukemia by unlinking the RING-PCGF1 enzymatic core from target genes

BCOR and its paralog BCORL1 encode subunits of the Polycomb repressive complex 1.1 (PRC1.1) and are recurrently mutated in myeloid malignancies. We show that leukemia-associated BCOR/BCORL1 mutations unlink the PRC1.1 RING-PCGF enzymatic core from the KDM2B-containing chromatin targeting auxiliary subcomplex, either by causing complete protein loss or expression of a C-terminally truncated protein lacking the PCGF Ub-like fold discriminator (PUFD) domain. By uncoupling PRC1.1 repressive function from target genes, BCOR/BCORL1 mutations activate aberrant cell signaling programs that confer acquired resistance to treatment. This study provides a mechanistic basis for Polycomb repressive dysfunction as a key oncogenic driver in myeloid malignancies and identifies a potential strategy for targeted therapy in BCOR-mutated cancer.

cancer biology↗

bioRxiv2020

Functional loss of a non-canonical BCOR-PRC1.1 complex accelerates SHH-driven medulloblastoma formation

Medulloblastoma is a childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH)-subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs) and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional co-repressor BCOR are recurrent and highly enriched in SHH-medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a germline genetically engineered mouse model to delete exons 9/10 of Bcor (Bcor{Delta}E9-10) in GNPs during development. This leads to reduced expression of C-terminally truncated BCOR (BCOR{Delta}E9-10). While Bcor{Delta}E9-10 alone did not promote tumorigenesis or affect GNP differentiation, Bcor{Delta}E9-10 combined with loss of the SHH-receptor gene Ptch1 resulted in highly penetrant medulloblastomas. In Ptch1+/-;Bcor{Delta}E9-10 tumors, the growth factor gene Igf2 was aberrantly upregulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/- GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/-;Bcor{Delta}E9-10 tumors. Overall, our data suggests that BCOR-PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.

cancer biology↗

bioRxiv2019

Insights into the involvement of spliceosomal mutations in myelodysplastic disorders from an analysis of SACY-1/DDX41 in Caenorhabditis elegans

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase found to be recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal general essential functions for SACY-1 in both the germline and the soma, as well as specific functions affecting germline sex determination and cell cycle control. Certain sacy-1/DDX41 mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that they compromise the function of the spliceosome. Consistent with these findings, sacy-1 exhibits synthetic lethal interactions with several spliceosomal components, and biochemical analyses suggest that SACY-1 is a component of the C. elegans spliceosome. We used the auxin-inducible degradation system to analyze the impact of SACY-1 on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-independent and splicing-dependent mechanisms. The observed transcriptome changes suggest that disruption of spliceosomal function induces a stress response. Altered 3 splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 as a second-step splicing factor. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. Author SummaryMutations affecting spliceosomal proteins are frequently found in hematological malignancies. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase recurrently mutated in inherited and relapsing myelodysplastic syndromes and acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing mutations in spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal that multiple sacy-1/DDX41 missense mutations, including the R525H human oncogenic variant, exhibit antimorphic activity that likely compromises the function of the spliceosome. The genomic consequences of SACY-1 depletion include splicing-splicing-independent and splicing-dependent alterations in the transcriptome.

genetics↗