Publications

One third of women in the United States are affected by obesity during pregnancy. Maternal obesity (MO) is associated with an increased risk of neurodevelopmental and metabolic disorders in the offspring. The placenta, located at the maternal-fetal interface, is a key organ determining fetal development and likely contributes to programming of long-term offspring health. We profiled the term placental transcriptome in humans (pre-pregnancy BMI 35+ [MO condition] or 18.5-25 [lean condition]) using single-nucleus RNA-seq to compare expression profiles in MO versus lean conditions, and to reveal potential mechanisms underlying offspring disease risk. We recovered 62,864 nuclei of high quality from 10 samples each from the maternal-facing and fetal-facing sides of the placenta. On both sides in several cell types, MO was associated with upregulation of hypoxia response genes. On the maternal-facing side only, hypoxia gene expression was associated with offspring neurodevelopmental measures, in Gen3G, an independent pregnancy cohort with bulk placental tissue RNA-seq. We leveraged Gen3G to determine genes that correlated with impaired neurodevelopment and found these genes to be most highly expressed in extravillous trophoblasts (EVTs). EVTs further showed the strongest correlation between neurodevelopment impairment gene scores (NDIGSs) and the hypoxia gene score. We reanalyzed gene expression of cultured EVTs, and found increased NDIGSs associated with exposure to hypoxia. Among EVTs, accounting for the hypoxia gene score attenuated 44% of the association between BMI and NDIGSs. These data suggest that hypoxia in EVTs may be a key process in the neurodevelopmental programming of fetal exposure to MO.

Genetic parasites, including viruses and transposons, exploit components from the host for their own replication. However, little is known about virus-transposon interactions within host cells. Here, we discover a strategy where human cytomegalovirus (HCMV) hijacks L1 retrotransposon encoded protein during its replication cycle. HCMV infection upregulates L1 expression by enhancing both the expression of L1-activating transcription factors, YY1 and RUNX3, and the chromatin accessibility of L1 promoter regions. Increased L1 expression, in turn, promotes HCMV replicative fitness. Affinity proteomics reveals UL44, HCMV DNA polymerase subunit, as the most abundant viral binding protein of the L1 ribonucleoprotein (RNP) complex. UL44 directly interacts with L1 ORF2p, inducing DNA damage responses in replicating HCMV compartments. While increased L1-induced mutagenesis is not observed in HCMV for genetic adaptation, the interplay between UL44 and ORF2p accelerates viral DNA replication by alleviating replication stress. Our findings shed light on how HCMV exploits host retrotransposons for enhanced viral fitness.

BACKGROUND: Repressor element 1 (RE1) silencing transcription factor (REST) is a transcriptional repressor abundantly expressed in aging human brains. It is known to regulate genes associated with oxidative stress, inflammation, and neurological disorders by binding to a canonical form of sequence motif and its non-canonical variations. Although analysis of genomic sequence motifs is crucial to understand transcriptional regulation by transcription factors (TFs), a comprehensive characterization of various forms of RE1 motifs in human cell lines has not been performed.

RESULTS: Here, we analyzed 23 ENCODE REST ChIP-seq datasets from diverse human cell lines and identified a non-redundant set of 68,975 loci with ChIP-seq peaks. Our systematic characterization of these binding sites revealed that the canonical form of REST binding motif was found primarily in ChIP-seq peaks shared across multiple cell lines, while non-canonical forms of motifs were identified in both cell-line-specific binding sites and those shared across cell lines. Remarkably, we observed a notable prevalence of non-canonical motifs that corresponded to half segments of the canonical motif. Furthermore, our analysis unveiled the presence of cell-line-specific REST binding patterns, as evidenced by the clustering of ChIP-seq experiments according to their respective cell lines. This observation underscores the cell-line specificity of REST binding at certain genomic loci, implying intricate cell-line-specific regulatory mechanisms.

CONCLUSIONS: Overall, our study provides a comprehensive characterization of REST binding motifs in human cell lines and genome-wide RE1 motif profiles. These findings contribute to a deeper understanding of REST-mediated transcriptional regulation and highlight the importance of considering cell-line-specific effects in future investigations.

Alzheimer's disease (AD) is an age-associated neurodegenerative disorder characterized by progressive neuronal loss and pathological accumulation of the misfolded proteins amyloid-β and tau1,2. Neuroinflammation mediated by microglia and brain-resident macrophages plays a crucial role in AD pathogenesis1-5, though the mechanisms by which age, genes, and other risk factors interact remain largely unknown. Somatic mutations accumulate with age and lead to clonal expansion of many cell types, contributing to cancer and many non-cancer diseases6,7. Here we studied somatic mutation in normal aged and AD brains by three orthogonal methods and in three independent AD cohorts. Analysis of bulk RNA sequencing data from 866 samples from different brain regions revealed significantly higher ( two-fold) overall burdens of somatic single-nucleotide variants (sSNVs) in AD brains compared to age-matched controls. Molecular-barcoded deep (>1000X) gene panel sequencing of 311 prefrontal cortex samples showed enrichment of sSNVs and somatic insertions and deletions (sIndels) in cancer driver genes in AD brain compared to control, with recurrent, and often multiple, mutations in genes implicated in clonal hematopoiesis (CH)8,9. Pathogenic sSNVs were enriched in CSF1R+ microglia of AD brains, and the high proportion of microglia (up to 40%) carrying some sSNVs in cancer driver genes suggests mutation-driven microglial clonal expansion (MiCE). Analysis of single-nucleus RNA sequencing (snRNAseq) from temporal neocortex of 62 additional AD cases and controls exhibited nominally increased mosaic chromosomal alterations (mCAs) associated with CH10,11. Microglia carrying mCA showed upregulated pro-inflammatory genes, resembling the transcriptomic features of disease-associated microglia (DAM) in AD. Our results suggest that somatic driver mutations in microglia are common with normal aging but further enriched in AD brain, driving MiCE with inflammatory and DAM signatures. Our findings provide the first insights into microglial clonal dynamics in AD and identify potential new approaches to AD diagnosis and therapy.

While ATM loss of function has long been identified as the genetic cause of ataxia-telangiectasia (A-T), how it leads to selective and progressive degeneration of cerebellar Purkinje and granule neurons remains unclear. ATM expression is enriched in microglia throughout cerebellar development and adulthood. Here, we find evidence of microglial inflammation in the cerebellum of patients with A-T using single-nucleus RNA sequencing. Pseudotime analysis revealed that activation of A-T microglia preceded upregulation of apoptosis-related genes in granule and Purkinje neurons and that microglia exhibited increased neurotoxic cytokine signaling to granule and Purkinje neurons in A-T. To confirm these findings experimentally, we performed transcriptomic profiling of A-T induced pluripotent stem cell (iPSC)-derived microglia, which revealed cell-intrinsic microglial activation of cytokine production and innate immune response pathways compared to controls. Furthermore, A-T microglia co-culture with either control or A-T iPSC-derived neurons was sufficient to induce cytotoxicity. Taken together, these studies reveal that cell-intrinsic microglial activation may promote neurodegeneration in A-T.

Neurodevelopmental changes and impaired stress resistance have been implicated in the pathogenesis of bipolar disorder (BD), but the underlying regulatory mechanisms are unresolved. Here we describe a human cerebral organoid model of BD that exhibits altered neural development, elevated neural network activity, and a major shift in the transcriptome. These phenotypic changes were reproduced in cerebral organoids generated from iPS cell lines derived in different laboratories. The BD cerebral organoid transcriptome showed highly significant enrichment for gene targets of the transcriptional repressor REST. This was associated with reduced nuclear REST and REST binding to target gene recognition sites. Reducing the oxygen concentration in organoid cultures to a physiological range ameliorated the developmental phenotype and restored REST expression. These effects were mimicked by treatment with lithium. Reduced nuclear REST and derepression of REST targets genes were also observed in the prefrontal cortex of BD patients. Thus, an impaired cellular stress response in BD cerebral organoids leads to altered neural development and transcriptional dysregulation associated with downregulation of REST. These findings provide a new model and conceptual framework for exploring the molecular basis of BD.

OBJECTIVES: Brain-limited pathogenic somatic variants are associated with focal pediatric epilepsy, but reliance on resected brain tissue samples has limited our ability to correlate epileptiform activity with abnormal molecular pathology. We aimed to identify the pathogenic variant and map variant allele fractions (VAFs) across an abnormal region of epileptogenic brain in a patient who underwent stereoelectroencephalography (sEEG) and subsequent motor-sparing left frontal disconnection.

METHODS: We extracted genomic DNA from peripheral blood, brain tissue resected from peri-sEEG electrode regions, and microbulk brain tissue adherent to sEEG electrodes. Samples were mapped based on an anatomic relationship with the presumed seizure onset zone (SOZ). We performed deep panel sequencing of amplified and unamplified DNA to identify pathogenic variants with subsequent orthogonal validation.

RESULTS: We detect a pathogenic somatic PIK3CA variant, c.1624G>A (p.E542K), in the brain tissue samples, with VAF inversely correlated with distance from the SOZ. In addition, we identify this variant in amplified electrode-derived samples, albeit with lower VAFs.

DISCUSSION: We demonstrate regional mosaicism across epileptogenic tissue, suggesting a correlation between variant burden and SOZ. We also validate a pathogenic variant from individual amplified sEEG electrode-derived brain specimens, although further optimization of techniques is required.

The conference "Transposable Elements at the Crossroads of Evolution, Health and Disease" was hosted by Keystone Symposia in Whistler, British Columbia, Canada, on September 3-6, 2023, and was organized by Kathleen Burns, Harmit Malik and Irina Arkhipova. The central theme of the meeting was the incredible diversity of ways in which transposable elements (TEs) interact with the host, from disrupting the existing genes and pathways to creating novel gene products and expression patterns, enhancing the repertoire of host functions, and ultimately driving host evolution. The meeting was organized into six plenary sessions and two afternoon workshops with a total of 50 invited and contributed talks, two poster sessions, and a career roundtable. The topics ranged from TE roles in normal and pathological processes to restricting and harnessing TE activity based on mechanistic insights gained from genetic, structural, and biochemical studies.

Although mutations in dozens of genes have been implicated in familial forms of amyotrophic lateral sclerosis (fALS) and frontotemporal degeneration (fFTD), most cases of these conditions are sporadic (sALS and sFTD), with no family history, and their etiology remains obscure. We tested the hypothesis that somatic mosaic mutations, present in some but not all cells, might contribute in these cases, by performing ultra-deep, targeted sequencing of 88 genes associated with neurodegenerative diseases in postmortem brain and spinal cord samples from 404 individuals with sALS or sFTD and 144 controls. Known pathogenic germline mutations were found in 20.6% of ALS, and 26.5% of FTD cases. Predicted pathogenic somatic mutations in ALS/FTD genes were observed in 2.7% of sALS and sFTD cases that did not carry known pathogenic or novel germline mutations. Somatic mutations showed low variant allele fraction (typically <2%) and were often restricted to the region of initial discovery, preventing detection through genetic screening in peripheral tissues. Damaging somatic mutations were preferentially enriched in primary motor cortex of sALS and prefrontal cortex of sFTD, mirroring regions most severely affected in each disease. Somatic mutation analysis of bulk RNA-seq data from brain and spinal cord from an additional 143 sALS cases and 23 controls confirmed an overall enrichment of somatic mutations in sALS. Two adult sALS cases were identified bearing pathogenic somatic mutations in DYNC1H1 and LMNA, two genes associated with pediatric motor neuron degeneration. Our study suggests that somatic mutations in fALS/fFTD genes, and in genes associated with more severe diseases in the germline state, contribute to sALS and sFTD, and that mosaic mutations in a small fraction of cells in focal regions of the nervous system can ultimately result in widespread degeneration.

Transposon-derived transcripts are abundant in RNA sequences, yet their landscape and function, especially for fusion transcripts derived from unannotated or somatically acquired transposons, remains underexplored. Here, we developed a new bioinformatic tool to detect transposon-fusion transcripts in RNA-sequencing data and performed a pan-cancer analysis of 10,257 cancer samples across 34 cancer types as well as 3,088 normal tissue samples. We identified 52,277 cancer-specific fusions with  30 events per cancer and hotspot loci within transposons vulnerable to fusion formation. Exonization of intronic transposons was the most prevalent genic fusions, while somatic L1 insertions constituted a small fraction of cancer-specific fusions. Source L1s and HERVs, but not Alus showed decreased DNA methylation in cancer upon fusion formation. Overall cancer-specific L1 fusions were enriched in tumor suppressors while Alu fusions were enriched in oncogenes, including recurrent Alu fusions in EZH2 predictive of patient survival. We also demonstrated that transposon-derived peptides triggered CD8+ T-cell activation to the extent comparable to EBV viruses. Our findings reveal distinct epigenetic and tumorigenic mechanisms underlying transposon fusions across different families and highlight transposons as novel therapeutic targets and the source of potent neoantigens.