Biology subjects

Raman, P.

Publications and source records attributed to Raman, P..

14 recordsLinked to original sources

bioRxiv2025

Dynamic evolution of EZHIP, an inhibitor of the Polycomb Repressive Complex 2 in mammals

The Polycomb Repressive Complex 2 (PRC2) is an ancient, conserved chromatin-interacting complex that controls gene expression, facilitating differentiation and cellular identity during development. Its regulation is critical in most eukaryotes. EZHIP was recently characterized as a PRC2 inhibitor and oncohistone mimic in mammals. Although EZHIP expression is typically restricted to the germline, its aberrant expression in pediatric brain tumors inhibits PRC2-mediated H3K27 methylation, driving disease progression. To gain a deeper understanding of its normal functions, we systematically examined EZHIP evolution across mammals using comparative genomics, synteny analyses, and motif discovery. Extending previous work, we find that EZHIP originated on the X chromosome and was retained there in most placental mammals, except in Afrotheria. In addition to the highly conserved H3K27M-like histone mimic motif, our motif analyses reveal six previously unidentified EZHIP motifs, including a putative nuclear localization signal, a serine-enriched region, and tandem repeats that are largely well-conserved in placental mammals. We hypothesize that these motifs are also critical to EZHIPs functions, including in PRC2 interaction and inhibition. We show that EZHIP has evolved under strong diversifying selection in primates and underwent dynamic expansions and losses across species. Some paralogs, such as EZHIP2 in primates, also evolved under positive selection. Based on its evolutionary attributes and germ-cell expression, we propose that EZHIP arose and evolved rapidly due to inter-parental conflict over fetal development in utero in placental mammals. Our work provides a foundation for further investigations into EZHIPs multifaceted roles in mammalian reproduction and disease. SignificanceHistone modifications regulate genes to orchestrate cell differentiation and development. Their precise regulation is essential for normal development and growth. The Polycomb Repressive Complex 2 (PRC2) deposits the repressive H3K27 methylation mark, whereas EZHIP antagonizes PRC2 to de-repress gene silencing mediated by H3K27 methylation. While aberrant EZHIP activity in cancers is well documented, its normal germline functions, evolutionary origins, and divergence remain unclear. We show that EZHIP is a placental, X-linked innovation that diversified through positive selection and recurrent gene duplications, yielding germline-specific paralogs. We discover conserved motifs that may have critical roles in PRC2 control. Our findings provide an evolutionary model - parental control over in utero reproduction - that drove the origin and innovation of EZHIPs in placental mammals.

evolutionary biology↗

bioRxiv2025

Administration of barcoded AAV capsid library to the putamen of non-human primates identifies variants with efficient retrograde transport

Adeno-associated viral vectors have become a leading choice for gene therapy in the central nervous system due to their safety profile, efficient neuronal transduction, and capacity for sustained transgene expression. We previously reported that AAV2-derived capsids developed using the BRAVE (Barcoded Rational AAV Vector Evolution) approach have enhanced retrograde transport properties in the rodent brain, compared to parental AAV2. Retrograde transport enables broader coverage of connected brain regions after a single focal intraparenchymal brain injection and is therefore a powerful tool for delivery of vectors to distant sites with potentially higher specificity, transduction efficacy and safety. Because transport properties can vary among species, we further characterized a barcoded library of 25 BRAVE-derived AAV2 capsid variants, along with the parental AAV2 serotype and benchmark AAV capsids, in brains of adult cynomolgus monkeys after intraputaminal dosing. Based on RNA and DNA amplicon sequencing, single-nucleus RNA sequencing, and histological assessment, we report here capsid variants with enhanced retrograde transport and expression compared to the parental AAV2 capsid. These properties make them potentially useful for disease indications in which broader brain coverage is desirable beyond the injection site.

neuroscience↗

bioRxiv2024

Evidence for multiple forms of heritable RNA silencing

Heritable gene silencing has been proposed to rely on DNA methylation, histone modifications, and/or non-coding RNAs in different organisms. Here we demonstrate that multiple RNA-mediated mechanisms with distinct and easily detectable molecular signatures can underlie heritable silencing of the same open-reading frame in the nematode C. elegans. Using two-gene operons, we reveal three cases of gene-selective silencing that provide support for the transmission of heritable epigenetic changes through different mechanisms of RNA silencing independent of changes in chromatin that would affect all genes of an operon equally. Different heritable epigenetic states of a gene were associated with distinct populations of stabilized mRNA fragments with untemplated poly-UG (pUG) tails, which are known intermediates of RNA silencing. These pUG signatures provide a way to distinguish the multiple mechanisms that can drive heritable RNA silencing of a single gene.

genetics↗

bioRxiv2023

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma

Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma. Plasma membrane-enriched mass spectrometry identified 1,461 cell surface proteins in cell lines and 1,401 in xenograft models, respectively. Additional proteogenomic analyses revealed 60 high-confidence candidate immunotherapeutic targets and we prioritized Delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlated with the presence of a super-enhancer spanning the DLK1 locus. Robust cell surface expression of DLK1 was validated by immunofluorescence, flow cytometry, and immunohistochemistry. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells resulted in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), showed potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Moreover, DLK1 is highly expressed in several adult cancer types, including adrenocortical carcinoma (ACC), pheochromocytoma/paraganglioma (PCPG), hepatoblastoma, and small cell lung cancer (SCLC), suggesting potential clinical benefit beyond neuroblastoma. Taken together, our study demonstrates the utility of comprehensive cancer surfaceome characterization and credentials DLK1 as an immunotherapeutic target. HighlightsO_LIPlasma membrane enriched proteomics defines surfaceome of neuroblastoma C_LIO_LIMulti-omic data integration prioritizes DLK1 as a candidate immunotherapeutic target in neuroblastoma and other cancers C_LIO_LIDLK1 expression is driven by a super-enhancer C_LIO_LIDLK1 silencing in neuroblastoma cells results in cellular differentiation C_LIO_LIADCT-701, a DLK1-targeting antibody-drug conjugate, shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma preclinical models C_LI

cancer biology↗

bioRxiv2023

Deletion of smooth muscle O-GlcNAc transferase prevents development of atherosclerosis in western diet-fed hyperglycemic ApoE-/- mice in vivo

Accumulating evidence highlights protein O-GlcNAcylation as a putative pathogenic contributor of diabetic vascular complications. We previously reported that elevated protein O-GlcNAcylation correlates with increased atherosclerotic lesion formation and VSMC proliferation in response to hyperglycemia. However, the role of O-GlcNAc transferase (OGT), regulator of O-GlcNAc signaling, in evolution of diabetic atherosclerosis remains elusive. The goal of this study was to determine whether smooth muscle OGT (smOGT) plays a direct role in hyperglycemia-induced atherosclerotic lesion formation and SMC de-differentiation. Using tamoxifen-inducible Myh11-CreERT2 and OGTfl/fl mice, we generated smOGTWT and smOGTKO mice, with and without ApoE-null backgrounds. Following STZ-induced hyperglycemia, smOGTWT and smOGTKO mice were kept on standard laboratory diet for study duration. In a parallel study, smOGTWTApoE-/- and smOGTKOApoE-/- were kept on Western diet beginning 8-wks-age. Animals harvested at 14-16-wks-age were used for plasma and tissue collection. Loss of smOGT augmented SM contractile marker expression in aortic vessels of STZ-induced hyperglycemic smOGTKO mice. Consistently, smOGT deletion attenuated atherosclerotic lesion lipid burden (Oil red O), plaque area (H&E), leukocyte (CD45) and smooth muscle cell (ACTA2) abundance in Western diet-induced hyperglycemic smOGTKOApoE-/- mice. This was accompanied with increased SM contractile markers, and reduced inflammatory and proliferative marker expression. Further, smOGT deletion attenuated YY1 and SRF expression (transcriptional regulators of SM contractile genes) in hyperglycemic smOGTKOApoE-/- and smOGTKO mice. These data uncover an atheroprotective outcome of smOGT loss-of-function and suggest a direct regulatory role of OGT-mediated O-GlcNAcylation in VSMC de-differentiation in hyperglycemia.

pathology↗

bioRxiv2023

Target-specific requirements for RNA interference can be explained by a single regulatory network

Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematode C. elegans can selectively impair the silencing of some genes. Here we show that such selective requirements can be explained by an intersecting network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one somatic gene, but each is singly required for silencing another somatic gene, where only the requirement for NRDE-3 can be overcome by enhanced dsRNA processing. Quantitative models and their exploratory simulations led us to find that (1) changing cis-regulatory elements of the target gene can reduce the dependence on NRDE-3, (2) animals can recover from silencing in non-dividing cells and (3) cleavage and tailing of mRNAs with UG dinucleotides, which makes them templates for amplifying small RNAs, is enriched within pUG zones matching the dsRNA. Similar crosstalk between pathways and restricted amplification could result in apparently selective silencing by endogenous RNAs.

genetics↗

bioRxiv2022

Transcriptional immunogenomic analysis reveals distinct immunological clusters in pediatric nervous system tumours

To inform immunotherapy approaches in children, we performed an immunogenomic analysis of RNA-seq data from 925 treatment-naive pediatric nervous system tumours (pedNST) spanning 12 cancer types from three public data sets. Within pedNST, we uncovered four broad immune clusters: Pediatric Inflamed (10%), Myeloid Predominant (30%), Immune Neutral (43%) and Immune Excluded (17%). We validated these clusters using immunohistochemistry, methylation immune inference, and segmentation analysis of tissue images. We report shared biology of these immune clusters within and across cancer types, and characterization of specific immune-cell frequencies as well as T- and B-cell repertoires. We found no associations between immune infiltration levels and tumour mutational burden, although molecular cancer entities were enriched within specific immune clusters. Given the heterogeneity within pedNST, our findings suggest personalized immunogenomic profiling is needed to guide selection of immunotherapeutic strategies.

genomics↗

bioRxiv2022

OpenPBTA: An Open Pediatric Brain Tumor Atlas

SummaryPediatric brain and spinal cancer are the leading disease-related cause of death in children, thus we urgently need curative therapeutic strategies for these tumors. To accelerate such discoveries, the Childrens Brain Tumor Network and Pacific Pediatric Neuro-Oncology Consortium created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to create OpenPBTA, an open collaborative project which establishes over 40 scalable analysis modules to genomically characterize 1,074 pediatric brain tumors. Transcriptomic classification reveals that TP53 loss is a significant marker for poor overall survival in ependymomas and H3 K28-altered diffuse midline gliomas and further identifies universal TP53 dysregulation in mismatch repair-deficient hypermutant high-grade gliomas. OpenPBTA is a foundational analysis platform actively being applied to other pediatric cancers and inform molecular tumor board decision-making, making it an invaluable resource to the pediatric oncology community. In BriefThe OpenPBTA is a global, collaborative open-science initiative which brought together researchers and clinicians to genomically characterize 1,074 pediatric brain tumors and 22 patient-derived cell lines. Shapiro, et. al create over 40 open-source, scalable modules to perform cancer genomics analyses and provide a richly-annotated somatic dataset across 58 brain tumor histologies. The OpenPBTA framework can be used as a model for large-scale data integration to inform basic research, therapeutic target identification, and clinical translation. HighlightsOpenPBTA collaborative analyses establish resource for 1,074 pediatric brain tumors NGS-based WHO-aligned integrated diagnoses generated for 641 of 1,074 tumors RNA-Seq analysis infers medulloblastoma subtypes, TP53 status, and telomerase activity OpenPBTA will accelerate therapeutic translation of genomic insights

genomics↗

bioRxiv2022

Recurrent but short-lived duplications of centromeric proteins in holocentric Caenorhabditis species

Centromeric histones (CenH3s) are essential for chromosome inheritance during cell division in most eukaryotes. CenH3 genes have rapidly evolved and undergone repeated gene duplications and diversification in many plant and animal species. In Caenorhabditis, two independent duplications of CenH3 (named hcp-3 for HoloCentric chromosome-binding Protein 3) have been previously identified: in C. elegans and C. remanei. Here, using phylogenomic analyses in Caenorhabditis, we find strict retention of the ancestral hcp-3 gene and eight additional independent hcp-3 duplications, most of which are only found in one or two species. hcp-3L (hcp-3-like) paralogs are expressed in both sexes (males and females/ hermaphrodites) and have a conserved histone fold domain. We identified novel N-terminal protein motifs, including putative kinetochore protein-interacting motifs and a potential separase cleavage site, which are well-conserved across Caenorhabditis HCP-3 proteins. Other N-terminal motifs vary in their retention across paralogs or species, revealing potential sub-functionalization or functional loss following duplication. C. afra encodes an unprecedented protein fusion, where the hcp-3 paralog fused to duplicated segments from hcp-4 (nematode CENP-C). Extending our analyses beyond CenH3, we found gene duplications of six inner and outer kinetochore genes in Caenorhabditis, including co-retention of different kinetochore protein paralogs in a few species. Our findings suggest that centromeric protein duplications occur frequently in Caenorhabditis nematodes, are selectively retained under purifying selection but only for short evolutionary periods, then degenerate or are lost entirely. We hypothesize that unique challenges associated with holocentricity in Caenorhabditis may lead to this rapid revolving door of kinetochore protein paralogs.

evolutionary biology↗

bioRxiv2021

Novel classes and evolutionary turnover of histone H2B variants in the mammalian germline

Histones and their post-translational modifications facilitate diverse chromatin functions in eukaryotes. Core histones (H2A, H2B, H3, and H4) package genomes after DNA replication. In contrast, variant histones promote specialized chromatin functions, including DNA repair, genome stability, and epigenetic inheritance. Previous studies have identified only a few H2B variants in animals; their roles and evolutionary origins remain largely unknown. Here, using phylogenomic analyses, we reveal the presence of five H2B variants broadly present in mammalian genomes. In addition to three previously described variants (H2B.1, subH2B, and H2B.W), we identify and describe two new variants, H2B.L and H2B.N. Four of these five H2B variants originated in mammals, whereas H2B.L arose prior to the last common ancestor of bony vertebrates. We find that though mammalian H2B variants are subject to high gene turnover, most are broadly retained in mammals, including humans. Despite an overall signature of purifying selection, H2B variants evolve more rapidly than core H2B with considerable divergence in sequence and length. All five H2B variants are expressed in the germline. H2B.L and H2B.N are predominantly expressed in oocytes, an atypical expression site for mammalian histone variants. Our findings suggest that H2B variants likely encode potentially redundant but vital functions via unusual chromatin packaging or non-chromatin functions in mammalian germline cells. Our discovery of novel histone variants highlights the advantages of comprehensive phylogenomic analyses and provides unique opportunities to study how innovations in chromatin function evolve.

evolutionary biology↗

bioRxiv2021

MYC controls metastatic heterogeneity in pancreatic cancer

The degree of metastatic disease varies widely amongst cancer patients and impacts clinical outcomes. However, the biological and functional differences that drive the extent of metastasis are poorly understood. We analyzed primary tumors and paired metastases using a multi-fluorescent lineage-labeled mouse model of pancreatic ductal adenocarcinoma (PDAC) - a tumor type where most patients present with metastases. Genomic and transcriptomic analysis revealed an association between metastatic burden and gene amplification or transcriptional upregulation of MYC and its downstream targets. Functional experiments showed that MYC promotes metastasis by recruiting tumor associated macrophages (TAMs), leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was associated with activation of MYC signaling pathways and enrichment for MYC amplifications specifically in metastatic patients. Collectively, these results implicate MYC activity as a major determinant of metastatic burden in advanced PDAC.

cancer biology↗

bioRxiv2020

Single-cell transcriptomic profile reveals macrophage heterogeneity in medulloblastoma and their treatment-dependent recruitment

The role of macrophages in medulloblastoma, the most common malignant pediatric brain tumor, is unclear. Using single-cell RNA sequencing in a mouse model of sonic hedgehog medulloblastoma and analysis of bulk RNA sequencing of human medulloblastoma, we investigated macrophage heterogeneity. Our findings reveal differential recruitment of macrophages with molecular-targeted versus radiation therapy and identify an immunosuppressive monocyte-derived macrophages following radiation treatment of mouse medulloblastoma, uncovering potential strategies for immunomodulation as adjunctive therapy.

cancer biology↗

bioRxiv2019

annoFuse: an R Package to annotate and prioritize putative oncogenic RNA fusions

BackgroundGene fusion events are a significant source of somatic variation across adult and pediatric cancers and are some of the most clinically-effective therapeutic targets, yet low consensus of RNA-Seq fusion prediction algorithms makes therapeutic prioritization difficult. In addition, events such as polymerase read-throughs, mis-mapping due to gene homology, and fusions occurring in healthy normal tissue require informed filtering, making it difficult for researchers and clinicians to rapidly discern gene fusions that might be true underlying oncogenic drivers of a tumor and in some cases, appropriate targets for therapy. ResultsWe developed annoFuse, an R package, and shinyFuse, a companion web application, to annotate, prioritize, and explore biologically-relevant expressed gene fusions, downstream of fusion calling. We validated annoFuse using a random cohort of TCGA RNA-Seq samples (N = 160) and achieved a 96% sensitivity for retention of high-confidence fusions (N = 603). annoFuse uses FusionAnnotator annotations to filter non-oncogenic and/or artifactual fusions. Then, fusions are prioritized if previously reported in TCGA and/or fusions containing gene partners that are known oncogenes, tumor suppressor genes, COSMIC genes, and/or transcription factors. We applied annoFuse to fusion calls from pediatric brain tumor RNA-Seq samples (N = 1,028) provided as part of the Open Pediatric Brain Tumor Atlas (OpenPBTA) Project to determine recurrent fusions and recurrently-fused genes within different brain tumor histologies. annoFuse annotates protein domains using the PFAM database, assesses reciprocality, and annotates gene partners for kinase domain retention. As a standard function, reportFuse enables generation of a reproducible R Markdown report to summarize filtered fusions, visualize breakpoints and protein domains by transcript, and plot recurrent fusions within cohorts. Finally, we created shinyFuse for algorithm-agnostic interactive exploration and plotting of gene fusions. ConclusionsannoFuse provides standardized filtering and annotation for gene fusion calls from STARFusion and Arriba by merging, filtering, and prioritizing putative oncogenic fusions across large cancer datasets, as demonstrated here with data from the OpenPBTA project. We are expanding the package to be widely-applicable to other fusion algorithms and expect annoFuse to provide researchers a method for rapidly evaluating, prioritizing, and translating fusion findings in patient tumors.

bioinformatics↗

bioRxiv2019

Pediatric High Grade Glioma Resources From the Children’s Brain Tumor Tissue Consortium (CBTTC) and Pediatric Brain Tumor Atlas (PBTA)

BackgroundPediatric high grade glioma (pHGG) remains a fatal disease. Increased access to richly annotated biospecimens and patient derived tumor models will accelerate pHGG research and support translation of research discoveries. This work describes the pediatric high grade glioma set of the Childrens Brain Tumor Tissue Consortium (CBTTC) from the first release (October 2018) of the Pediatric Brain Tumor Atlas (PBTA).\n\nMethodspHGG tumors with associated clinical data and imaging were prospectively collected through the CBTTC and analyzed as the Pediatric Brain Tumor Atlas (PBTA) with processed genomic data deposited into PedcBioPortal for broad access and visualization. Matched tumor was cultured to create high grade glioma cell lines analyzed by targeted and WGS and RNA-seq. A tissue microarray (TMA) of primary pHGG tumors was also created.\n\nResultsThe pHGG set included 87 collection events (73 patients, 60% at diagnosis, median age of 9 yrs, 55% female, 46% hemispheric). Analysis of somatic mutations and copy number alterations of known glioma genes were of expected distribution (36% H3.3, 47% TP53, 24% ATRX and 7% BRAF V600E variants). A pHGG TMA (n=77), includes 36 (53%) patient tumors with matched sequencing. At least one established glioma cell line was generated from 23 patients (32%). Unique reagents include those derived from a H3.3 G34R glioma and from tumors with mismatch repair deficiency.\n\nConclusionThe CBTTC and PBTA have created an openly available integrated resource of over 2,000 tumors, including a rich set of pHGG primary tumors, corresponding cell lines and archival fixed tissue to advance translational research for pHGG.\n\nIMPORTANCE OF STUDYHigh-grade gliomas (HGG) remain the leading cause of cancer death in children. Since molecularly heterogeneous, preclinical studies of pediatric HGG will be most informative if able to compare across groups. Given their relatively rarity, there are few readily available biospecimens and cellular models to inform preclinical laboratory and genomic translational research. Therefore, the aim of this CBTTC study was to highlight the panel of pediatric HGG cases whose primary tumors have undergone extensive genomic analysis, have clinical data, available imaging and additional biospecimens, including tumor, nucleic acids, cell lines and FFPE tissue on a tissue microarray (TMA).

cancer biology↗