Biology subjects

Nagarajan, P.

Publications and source records attributed to Nagarajan, P..

11 recordsLinked to original sources

bioRxiv2026

Integration of spatial single-cell proteomics and spatial metabolomics reveals tumor microenvironment predictive of immunotherapy response in mucosal melanoma

Mucosal melanoma (MuM) is a rare but aggressive malignancy with limited benefit from immune checkpoint inhibition and few predictive biomarkers. We integrated single-cell spatial proteomics (COMET) and spatial metabolomics (MALDI-IMS) to profile 97 tissue cores from 26 patients treated with PD-1/PD-L1 and/or CTLA-4 inhibitors. We profiled 695,444 cells and resolved 25 cell states across eight major cell types. Cellular neighborhood (CN) analysis revealed distinct tumor- and stromal-associated spatial architectures. Responders were enriched for tumor-associated CNs (invasive tumor and tumor boundary) with close spatial proximity among Ki67 tumor cells, CD163 macrophages, and CD11c dendritic cells (DCs), and increased proliferating/cytotoxic CD8+ T-cell subsets. Non-responders showed stromal CN dominance with reduced immune infiltration. Spatial metabolomics identified lower abundance of indole-derived metabolites and reduced indole/tryptophan pathway activity in responders within tumor and TME regions that tracked with DC/macrophage-enriched spatial contexts. This study advances MuM spatial biology and provides a framework for biomarker-driven immunotherapy strategies. Statement of significanceMucosal melanoma (MuM) responds poorly to immune checkpoint blockade, and predictive biomarkers are limited. Integrated spatial proteomics and metabolomics reveal response-associated tumor-immune neighborhood architecture, stromal contexts linked to immune exclusion, and altered indole/tryptophan metabolism in the microenvironment. These spatial features nominate biomarkers and therapeutic hypotheses to improve immunotherapy for MuM.

cancer biology↗

bioRxiv2026

Hat1 Orchestrates Heterochromatin Inheritance by Regulating Localization of H3K9 Methyltransferases

Many regions of heterochromatin associate with the nuclear periphery and are known as Lamin-associated domains (LADs). Histone acetyltransferase 1 (Hat1) is a highly conserved enzyme which acetylates newly synthesized histones H4 on lysines 5 and 12 prior to their deposition on chromatin. Hat1 is required to preserve chromatin accessibility within a subset of LADs called Hat1-dependent accessibility domains (HADs). Here we profile a diverse set of histone modifications in Hat1 KO and WT immortalized mouse embryonic fibroblasts (iMEFs) and find that Hat1 regulates diverse aspects of the structure of HADs and non-HAD LADs (nhLADS). In HADs, these changes include the conversion of H3K9me2 to H3K9me3. Analysis of H3K9-specific histone methyltransferases (HMTs) shows that that Suv39h1 and Suv39h2 have distinct localization patterns, where only Suv39h2 localizes to LADs. G9a only localizes to LADs in regions enriched for H3K9me2. We find that Hat1 loss results in a redistribution of these HMTs in both HADs and nh LADs. There is a decrease in the levels of G9a with a concomitant increase in Suv39h2. These results suggest Hat1 functions to restrain the formation of a more strongly heterochromatic state and highlight a role for Hat1 as an essential regulator of heterochromatin inheritance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/713225v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@9f6238org.highwire.dtl.DTLVardef@1e95415org.highwire.dtl.DTLVardef@18f3e0aorg.highwire.dtl.DTLVardef@1322426_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

HAT1 Regulates Intestinal Stem Cell Proliferation and Differentiation

Stem cells are critical for the development and maintenance of tissue integrity. An important example is intestinal stem cells (ISCs) that generate all epithelial cell types necessary for formation of the intestinal lining. HAT1, a histone acetyltransferase that acetylates newly synthesized histone H4 molecules on lysine residues 5 and 12 during replication-coupled chromatin assembly, is specifically expressed in intestinal stem and progenitor cells located in intestinal crypts. To determine if HAT1 is important for intestinal stem and progenitor cell function, we generated an inducible deletion of the HAT1 gene in intestinal epithelial cells. Loss of HAT1 resulted in morphological defects in the proximal end of the small intestine. Following loss of HAT1, intestinal crypts became elongated, with an increase in stem and progenitor cell proliferation and an increase in the population of OLFM+ cells. Loss of HAT1 also resulted in alterations in intestinal stem cell differentiation, including an increase in the number of Goblet cells and the mislocalization of Paneth cells into villi. HAT1 is specifically responsible for the acetylation of histone H4 lysine 5 (H4K5ac) in intestinal stem cells. Genome-wide characterization of HAT1-dependent H4K5ac in intestinal crypt cells indicates that the most significant loss of H4K5ac occurs in lamina-associated domains (LADs). Loss of H4K5ac in LADs is accompanied by an increase in histone H3 K9 tri-methylation indicating that HAT1 regulates LAD chromatin structure in intestinal crypt cells. A direct role for HAT1 in intestinal stem cell function was demonstrated using organoids in culture. HAT1 is required for differentiation in organoids and for the maintenance of Lgr5+ stem cells. These results indicate that HAT1 is required for the proper regulation of intestinal stem cell renewal and differentiation.

cell biology↗

bioRxiv2026

Inhibition of mucin-type O-glycosylation impairs melanogenesis, melanoma growth, and metastatic capacity

Tumor-associated glycans are critical regulators of immune evasion in melanoma. Despite the success of immune checkpoint blockade, resistance and relapse remain a challenge, highlighting the need to therapeutically target additional immunosuppressive axes. One such axis involves Siglec-sialoglycan interactions that facilitate tumor immune evasion. The melanoma-associated antigen Pmel17/gp100 is a melanosomal glycoprotein overexpressed in tumor cells and essential for melanosomal architecture. Pmel17/gp100 forms amyloid fibrils within melanosomes, and its extensive O-glycosylation contributes to melanin biosynthesis and melanoma progression. We show that pharmacological inhibition of O-glycosylation using peracetyl N-thioglycolyl-D-galactosamine (Ac5GalNTGc, 1a) decreases Pmel17/gp100 glycosylation and melanin synthesis in B16F10 melanoma cells, induces tumor surface hyposialylation, diminishes Siglec-E engagement, and disrupts the Siglec-sialoglycan immune checkpoint. In C57BL/6J mice, 1a significantly delayed tumor growth, prolonged survival, and reduced lung metastases relative to controls. These findings identify O-glycosylation as a dual-mode therapeutic vulnerability, linking melanosome dysfunction to attenuation of Siglec-mediated immune evasion in melanoma. SummaryMucin-type O-glycans on melanoma cell surfaces and the melanosomal protein, Pmel17/gp100, regulate melanogenesis and melanoma metastasis. The O-glycosylation inhibitor, peracetyl N-thioglycolyl-D-galactosamine (Ac5GalNTGc, 1a), suppressed melanin synthesis and induced hyposialylation, thereby disrupting Siglec-mediated immune evasion and retarding tumor growth and metastasis.

cancer biology↗

bioRxiv2024

Distinct Classes of Lamin-Associated Domains are Defined by Differential Patterns of Repressive Histone Methylation

A large fraction of the genome interacts with the nuclear periphery through lamina-associated domains (LADs), repressive regions which play an important role in genome organization and gene regulation across development. Despite much work, LAD structure and regulation are not fully understood, and a mounting number of studies have identified numerous genetic and epigenetic differences within LADs, demonstrating they are not a uniform group. Here we profile Lamin B1, HP1{beta}, H3K9me3, H3K9me2, H3K27me3, H3K14ac, H3K27ac, and H3K9ac in MEF cell lines derived from the same mouse colony and cluster LADs based on the abundance and distribution of these features across LADs. We find that LADs fall into 3 groups, each enriched in a unique set of histone modifications and genomic features. Each group is defined by a different heterochromatin modification (H3K9me3, H3K9me2, or H3K27me3), suggesting that all three of these marks play important roles in regulation of LAD chromatin and potentially of lamina association. We also discover unique features of LAD borders, including a LAD border-specific enrichment of H3K14ac. These results reveal important distinctions between LADs and highlight the rich diversity and complexity in LAD structure and regulatory mechanisms.

genomics↗

bioRxiv2023

A hepatocyte-specific transcriptional program driven by Rela and Stat3 exacerbates experimental colitis in mice by modulating bile synthesis

Hepatic factors secreted by the liver promote homeostasis and are pivotal for maintaining the liver-gut axis. Bile acid metabolism is one such example wherein, bile acid synthesis occurs in the liver and its biotransformation happens in the intestine. Dysfunctional interactions between the liver and the intestine stimulate varied pathological outcomes through its bidirectional portal communication. Indeed, aberrant bile acid metabolism has been reported in inflammatory bowel disease (IBD). However, the molecular mechanisms underlying these crosstalks that perpetuate intestinal permeability and inflammation remain obscure. Here, we identify a novel hepatic gene program regulated by Rela and Stat3 that accentuates the inflammation in an acute experimental colitis model. Hepatocyte-specific ablation of Rela and Stat3 reduces the levels of primary bile acids in both the liver and the gut and shows a restricted colitogenic phenotype. On supplementation of chenodeoxycholic acid (CDCA), knock-out mice exhibit enhanced colitis-induced alterations. This study provides persuasive evidence for the development of multi-organ strategies for treating IBD and identifies a hepatocyte-specific rela-stat3 network as a promising therapeutic target.

immunology↗

bioRxiv2022

Dynamic Reassociation of the Nuclear Lamina with Newly Replicated DNA

The physical association of specific regions of chromatin with components of the nuclear lamina provides the framework for the 3-dimensionl architecture of the genome. The regulation of these interactions plays a critical role in the maintenance of gene expression patterns and cell identity. The breakdown and reassembly of the nuclear membrane as cells transit mitosis plays a central role in the regulation of the interactions between the genome and the nuclear lamina. However, other nuclear processes, such as transcription, have emerged as regulators of the association of DNA with the nuclear lamina. To determine whether DNA replication also has the potential to regulate DNA-nuclear lamina interactions, we adapted proximity ligation-based chromatin assembly assays to analyze the dynamics of nuclear lamina association with newly replicated DNA. We observe that lamin A/C and lamin B, as well as inner nuclear membrane proteins LBR and emerin, are found in proximity to newly replicated DNA. While core histones rapidly reassociate with DNA following passage of the replication fork, the complete reassociation of nuclear lamina components with newly replicated DNA occurs over a period of approximately 30 minutes. We propose models to describe the disassembly and reassembly of nascent chromatin with the nuclear lamina.

biochemistry↗

bioRxiv2022

A Novel congenic Mouse Model to study Obesity, Diabetes and NAFLD

The genetic background of the mutant mouse strain plays an important role in disease development. We investigated whether Lepr mutation on different genetic background (WSB/EiJ) influences the development of obesity, diabetes and non-alcoholic fatty liver diseases (NAFLD). For this study heterozygote (db/+) mice were backcrossed with WSB/EiJ strain (that is resistant to obesity and NAFLD) for more than 10 generations and intercrossed for five generations having Lepr mutated gene incorporated in WSB/EiJ background. The newly derived congenic strain was assessed and related with the B6.db strain and WSB/EiJ strain for the development of obesity, NAFLD. The Lepr mutated congenic strain on the WSB/EiJ background gained significant body weight and showed other characteristic features of obesity. However, the incidence of diabetes, NAFLD, and other kidney complications were not observed in this strain. The novel congenic mouse strain having Lepr gene on WSB/EiJ background would be a promising model for the study of obesity without NAFLD and will also help to understand the various mechanisms involved in the development of NAFLD.

physiology↗

bioRxiv2021

Selective estrogen receptor modulators limit alphavirus infection by targeting the viral capping enzyme nsP1

Alphaviruses cause animal or human diseases that are characterized by febrile illness, debilitating arthralgia, or encephalitis. Selective estrogen receptor modulators (SERMs), a class of FDA-approved drugs, have been shown to possess antiviral activities against multiple viruses, including Hepatitis C virus, Ebola virus, dengue virus, and vesicular stomatitis virus. Here, we evaluated three SERM compounds, namely 4-hydroxytamoxifen, tamoxifen, and clomifene, for plausible antiviral properties against two medically important alphaviruses, chikungunya virus (CHIKV) and Sindbis virus (SINV). In cell culture settings, these SERMs displayed potent activity against CHIKV and SINV at non-toxic concentrations with EC50 values ranging between 400 nM and 3.9 M. Further studies indicated that these compounds inhibit a post-entry step of the alphavirus life cycle, while enzymatic assays involving purified recombinant proteins confirmed that these SERMs target the enzymatic activity of non-structural protein 1 (nsP1), the capping enzyme of alphaviruses. Finally, tamoxifen treatment restrained CHIKV growth in the infected mice and diminished musculoskeletal pathologies. Combining biochemical, cell culture-based studies, and in vivo analyses, we strongly argue that SERM compounds, or their derivatives, may provide for attractive therapeutic options against alphaviruses.

immunology↗

bioRxiv2021

Epigenetic Regulation of Nuclear Lamina-Associated Heterochromatin by HAT1 and the Acetylation of Newly Synthesized Histones

During S phase, eukaryotic cells must faithfully duplicate both the sequence of the genome and the regulatory information found in the epigenome. A central component of the epigenome is the pattern of histone post-translational modifications that play a critical role in the formation of specific chromatin states. During DNA replication, parental nucleosomes are disrupted and re-deposited on the nascent DNA near their original location to preserve the spatial memory of the epigenetic modifications. Newly synthesized histones must also be incorporated into the nascent chromatin to maintain nucleosome density. Transfer of modification patterns from parental histones to new histones is a fundamental step in epigenetic inheritance. Whether new histones play an active or passive role in epigenetic inheritance is unknown. Here we report that HAT1, which acetylates lysines 5 and 12 of newly synthesized histone H4 during replication-coupled chromatin assembly, regulates the epigenetic inheritance of chromatin states. HAT1 regulates the accessibility of large domains of heterochromatin termed HAT1-dependent Accessibility Domains (HADs). HADs are mega base-scale domains that comprise ~10% of the mouse genome. HAT1 functions as a global negative regulator of H3 K9me2/3 and HADs correspond to the regions of the genome that display HAT1-dependent increases in H3 K9me3 peak density. HADs display a high degree of overlap with a subset of Lamin-Associated Domains (LADs). HAT1 is required to maintain nuclear structure and integrity. These results indicate that HAT1 and the acetylation of newly synthesized histones are critical regulators of the epigenetic inheritance of heterochromatin and suggest a new mechanism for the epigenetic regulation of nuclear lamina-heterochromatin interactions.

genomics↗

bioRxiv2020

Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

The replisome functions in a dynamic environment that is at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the action of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (Hat1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12 that accompanies replication-coupled chromatin assembly. Analysis of the role of Hat1 in replication-coupled chromatin assembly demonstrates that Hat1 also physically associates with chromatin near sites of DNA replication. The association of Hat1 with newly replicated DNA is transient but can be stabilized by replication fork stalling. The association of Hat1 with nascent chromatin may be functionally relevant as loss of Hat1 results in a decrease in replication fork progression and an increase in replication fork stalling. In addition, in the absence of Hat1, stalled replication forks are unstable and newly synthesized DNA becomes susceptible to Mre11-dependent degradation. These results suggest that Hat1 links replication fork function to the proper processing and assembly of newly synthesized histones.

biochemistry↗