Biology subjects

Ahmad, K.

Publications and source records attributed to Ahmad, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Elevated polyadenylated histone gene transcription in human cancer

Histone production is tightly restricted to S-phase to prevent genomic instability, yet human cancers with an overabundance of RNA Polymerase II (RNAPII) at histone genes are associated with aggressive cancers. To better understand histone gene overexpression in cancer, we applied a quantitative PCR assay to measure total replication-coupled histone output across the histone gene clusters using cell line models. We observed pervasive accumulation of polyadenylated histone transcripts, supported by analysis of >1,400 RNA-sequencing datasets in human cancers, with more than 70% of patients showing tumor specific increases. This alternative processing preferentially enriches for histone H2B, which lacks replication independent variants, across >8,300 tumors comprising diverse cancer types and transcriptional subtypes. We also found that disruption of nuclear components required to maintain RNAPII levels at the histone genes increased polyadenylated histone transcripts. Our results suggest that S-phase-dependent histone genes are subject to unscheduled gap phase transcription in human cancer.

cancer biology↗

bioRxiv2026

Neurite Complexity and Plasticity Markers Track Synaptic Vesicle Density in the Living Human Brain: A Multimodal PET-MRI-EEG Study

Neuroplasticity, the brain's capacity to reorganise its structure and function, is a transdiagnostic feature across psychiatric and neurodegenerative diseases and a putative mechanism of action for pharmacological interventions. [11C]-UCBJ positron emission tomography (PET), which quantifies synaptic vesicle glycoprotein 2A (SV2A) density, offers the most direct in vivo measure of human synaptic density, but its relationship to complementary, more scalable neuroimaging measures of structural and functional plasticity remains uncharacterised. We combined [11C]-UCBJ PET, diffusion weighted imaging (DWI/NODDI), resting-state functional magnetic resonance imaging (rs-fMRI), and electroencephalography (EEG)-based sensory tasks (roving mismatch negativity, MMN; visual long-term potentiation, vLTP) in 18 healthy volunteers to test the relationship between synaptic terminal SV2A density and complementary structural and functional neuroplasticity measures, and whether these relationships were reproducible following pharmacological challenge with ketamine (n = 11) or N,N-dimethyltryptamine (DMT; n = 7). There were no statistically significant differences in PET-derived SV2A binding potential (BPND), DWI-derived neurite orientation dispersion (ODI) and structural weighted degree (SWD), EEG-derived MMN repetition suppression, and late-phase vLTP P2 potentiation, 1 to 8 days following DMT and ketamine exposure compared to baseline. BPND was coupled with ODI across cortical regions before and after pharmacological challenge, and this relationship was tighter within visual, attentional, and frontoparietal control brain networks. SWD showed a weaker, more spatially restricted association with BPND, primarily in regions belonging to the limbic and visual brain networks, but was not replicated post-drug. No consistent association was found between BPND and functional weighted degree (FWD), derived from rs-fMRI, at baseline or after pharmacological challenge. Frontotemporal BPND was positively associated with MMN repetition suppression (r = 0.61), and occipital BPND with late-phase vLTP P2 potentiation (r = 0.54), with the MMN-BPND association replicating after pharmacological challenge (r = 0.49). These findings link synaptic terminal SV2A density in the living human brain to neurite architecture and the capacity for short-term and Hebbian-like long-term synaptic plasticity, while dissociating these relationships from generic functional connectivity and magnitude of sensory-evoked responses. These data provide cross-modal validation for DWI- and EEG-derived measures as biological indices and surrogate measures of human synaptic plasticity.

neuroscience↗

bioRxiv2026

FFPE-CUTAC: A Single Assay, Multiple Layers

Background: Archival biobanks of formalin-fixed paraffin-embedded (FFPE) specimens represent a vast and underused resource for retrospective molecular studies linked to clinical follow-up. However, fixation-induced chemical modification and nucleic acid fragmentation limit scalable genomic and transcriptomic profiling. FFPE-CUTAC (Cleavage Under Targeted Accessible Chromatin) is an RNA polymerase II-targeted DNA assay specifically designed to profile regulatory activity in FFPE tissue. Here, we assess its archival robustness, information content, compatibility with RNA-seq, and ability to recover copy-number alterations. Results: In meningioma and breast cancer cohorts, we benchmarked FFPE-CUTAC against matched fresh-frozen RNA-seq, FFPE RNA-seq and FFPE whole-genome sequencing. For FFPE specimens spanning up to 25 years, FFPE-CUTAC showed no systematic collection-year-associated decline in library yield or signal-quality metrics. In contrast, FFPE RNA-seq is more affected by the FFPE tissue degradation and specimen age on read composition, gene-body coverage, and gene detection. By measuring RNA polymerase II occupancy on chromatin rather than mature RNA abundance, FFPE-CUTAC is less constrained by transcript half-life, polyadenylation or transcript-capture design. FFPE-CUTAC extends beyond gene regions to non-coding regulatory elements, while the shared gene-level signals supported its integration with existing RNA-seq cohorts for large-scale, long-term clinical association study. FFPE-CUTAC libraries also retain DNA dosage information and can be used to recover chromosome-arm gain, loss and intact states with 96% concordance to matched whole-genome sequencing. Conclusions: By converting routinely preserved pathology sections into integrated regulatory and copy-number profiles, FFPE-CUTAC provides a practical foundation for constructing clinically annotated disease maps to support molecular stratification, prognostic modeling and treatment-association studies.

cancer biology↗

bioRxiv2026

Aclarubicin disrupts RNA polymerase II progression at replication-coupled histone genes

Anthracyclines are highly effective chemotherapeutic agents that cause DNA and chromatin damage. One member of the anthracyclines, aclarubicin, has recently gained therapeutic interest due to its ability to kill cancer cells through chromatin-based mechanisms, thus avoiding the off-target effects associated with DNA damage. Despite this, the molecular mechanism of action leading to aclarubicin-induced chromatin damage remains elusive. Here we performed Cleavage Under Targets and Tagmentation (CUT&Tag) of RNA polymerase II (Pol II) and other transcriptional regulators in human cells during aclarubicin treatment. We found that aclarubicin strongly disrupts the replication-coupled histone genes, resulting in a nonproductive accumulation of Pol II-transcription machinery here beyond the levels at other genes. We attribute this sensitivity to the dense Pol II loading and rapid transcription of the histone genes, which intensify the chromatin-disrupting effects of aclarubicin at these loci. Together, our findings support the effectiveness of aclarubicin as an anticancer drug and point to the histone gene cluster as a promising target for therapeutic intervention.

molecular biology↗

bioRxiv2026

KAS-CUT&Tag for direct mapping of transcription bubbles

Transcription by RNA Polymerase II (Pol II) generates dynamic transcription bubbles as it moves forward, but existing methods map transcriptional activity in vivo only indirectly. We introduce KAS-CUT&Tag, a method that combines N3-kethoxal labeling of exposed guanines with CUT&Tag to directly map transcription bubbles. We find that bubble density varies across Pol II-bound genes at transcription start sites, gene bodies, and termination sites. Transcription bubbles are enriched at genes marked by the H3K36me3 histone modification and chromatin-bound splicing factor U2AF2, but the highest enrichment is at replication-coupled histone genes throughout the cell cycle. KAS-CUT&Tag also detects colocalization of the histone gene transcription factor NPAT with Pol II at the Histone Locus Body, suggesting NPAT is juxtaposed to transcription bubbles via interaction with Pol II. Together, KAS-CUT&Tag enables direct mapping of Pol II and regulatory interactions at transcription bubbles, providing a powerful tool for precise analysis of transcriptional activity.

genomics↗

bioRxiv2026

Histone H3K27 methylation states are sequentially catalyzed in cycling cells

Polycomb domains form at silenced genes and are marked by histone H3 lysine 27 tri-methylation, but it is unclear how this methylation is maintained following DNA replication in proliferating cells. Here, we use CUT&Tag chromatin profiling to track the dynamics of H3K27 methylation in human cells. We find that tri-methylation in Polycomb domains is maintained by stepwise methylation after DNA replication. Outside of Polycomb domains, thousands of inactive genes gain histone H3K27 di-methylation hours after DNA replication. Acute treatment with small-molecule inhibitors of the Polycomb Repressive Complex 2 (PRC2) histone methyltransferase slows H3K27 methylation during S-phase of the cell cycle and increases acetylation of the residue. This effect is most pronounced at Polycomb domains replicating in early S-phase, suggesting that early replication and slow histone methylation confers plasticity to developmentally silenced genes. Together, our results explain how H3K27 modification patterns are faithfully duplicated in rapidly proliferating cells.

genomics↗

bioRxiv2025

Chromatin profiling for everyone: FFPE-CUTAC for the theory and practice of modern molecular biology

In 2025, together with the Fred Hutch Summer Undergraduate Research and Summer High School Internship programs, we developed and implemented a laboratory genomics research experience to introduce students to modern molecular biology techniques and bioinformatics. The course centered around using a new method we had developed in 2023 that uses readily available fixed tissue sections on glass slides. Students performed a series of steps to tagment genomic locations of RNA Polymerase II and then used PCR to enrich libraries for next-generation sequencing in a core facility. Students then visualized their data in genomic browser tracks and assessed the results. At the end of the summer, students prepared and presented their work and experiences in seminar format to their cohorts. Overall, the technical simplicity of on-slide chromatin profiling introduced the students to laboratory practice and current techniques in genomics, bioinformatics, and medical sciences.

scientific communication and education↗

bioRxiv2025

Nascent CUT&Tag captures transcription factor binding after chromatin duplication

DNA replication strips off all chromatin proteins, which must be reassembled behind the replication fork. To track chromatin reassembly on newly synthesized DNA, we developed Nascent CUT&Tag, a chromatin profiling method that directly measures transcription factor (TF) binding on nascent chromatin. We tracked the recovery of GAGA factor (GAF) and Pleiohomeotic (PHO) in Drosophila Kc167 cells. Whereas both TFs are evicted following passage of the replication fork, GAF recovers on newly synthesized DNA over a minutes-to-hours range, whereas PHO generally requires hours to fully reestablish binding. Early recovering GAF peaks are characterized by shorter GAF motifs and are associated with functions related to cell cycle progression. Conversely, late recovering peaks are characterized by longer, degenerate GAF motifs and are associated with developmental functions. GAF recovery on newly synthesized DNA requires chromatin remodeling by Brahma Associated Factor (BAF), implying that nucleosome turnover is critical to fully reestablish GAF binding.

genomics↗

bioRxiv2025

Detecting neuroplastic effects induced by ketamine in healthy human subjects: a multimodal approach

We investigated ketamines neuroplastic effects in healthy human subjects using integrated Positron Emission Tomography (PET)/Magnetic Resonance Imaging (MRI) measures before and 1-8 days after a single psychedelic dose of ketamine (1 mg/kg, intravenous). Eleven participants underwent two PET/MRI scans with [11C]-UCBJ (synaptic density/plasticity), 1H-MRS (Glutamate and GABA), and resting-state fMRI (intrinsic brain activity, functional connectivity, graph-theoretic metrics), before and after ketamine. While group-level analyses showed only trend-level increases in PET synaptic markers, we observed significantly elevated Anterior Cingulate Cortex (ACC) glutamate levels post-ketamine. Functional connectivity analyses revealed decreased within-network integrity, particularly in high-order networks like the default mode network (DMN), alongside increased low-to-high-order network integration. Our multimodal analysis showed that increased [11C]-UCBJ volume distribution (VT), a putative index of synaptic plasticity, correlated with reduced intrinsic activity in DMN regions and decreased influence of the posterior cingulate cortex (PCC) in global network dynamics. By linking molecular and network-level changes, our results point to the PCC as a central hub where ketamine may reshape brain hierarchies in the long term, providing new directions for understanding its therapeutic mechanisms and developing targeted treatments.

neuroscience↗

bioRxiv2025

Total whole-arm chromosome losses predict malignancy in human cancer

Aneuploidy is observed as gains or losses of whole chromosomes or chromosome arms and is a common hallmark of cancer. Whereas models for the generation of aneuploidy in cancer invoke mitotic chromosome segregation errors, whole-arm losses might occur simply as a result of centromere breakage. We recently showed that elevated RNA Polymerase II (RNAPII) level over S-phase-dependent histone genes predicts rapid recurrence of human meningioma and is correlated with total whole-arm losses relative to gains. To explain this imbalance in arm losses over gains, we have proposed that histone overexpression at S-phase competes with the histone H3 variant CENP-A, resulting in centromere breaks and whole-arm losses. To test whether centromere breaks alone can drive aneuploidy, we ask whether total whole-arm aneuploids can predict outcome across different cancer types in large RNA and whole-genome sequencing databanks. We find that total whole-arm losses generally predict outcome, suggesting that centromere breakage is a major initiating factor leading to aneuploidy and the resulting changes in the selective landscape that drive most cancers. We also present evidence that centromere breakage alone is sufficient to account for whole-arm losses and gains, contrary to mitotic spindle error models for generation of aneuploidy. Our results suggest that therapeutic intervention targeting histone overexpression has the potential of reducing aneuploidy and slowing cancer progression. Significance StatementGain or loss of whole chromosome arms following centromere breaks is frequent in cancer, but whether or not there is a common initiating event is unknown. Here we show that the total number of whole-arm losses predicts patient outcomes across cancer types, suggesting a causal relationship. This general excess of losses over gains is not predicted by mitotic error models of aneuploidy but rather suggests that centromere breaks themselves initiate whole-arm aneuploidies. Insofar as aneuploidy reshapes the selective landscapes that drive most cancers, our results have potential clinical implications.

cancer biology↗

bioRxiv2024

Histone H4 limits transcription of the histone locus in Drosophila

In all eukaryotes DNA replication is coupled to histone synthesis to coordinate chromatin packaging of the genome. Canonical histone genes coalesce in the nucleus into the Histone Locus Body (HLB), where gene transcription and 3 mRNA processing occurs. Both histone gene transcription and mRNA stability are reduced when DNA replication is inhibited, implying that the Histone Locus Body senses the rate of DNA synthesis. In Drosophila melanogaster, the S-phase-induced histone genes are tandemly repeated in an [~]100 copy array, whereas in humans, these histone genes are scattered. In both organisms these genes coalesce into Histone Locus Bodies. We used a transgenic histone gene reporter and RNAi in Drosophila to identify canonical H4 histone as a unique repressor of histone synthesis during the G2 phase in germline cells. Using cytology and CUT&Tag chromatin profiling, we find that histone H4 uniquely occupies histone gene promoters in both Drosophila and human cells. Our results suggest that repression of histone genes by soluble histone H4 is a conserved mechanism that coordinates DNA replication with histone synthesis in proliferating cells.

genomics↗

bioRxiv2024

Nanobactericides Derived from Cinnamon Bark Extract: Phytochemical Profiling and Antibacterial Efficacy Against Bacterial Panicle Blight in Rice

Bacterial panicle blight (BPB), caused by the Gram-negative aerobic bacterium Burkholderia glumae (B.glumae), poses a significant threat to global rice production. Cinnamon bark extract (CBE) has demonstrated potent antioxidant and antimicrobial properties due to its high concentration of bioactive compounds, including eugenol and cinnamaldehyde. To enhance the efficacy and stability of these volatile compounds, this study employed nanotechnology and encapsulation techniques. The objective was to develop a CBE-based nanoformulation to inhibit B. glumae and control BPB in rice. CBE-chitosan (CBE-CS) nanoformulations were synthesized using ionic cross-linking between chitosan and trisodium phosphate (TPP) at various concentrations (0%, 0.5%, 1%, 2%, and 4% TPP). More than 15 active compounds were identified in CBE, including (Z)-3-Phenylacrylaldehyde, 2-Propenoic acid, 3-(2-hydroxyphenyl), cinnamaldehyde dimethyl acetal, and hexadecanoic acid. Bacterial membrane damage was significantly greater in treatments with CBE compared to untreated controls. The synthesized nanoparticles ranged in size from 43.66 nm to 106.1 nm, with encapsulation efficiencies between 48.65% and 48.78%, and loading capacities between 25.65% and 33.9%. Scanning electron microscopy (SEM) revealed spherical and homogeneous nanoparticles, while FTIR and XRD analysis confirmed the successful encapsulation of CBE in the chitosan nanoparticles. The antibacterial activity of the nanoformulations showed inhibition zones ranging from 7.5 to 11.8 mm, with the CBE-CS formulation containing 0.5% TPP demonstrating the highest efficacy (MIC = 15.6 mol/ml; MBC = 31.25 mol/ml).

plant biology↗

bioRxiv2024

Understanding genetic diversity and phylogeography of Common Teal and its phylogenetic relationship with other water bird species in the wetlands of Kashmir Himalayas.

Understanding the genetic diversity and phylogeography of migratory species is critical for biodiversity conservation and the effective management of wetland ecosystems. The Kashmir Himalayas, an integral part of the Central Asian Flyway, host several key wetlands that provide critical wintering grounds for a variety of migratory birds. This study focuses on assessing the genetic characteristics and phylogenetic relationships of the Common Teal (Anas crecca) in comparison to other species within the families Anatidae and Rallidae. We analysed 149 blood samples, including 71 from A. crecca and 78 from other species in the two families, collected from wetlands in the Kashmir region. Using four mitochondrial markers--cytochrome oxidase subunit I (COI), cytochrome b (Cyt b), 16S rRNA, and the control region--we evaluated the genetic diversity and lineage connectivity of these species. Our findings reveal that the mitochondrial DNA haplotypes of A. crecca in the Kashmir Himalayas are shared with European populations, indicating strong maternal gene flow and connectivity between distant populations. A minimum spanning haplotype network analysis showed minimal nucleotide differences among haplotypes, particularly in the Cyt b and control regions, suggesting low genetic differentiation and a high degree of similarity among individuals. Notably, we identified at least four distinct maternal lineages of A. crecca in the Kashmir wetlands, reflecting diverse migratory sources. Our results also highlight that DNA barcoding using COI exhibited both high and low species resolution, with significant intraspecific variation, making it a valuable tool for further phylogeographic studies. The observed genetic diversity and haplotype sharing with distant populations underscore the ecological importance of Kashmirs wetlands as crucial habitats for migratory species. Our study emphasizes the need for targeted conservation and management strategies to preserve these vital ecosystems and the biodiversity they support.

genetics↗

bioRxiv2024

Direct measurement of RNA Polymerase II hypertranscription in cancer FFPE samples

Genome-wide hypertranscription is common in hu-man cancer and predicts poor prognosis. To under-stand how hypertranscription might drive cancer, we applied our FFPE-CUTAC method for mapping RNA Polymerase II (RNAPII) genome-wide in formalin-fixed paraffin-embedded (FFPE) sections. We demonstrate global RNAPII elevations in mouse gliomas and assort-ed human tumors in small clinical samples and discov-er regional elevations corresponding to de novo HER2 amplifications punctuated by likely selective sweeps. RNAPII occupancy at replication-coupled histone genes correlated with WHO grade in meningiomas, ac-curately predicted rapid recurrence, and corresponded to whole-arm chromosome losses. Elevated RNAPII at histone genes in meningiomas and diverse breast cancers is consistent with histone production being rate-limiting for S-phase progression and histone gene hypertranscription driving overproliferation and aneu-ploidy in cancer, with general implications for precision oncology.

cancer biology↗

bioRxiv2023

KMT2A oncoproteins induce epigenetic resistance to targeted therapies

Chromosomal translocations involving the Lysine-Methyl-Transferase-2A (KMT2A) locus generate potent oncogenic fusion proteins (oncoproteins) that disrupt regulation of developmental gene expression. By profiling the oncoprotein-target sites of 36 broadly representative KMT2A-rearranged leukemia samples, including three samples that underwent a lymphoid-to-myeloid lineage-switching event in response to therapy, we find the genomic enrichment of the oncoprotein is highly variable between samples and subject to dynamic regulation. At high levels of expression, the oncoproteins preferentially activate either an acute lymphoblastic leukemia (ALL) program, enriched for pro-B-cell genes, or an acute myeloid leukemia (AML) program, enriched for hematopoietic-stem-cell genes. The fusion-partner-specific-binding patterns over these gene sets are highly correlated with the prevalence of each mutation in ALL versus AML. In lineage-switching samples the oncoprotein levels are reduced and the oncoproteins preferentially activate granulocyte-monocyte progenitor (GMP) genes. In a sample that lineage switched during treatment with the menin inhibitor revumenib, the oncoprotein and menin are reduced to undetectable levels, but ENL, a transcriptional cofactor of the oncoprotein, persists on numerous oncoprotein-target loci, including genes in the GMP-like lineage-switching program. We propose KMT2A oncoproteins promote lineage-switching events through dynamic chromatin binding and can induce epigenetic lesions, marked by ENL, that support resistance to targeted therapies.

cancer biology↗

bioRxiv2023

Epigenomic analysis of Formalin-Fixed Paraffin-Embedded samples by CUT&Tag

For more than a century, Formalin Fixed Paraffin Embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our single-tube CUTAC protocol are sufficient to produce high-resolution maps of paused RNA Polymerase II (RNAPII) at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumor specimens and identify regulatory element markers with high confidence and precision. Our simple work-flow is suitable for automation, making possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.

cancer biology↗

bioRxiv2023

Chromosome-specific maturation of the epigenome in the Drosophila male germline

Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome, and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, we profiled active chromatin features in the testes from wildtype and meiotic arrest mutants and integrate this with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, we detail widespread patterns associated with regulation of the sex chromosomes. Our results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome.

developmental biology↗

bioRxiv2023

Aclarubicin stimulates RNA polymerase II elongation at closely spaced divergent promoters

Anthracyclines are a class of widely prescribed anti-cancer drugs that disrupt chromatin by intercalating into DNA and enhancing nucleosome turnover. To understand the molecular consequences of anthracycline-mediated chromatin disruption, we utilized CUT&Tag to profile RNA polymerase II during anthracycline treatment in Drosophila cells. We observed that treatment with the anthracycline aclarubicin leads to elevated levels of elongating RNA polymerase II and changes in chromatin accessibility. We found that promoter proximity and orientation impacts chromatin changes during aclarubicin treatment, as closely spaced divergent promoter pairs show greater chromatin changes when compared to codirectionally-oriented tandem promoters. We also found that aclarubicin treatment changes the distribution of non-canonical DNA G-quadruplex structures both at promoters and at G-rich pericentromeric repeats. Our work suggests that the anti-cancer activity of aclarubicin is driven by the effects of nucleosome disruption on RNA polymerase II, chromatin accessibility and DNA structures.

genomics↗