Biology subjects

Ha, A.

Publications and source records attributed to Ha, A..

8 recordsLinked to original sources

bioRxiv2026

Genomic imprinting of the metabolic regulator gene Klf14 is regulated by a paternal sub-TAD anchored at Mest and a shared enhancer in mice.

KLF14 acts as a master regulator of gene expression in adipose tissue and variants at the human KLF14 gene show strong and reproducible association with type 2 diabetes and metabolic syndrome. Risk alleles are only pathologic when maternally inherited, consistent with the observation that KLF14/Klf14 is a maternally-expressed imprinted gene in human and mouse. However, how genomic imprinting is regulated at this important locus is currently unknown. In both species, KLF14/Klf14 is located [~]200 kb away from the paternally imprinted gene MEST/Mest, which is regulated by a maternal gametic differentially methylated region (gDMR) at its CpG island (CGI) promoter. Although the Klf14 CGI is kept unmethylated in most tissues, maternally-inherited DNA methylation (DNAme) marks are paradoxically required for Klf14 expression in mouse. Here, we show that Mest and Klf14 reside within the same topologically associating domain (TAD) in both mouse embryonic stem cells (ESCs) and differentiated cells, defined by sites of biallelic CTCF binding at the boundaries. Using allele-specific 4C-seq in F1 hybrid ESCs, we show that CTCF binding specifically to the unmethylated Mest gDMR generates a paternal allele-specific sub-TAD encompassing Klf14. We further show through CRISPR-Cas9-mediated mutagenesis, that deletions of the paternal Mest promoter region in ESCs as well as in vivo in mutant mice, results in both loss of Mest expression and acquisition of biallelic expression at Klf14. By analyzing epigenetic marks and chromatin looping in the Klf14-expressing pituitary cell line AtT-20, we identify a putative enhancer element shared by Mest and Klf14, providing a mechanistic model for the regulation of Klf14 imprinting. This work defines a new role for the Mest maternally methylated gDMR, revealing that it exerts long-range effects via allele-specific modulation of TAD structures and consequently act as an imprinting control region (ICR) in the regulation of Klf14 imprinting. Conservation of this TAD organisation at the human locus suggests that a similar regulatory mechanism operates at KLF14.

developmental biology↗

bioRxiv2026

Ultra-large targeted DNA integrations in primary human cells

Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cells genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond [~]5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large--up to 10 kb DNA--integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo. Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.

genetics↗

bioRxiv2026

3D Droplet-Based Bioprinting of Customized In Vitro Head and Neck Cancer Tumor Microenvironment Models

In vitro models are increasingly critical for interrogating cancer biology and therapeutic response, however, accurately recapitulating the tumor microenvironment (TME) remains a persistent challenge, particularly in head and neck cancers (HNC) characterized by complex cell-matrix interactions and heterogeneity. Current models often lack independent tunability of biochemical and biophysical cues, limiting systematic investigation of microenvironmental cues in a high-throughput format. Here, we establish a 3D droplet-based bioprinting platform for the fabrication of customizable in vitro TME models using poly(ethylene glycol) (PEG) hydrogels. Human HNC cell lines (FaDu and 2A3) with differing HPV statuses were bioprinted into PEG matrices spanning physiologically relevant stiffnesses (0.7-4.8 kPa) and compositions, including non-functionalized PEG and peptide-functionalized PEG (PEGfnc: RGD, YIGSR, CNYYSNS) and cultured for 7 days. Cluster growth, cell viability, and cluster morphology were assessed across multiple time points, matrix compositions, and matrix stiffnesses. Proliferation and endpoint phenotype expression were visualized using confocal microscopy through immunofluorescence. Results indicated enhanced cell viability in PEGfnc matrices, compared to non-functionalized matrices, while effect of matrix stiffness was less prominent. Median cluster size reached 40-50 m by day 7, and linear mixed-effects modeling identified how changes in cluster surface area, volume, and tumoroid complexity varied with cell type, matrix, and stiffness. By decoupling and systematically varying key TME parameters, this approach provides a robust and scalable framework for dissecting tumor-matrix interactions and advancing physiologically relevant in vitro models for cancer research and therapeutic screening.

bioengineering↗

bioRxiv2026

The elongation of Mest transcript into MestXL sustains, but does not initiate, the maternal allele bias of its convergent gene Copg2 during neurogenesis

Precise gene dosage control is critical for establishing cellular identity and development, especially for imprinted genes, where dosage imbalances are linked to severe pathologies such as neurodevelopmental disorders. The Mest/Copg2 imprinted locus is a paradigm for this fine-tuned regulation. While Mest is constitutively expressed from the paternal allele, Copg2 expression shifts from biallelic to a maternal allele bias specifically during neural differentiation, a transition proposed to involve transcriptional interference mediated by the long Mest isoform, MestXL, which extends into the Copg2 locus. However, the mechanisms underlying this allelic switch, and whether factors beyond MestXL contribute, are elusive. To address this, we employed a stem cell-based brain organoid model, integrating multi-omic analyses, 3D chromatin structure mapping, and functional approaches to dissect the regulatory events governing the induction and maintenance of Copg2 maternal allele bias throughout neural lineage specification. Our findings challenge the prevailing model by demonstrating that the maternal allele bias of Copg2 during neural differentiation is not solely driven by MestXL-mediated transcriptional interference. Instead, our data support a temporal and neural stage-specific two-step mechanism: putative enhancer-driven activation of the maternal allele in neural progenitor cells is followed by MestXL-dependent repression of the paternal allele in neuron-enriched stages. This uncovers an unexpected layer of complexity in the regulation of imprinted gene dosage during brain development, with profound implications for understanding the molecular underpinnings of neurodevelopmental disorders.

molecular biology↗

bioRxiv2025

Ancestry-Dependent Immunologic and Prognostic Effects Characterize the Prostate Cancer Urinary Proteome

Urine is an attractive biomarker analyte for non-invasive longitudinal monitoring of health and disease, particularly for diseases of the genitourinary tract, like prostate and bladder cancer. The composition of an individuals urine reflects both genetic and lifestyle characteristics that differ across geographies and populations, like diet, hydration and other socio-economic factors. While men of African ancestry have elevated prostate cancer risk, it is unclear to what extent this influences urinary biomarkers. We therefore quantified the urinary proteomes of 329 localized prostate cancer patients: 135 self-identifying as White and 194 self-identifying as Black. We identified 110 proteins that significantly differed between these groups after controlling for age, PSA, and cISUP. Immune pathways were particularly dysregulated. The urinary proteome of Black patients harboured more features of aggressive cancers than those of grade- and PSA-matched White patients. These observations highlight the importance of controlling for race- and ancestry-associated differences in the development of urinary biomarkers.

cancer biology↗

bioRxiv2024

Engineering an Escherichia coli strain for production of long single-stranded DNA

Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA- guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered E. coli "helper" strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20,724 nucleotides with titers up to 250 g/L following alkaline-lysis purification. The efficacy of our system was confirmed through its application in primary T cell genome modifications and DNA origami folding. The reliability, scalability, and ease of our approach promises to unlock new experimental applications requiring large quantities of long ssDNA.

bioengineering↗

bioRxiv2021

Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing

CRISPR-Cas9 offers unprecedented opportunities to modify genome sequences in primary human cells to study disease variants and reprogram cell functions for next-generation cellular therapies. CRISPR has several potential advantages over widely used retroviral vectors including: 1) site-specific transgene insertion via homology directed repair (HDR), and 2) reductions in the cost and complexity of genome modification. Despite rapid progress with ex vivo CRISPR genome engineering, many novel research and clinical applications would be enabled by methods to further improve knock-in efficiency and the absolute yield of live knock-in cells, especially with large HDR templates (HDRT). We recently reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDRTs to improve knock-in, but yields and efficiencies were limited by toxicity at high HDRT concentrations. Here we developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA). We designed hybrid ssDNA HDRTs that incorporate CTS sites and were able to boost knock-in percentages by >5-fold and live cell yields by >7-fold relative to dsDNA HDRTs with CTS. Knock-in efficiency and yield with ssCTS HDRTs were increased further with small molecule inhibitor combinations to improve HDR. We demonstrate application of these methods across a variety of target loci, knock-in constructs, and primary human cell types to reach ultra-high HDR efficiencies (>80-90%) which we use for pathogenic gene variant modeling and universal gene replacement strategies for IL2RA and CTLA4 mutations associated with mendelian immune disorders. Finally, we develop a GMP-compatible method for fully non-viral CAR-T cell manufacturing, demonstrating knock-in efficiencies of 46-62% and generating yields of >1.5 x 109 CAR+ T cells, well above current doses for adoptive cellular therapies. Taken together, we present a comprehensive non-viral approach to model disease associated mutations and re-write targeted genome sequences to program immune cell therapies at a scale compatible with future clinical application.

synthetic biology↗

bioRxiv2019

Parallel accumulation - serial fragmentation combined with data-independent acquisition (diaPASEF): Bottom-up proteomics with near optimal ion usage

Data independent acquisition (DIA) modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall only a few percent of all incoming ions are sampled. Making use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro), we here devise a novel scan mode that samples up to 100% of the peptide precursor ion current. We extend an established targeted data extraction workflow by including the ion mobility dimension for both signal extraction and scoring, thereby increasing the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a very high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.

systems biology↗