Biology subjects

Husain, A.

Publications and source records attributed to Husain, A..

6 recordsLinked to original sources

bioRxiv2025

Mechanosensitive Endothelial METTL7A Regulates Internal m7G mRNA Methylation and Protects Against Atherosclerosis

BACKGROUNDInternal N7-methylguanosine (m7G) is a recently identified chemical modification of mammalian mRNA and a component of the epitranscriptome. While the epitranscriptome plays a key role in regulating RNA metabolism and cellular function, the specific contribution of internal m7G to cardiovascular health and disease remains unknown. Atherosclerosis preferentially develops at sites where disturbed blood flow activates endothelial cells, but whether internal m7G and its regulatory machinery influence endothelial mechanotransduction and atherogenesis is unclear. METHODSWe integrated epitranscriptomic profiling, human tissue analysis, genetically modified mouse models, and targeted nanomedicine approaches to investigate the role of Methyltransferase-like protein 7A (METTL7A), a putative internal m7G methyltransferase, in regulating the flow-sensitive endothelial transcriptome and atherosclerosis. Vascular endothelial cells were subjected to well-defined athero-protective and athero-prone flow waveforms in vitro and in vivo. METTL7A function was assessed using RNA sequencing (RNA-seq), liquid chromatography-tandem mass spectrometry (LC-MS/MS), crosslinking immunoprecipitation sequencing (CLIP-seq), RNA stability assays, and a CRISPR-Cas-inspired RNA targeting system (CIRTS). METTL7A expression in human coronary arteries with and without atherosclerosis was evaluated by RNA-seq and immunostaining. In vivo atherosclerosis studies were conducted in both global and endothelial-specific Mettl7a1 knockout mice. Endothelial METTL7A expression was restored using cationic polymer-based nanoparticles delivering CDH5 promoter-driven METTL7A plasmids or VCAM1-targeted lipid nanoparticles delivering N1-methylpseudouridine (m{superscript 1}{Psi})-modified METTL7A mRNA. RESULTSAthero-protective unidirectional flow significantly induced METTL7A expression, which promoted internal m7G methylation of endothelial transcripts, while other major epitranscriptomic marks and cap-associated m7G were not affected by METTL7A. METTL7A preferentially binds to AG-enriched motifs in protein-coding mRNAs and plays a key role in regulating KLF4 and NFKBIA transcripts, enhancing their internal m7G and stability and supporting vascular homeostasis. In contrast, endothelial METTL7A expression was significantly reduced by disturbed blood flow and in human atherosclerotic lesions. Global or endothelial-specific loss of METTL7A exacerbated disturbed flow-induced atherosclerosis in mice, independent of serum lipid levels. Restoration of endothelial METTL7A, via nanoparticle-mediated plasmid or m1{Psi} mRNA delivery, markedly reduced lesion formation in Mettl7a1-/- and ApoE-/- mice. CONCLUSIONSThese findings establish METTL7A as a previously unrecognized mechanosensitive methyltransferase that maintains endothelial homeostasis by stabilizing key anti-inflammatory transcripts, KLF4 and NFKBIA, through internal m7G methylation. Loss of METTL7A disrupts endothelial function and accelerates atherogenesis in response to disturbed flow. Therapeutic restoration of endothelial METTL7A, via targeted nanoparticle-mediated gene or m1{Psi} mRNA delivery, significantly lessens atherosclerosis. Collectively, these results uncover a novel epitranscriptomic mechanism governing vascular health and position METTL7A as a promising target for precision nanomedicine in atherosclerotic cardiovascular disease.

molecular biology↗

bioRxiv2021

A standardized method to purify cardiomyocytes from individual mouse hearts of any age

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different agedependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2x106 cells/heart) and viability (~70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell-type specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR<0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rodshaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

cell biology↗

bioRxiv2021

Polycomb group protein CBX7 represses cardiomyocyte proliferation via modulation of the TARDBP/Rbm38 axis

Cardiomyocyte (CM) proliferation notably decreases during the perinatal period. At present, regulatory mechanisms for this loss of proliferative capacity is poorly understood. CBX7, a polycomb group (PcG) protein, regulates the cell cycle but its role in CM proliferation is unknown. Here, we report that CBX7 inhibits proliferation of perinatal CMs by controlling TARDBP/Rbm38 pathway. Gene expression profiling demonstrated that CBX7 expression in the heart was low during the prenatal period, abruptly increased during the perinatal period, and sustained constantly throughout the adulthood. CBX7, when overexpressed via adenoviral transduction in neonatal CMs, reduced proliferation and promoted multinucleation of the CMs. Mutant mice carrying targeted inhibition of CBX7 in CMs exhibited cardiomegaly with increased proliferation of CMs at postnatal stages. Mechanistically, CBX7 interacted with TAR DNA-binding protein 43 (TARDBP) and positively regulated its downstream target, RNA Binding Motif Protein 38 (RBM38). Rbm38 was upregulated in the postnatal hearts and overexpression of RBM38 reduced proliferation of neonatal CMs. Together, this study provides a novel insight into the role of CBX7 in regulation of CM proliferation during the perinatal period.

developmental biology↗

bioRxiv2020

Poised cell circuits in human skin are activated in disease

The human skin confers biophysical and immunological protection through a complex cellular network that is established early in development. We profiled ~500,000 single cells using RNA-sequencing from healthy adult and developing skin, and skin from patients with atopic dermatitis and psoriasis. Our findings reveal a predominance of innate lymphoid cells and macrophages in developing skin in contrast to T cells and migratory dendritic cells in adult skin. We demonstrate dual keratinocyte differentiation trajectories and activated cellular circuits comprising vascular endothelial cells mediating immune cell trafficking, disease-specific clonally expanded IL13/IL22 and IL17A/F-expressing lymphocytes, epidermal IL23-expressing dendritic cells and inflammatory keratinocytes in disease. Our findings provide key insights into the dynamic cellular landscape of human skin in health and disease. One Sentence SummarySingle cell atlas of human skin reveals cell circuits which are quantitatively and qualitatively reconfigured in inflammatory skin disease.

immunology↗

bioRxiv2019

Epigenetic dysregulation underpins tumorigenesis in a cutaneous tumor syndrome.

Patients with CYLD cutaneous syndrome (CCS; syn. Brooke-Spiegler syndrome) carry germline mutations in the tumor suppressor CYLD and develop multiple skin tumors with diverse histophenotypes 1,2. We comprehensively profiled the genomic landscape of 42 benign and malignant tumors across 13 individuals from four multigenerational families. Novel driver mutations were found in epigenetic modifiers DNMT3A and BCOR in 29% of benign tumors. Multi-level and microdissected sampling strikingly reveal that many clones with different DNMT3A mutations exist in these benign tumors, suggesting that intra-tumor heterogeneity is common. Integrated genomic and methylation profiling suggest that mutated DNMT3A drives tumorigenesis mechanistically through Wnt/{beta}-catenin pathway signaling. Phylogenetic and mutational signature analyses confirm the phenomenon of benign pulmonary metastases from primary skin lesions. In malignant tumors, additional epigenetic modifiers MBD4, CREBBP, KDM6A and EP300 were mutated. We thus present epigenetic dysregulation as a driver in CCS tumorigenesis and propose this may account for the diverse histophenotypic patterns despite the paucity of mutations seen. These findings add novel dimensions to existing paradigms of cutaneous tumorigenesis and metastasis.

cancer biology↗

bioRxiv2017

Traction force screening enabled by compliant PDMS elastomers

Acto-myosin contractility is an essential element of many aspects of cellular biology, and manifests as traction forces that cells exert on their surroundings. The central role of these forces makes them a novel principal therapeutic target in diverse diseases. This requires accurate and higher capacity measurements of traction forces; however, existing methods are largely low throughput, limiting their utility in broader applications. To address this need, we employ Fourier-transform traction force microscopy in a parallelized 96-well format, which we refer to as contractile force screening (CFS).Critically, rather than the frequently employed hydrogel polyacrylamide (PAA), we fabricate these plates using polydimethylsiloxane (PDMS) rubber. Key to this approach is that the PDMS used is very compliant, with a lower-bound Youngs modulus of approximately 0.7 kPa. We subdivide these monolithic substrates spatially into biochemically independent wells, creating a uniform multiwell platform for traction force screening. We demonstrate the utility and versatility of this platform by quantifying the compound and dose-dependent contractility responses of human airway smooth muscle cells and retinal pigment epithelial cells.

biophysics↗