Biology subjects

Daneshvar, K.

Publications and source records attributed to Daneshvar, K..

5 recordsLinked to original sources

bioRxiv2025

T-PGI: an engineered STITCHR system for scarless, programmable genome integration

Scarless, programmable insertion of defined DNA remains a central goal for therapeutic genome editing. We introduce T-PGI (Transposon-mediated programmable genomic integration), an engineered implementation of STITCHR that preserves target-primed reverse transcription (TPRT) while substantially improving efficiency, specificity, and modularity. T-PGI uses R2Tocc, a low-background ortholog that we further engineered through defined deletions, rational point mutations, and modular domain insertions to enhance performance. The system employs paired nCas9 nicks flanking homology arms and bicistronic co-expression with dual NLSs, together with optimized RNA donor designs. Using combinatorial guide-template screening, T-PGI achieves robust integration across diverse cargos, including precise cassette insertion following multi-kilobase deletions. Short- and long-read sequencing confirm high-fidelity insertion with minimal local indels or structural variants. Collectively, these advances establish T-PGI as a practical and adaptable platform for accurate, scarless genome integration and provide concise design principles spanning enzyme architecture, donor configuration, and guide pairing for next-generation therapeutic editing.

bioengineering↗

bioRxiv2024

Epigenetic silencing and blockade of latency reversal by an HIV-1 encoded antisense transcript

The mechanisms that regulate human immunodeficiency virus 1 (HIV-1) latency are not fully elucidated. We reported that an HIV-1 antisense transcript (AST) induces epigenetic modifications at the HIV-1 promoter causing a closed chromatin state that suppresses viral transcription. Here we show that ectopic expression of AST in CD4+ T-cells from people with HIV-1 under antiretroviral therapy blocks latency reversal in response to pharmacologic and T-cell receptor stimulation, enforcing transcriptional silencing. We define structural domains and sequence motifs of AST contributing to its latency-promoting functions. Finally, we report an unbiased proteomics screen of AST interactors that revealed an array of previously known and potential new HIV-1-suppressive factors. Our studies identify AST as a first-in-class biological molecule capable of enforcing HIV-1 latency and with actionable curative potential.

molecular biology↗

bioRxiv2023

Conserved long noncoding RNA TILAM promotes liver fibrosis through interaction with PML in hepatic stellate cells

Background & AimsFibrosis is the common endpoint for all forms of chronic liver injury, and progression of fibrosis leads to the development of end-stage liver disease. Activation of hepatic stellate cells (HSCs) and their transdifferentiation to myofibroblasts results in the accumulation of extracellular matrix (ECM) proteins that form the fibrotic scar. Long noncoding (lnc) RNAs regulate the activity of HSCs and may provide targets for fibrotic therapies. MethodsWe identified lncRNA TILAM as expressed near COL1A1 in human HSCs and performed loss-of-function studies in human HSCs and liver organoids. Transcriptomic analyses of HSCs isolated from mice defined the murine ortholog of TILAM. We then generated Tilam-deficient GFP reporter mice and quantified fibrotic responses to carbon tetrachloride (CCl4) and choline-deficient L-amino acid defined high fat diet (CDA-HFD). Co-precipitation studies, mass spectrometry, and gene expression analyses identified protein partners of TILAM. ResultsTILAM is conserved between human and mouse HSCs and regulates expression of ECM proteins, including collagen. Tilam is selectively induced in HSCs during the development of fibrosis in vivo. In both male and female mice, loss of Tilam results in reduced fibrosis in the setting of CCl4 and CDA-HFD injury models. TILAM interacts with promyelocytic leukemia protein (PML) to stabilize PML protein levels and promote the fibrotic activity of HSCs. ConclusionTILAM is activated in HSCs and interacts with PML to drive the development of liver fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end stage liver disease.

cell biology↗

bioRxiv2019

lncRNA DIGIT and BRD3 protein form phase-separated condensates to regulate endoderm differentiation

Gene programs that control differentiation are regulated through the interplay between DNA, RNA, and protein. Cooperation among these fundamental cellular components can occur through highly structured interactions connecting domains formed by specific sequences of nucleotides, ribonucleotides, and/or amino acids and also through the assembly of biomolecular condensates. Here, we show that endoderm differentiation is regulated through the interaction of the long noncoding (lnc) RNA DIGIT and the bromodomain and extra-terminal (BET) domain family protein BRD3. BRD3 forms phase-separated condensates that contain DIGIT, occupies enhancers of endoderm transcription factors, and is required for endoderm differentiation. Purified BRD3 binds to acetylated histone H3 lysine 18 (H3K18ac) in vitro and occupies regions of the genome enriched in H3K18ac during endoderm differentiation, including the key transcription factors that regulate endoderm differentiation. DIGIT is also enriched in regions of H3K18ac, and depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings support a model where cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest a broader role for protein-lncRNA phase-separated condensates as regulators of transcription in development.

molecular biology↗

bioRxiv2017

Identification and characterization of m6A circular RNA epitranscriptomes

This study brings together the expanding fields of RNA modifications and circular (circ) RNAs. We find that cells express thousands of m6A methylated circRNAs, with cell-type specificity observed between human embryonic stem cells and HeLa cells. m6A-circRNAs were identified by RNA sequencing of total RNA following ribosome depletion and m6A immunoprecipitation. The presence of m6A-circRNAs is corroborated by the identification of complexes between circRNAs and YTHDF1 and YTHDF2, proteins that \"read\" m6A sites in mRNAs.\n\nFurthermore, m6A modifications on non-linear RNAs depend on METTL3 and METTL14, the known m6A methyltransferase \"writer\" complex components, suggesting that circRNAs are methylated by the same complexes responsible for m6A modification of linear RNAs. Despite sharing m6A readers and writers, m6A-circRNAs are frequently derived from exons not methylated in mRNAs. Nevertheless, m6A-mRNAs that are methylated on the same exons as those composing m6A-circRNAs exhibit less stability than other m6A-mRNA, and this circRNA-mRNA cross-talk is regulated by YTHDF2. Thus, our results expand the m6A regulatory code through identification of the first circRNA epitranscriptome.

bioinformatics↗