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Derek M Dykxhoorn

Publications and source records attributed to Derek M Dykxhoorn.

14 recordsLinked to original sources
PubMed2026

Distal enhancer-insulator module of GDF6 is essential for cochlear formation.

Several genes guide inner ear development, and mutations in these genes can cause malformations that result in congenital hearing loss. However, the contribution of noncoding regulatory elements remains largely unclear. This study investigates the function of distal enhancer elements in the transcriptional regulation of GDF6, a gene implicated in cochlear development. Using mouse models with targeted deletions, human inner ear organoids, and CRISPR interference (CRISPRi), we identified a downstream regulatory interval harboring a developmental enhancer required to maintain GDF6 expression during otic epithelial maturation and cochlear morphogenesis. Deletion of this regulatory region or targeting of CRISPRi-based repressors to these regions resulted in decreased GDF6 expression, failure of otic-epithelium development, and prevention of hair cell-like differentiation, reflecting cochlear aplasia observed in patients with corresponding genomic deletions. These findings highlight the contribution of long-range regulatory elements to auditory development and illustrate how their disruption contributes to human deafness.

Animals↗
PubMed2025

Integrator promotes the association of TFIID and RNA polymerase II to maintain pluripotency during development.

The mechanisms by which the expression of pluripotency and Polycomb networks are harmonized to allow the transition from pluripotency to a differentiated state have not been fully elucidated. Integrator complex regulates transcription pause release and RNA processing in metazoans. We show that Integrator is required for stemness and plays a critical role as early as day 2 in embryonic development. While the catalytic endonuclease activity enhances cellular reprogramming, Integrator recruits RNA polymerase II (RNAPII) to promoters and super enhancers of pluripotency and Polycomb genes. Integrator coordinates expression of pluripotency and Polycomb networks by fostering the association of RNAPII and basal transcription factors. We pinpoint a critical role for TATA-binding protein-associated factors (TAFs) in Integrator entry into the preinitiation complex. Taken together, beyond its role in RNAPII pause release, Integrator recruitment of RNAPII ensures an orderly cellular differentiation during development.

RNA Polymerase II↗
PubMed2006

Knocking down disease with siRNAs.

Knocking down expression of disease-related genes using small interfering RNAs (siRNAs) has potential for treating a variety of illnesses. This Essay will examine the opportunities for harnessing RNA interference (RNAi) for therapy, as well as the obstacles and possible ways to circumvent them.

Animals↗
PubMed2006

Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism.

A single nucleotide polymorphism (SNP) in the sickle beta-globin gene (beta(S)) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal gamma-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A beta(S) siRNA with position 10 of the guide strand designed to align with the targeted beta(S) SNP specifically silences beta(S) gene expression without affecting the expression of the gamma-globin or normal beta-globin (beta(A)) genes. Silencing is increased by altering the 5' end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific beta(S) silencing was demonstrated by using a luciferase reporter and full-length beta(S) cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous beta-globin gene promoter and the locus control region enhancers. When this strategy was used to target beta(E), silencing was not limited to the mutant gene but also targeted the normal beta(A) gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the beta(S)-globin gene, as compared with beta(E), as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.

Base Sequence↗
PubMed2006

Running interference: prospects and obstacles to using small interfering RNAs as small molecule drugs.

RNA interference (RNAi) is a well-conserved, ubiquitous, endogenous mechanism that uses small noncoding RNAs to silence gene expression. The endogenous small RNAs, called microRNAs, are processed from hairpin precursors and regulate important genes involved in cell death, differentiation, and development. RNAi also protects the genome from invading genetic elements, encoded by transposons and viruses. When small double-stranded RNAs, called small interfering (si)RNAs, are introduced into cells, they bind to the endogenous RNAi machinery to disrupt the expression of mRNAs containing complementary sequences with high specificity. Any disease-causing gene and any cell type or tissue can potentially be targeted. This technique has been rapidly utilized for gene-function analysis and drug-target discovery and validation. Harnessing RNAi also holds great promise for therapy, although introducing siRNAs into cells in vivo remains an important obstacle. Pilot siRNA clinical studies began just three years after the discovery that RNAi works in mammalian cells. This review discusses recent progress and obstacles to using siRNAs as small molecule drugs.

Animals↗
PubMed2005

Genetic analysis of the subunit organization and function of the conserved oligomeric golgi (COG) complex: studies of COG5- and COG7-deficient mammalian cells.

The conserved oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in Golgi-associated membrane trafficking and glycoconjugate synthesis. We have analyzed the structure and function of COG using Cog1 or Cog2 null Chinese hamster ovary cell mutants, fibroblasts from a patient with Cog7-deficient congenital disorders of glycosylation, and stable Cog5-deficient HeLa cells generated by RNA interference. Although the dilation of some Golgi cisternae in Cog5-deficient cells resembled that observed in Cog1- or Cog2-deficient cells, their global glycosylation defects (less severe) and intracellular processing and function of low density lipoprotein receptors (essentially normal) differed from Cog1- and Cog2-deficient cells. Immunoblotting, gel filtration, and immunofluorescence microscopy analyses of the COG-deficient cells and cell extracts indicated that 1) Cog2-4 and Cog5-7 form stable subcomplexes, 2) Cog1 mediates Golgi association of a Cog2-4 plus Cog8 subcomplex, 3) Cog8 associates stably with both Cog5-7 and Cog1-4 subcomplexes, and thus 4) Cog8 helps assemble the Cog1-4 and Cog5-7 subcomplexes into the complete COG complex. This model of the subunit organization of COG is in excellent agreement with in vitro data presented in an accompanying paper (Ungar, D., Oka, T., Vasile, E., Krieger, M., and Hughson, F. M. (2005) J. Biol. Chem. 280, 32729-32735). Only one or two of the seven Cog1- or Cog2-dependent Golgi membrane proteins called GEARs are also sensitive to Cog5 or Cog7 deficiency, indicating that the COG subunits play distinctive roles in controlling Golgi structure and function.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.

Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

Animals↗
PubMed2005

Lentiviral delivery of short hairpin RNAs protects CD4 T cells from multiple clades and primary isolates of HIV.

Viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference (RNAi) against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested 3 viral target sites with differing propensity for mutations: a highly variable rev sequence, a gag sequence conserved only among clade B isolates, and a vif sequence highly conserved across clades. Lentiviral expression of all 3 shRNAs inhibited replication of the homologous HIV(IIIB) strain. However, they differed in their ability to protect primary CD4 T cells against multiple isolates within and across HIV clades. The least conserved rev sequence inhibited only 2 of 5 clade B isolates. The gag sequence (conserved within clade B) protected 5 of 5 clade B isolates but not other clade viruses with 2 or 3 mutations in the central region. In contrast, the vif sequence, which was conserved across clades except for single mutations at positions 14 and 17, inhibited viruses from 5 different clades. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas mutations in vif siRNA only modestly decreased silencing. Thus, although 1 or 2 mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.

Antiviral Agents↗
PubMed2005

The silent revolution: RNA interference as basic biology, research tool, and therapeutic.

RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression. In primitive organisms, RNAi protects the genome from viruses and other insertable genetic elements and regulates gene expression during development. The antisense (guide) strand of short double-stranded RNAs is incorporated into an RNA-induced silencing complex that can either suppress protein expression or direct degradation of messenger RNAs that contain homologous sequence(s). The discovery that RNAi works in mammalian cells has sparked intense investigation into its role in normal mammalian cell function, its use as a tool to understand or screen for genes functioning in cellular pathways in healthy and diseased cells and animals, and its potential for therapeutic gene silencing. RNAi may provide an important new therapeutic modality for treating infection, cancer, neurodegenerative disease, and other illnesses, although in vivo delivery of small interfering RNAs into cells remains a significant obstacle.

Animals↗
PubMed2003

Killing the messenger: short RNAs that silence gene expression.

Short interfering RNAs can be used to silence gene expression in a sequence-specific manner in a process that is known as RNA interference. The application of RNA interference in mammals has the potential to allow the systematic analysis of gene expression and holds the possibility of therapeutic gene silencing. Much of the promise of RNA interference will depend on the recent advances in short-RNA-based silencing technologies.

Animals↗
PubMed2003

Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.

Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

Antiviral Agents↗
PubMed2003

Lentivirus-delivered stable gene silencing by RNAi in primary cells.

Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.

Dendritic Cells↗
PubMed2002

siRNA-directed inhibition of HIV-1 infection.

RNA interference silences gene expression through short interfering 21 23-mer double-strand RNA segments that guide mRNA degradation in a sequence-specific fashion. Here we report that siRNAs inhibit virus production by targeting the mRNAs for either the HIV-1 cellular receptor CD4, the viral structural Gag protein or green fluorescence protein substituted for the Nef regulatory protein. siRNAs effectively inhibit pre- and/or post-integration infection events in the HIV-1 life cycle. Thus, siRNAs may have potential for therapeutic intervention in HIV-1 and other viral infections.

Base Sequence↗