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Biomedical subjects

Frank Buchholz

Publications and source records attributed to Frank Buchholz.

18 recordsLinked to original sources
PubMed2025

The histone modifier KAT2A presents a selective target in a subset of well-differentiated microsatellite-stable colorectal cancers.

Lysine acetyltransferase 2 A (KAT2A) plays a pivotal role in epigenetic gene regulation across various types of cancer. In colorectal cancer (CRC), increased KAT2A expression is associated with a more aggressive phenotype. Our study aims to elucidate the molecular underpinnings of KAT2A dependency in CRC and assess the consequences of KAT2A depletion. We conducted a comprehensive analysis by integrating CRISPR-Cas9 screening data with genomics, transcriptomics, and global acetylation patterns in CRC cell lines to pinpoint molecular markers indicative of KAT2A dependency. Additionally, we characterized the phenotypic effect of a CRISPR-interference-mediated KAT2A knockdown in CRC cell lines and patient-derived 3D spheroid cultures. Moreover, we assessed the effect of KAT2A depletion within a patient-derived xenograft mouse model in vivo. Our findings reveal that KAT2A dependency is closely associated with microsatellite stability, lower mutational burden, and increased molecular differentiation signatures in CRC, independent of the KAT2A expression levels. KAT2A-dependent CRC cells display higher gene expression levels and enriched H3K27ac marks at gene loci linked to enterocytic differentiation. Furthermore, loss of KAT2A leads to decreased cell growth and viability in vitro and in vivo, downregulation of proliferation- and stem cell-associated genes, and induction of differentiation markers. Altogether, our data show that a specific subset of CRCs with a more differentiated phenotype relies on KAT2A. For these CRC cases, KAT2A might represent a promising novel therapeutic target.

Humans↗
PubMed2023

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma.

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations.

Child↗
PubMed2006

Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I.

Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres.

Animals↗
PubMed2006

RNAi in embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells that can be isolated and grown in vitro from the inner cell mass of blastocysts. Their potential to differentiate into any cell of the body makes them a promising starting material for cell therapy. Much progress has been made in recent years to develop ES cell differentiation protocols employing cocktails of certain growth factors or by using cell-type-restricted promoters driving the expression of selection markers or fluorescent proteins. However, little is known about the molecular details underlying the earliest processes of mammalian development. Genetic tools that provide novel insight into these processes would be very helpful to gain a better molecular understanding and to design better differentiation protocols. Recently, RNAi has emerged as a powerful technology to perform loss-of-function studies in mammalian cells. This technology should be ideal to identify and study genes required for ES cell self-renewal and differentiation. Here, we review the recent advances and challenges of RNAi research in ES cells and we provide a perspective on possible applications to enhance our understanding of ES cell self-renewal and early differentiation.

Animals↗
PubMed2006

Minimizing the risk of reporting false positives in large-scale RNAi screens.

Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.

Databases, Genetic↗
PubMed2006

Enzymatically prepared RNAi libraries.

Large-scale RNA interference (RNAi) screens in mammalian cells have mainly used synthetic small interfering RNA (siRNA) or short hairpin RNA (shRNA) libraries. The RNAi triggers for both of these approaches were designed with algorithm-based predictions to identify single sequences for mRNA knockdown. Alternatives to these approaches have recently been developed using enzymatic methods. Here we describe the concepts of enzymatically prepared shRNA and siRNA libraries, and discuss their strengths and limitations.

Animals↗
PubMed2005

Selective expression of the Cre recombinase in late-stage thymocytes using the distal promoter of the Lck gene.

Transgenic mouse lines were generated that express the Cre recombinase under the control of the distal promoter of the mouse Lck gene. Cre recombination in four of these lines of transgenic mice was characterized at the single cell level using ROSA26-regulated loxP-Stop-loxP-betageo and loxP-Stop-loxP-YFP reporter mouse lines. Two of the lines showed T cell-restricted Cre recombination, whereas the other two also expressed Cre in B cells, NK cells, and monocytes. Cre recombination began at a late stage of T cell development (at or after up-regulation of the TCR during positive selection) in the two T cell-restricted lines. Lines of mice that express the Cre recombinase at late stages of thymocyte development are of value for determining the impact of mutations on T cell function in the absence of complicating effects on early thymocyte selection.

Animals↗
PubMed2005

Functional genomic analysis of cell division by endoribonuclease-prepared siRNAs.

The emergence of RNA interference (RNAi) technology has revolutionized functional genomic analyses in cell biology, including the study of cell division. In particular, the introduction of short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has facilitated loss-of-function studies in mammalian cell lines. We have pioneered the rapid and cost-efficient generation of libraries of endoribonuclease-prepared short interfering RNAs (esiRNAs) for large-scale genetic screens in mammalian tissue culture cells. Our first pilot screen of 5305 genes in human HeLa cells identified novel genes required for cell division, establishing esiRNA as a sophisticated method for loss-of-function screens. Future genome-wide studies with esiRNA, using image-based assays and video microscopy, will help provide novel insights on the molecular mechanisms that govern cell division in mammalian cells.

Animals↗
PubMed2005

RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells.

RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function.

Alternative Splicing↗
PubMed2004

An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division.

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.

Cell Division↗
PubMed2004

DEQOR: a web-based tool for the design and quality control of siRNAs.

RNA interference (RNAi) is a powerful tool for inhibiting the expression of a gene by mediating the degradation of the corresponding mRNA. The basis of this gene-specific inhibition is small, double-stranded RNAs (dsRNAs), also referred to as small interfering RNAs (siRNAs), that correspond in sequence to a part of the exon sequence of a silenced gene. The selection of siRNAs for a target gene is a crucial step in siRNA-mediated gene silencing. According to present knowledge, siRNAs must fulfill certain properties including sequence length, GC-content and nucleotide composition. Furthermore, the cross-silencing capability of dsRNAs for other genes must be evaluated. When designing siRNAs for chemical synthesis, most of these criteria are achievable by simple sequence analysis of target mRNAs, and the specificity can be evaluated by a single BLAST search against the transcriptome of the studied organism. A different method for raising siRNAs has, however, emerged which uses enzymatic digestion to hydrolyze long pieces of dsRNA into shorter molecules. These endoribonuclease-prepared siRNAs (esiRNAs or 'diced' RNAs) are less variable in their silencing capabilities and circumvent the laborious process of sequence selection for RNAi due to a broader range of products. Though powerful, this method might be more susceptible to cross-silencing genes other than the target itself. We have developed a web-based tool that facilitates the design and quality control of siRNAs for RNAi. The program, DEQOR, uses a scoring system based on state-of-the-art parameters for siRNA design to evaluate the inhibitory potency of siRNAs. DEQOR, therefore, can help to predict (i) regions in a gene that show high silencing capacity based on the base pair composition and (ii) siRNAs with high silencing potential for chemical synthesis. In addition, each siRNA arising from the input query is evaluated for possible cross-silencing activities by performing BLAST searches against the transcriptome or genome of a selected organism. DEQOR can therefore predict the probability that an mRNA fragment will cross-react with other genes in the cell and helps researchers to design experiments to test the specificity of esiRNAs or chemically designed siRNAs. DEQOR is freely available at http://cluster-1.mpi-cbg.de/Deqor/deqor.html.

Internet↗
PubMed2004

Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture.

In mammals, embryonic development is more difficult to analyze than in non-mammalian species because this development occurs in utero. Interestingly, whole embryo culture allows the normal development of mouse post-implantation embryos for up to 2 days in vitro. One limitation of this technology has been the difficulty of performing loss-of-gene function studies in this system. RNA interference (RNAi), whereby double-stranded RNA molecules suppress the expression of complementary genes, has rapidly become a widely used tool for gene function analyses. We have combined the technologies of mouse whole embryo culture and RNAi to allow the molecular dissection of developmental processes. Here, we review the manipulation by topical injection followed by directional electroporation of endoribonuclease-prepared siRNA to demonstrate that this technology may be useful to knock down genes in a tissue- and region-specific manner in several organs of the developing mouse embryo.

Animals↗
PubMed2004

Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs.

Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It has recently been shown that HCV RNA replication is susceptible to small interfering RNAs (siRNAs), but the antiviral activity of siRNAs depends very much on their complementarity to the target sequence. Thus, the high degree of sequence diversity between different HCV genotypes and the rapid evolution of new quasispecies is a major problem in the development of siRNA-based gene therapies. For this study, we developed two alternative strategies to overcome these obstacles. In one approach, we used endoribonuclease-prepared siRNAs (esiRNAs) to simultaneously target multiple sites of the viral genome. We show that esiRNAs directed against various regions of the HCV coding sequence as well as the 5' nontranslated region (5' NTR) efficiently block the replication of subgenomic and genomic HCV replicons. In an alternative approach, we generated pseudotyped retroviruses encoding short hairpin RNAs (shRNAs). A total of 12 shRNAs, most of them targeting highly conserved sequence motifs within the 5' NTR or the early core coding region, were analyzed for their antiviral activities. After the transduction of Huh-7 cells containing a subgenomic HCV replicon, we found that all shRNAs targeting sequences in domain IV or nearby coding sequences blocked viral replication. In contrast, only one of seven shRNAs targeting sequences in domain II or III had a similar degree of antiviral activity, indicating that large sections of the NTRs are resistant to RNA interference. Moreover, we show that naive Huh-7 cells that stably expressed certain 5' NTR-specific shRNAs were largely resistant to a challenge with HCV replicons. These results demonstrate that the retroviral transduction of HCV-specific shRNAs provides a new possibility for antiviral intervention.

Antiviral Agents↗
PubMed2003

Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation.

Escherichia coli phage P1 Cre recombinase catalyzes the site-specific recombination of DNA containing loxP sites. We report here two crystal structures of a wild-type Cre recombinase-loxP synaptic complex corresponding to two distinct reaction states: an initial pre-cleavage complex, trapped using a phosphorothioate modification at the cleavable scissile bond that prevents the recombination reaction, and a 3'-phosphotyrosine protein-DNA intermediate resulting from the first strand cleavage. In contrast to previously determined Cre complexes, both structures contain a full tetrameric complex in the asymmetric unit, unequivocally showing that the anti-parallel arrangement of the loxP sites is an intrinsic property of the Cre-loxP recombination synapse. The conformation of the spacer is different to the one observed for the symmetrized loxS site: a kink next to the scissile phosphate in the top strand of the pre-cleavage complex leads to unstacking of the TpG step and a widening of the minor groove. This side of the spacer is interacting with a 'cleavage-competent' Cre subunit, suggesting that the first cleavage occurs at the ApT step in the top strand. This is further confirmed by the structure of the 3'-phosphotyrosine intermediate, where the DNA is cleaved in the top strands and covalently linked to the 'cleavage-competent' subunits. The cleavage is followed by a movement of the C-terminal part containing the attacking Y324 and the helix N interacting with the 'non-cleaving' subunit. This rearrangement could be responsible for the interconversion of Cre subunits. Our results also suggest that the Cre-induced kink next to the scissile phosphodiester activates the DNA for cleavage at this position and facilitates strand transfer.

Base Sequence↗
PubMed2003

RNA interference: gene silencing in the fast lane.

Sequencing of whole genomes has provided new perspectives into the blueprints of diverse organisms. Knowing the sequences, however, does not always tell us much about the function of the genes that regulate development and homeostasis. RNA interference (RNAi) is becoming the method of choice for gene function analysis in cells and whole organisms. Here we review the approaches available to perform RNAi experiments in mammalian cells and in mice. We discuss usage of RNAi in cancer research and as a possible therapeutic tool for cancer treatment.

Animals↗
PubMed2002

Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA.

RNA interference (RNAi) using double-stranded RNA has been used for the systematic analysis of gene function in invertebrate organisms. Here we have explored the use of short interfering RNA (siRNA) to knock down gene expression during the development of mammalian postimplantation embryos. The developing CNS system of embryonic day 10 mouse embryos was used as a model tissue. siRNA prepared by endoribonuclease digestion (esiRNA) was injected into the lumen of the neural tube at specific regions and delivered into neuroepithelial cells by directed electroporation. Injected and electroporated embryos were grown for 1 day in whole-embryo culture and the effects of RNAi were examined. esiRNA directed against beta-galactosidase (beta-gal), coelectroporated into neuroepithelial cells together with reporter plasmids expressing GFP and beta-gal, abolished expression of beta-gal but not GFP, showing the specificity of the esiRNA-mediated RNAi. To demonstrate RNAi of endogenous gene expression, we used heterozygous embryos of a knock-in mouse line expressing GFP from the Tis21 locus, a gene turned on in neuroepithelial cells that switch from proliferation to neurogenesis. GFP-directed esiRNA electroporated into neuroepithelial cells of such embryos blocked the GFP expression normally occurring on the onset of neurogenesis. Taken together, our data indicate that esiRNA delivered in a tissue-specific manner by topical injection followed by directed electroporation can efficiently silence endogenous gene expression in mammalian postimplantation embryos.

Animals↗
PubMed2002

Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells.

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of Escherichia coli RNase III to cleave double-stranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in Drosophila S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells. esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.

Animals↗