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Hendrik G Stunnenberg

Publications and source records attributed to Hendrik G Stunnenberg.

18 recordsLinked to original sources
PubMed2026

Methylome profiling of SetDB1-deficient ESCs reveals coordinated epigenetic cross-talk during pluripotency.

SetDB1 is best known for catalyzing H3K9me3, but it also influences H3K27me3 deposition, CTCF-binding, and DNA methylation (DNAme). Given the interplay between DNAme and the other epigenetic features, we profiled DNAme following Setdb1 knockout (KO) in ground-state and serum-grown mouse embryonic stem cells (ESCs) to illuminate DNAme-dependent and -independent functions of SetDB1. Time-course whole-genome bisulfite sequencing of serum-grown ESCs shows that nearly half of SetDB1 binding sites are enriched with DNAme and H3K9me3, primarily at retrotransposons. Upon Setdb1 KO, both H3K9me3 and DNAme are reduced, with DNAme rapidly removed at many sites by TET enzymes. Some retrotransposons, primarily IAPs, are TET-resistant and lose DNAme slowly via passive dilution. Notably, SetDB1-mediated regulation of H3K27me3, CTCF-binding, and SMAD3 are uncoupled from the DNAme-H3K9me3 axis, and from each other. AlphaFold modeling and co-immunoprecipitation mass spectrometry suggest this uncoupling involves competitive binding to distinct SetDB1 protein domains, highlighting the complex coordination underlying SetDB1 functions.

AlphaFold modeling↗
PubMed2007

DNA methylation immediately adjacent to active histone marking does not silence transcription.

Active promoters generally contain histone H3/H4 hyperacetylation and tri-methylation at H3 lysine 4, whereas repressed promoters are associated with DNA methylation. Here we show that the repressed erythroid-specific carbonic anhydrase II (CAII) promoter has active histone modifications localized around the transcription start, while high levels of CpG methylation are present directly upstream from these active marks. Despite the presence of active histone modifications, the repressed promoter requires hormone-induced activation for efficient preinitiation complex assembly. Transient and positional changes in histone H3/H4 acetylation and local changes in nucleosome density are evident during activation, but the bipartite epigenetic code is stably maintained. Our results suggest that active histone modifications may prevent spreading of CpG methylation towards the promoter and show that repressive DNA methylation immediately adjacent to a promoter does not necessarily repress transcription.

Acetylation↗
PubMed2006

Molecular characterization of Sin3 PAH-domain interactor specificity and identification of PAH partners.

Sin3 is the central component of a multisubunit co-repressor complex. A number of DNA-binding proteins are targeted by the Sin3 complex to chromatin through association with its paired amphipathic helix (PAH) domains. Here, we performed a yeast two-hybrid screening using a peptide aptamer library and identified peptides that interact with either PAH1 or PAH2. Analysis of PAH2 interacting peptides uncovered motifs similar to previously characterized PAH2 interacting proteins, Mad, Ume6 and kruppel-like members, while analysis of PAH1 interacting peptides revealed an LXXLL motif. In addition, a tandem affinity purification (TAP)-tagging approach of Sin3b resulted in the isolation of known and novel interactors amongst which neural retina leucine (NRL) zipper. Strikingly, one of the identified PAH2 interacting peptide showed strong resemblance to the NRL region amino acids 125-150. Direct association between PAH2 and NRL was shown and NRL(125-150) mediated transcriptional repression in reporter assays. Finally, we reveal that PAH1 and PAH2 amino acids 7, 14 and 39 shown previously to be important for Mad-PAH2 interaction, also play an important role in the specificity of interaction between PAH1, PAH2 and identified aptamers. Our results provide novel insights into the molecular determinant of the specificity of PAH1 and PAH2 for their interacting partners.

Amino Acid Motifs↗
PubMed2006

Histone modification patterns associated with the human X chromosome.

X inactivation is associated with chromosome-wide establishment of inactive chromatin. Although this is classically regarded as facultative heterochromatin that is uniform in nature, the exact distribution of associated epigenetic marks is not well defined. Here we have analysed histone modifications in human somatic cells within two selected regions of the X chromosome. Intergenic, coding and promoter regions are segregated into differentially marked chromatin. H3K27me3 is most prominent in intergenic and silenced coding regions, but is associated with some active coding regions as well. Histone H3/H4 acetylation and H3K4me3 are locally enriched at promoter regions but do not necessarily mark continuing transcription. Remarkably, H3K9me3 is predominant in coding regions of active genes, a phenomenon that is not restricted to the X chromosome. These results argue against the exclusiveness of individual marks to heterochromatin or euchromatin, but rather suggest that composite patterns of interdependent or mutually exclusive modifications together signal the gene expression status.

Acetylation↗
PubMed2006

A feed-forward repression mechanism anchors the Sin3/histone deacetylase and N-CoR/SMRT corepressors on chromatin.

Transcription in eukaryotes is governed in part by histone acetyltransferase (HAT)- and histone deacetylase (HDAC)-containing complexes that are recruited via activators and repressors, respectively. Here, we show that the Sin3/HDAC and N-CoR/SMRT corepressor complexes repress transcription from histone H3- and/or H4-acetylated nucleosomal templates in vitro. Repression of histone H3-acetylated templates was completely dependent on the histone deacetylase activity of the corepressor complexes, whereas this activity was not required to repress H4-acetylated templates. Following deacetylation, both complexes become stably anchored in a repressor-independent manner to nucleosomal templates containing hypoacetylated histone H3, but not H4, resulting in dominance of repression over activation. The observed stable anchoring of corepressor complexes casts doubt on the view of a dynamic balance between readily exchangeable HAT and HDAC activities regulating transcription and implies that pathways need to be in place to actively remove HDAC complexes from hypoacetylated promoters to switch on silent genes.

Acetylation↗
PubMed2006

Peroxisome proliferator-activated receptor subtype- and cell-type-specific activation of genomic target genes upon adenoviral transgene delivery.

Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes. We demonstrate that PPARgamma2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARgamma2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (alpha, gamma, and beta/delta), we show that the subtypes have very different abilities to gain access to target sites and that in general the genomic occupancy correlates with the ability to activate the corresponding target gene. In addition, the specificity and potency of activation by PPAR subtypes are highly dependent on the cell type. Thus, PPAR subtype-specific activation of genomic target genes involves an intricate interplay between the properties of the subtype- and cell-type-specific settings at the individual target loci.

Adenoviridae↗
PubMed2006

MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties.

The human genome contains a number of methyl CpG binding proteins that translate DNA methylation into a physiological response. To gain insight into the function of MBD2 and MBD3, we first applied protein tagging and mass spectrometry. We show that MBD2 and MBD3 assemble into mutually exclusive distinct Mi-2/NuRD-like complexes, called MBD2/NuRD and MBD3/NuRD. We identified DOC-1, a putative tumor suppressor, as a novel core subunit of MBD2/NuRD as well as MBD3/NuRD. PRMT5 and its cofactor MEP50 were identified as specific MBD2/NuRD interactors. PRMT5 stably and specifically associates with and methylates the RG-rich N terminus of MBD2. Chromatin immunoprecipitation experiments revealed that PRMT5 and MBD2 are recruited to CpG islands in a methylation-dependent manner in vivo and that H4R3, a substrate of PRMT, is methylated at these loci. Our data show that MBD2/NuRD and MBD3/NuRD are distinct protein complexes with different biochemical and functional properties.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Uncleaved TFIIA is a substrate for taspase 1 and active in transcription.

In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by the single TFIIAalphabeta gene and posttranslationally cleaved into alpha and beta subunits. The molecular mechanisms and biological significance of this proteolytic process have remained obscure. Here, we show that TFIIA is a substrate of taspase 1 as reported for the trithorax group mixed-lineage leukemia protein. We demonstrate that recombinant taspase 1 cleaves TFIIA in vitro. Transfected taspase 1 enhances cleavage of TFIIA, and RNA interference knockdown of endogenous taspase 1 diminishes cleavage of TFIIA in vivo. In taspase 1-/- MEF cells, only uncleaved TFIIA is detected. In Xenopus laevis embryos, knockdown of TFIIA results in phenotype and expression defects. Both defects can be rescued by expression of an uncleavable TFIIA mutant. Our study shows that uncleaved TFIIA is transcriptionally active and that cleavage of TFIIA does not serve to render TFIIA competent for transcription. We propose that cleavage fine tunes the transcription regulation of a subset of genes during differentiation and development.

Amino Acid Sequence↗
PubMed2005

Characterization of lysine 56 of histone H3 as an acetylation site in Saccharomyces cerevisiae.

Post-translational histone modifications abound and regulate multiple nuclear processes. Most modifications are targeted to the amino-terminal domains of histones. Here we report the identification and characterization of acetylation of lysine 56 within the core domain of histone H3. In the crystal structure of the nucleosome, lysine 56 contacts DNA. Phenotypic analysis suggests that lysine 56 is critical for histone function and that it modulates formamide resistance, ultraviolet radiation sensitivity, and sensitivity to hydroxyurea. We show that the acetylated form of histone H3 lysine 56 (H3-K56) is present during interphase, metaphase, and S phase. Finally, reverse genetic analysis indicates that none of the known histone acetyltransferases is solely responsible for H3-K56 acetylation in Saccharomyces cerevisiae.

Alleles↗
PubMed2004

Cleavage and proteasome-mediated degradation of the basal transcription factor TFIIA.

The transcription factor TFIIA is encoded by two genes, TFIIAalphabeta and TFIIAgamma. In higher eukaryotes, the TFIIAalphabeta is translated as a precursor and undergoes proteolytic cleavage; the regulation and biological implications of the cleavage have remained elusive. We determined by Edman degradation that the TFIIAbeta subunit starts at Asp 278. We found that a cleavage recognition site (CRS), a string of amino acids QVDG at positions -6 to -3 from Asp 278, is essential for cleavage. Mutations in the CRS that prevent cleavage significantly prolong the half-life of TFIIA. Consistently, the cleaved TFIIA is a substrate for the ubiquitin pathway and proteasome-mediated degradation. We show that mutations in the putative phosphorylation sites of TFIIAbeta greatly affect degradation of the beta-subunit. We propose that cleavage and subsequent degradation fine-tune the amount of TFIIA in the cell and consequently the level of transcription.

Amino Acid Sequence↗
PubMed2004

Molecular determinants of the interaction of Mad with the PAH2 domain of mSin3.

The Sin3 co-repressor acts as a protein scaffold to recruit transcription factors via its four highly homologous paired amphipathic helix (PAH) domains. PAH2 has been shown to interact strongly with the Sin3 interacting domain (SID) of the tumor suppressor Mad. This PAH2/Mad complex has been studied extensively by NMR, but the molecular determinants that dictate the specificity of interaction remain to be elucidated. To uncover residues that convey the specificity of interaction between PAH2 and Mad, PAH2 residues contacted by the Mad-SID were introduced into the PAH1 domain of mSin3b and tested for gain-of-interaction in vivo in a yeast two-hybrid setting and further confirmed in a cell-free system. This approach led to the identification of PAH2-Phe-7 as a critical residue. Stabilization of the interaction between PAH1-Phe-7 and the Mad-SID was achieved by introducing Val-14 and Gln-39 into PAH1. Substitution of PAH2 residues contacted by the Mad-SID with their respective residues in PAH1 corroborated and extended the critical role of Phe-7 and the stabilizing role of Val-14 and Gln-39. We conclude that Phe-7 is the critical determinant and provides the molecular specificity for the association between Sin3 and Mad in regulating cell growth and differentiation.

Amino Acid Sequence↗
PubMed2004

In vitro targeting reveals intrinsic histone tail specificity of the Sin3/histone deacetylase and N-CoR/SMRT corepressor complexes.

The histone code is among others established via differential acetylation catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). To unambiguously determine the histone tail specificity of HDAC-containing complexes, we have established an in vitro system consisting of nucleosomal templates reconstituted with hyperacetylated histones or recombinant histones followed by acetylation with native SAGA or NuA4. Selective targeting of the mammalian Sin3/HDAC and N-CoR/SMRT corepressor complexes by using specific chimeric repressors created a near physiological setting to assess their histone tail specificity. Recruitment of the Sin3/HDAC complex to nucleosomal templates preacetylated with SAGA or NuA4 resulted in deacetylation of histones H3 and H4, whereas recruitment of N-CoR/SMRT resulted in deacetylation of histone H3 only. These results provide solid evidence that HDAC-containing complexes display distinct, intrinsic histone tail specificities and hence may function differently to regulate chromatin structure and transcription.

Animals↗
PubMed2003

p300 is involved in formation of the TBP-TFIIA-containing basal transcription complex, TAC.

We have recently identified a novel basal transcription complex, TAC, that is present and active in embryonal carcinoma (EC) cells but not in other adult cells such as COS7. In the search for factors involved in TAC formation, we found that expression of the adenoviral 12S E1A oncoprotein abolishes TAC formation in EC cells. This effect of E1A depends on its N-terminal domain that is essential for cell differentiation and that targets the transcriptional coactivators p300 and PCAF. Expression of p300 lacking its major E1A interaction domain, CH3, restores TAC formation in the presence of E1A, in a bromodomain- and HAT domain-dependent manner. Consistently, the unprocessed TFIIAalphabeta precursor that is selectively assembled into TAC is acetylated preferentially compared with the processed subunits present in 'free' TFIIA. Intriguingly, expression of p300 in COS7 cells that do not contain detectable levels of TAC instigates formation of TAC from endogenous components. Our data suggest that p300 plays a role in formation of the TBP-TFIIA-containing basal transcription complex, TAC.

Acetylation↗
PubMed2002

Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry.

The annotated genomes of organisms define a 'blueprint' of their possible gene products. Post-genome analyses attempt to confirm and modify the annotation and impose a sense of the spatial, temporal and developmental usage of genetic information by the organism. Here we describe a large-scale, high-accuracy (average deviation less than 0.02 Da at 1,000 Da) mass spectrometric proteome analysis of selected stages of the human malaria parasite Plasmodium falciparum. The analysis revealed 1,289 proteins of which 714 proteins were identified in asexual blood stages, 931 in gametocytes and 645 in gametes. The last two groups provide insights into the biology of the sexual stages of the parasite, and include conserved, stage-specific, secreted and membrane-associated proteins. A subset of these proteins contain domains that indicate a role in cell-cell interactions, and therefore can be evaluated as potential components of a malaria vaccine formulation. We also report a set of peptides with significant matches in the parasite genome but not in the protein set predicted by computational methods.

Animals↗
PubMed2002

The gene encoding the Acyl-CoA-binding protein is activated by peroxisome proliferator-activated receptor gamma through an intronic response element functionally conserved between humans and rodents.

The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular protein that specifically binds acyl-CoA esters with high affinity and is structurally and functionally conserved from yeast to mammals. In vitro studies indicate that ACBP may regulate the availability of acyl-CoA esters for various metabolic and regulatory purposes. The protein is particularly abundant in cells with a high level of lipogenesis and de novo fatty acid synthesis and is significantly induced during adipocyte differentiation. However, the molecular mechanisms underlying the regulation of ACBP expression in mammalian cells have remained largely unknown. Here we report that ACBP is a novel peroxisome proliferator-activated receptor (PPAR)gamma target gene. The rat ACBP gene is directly activated by PPARgamma/retinoid X receptor alpha (RXRalpha) and PPARalpha/RXRalpha, but not by PPARdelta/RXRalpha, through a PPAR-response element in intron 1, which is functionally conserved in the human ACBP gene. The intronic PPAR-response element (PPRE) mediates induction by endogenous PPARgamma in murine adipocytes and confers responsiveness to the PPARgamma-selective ligand BRL49653. Finally, we have used chromatin immunoprecipitation to demonstrate that the intronic PPRE efficiently binds PPARgamma/RXR in its natural chromatin context in adipocytes. Thus, the PPRE in intron 1 of the ACBP gene is a bona fide PPARgamma-response element.

3T3 Cells↗
PubMed2002

In vivo repression of an erythroid-specific gene by distinct corepressor complexes.

To assess the mechanisms of repression of the erythroid-specific carbonic anhydrase II (CAII) locus we used chromatin immunoprecipitation and show that an NCoR-histone deacetylase (HDAC)3 complex is recruited by the nuclear receptor v-ErbA to the intronic HS2 enhancer turning it into a potent silencer. Furthermore we demonstrate that efficient CAII silencing requires binding of a MeCP2-targeted HDAC-containing corepressor complex to the hypermethylated CpG-island at the promoter. Activation of transcription by either AZAdC or thyroid hormone results in loss of one of the two corepressor complexes. Thyroid hormone further replaces the enhancer-bound NCoR-corepressor complex by the TRAP220 coactivator. Treatment with the HDAC inhibitor trichostatin A (TSA) causes activation of CAII transcription and histone H3 and H4 hyperacetylation at the enhancer, apparently without affecting binding of the two corepressor complexes. Unexpectedly, histone H3 and H4 at the fully repressed promoter are already hyperacetylated despite the close apposition of the MeCP2-targeted HDAC complex. Acetylation of histone H4, but not H3, at the promoter is moderately increased following TSA treatment. Our data suggest that the hyperacetylated but repressed CAII promoter is (partially) remodeled and primed for activation in v-ErbA-transformed cells.

Acetylation↗
PubMed2002

E2F transcriptional repressor complexes are critical downstream targets of p19(ARF)/p53-induced proliferative arrest.

The p16(INK4a)/pRB/E2F and p19(ARF)/p53 tumor suppressor pathways are disrupted in most human cancers. Both p19(ARF) and p53 are required for the induction of senescence in primary mouse embryonic fibroblasts (MEFs), but little is known about their downstream targets. Disruption of E2F-mediated transcriptional repression in MEFs caused a general increase in the expression of E2F target genes, including p19ARF. We detected no contribution of E2F-mediated transactivation in this setting, indicating that a predominant role of endogenous E2F in asynchronously growing primary MEFs is to repress its target genes. Moreover, relief of transcriptional repression by E2F rendered MEFs resistant to senescence induced by either p19(ARF), p53, or RAS(V12). Thus, E2F transcriptional repressor complexes are critical downstream targets of antiproliferative p19(ARF)/p53 signaling.

3T3 Cells↗