Helen Hoffmeister

Polycystin-2 takes different routes to the somatic and ciliary plasma membrane

Polycystin-2 goes through the Golgi apparatus when going to the plasma membrane, but bypasses it en route to the ciliary membrane.

Mechanisms of in vivo binding site selection of the hematopoietic master transcription factor PU.1

The transcription factor PU.1 is crucial for the development of many hematopoietic lineages and its binding patterns significantly change during differentiation processes. However, the ‘rules’ for binding or not-binding of potential binding sites are only partially understood. To unveil basic characteristics of PU.1 binding site selection in different cell types, we studied the binding properties of PU.1 during human macrophage differentiation. Using in vivo and in vitro binding assays, as well as computational prediction, we show that PU.1 selects its binding sites primarily based on sequence affinity, which results in the frequent autonomous binding of high affinity sites in DNase I inaccessible regions (25–45% of all occupied sites). Increasing PU.1 concentrations and the availability of cooperative transcription factor interactions during lineage differentiation both decrease affinity thresholds for in vivo binding and fine-tune cell type-specific PU.1 binding, which seems to be largely independent of DNA methylation. Occupied sites were predominantly detected in active chromatin domains, which are characterized by higher densities of PU.1 recognition sites and neighboring motifs for cooperative transcription factors. Our study supports a model of PU.1 binding control that involves motif-binding affinity, PU.1 concentration, cooperativeness with neighboring transcription factor sites and chromatin domain accessibility, which likely applies to all PU.1 expressing cells.

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo.

Ca2+-dependent Conformational Changes in a C-terminal Cytosolic Domain of Polycystin-2

The PKD1 and PKD2 genes are the genes that are mutated in patients suffering from autosomal dominant polycystic kidney disease. The human PKD2 gene codes for a 968-amino acid long membrane protein called polycystin-2 that represents a cation channel whose activity can be regulated by Ca2+ ions. By CD, fluorescence, and NMR spectroscopy, we have studied a 117-amino acid-long fragment of the cytoplasmic domain of polycystin-2, polycystin-2-(680–796) that was proposed to contain a Ca2+-binding site. NMR structure determination reveals the existence of two Ca2+-binding sites in polycystin-2-(680–796) arranged in a typical and an atypical EF-hand motif. In the absence of Ca2+ the protein forms a dimer that is dissociated by Ca2+ binding. This dissociation may be related to the Ca2+ inactivation observed earlier. The calcium affinity of the protein was determined by fluorescence and NMR spectroscopy. At 293 K, the KD values for the high and low affinity sites are 55 μm and 179 μm, respectively.

Quantifying the interaction of the C-terminal regions of polycystin-2 and polycystin-1 attached to a lipid bilayer by means of QCM

The pkd1 and pkd2 genes encode for the proteins polycystin-1 (PC1) and polycystin-2 (PC2). These genes are mutated in patients diagnosed with autosomal dominant polycystic kidney disease. PC1 and PC2 interact via their C-terminal, cytosolic regions, which is an essential step in the regulation of cell proliferation and differentiation. Here, we developed an assay that allowed us to quantitatively monitor the interaction of the C-terminal region of PC1 (cPC1) with that of PC2 (cPC2) to be able to answer the question of how Ca(2+) influences the PC1/PC2 complex formation. By means of the quartz crystal microbalance (QCM) technique, we were able to determine binding affinities and kinetic constants of the cPC1/cPC2 interaction using a model based on the scaled particle theory. The results suggest that cPC2 forms trimers in solution in the absence of Ca(2+), which bind in a one step process to cPC1.

The human polycystin-2 protein represents an integral membrane protein with six membrane-spanning domains and intracellular N- and C-termini.

PKD2 is one of the two genes mutated in ADPKD (autosomal-dominant polycystic kidney disease). The protein product of PKD2, polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP (transient receptor potential) family of cation channels suggest that polycystin-2 contains six transmembrane domains and that both the N- and C-termini extend into the cytoplasm. However, no experimental evidence for this model has so far been provided. To determine the orientation of the different loops of polycystin-2, we truncated polycystin-2 within the predicted loops 1-5 and tagged the constructs at the C-terminus with an HA (haemagglutinin) epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, whereas loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the N- and C-termini of polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when polycystin-2 was truncated in loops 1, 3 and 5, whereas no protected fragments could be detected when polycystin-2 was truncated in loops 2 and 4. The results of the present study therefore provide the first experimental evidence for the topological orientation of polycystin-2.

CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality

Abstract CHD3 and CHD4 (Chromodomain Helicase DNA binding protein), two highly similar representatives of the Mi-2 subfamily of SF2 helicases, are coexpressed in many cell lines and tissues and have been reported to act as the motor subunit of the NuRD complex (nucleosome remodeling and deacetylase activities). Besides CHD proteins, NuRD contains several repressors like HDAC1/2, MTA2/3 and MBD2/3, arguing for a role as a transcriptional repressor. However, the subunit composition varies among cell- and tissue types and physiological conditions. In particular, it is unclear if CHD3 and CHD4 coexist in the same NuRD complex or whether they form distinct NuRD complexes with specific functions. We mapped the CHD composition of NuRD complexes in mammalian cells and discovered that they are isoform-specific, containing either the monomeric CHD3 or CHD4 ATPase. Both types of complexes exhibit similar intranuclear mobility, interact with HP1 and rapidly accumulate at UV-induced DNA repair sites. But, CHD3 and CHD4 exhibit distinct nuclear localization patterns in unperturbed cells, revealing a subset of specific target genes. Furthermore, CHD3 and CHD4 differ in their nucleosome remodeling and positioning behaviour in vitro. The proteins form distinct CHD3- and CHD4-NuRD complexes that do not only repress, but can just as well activate gene transcription of overlapping and specific target genes.

NMR-assignments of a cytosolic domain of the C-terminus of polycystin-2

Mutations in the PKD2 gene lead to the development of polycystic kidney disease (PKD). The PKD2 gene codes for polycystin-2, a cation channel with unknown function. The cytoplasmic, C-terminal domain interacts with a large number of proteins including mDia1, α-actinin, PIGEA-14, troponin, and tropomyosin. The C-terminal fragment polycystin-2 (680–796) consisting of 117 amino acids contains a putative calcium binding EF-hand. It was produced in Escherichia coli and enriched uniformly with 13C and 15N. The backbone and side chain resonances were assigned by multidimensional NMR methods, the obtained chemical shifts are typical for a partially folded protein. The chemical shifts obtained are in line with the existence of two paired helix-loop-helix (HLH) motifs.

Phosphorylation of C-terminal polycystin-2 influences the interaction with PIGEA14: A QCM study based on solid supported membranes

Polycystin-2 (PC2) trafficking has been proposed to be a result of the interaction of PIGEA14 with PC2 as a function of the phosphorylation state of PC2. Here, we investigated the interaction of PIGEA14 with the C-terminal part of polycystin-2 wild type (cPC2wt) and the pseudophosphorylated mutant (cPC2S812D) to first, quantify the binding affinity between cPC2 and PIGEA14 and second, to elucidate the influence of PC2 phosphorylation on PIGEA14 binding. Solid supported membranes composed of octanethiol/1,2-dioleoyl-sn-glycero-3-phosphocholine doped with the receptor lipid DOGS-NTA-Ni were used to attach PIGEA14 to the membrane via its hexahistidine tag. By means of the quartz crystal microbalance technique, binding affinities as well as kinetic constants of the interaction were extracted in a label-free manner by applying the scaled particle theory. The results show that the dissociation constant of cPC2 to PIGEA14 is in the 10 nM regime providing strong evidence of a very specific interaction of cPC2 with PIGEA14. The interaction of cPC2wt is twofold larger than that of cPC2S812D. The moderate higher binding affinity of cPC2wt to PIGEA14 is discussed in light of PC2 trafficking to the plasma membrane.

Tumour-associated missense mutations in the dMi-2 ATPase alters nucleosome remodelling properties in a mutation-specific manner

ATP-dependent chromatin remodellers are mutated in more than 20% of human cancers. The consequences of these mutations on enzyme function are poorly understood. Here, we characterise the effects of CHD4 mutations identified in endometrial carcinoma on the remodelling properties of dMi-2, the highly conserved Drosophila homologue of CHD4. Mutations from different patients have surprisingly diverse defects on nucleosome binding, ATPase activity and nucleosome remodelling. Unexpectedly, we identify both mutations that decrease and increase the enzyme activity. Our results define the chromodomains and a novel regulatory region as essential for nucleosome remodelling. Genetic experiments in Drosophila demonstrate that expression of cancer-derived dMi-2 mutants misregulates differentiation of epithelial wing structures and produces phenotypes that correlate with their nucleosome remodelling properties. Our results help to define the defects of CHD4 in cancer at the mechanistic level and provide the basis for the development of molecular approaches aimed at restoring their activity.ATP-dependent chromatin remodelers are often found mutated in human cancers. Here, the authors characterize the nucleosome remodelling properties of cancer-associated mutants of the Drosophila Chd4 homolog dMi-2.