The Impact of Lens Epithelium-Derived Growth Factor p75 Dimerization on Its Tethering Function.

The transcriptional co-activator lens epithelium-derived growth factor/p75 (LEDGF/p75) plays an important role in the biology of the cell and in several human diseases, including MLL-rearranged acute leukemia, autoimmunity, and HIV-1 infection. In both health and disease, LEDGF/p75 functions as a chromatin tether that interacts with proteins such as MLL1 and HIV-1 integrase via its integrase-binding domain (IBD) and with chromatin through its N-terminal PWWP domain. Recently, dimerization of LEDGF/p75 was shown, mediated by a network of electrostatic contacts between amino acids from the IBD and the C-terminal α6-helix. Here, we investigated the functional impact of LEDGF/p75 variants on the dimerization using biochemical and cellular interaction assays. The data demonstrate that the C-terminal α6-helix folds back in cis on the IBD of monomeric LEDGF/p75. We discovered that the presence of DNA stimulates LEDGF/p75 dimerization. LEDGF/p75 dimerization enhances binding to MLL1 but not to HIV-1 integrase, a finding that was observed in vitro and validated in cell culture. Whereas HIV-1 replication was not dependent on LEDGF/p75 dimerization, colony formation of MLLr-dependent human leukemic THP-1 cells was. In conclusion, our data indicate that intricate changes in the quaternary structure of LEDGF/p75 modulate its tethering function.

High-yield ligation-free assembly of DNA constructs with nucleosome positioning sequence repeats for single-molecule manipulation assays.

Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCRs: first ∼380 bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR to generate ∼kbp long double-stranded DNA constructs with multiple labels at the respective ends. To achieve high-force stability, we use dibenzocyclooctyne-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under load, with an average lifetime of 70 ± 3 h at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA, and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.

LEDGF/p75-mediated chemoresistance of mixed-lineage leukemia involves cell survival pathways and super enhancer activators.

MLL is an aggressive subtype of leukemia with a poor prognosis that mostly affects pediatric patients. MLL-rearranged fusion proteins (MLLr) induce aberrant target gene expression resulting in leukemogenesis. MLL and its fusions are tethered to chromatin by LEDGF/p75, a transcriptional co-activator that specifically recognizes H3K36me2/3. LEDGF/p75 is ubiquitously expressed and associated with regulation of gene expression, autoimmune responses, and HIV replication. LEDGF/p75 was proven to be essential for leukemogenesis in MLL. Apart from MLL, LEDGF/p75 has been linked to lung, breast, and prostate cancer. Intriguingly, LEDGF/p75 interacts with Med-1, which co-localizes with BRD4. Both are known as co-activators of super-enhancers. Here, we describe LEDGF/p75-dependent chemoresistance of MLLr cell lines. Investigation of the underlying mechanism revealed a role of LEDGF/p75 in the cell cycle and in survival pathways and showed that LEDGF/p75 protects against apoptosis during chemotherapy. Remarkably, LEDGF/p75 levels also affected expression of BRD4 and Med1. Altogether, our data suggest a role of LEDGF/p75 in cancer survival, stem cell renewal, and activation of nuclear super enhancers.

High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes.

Nucleosomes, the fundamental units of chromatin, regulate readout and expression of eukaryotic genomes. Single-molecule experiments have revealed force-induced nucleosome accessibility, but a high-resolution unwrapping landscape in the absence of external forces is currently lacking. Here, we introduce a high-throughput pipeline for the analysis of nucleosome conformations based on atomic force microscopy and automated, multi-parameter image analysis. Our data set of ∼10 000 nucleosomes reveals multiple unwrapping states corresponding to steps of 5 bp DNA. For canonical H3 nucleosomes, we observe that dissociation from one side impedes unwrapping from the other side, but in contrast to force-induced unwrapping, we find only a weak sequence-dependent asymmetry. Notably, centromeric CENP-A nucleosomes do not unwrap anti-cooperatively, in stark contrast to H3 nucleosomes. Finally, our results reconcile previous conflicting findings about the differences in height between H3 and CENP-A nucleosomes. We expect our approach to enable critical insights into epigenetic regulation of nucleosome structure and stability and to facilitate future high-throughput AFM studies that involve heterogeneous nucleoprotein complexes.

Molecular Mechanism of LEDGF/p75 Dimerization.

Dimerization of many eukaryotic transcription regulatory factors is critical for their function. Regulatory role of an epigenetic reader lens epithelium-derived growth factor/p75 (LEDGF/p75) requires at least two copies of this protein to overcome the nucleosome-induced barrier to transcription elongation. Moreover, various LEDGF/p75 binding partners are enriched for dimeric features, further underscoring the functional regulatory role of LEDGF/p75 dimerization. Here, we dissected the minimal dimerization region in the C-terminal part of LEDGF/p75 and, using paramagnetic NMR spectroscopy, identified the key molecular contacts that helped to refine the solution structure of the dimer. The LEDGF/p75 dimeric assembly is stabilized by domain swapping within the integrase binding domain and additional electrostatic "stapling" of the negatively charged α helix formed in the intrinsically disordered C-terminal region. We validated the dimerization mechanism using structure-inspired dimerization defective LEDGF/p75 variants and chemical crosslinking coupled to mass spectrometry. We also show how dimerization might affect the LEDGF/p75 interactome.

The free energy landscape of retroviral integration.

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo.

Free Energy Landscape and Dynamics of Supercoiled DNA by High-Speed Atomic Force Microscopy.

DNA supercoiling fundamentally constrains and regulates the storage and use of genetic information. While the equilibrium properties of supercoiled DNA are relatively well understood, the dynamics of supercoils are much harder to probe. Here we use atomic force microscopy (AFM) imaging to demonstrate that positively supercoiled DNA plasmids, in contrast to their negatively supercoiled counterparts, preserve their plectonemic geometry upon adsorption under conditions that allow for dynamics and equilibration on the surface. Our results are in quantitative agreement with a physical polymer model for supercoiled plasmids that takes into account the known mechanical properties and torque-induced melting of DNA. We directly probe supercoil dynamics using high-speed AFM imaging with subsecond time and ∼nanometer spatial resolution. From our recordings we quantify self-diffusion, branch point flexibility, and slithering dynamics and demonstrate that reconfiguration of molecular extensions is predominantly governed by the bending flexibility of plectoneme arms. We expect that our methodology can be an asset to probe protein-DNA interactions and topochemical reactions on physiological relevant DNA length and supercoiling scales by high-resolution AFM imaging.

DNA Translocation through Nanopores at Physiological Ionic Strengths Requires Precise Nanoscale Engineering.

Many important processes in biology involve the translocation of a biopolymer through a nanometer-scale pore. Moreover, the electrophoretic transport of DNA across nanopores is under intense investigation for single-molecule DNA sequencing and analysis. Here, we show that the precise patterning of the ClyA biological nanopore with positive charges is crucial to observe the electrophoretic translocation of DNA at physiological ionic strength. Surprisingly, the strongly electronegative 3.3 nm internal constriction of the nanopore did not require modifications. Further, DNA translocation could only be observed from the wide entry of the nanopore. Our results suggest that the engineered positive charges are important to align the DNA in order to overcome the entropic and electrostatic barriers for DNA translocation through the narrow constriction. Finally, the dependencies of nucleic acid translocations on the Debye length of the solution are consistent with a physical model where the capture of double-stranded DNA is diffusion-limited while the capture of single-stranded DNA is reaction-limited.