An Associative Memory Hamiltonian for DNA.

(a) Comparison of an atomistic representation and a coarse-grained representation of a B-DNA dodecamer (PDB ID: 1BNA [55]). (b) The AMH recapitulates the energy landscape of the DNA molecule using a series of Gaussian wells. The parameters are determined using the distances measured in the structural memories, while the energy of interactions, λ and the scale of structural fluctuations, σ, are tuned to reproduce experimental observables. (c) Schematic representation of the energy terms in the model: connectivity (springs), excluded-volume terms (dashed circle), and AMH terms encoding the double helix structure (red dashed lines). An AMH term is applied between any pair of particles inside the box of 4bp. The box is then slid along the DNA, and the procedure is repeated.</p

Nucleosome model and unwrapping analysis.

(a) A detailed representation of the nucleosome core particle (PDB ID: 1KX5 [59]) (left) and the corresponding coarse-grained WEChroM representation (right). In the left figure, the upper half of the nucleosome particle is shown as solid while the lower half is transparent for clear visualization. The DNA nucleotides in close contact with amino acids are defined as “contact DNA” and subject to the AMH potential modeling histones-to-DNA interactions (black circle). In our model, contact DNA interacts with the histone octamer’s center of mass (Ucenter, represented by the red arrow in the figure). Contact DNA also interacts with neighboring contact DNA (Uneighbor, represented by the black arrows inside the indigo dashed circle). (b) Free energy of partial DNA unwrapping for a system composed of the histone octamer plus 223-bp of DNA; WEChroM model (blue) and 3SPN-AICG model (orange). Simulation data shown for the 3SPN-AICG mode are extracted from ref. [38], figure 8. Examples of nucleosome configurations are shown for end-to-end distances of roughly 45 Å (left), 107 Å (right), 330 Å (outside) (c) Free energy of partial nucleosome unwrapping for a system composed of the histone octamer plus 147-bp of DNA; WEChroM model. (d) Nucleosome AMH energy of the partial nucleosome unwrapping for the 147-bp WEChroM model. Example nucleosome configurations are shown for end-to-end distances of 76 Å (left), 142 Å (middle), 221 Å (right).</p

DNA Persistence Length and Parametrization.

(a)(b) These figures show how persistence lengths depend on the strength of the energy interactions, λ, and the scale of structural fluctuations, σ. The bending persistence length is shown in panel (a), while the twisting persistence length is shown in panel (b). The σ is in reduced units (r.u.) which is 0.241 nm as explained in the Appendix A in S1 Document. Dots indicate the data points obtained by simulation; colored surface indicate their linear interpolation. The horizontal gray plane in (a) shows the experimentally determined bending persistence length of DNA, 150bp. This persistence length is set as the target for the optimization of the parameters. The red line indicates the intersection of the two surfaces, where our model meets the target persistence length value. Similarly, the gray box in (b) is the experimentally determined twisting persistence length, 75-360bp, our target in this case. (c) The figure shows the temperature dependence of bending persistence length. The four colored lines show results obtained using the WEChroM model. Black lines show experimental results published by Gray et al. [61] and Geggier et al. [62], respectively. As shown, the proposed model agrees well with the experimental observations for several couples of parameters (λ, σ).</p

WEChroM simulation and software description.

Detailed description of the reduced unit system, force field setup, and mechanical property analysis for WEChroM systems.ik (DOCX)</p

Supercoiling behavior of DNA minicircles.

(a) Commonly observed shapes of DNA minicircles (b) Writhe analysis of DNA topoisomers -4 to 4. Positively supercoiled minicircles relax torsional stress forming plectonemes, which manifest in increasing writhe and are visible in the DNA conformations in (a). Moderately negatively supercoiled minicircles also form plectonemes, this time resulting in negative writhe. Strongly negatively supercoiled minicircles do not appear to form plectonemes. (c) Negatively supercoiled DNA relaxes torsional strain through local melting of the DNA double helix. The figure shows an example structure of a topoisomer -2, defects are indicated by the black and red arrows. (d) Bending angle along DNA for the minicircle structure in (c). Local defects are clearly visible at index 304 and 193, once again indicated by the black and red arrows.</p

NLK c-Myb expression and clinicopathological parameters in 62 breast cancer specimens.

<p>Statistical analyses were performed by the Pearson χ2 test.</p>*<p>P<0.05 is considered significant.</p

NLK induces c-Myb degradation in MCF-7 cells.

<p>(A). MCF-7 cells were transfected with the pcDNA3.1-Myc-NLK or pcDNA3.1-Myc expression vectors, and 32 hours posttransfection, 26S proteasome inhibitor MG132 and DMSO as control were added for 16 hours. At 48 hours posttransfection, whole cell lysates were prepared, and proteins were analyzed by immunoblotting. The data were representative of three experiments. (B). pcDNA3.1-Myc-NLK or empty vector was transfected into MCF-7 cells using Lipofectamine 2000. Western blotting was performed using antibodies against c-myc, Bcl-2 and β-actin. The bar chart demonstrates the ratio of c-myc and Bcl-2 proteins to β-actin as determined by densitometry. The data are represented as the mean ± SEM (n = 3, *, #, P<0.01, compared with control).</p

Expression of NLK and c-Myb in human breast cancer.

<p>Paraffin-embedded tissue sections were stained with antibodies for NLK, c-Myb and Ki-67 and then counterstained with hematoxylin. Fig. A–C, E–G High NLK expression was observed in benign breast disease and breast carcinoma specimens (grade I), whereas c-Myb and Ki67 levels were low in the same specimens (SP×400). Fig. I-K High levels of c-Myb and Ki67 were observed in grade III tumor cells. In contrast, NLK expression was low. Fig. 1D, H, and L show negative controls for the benign breast disease and the breast carcinoma specimens. Experimental details are described in the Materials and Methods section. (M) Expression of NLK and c-Myb in eight representative paired samples of breast carcinomas and adjacent normal tissues. (N) Western blot analysis of endogenous NLK and c-Myb in a normal human breast epithelial cell line (HBL-100) and two human breast cancer cell lines (MDA-MB-231 (ER–) and MCF-7 (ER+)). β-actin was used as a loading control. The experiment was repeated at least three times. (O) Quantification indicated that MDA-MB-231 cells displayed the highest levels of NLK and the lowest levels of c-Myb among the two tumor cell lines. In contrast, the lowest NLK and highest c-Myb expression were observed in MCF-7 cells.</p

Effect of NLK overexpression on apoptosis of MCF-7 cells.

<p>(A). MCF-7 cells were transfected with pcDNA3.1-EGFP, pcDNA3.1-EGFP-NLK or nothing (control). Western blotting was performed using anti-active-caspase-3 and anti-β-actin antibodies. The data are presented as the mean±standard error of three experiments. (B). The effect of NLK overexpression on apoptosis was assessed by Annexin V and PI staining; cells that were double stained with Annexin V and PI were considered to be apoptotic (lower right). Flow cytometry confirmed the induction of apoptosis in MCF-7 cells, for which overexpression of NLK caused an increase in apoptosis. (C). MCF-7 cells were transfected with pcDNA3.1-EGFP, pcDNA3.1-EGFP-NLK or nothing (control). Forty-eight hours after transfection, DAPI-stained nuclei were visualized by fluorescence microscopy, and images were taken. The bar chart shows the ratio of necrotic to transfected cells. The data are represented as the mean ± SEM (n = 3, *, #, P<0.01, compared with control).</p

Graphic representation of the relationship between NLK and Ki-67 expression in breast carcinoma.

<p>Scatterplot of NLK versus Ki-67 levels with a regression line showing the correlation between the two levels using the Spearman’s correlation coefficient (<i>P<</i>0.05).</p