Local DNA Sequence Controls Asymmetry of DNA Unwrapping from Nucleosome Core Particles

DNA is tightly wrapped around histone proteins in nucleosome core particles (NCPs) yet must become accessible for processing in the cell. This accessibility, a key component of transcription regulation, is influenced by the properties of both the histone proteins and the DNA itself. Small angle x-ray scattering with contrast variation is used to examine how sequence variations affect DNA unwrapping from NCPs at different salt concentrations. Salt destabilizes NCPs, populating multiple unwrapped states as many possible unwrapping pathways are explored by the complexes. We apply coarse-grained Monte Carlo methods to generate realistic sequence-dependent unwrapped structures for the nucleosomal DNA with thermal variations. An ensemble optimization method is employed to determine the composition of the overall ensemble as electrostatic interactions are weakened. Interesting DNA-sequence-dependent differences are revealed in the unwrapping paths and equilibrium constants. These differences are correlated with specific features within the nucleic acid sequences

Revealing transient structures of nucleosomes as DNA unwinds

The modulation of DNA accessibility by nucleosomes is a fundamental mechanism of gene regulation in eukaryotes. The nucleosome core particle (NCP) consists of 147 bp of DNA wrapped around a symmetric octamer of histone proteins. The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications. Although the structure of the intact NCP has been studied by crystallography at atomic resolution, little is known about the structures of the partially unwrapped, transient intermediates relevant to nucleosome dynamics in processes such as transcription, DNA replication and repair. We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly. We measure the structures of unwrapping DNA and monitor protein dissociation from Xenopus laevis histones reconstituted with two model NCP positioning constructs: the Widom 601 sequence and the sea urchin 5S ribosomal gene. Both constructs reveal asymmetric release of DNA from disrupted histone cores, but display different patterns of protein dissociation. These kinetic intermediates may be biologically important substrates for gene regulation

Succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5′-Phosphate (SAICAR) Activates Pyruvate Kinase Isoform M2 (PKM2) in Its Dimeric Form

Human pyruvate kinase isoform M2 (PKM2) is a glycolytic enzyme isoform implicated in cancer. Malignant cancer cells have higher levels of dimeric PKM2, which is regarded as an inactive form of tetrameric pyruvate kinase. This perceived inactivity has fueled controversy about how the dimeric form of pyruvate kinase might contribute to cancer. Here we investigate enzymatic properties of PKM2^G415R, a variant derived from a cancer patient, which we show by size-exclusion chromatography and small-angle X-ray scattering to be a dimer that cannot form a tetramer in solution. Although PKM2^G415R binds to fructose 1,6-bisphosphate (FBP), unlike the wild type this PKM2 variant shows no activation by FBP. In contrast, PKM2^G415R is activated by succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5′-phosphate (SAICAR), an endogenous metabolite that we previously showed correlates with an increased level of cell proliferation and promotes protein kinase activity of PKM2. Our results demonstrate an important and unexpected enzymatic activity of the PKM2 dimer that likely has a key role in cancer progression

Motivational disturbances and effects of L-dopa administration in neurofibromatosis-1 model mice.

Children with neurofibromatosis type 1 (NF1) frequently have cognitive and behavioral deficits. Some of these deficits have been successfully modeled in Nf1 genetically-engineered mice that develop optic gliomas (Nf1 OPG mice). In the current study, we show that abnormal motivational influences affect the behavior of Nf1 OPG mice, particularly with regard to their response to novel environmental stimuli. For example, Nf1 OPG mice made fewer spontaneous alternations in a Y-maze and fewer arm entries relative to WT controls. However, analysis of normalized alternation data demonstrated that these differences were not due to a spatial working memory deficit. Other reported behavioral results (e.g., open-field test, below) suggest that differential responses to novelty and/or other motivational influences may be more important determinants of these kinds of behavior than simple differences in locomotor activity/spontaneous movements. Importantly, normal long-term depression was observed in hippocampal slices from Nf1 OPG mice. Results from elevated plus maze testing showed that differences in exploratory activity between Nf1 OPG and WT control mice may be dependent on the environmental context (e.g., threatening or non-threatening) under which exploration is being measured. Nf1 OPG mice also exhibited decreased exploratory hole poking in a novel holeboard and showed abnormal olfactory preferences, although L-dopa (50 mg/kg) administration resolved the abnormal olfactory preference behaviors. Nf1 OPG mice displayed an attenuated response to a novel open field in terms of decreased ambulatory activity and rearing but only during the first 10 min of the session. Importantly, Nf1 OPG mice demonstrated investigative rearing deficits with regard to a novel hanging object suspended on one side of the field which were not rescued by L-dopa administration. Collectively, our results provide new data important for evaluating therapeutic treatments aimed at ameliorating NF1-associated cognitive/behavioral deficits

The investigative rearing deficit in male <i>Nf1</i> OPG mice is not rescued by L-dopa administration.

<p>(A–C) No significant overall effects of Group were found in the third cohort of mice for ambulatory activity (A), rearing frequency (B), or time spent rearing (C) during a 30-min habituation trial in the open-field apparatus, which was conducted the day before the test session and did not include any drug/vehicle injections. (D) In contrast, planned comparisons showed that the CON+SAL mice spent significantly more time rearing to investigate the hanging object (ball) compared to the time spent rearing in the same area at the opposite end of the field (BALL vs OPP; *p = 0.017), while the <i>Nf1</i> OPG+SAL group did not show significantly different rearing times with regard to the ball versus the opposite area. The <i>Nf1</i> OPG+LDOPA mice reared for substantially longer times in investigating the ball versus the opposite area but these differences were not statistically significant (p = 0.064). (E) Similar results were found for the rearing frequency data where planned comparisons revealed that the CON+SAL mice spent significantly more time rearing to investigate the hanging ball versus the time spent rearing in the opposite area of the field (BALL vs OPP; *p = 0.010), while the <i>Nf1</i> OPG+SAL group did not. Again, the <i>Nf1</i> OPG+LDOPA mice showed a trend toward greater investigative rearing toward the ball versus the opposite area but these differences were not statistically significant (p = 0.063). (F) Although the <i>Nf1</i> OPG+SAL mice tended to spend less time rearing in general throughout the field compared to the CON+SAL and <i>Nf1</i> OPG+LDOPA groups, no statistically significant effects were observed for this variable. The male mice in cohort 3 were 3.5–4.5 months old and the sample size for each of the three groups was the same (n = 12).</p

<i>Nf1</i> OPG mice exhibit an abnormal response to novel environmental stimuli in an open field.

<p>(A) In the cohort 2 mice, locomotor and exploratory activity were quantified over a 30-min period in an open field. An rmANOVA and pair-wise comparisons revealed that <i>Nf1</i> OPG mice showed significantly (beyond Bonferroni correction: p<0.017) reduced total ambulations (whole body movements) compared to WT littermate controls but only during the first 10-min block of the open-field test (*p = 0.015), although large differences were also observed during Block 2 (^†p = 0.038) (Genotype effect: ^††p = 0.027; Genotype by Time interaction: ^††p = 0.033). (B) Similarly, <i>Nf1</i> OPG mice exhibited significantly decreased numbers of vertical rearings during only the first 10-min time block as well (*p = 0.010). (Genotype by Time interaction: ^†p = 0.043). (C) The <i>Nf1</i> OPG mice also spent significantly less total time rearing in the open field compared to control mice (*p = 0.005) during the first time block with large differences also being observed for the second time block (^†p = 0.046), (Genotype effect: F(1,16) = 5.00, **p = 0.040). (D) <i>Nf1</i> OPG mice displayed significantly reduced rearing to investigate an object (ball) suspended on one side of the open field apparatus relative to control mice (*p = 0.014) although the groups did not differ in the time spent rearing in the same area on the opposite side of the field. In addition, the WT control mice showed significantly increased rearing times to investigate the ball relative to the amount of rearing time displayed on the opposite side of the field (BALL vs OPP; **p = 0.0001), while no significant differences were found in terms of the rearing times between the two areas in <i>Nf1</i> OPG mice. (E) <i>Nf1</i> OPG mice spent significantly less time rearing in the open field in general (*p = 0.014) compared to the control group. (F) When rearing to investigate the hanging object and rearing displayed in the same area on the opposite side of the field were calculated as percentages of the total rearing time, the WT control mice, but not the <i>Nf1</i> OPG mice, showed significant differences in rearing to investigate the ball versus rearing on the opposite side of the field (BALL vs OPP; **p = 0.001). During the open-field testing, the cohort 2 groups were 5.5 months old and consisted of the same sample sizes and sex distribution (n = 10: M = 4; F = 6).</p