Sequence Information Encoded in DNA that May Influence Long-Range Chromatin Structure Correlates with Human Chromosome Functions.

Little is known about the possible function of the bulk of the human genome. We have recently shown that long-range regular oscillation in the motif non-T, A/T, G (VWG) existing at ten-nucleotide multiples influences large-scale nucleosome array formation. In this work, we have determined the locations of all 100 kb regions that are predicted to form distinctive chromatin structures throughout each human chromosome (except Y). Using these data, we found that a significantly greater fraction of 300 kb sequences lacked annotated transcripts in genomic DNA regions ≥300 kb that contained nearly continuous chromatin organizing signals than in control regions. We also found a relationship between the meiotic recombination frequency and the presence of strong VWG chromatin organizing signals. Large (≥300 kb) genomic DNA regions having low average recombination frequency are enriched in chromatin organizing signals. As additional controls, we show using chromosome 1 that the VWG motif signals are not enriched in randomly selected DNA regions having the mean size of the recombination coldspots, and that non-VWG motif sets do not generate signals that are enriched in recombination coldspots. We also show that tandemly repeated alpha satellite DNA contains strong VWG signals for the formation of distinctive nucleosome arrays, consistent with the low recombination activity of centromeres. Our correlations cannot be explained simply by variations in the GC content. Our findings suggest that a specific set of periodic DNA motifs encoded in genomic DNA, which provide signals for chromatin organization, influence human chromosome function

Aspects of large-scale chromatin structures in mouse liver nuclei can be predicted from the DNA sequence

The large amount of non-coding DNA present in mammalian genomes suggests that some of it may play a structural or functional role. We provide evidence that it is possible to predict computationally, from the DNA sequence, loci in mouse liver nuclei that possess distinctive nucleosome arrays. We tested the hypothesis that a 100 kb region of DNA possessing a strong, in-phase, dinucleosome period oscillation in the motif period-10 non-T, A/T, G, should generate a nucleosome array with a nucleosome repeat that is one-half of the dinucleosome oscillation period value, as computed by Fourier analysis of the sequence. Ten loci with short repeats, that would be readily distinguishable from the pervasive bulk repeat, were predicted computationally and then tested experimentally. We estimated experimentally that less than 20% of the chromatin in mouse liver nuclei has a nucleosome repeat length that is 15 bp, or more, shorter than the bulk repeat value of 195 ± bp. All 10 computational predictions were confirmed experimentally with high statistical significance. Nucleosome repeats as short as 172 ± 5 bp were observed for the first time in mouse liver chromatin. These findings may be useful for identifying distinctive chromatin structures computationally from the DNA sequence

Long-range oscillation in a periodic DNA sequence motif may influence nucleosome array formation

We have experimentally examined the characteristics of nucleosome array formation in different regions of mouse liver chromatin, and have computationally analyzed the corresponding genomic DNA sequences. We have shown that the mouse adenosine deaminase (MADA) gene locus is packaged into an exceptionally regular nucleosome array with a shortened repeat, consistent with our computational prediction based on the DNA sequence. A survey of the mouse genome indicates that <10% of 70 kb windows possess a nucleosome-ordering signal, consisting of regular long-range oscillations in the period-10 triplet motif non-T, A/T, G (VWG), which is as strong as the signal in the MADA locus. A strong signal in the center of this locus, confirmed by in vitro chromatin assembly experiments, appears to cooperate with weaker, in-phase signals throughout the locus. In contrast, the mouse odorant receptor (MOR) locus, which lacks locus-wide signals, was representative of ∼40% of the mouse genomic DNA surveyed. Within this locus, nucleosome arrays were similar to those of bulk chromatin. Genomic DNA sequences which were computationally similar to MADA or MOR resulted in MADA- or MOR-like nucleosome ladders experimentally. Overall, we provide evidence that computationally predictable information in the DNA sequence may affect nucleosome array formation in animal tissue

Failure to normalize lymphopenia following trauma is associated with increased mortality, independent of the leukocytosis pattern

INTRODUCTION: Following trauma and systemic inflammatory response syndrome (SIRS), the typical response is an elevation of the total complete blood count (CBC) and a reduction of the lymphocyte count. This leukocytosis typically returns to normal within 48 hours. The persistence of a leukocytosis following trauma is associated with adverse outcomes. Although lymphocyte anergy and dysfunction following trauma is associated with increased risk for infection and sepsis, there is a paucity of data regarding the impact of a persistence of a low lymphocyte count in trauma patients. METHODS: This is a retrospective review of prospectively collected data from trauma patients collected over the 5 years of September 2003 to September 2008. Patients were included if the injury severity score (ISS) was >/=15, and they survived at least 3 days. Demographic data, mechanism and injury severity score, mortality, and length of stay were collected from the medical record. Laboratory values for the first 4 hospital days were collected. Leukocyte, neutrophil and lymphocyte counts were extracted from the daily complete blood count (CBC). Patients were then grouped based on response (elevation/depression) of each component of the CBC, and their return, or failure thereof, to normal. Proportional hazards regression with time-varying covariates as well as Kaplan-Meier curves were used to predict risk of death, time to death and time to healthy discharge based on fluctuations of the individual components of the CBC. RESULTS: There were 2448 patients admitted over the 5 years included in the analysis. When adjusting for age, gender and ISS the relative risk of death was elevated with a persistent leukocytosis (2.501 (95% CI = 1.477-4.235)) or failure to normalize lymphopenia (1.639 (95% CI = 10.17-2.643)) within the first 4 days following admission. Similar results were seen when Kaplan-Meier curves were created. Persistent lymphopenia was associated with shortest time to death. Paradoxically in survivors persistent lymphopenia was associated with the shortest time to discharge. CONCLUSIONS: Persistently abnormal CBC responses are associated with a higher mortality following trauma. This is the first report noting that a failure to normalize lymphopenia in severely injured patients is associated with significantly higher mortality

Identification and mechanistic basis of non-ACE2 blocking neutralizing antibodies from COVID-19 patients with deep RNA sequencing and molecular dynamics simulations

Variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continue to cause disease and impair the effectiveness of treatments. The therapeutic potential of convergent neutralizing antibodies (NAbs) from fully recovered patients has been explored in several early stages of novel drugs. Here, we identified initially elicited NAbs (Ig Heavy, Ig lambda, Ig kappa) in response to COVID-19 infection in patients admitted to the intensive care unit at a single center with deep RNA sequencing (&gt;100 million reads) of peripheral blood as a diagnostic tool for predicting the severity of the disease and as a means to pinpoint specific compensatory NAb treatments. Clinical data were prospectively collected at multiple time points during ICU admission, and amino acid sequences for the NAb CDR3 segments were identified. Patients who survived severe COVID-19 had significantly more of a Class 3 antibody (C135) to SARS-CoV-2 compared to non-survivors (15059.4 vs. 1412.7, p = 0.016). In addition to highlighting the utility of RNA sequencing in revealing unique NAb profiles in COVID-19 patients with different outcomes, we provided a physical basis for our findings via atomistic modeling combined with molecular dynamics simulations. We established the interactions of the Class 3 NAb C135 with the SARS-CoV-2 spike protein, proposing a mechanistic basis for inhibition via multiple conformations that can effectively prevent ACE2 from binding to the spike protein, despite C135 not directly blocking the ACE2 binding motif. Overall, we demonstrate that deep RNA sequencing combined with structural modeling offers the new potential to identify and understand novel therapeutic(s) NAbs in individuals lacking certain immune responses due to their poor endogenous production. Our results suggest a possible window of opportunity for administration of such NAbs when their full sequence becomes available. A method involving rapid deep RNA sequencing of patients infected with SARS-CoV-2 or its variants at the earliest infection time could help to develop personalized treatments using the identified specific NAbs

The VWG hypothesis: Predicting chromatin higher order structure from the DNA sequence

DNA has evolved in living organisms to function as chromatin, and the basic unit of chromatin is the nucleosome. It is known that the way nucleosomes are arrayed on the DNA molecule influences chromatin structure and function, and that nuclear factors, histone modifications and the replication machinery can determine where nucleosomes form. What is debated is whether there is any information in the DNA sequence that influences nucleosome array formation. This study addresses this question in three steps. First, experiments with a 4.5 kb fragment of the chicken ovalbumin gene established that the period-10 non-T, A/T, G (p-10 VWG) motif influences where nucleosomes form in chromatin reconstituted in vitro, tending to avoid p-10 VWG-poor regions. The nucleosome periodicity of this fragment was also predicted from the DNA sequence by Fourier analysis of the density of this motif. Small sequence alterations on the reconstituted chromatin of the gene confirmed that the observed periodicities were solely the result of the p-10 VWG signal. Second, our ability to predict nucleosome array periodicities solely from the DNA sequence was tested in mouse liver chromatin. Long-range array periodicities were predicted from the annotated sequence of the mouse genome. Probing the corresponding regions validated ten consecutive predictions (P = 10-7). Repeats as short as ∼20 bp less than the bulk repeat for mouse liver were found; the first time that such low values have been observed. Third, a plausible correlation was found between regions (∼100 kb range) of predicted ordered nucleosome arrays and regional recombination coldspots in the human genome (P = .01). Also, similar-size regions (∼300 kb range) of predicted ordered nucleosome arrays were found to contain significantly higher gene densities than in genome regions lacking these signals (P \u3c .01). This offers a functional explanation for why this motif has been reported to be overly abundant in the human genome. This is the first time that evidence is provided that the DNA sequence is implicated in containing information that may influence chromatin higher order structures. It is reasonable to expect that distinct chromatin structures play a role in gene regulation and, therefore, can also be a cause of miss-regulation and disease