Epigenetic dysregulation from chromosomal transit in micronuclei

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers [1-4], yet whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei [5, 6], and subsequent micronuclear envelope rupture [7] profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice as well as cancer and non-transformed cells. Some of the changes to histone PTMs occur due to micronuclear envelope rupture whereas others are inherited from mitotic abnormalities prior to micronucleus formation. Using orthogonal techniques, we show that micronuclei exhibit extensive differences in chromatin accessibility with a strong positional bias between promoters and distal or intergenic regions. Finally, we show that inducing CIN engenders widespread epigenetic dysregulation and that chromosomes which transit in micronuclei experience durable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, in addition to genomic copy number alterations, CIN can serve as a vehicle for epigenetic reprogramming and heterogeneity in cancer.

Integration of adeno-associated virus (AAV) into the genomes of most Thai and Mongolian liver cancer patients does not induce oncogenesis

Background Engineered versions of adeno-associated virus (AAV) are commonly used in gene therapy but evidence revealing a potential oncogenic role of natural AAV in hepatocellular carcinoma (HCC) has raised concerns. The frequency of potentially oncogenic integrations has been reported in only a few populations. AAV infection and host genome integration in another type of liver cancer, cholangiocarcinoma (CCA), has been studied only in one cohort. All reported oncogenic AAV integrations in HCC come from strains resembling the fully sequenced AAV2 and partly sequenced AAV13. When AAV integration occurs, only a fragment of the AAV genome is detectable in later DNA or RNA sequencing. The integrated fragment is typically from the 3’ end of the AAV genome, and this positional bias has been only partly explained. Three research groups searched for evidence of AAV integration in HCC RNAseq samples in the Cancer Genome Atlas (TCGA) but reported conflicting results. Results We collected and analyzed whole transcriptome and viral capture DNA sequencing in paired tumor and non-tumor samples from two liver cancer Asian cohorts from Thailand (N = 147, 47 HCC and 100 intrahepatic cholangiocarcinoma (iCCA)) and Mongolia (N = 70, all HCC). We found only one HCC patient with a potentially oncogenic integration of AAV, in contrast to higher frequency reported in European patients. There were no oncogenic AAV integrations in iCCA patients. AAV genomic segments are present preferentially in the non-tumor samples of Thai patients. By analyzing the AAV genome positions of oncogenic and non-oncogenic integrated fragments, we found that almost all the putative oncogenic integrations overlap the X gene, which is present and functional only in the strain AAV2 among all fully sequenced strains. This gene content difference could explain why putative oncogenic integrations from other AAV strains have not been reported. We resolved the discrepancies in previous analyses of AAV presence in TCGA HCC samples and extended it to CCA. There are 12 TCGA samples with an AAV segment and none are in Asian patients. AAV segments are present in preferentially in TCGA non-tumor samples, like what we observed in the Thai patients. Conclusions Our findings suggest a minimal AAV risk of hepatocarcinogenesis in Asian liver cancer patients. The partial genome presence and positional bias of AAV integrations into the human genome has complicated analysis of possible roles of AAV in liver cancer.

Obligate movements of an active site–linked surface domain control RNA polymerase elongation and pausing via a Phe pocket anchor

The catalytic trigger loop (TL) in RNA polymerase (RNAP) alternates between unstructured and helical hairpin conformations to admit and then contact the NTP substrate during transcription. In many bacterial lineages, the TL is interrupted by insertions of two to five surface-exposed, sandwich-barrel hybrid motifs (SBHMs) of poorly understood function. The 188-amino acid, two-SBHM insertion in Escherichia coli RNAP, called SI3, occupies different locations in elongating, NTP-bound, and paused transcription complexes, but its dynamics during active transcription and pausing are undefined. Here, we report the design, optimization, and use of a Cys-triplet reporter to measure the positional bias of SI3 in different transcription complexes and to determine the effect of restricting SI3 movement on nucleotide addition and pausing. We describe the use of H2O2 as a superior oxidant for RNAP disulfide reporters. NTP binding biases SI3 toward the closed conformation, whereas transcriptional pausing biases SI3 toward a swiveled position that inhibits TL folding. We find that SI3 must change location in every round of nucleotide addition and that restricting its movements inhibits both transcript elongation and pausing. These dynamics are modulated by a crucial Phe pocket formed by the junction of the two SBHM domains. This SI3 Phe pocket captures a Phe residue in the RNAP jaw when the TL unfolds, explaining the similar phenotypes of alterations in the jaw and SI3. Our findings establish that SI3 functions by modulating TL folding to aid transcriptional regulation and to reset secondary channel trafficking in every round of nucleotide addition.

Detecting Geo-Positional Bias in Imagery Collected Using Small UASs

Statistical methods for detecting bias in global positioning system (<small>GPS</small>) error are presented and applied to imagery collected using three common unmanned aerial systems (<small>UASs</small>). Imagery processed without ground control points (<small>GCPs</small>) had horizontal errors of 1.0–2.5 m; however, the errors had unequal variances, significant directional bias, and did not conform to the expected statistical distribution and so should be considered unreliable. When <small>GCPs</small>were used, horizontal errors decreased to less than 5 cm, and the errors had equal variances, directional uniformity, and they conformed to the expected distribution. The analysis identified a longitudinal bias in some of the reference data, which were subsequently excluded from the analysis. Had these data been retained, the estimates of positional accuracy would have been unreliable and inaccurate. These results strongly suggest that examining <small>GPS</small> data for bias should be a much more common practice.

Language transfer and positional bias in English stress

This paper shows that L1 transfer may not be effectively maintained in the interlanguage due to confounding factors in the L2. When two factors, A and B, are correlated in the L2, second language learners may only acquire B, even if A is present in the L1. Transfer may not be effective because B, being more robust in the input, conceals A. Native speakers, on the other hand, generalize A in spite of B. The variables in question are weight-sensitivity (A) and positional bias (B) in English, both of which can predict the location of stress in the language. I show that two seemingly target-like groups of second language learners of English (speakers of Mandarin and speakers Portuguese) fail to accurately generalize weight-sensitivity in the language, and instead display response patterns which are predictable given the existing positional bias in English stress.

On the NF-Y regulome as in ENCODE (2019)

NF-Y is a trimeric Transcription Factor -TF- which binds with high selectivity to the conserved CCAAT element. Individual ChIP-seq analysis as well as ENCODE have progressively identified locations shared by other TFs. Here, we have analyzed data introduced by ENCODE over the last five years in K562, HeLa-S3 and GM12878, including several chromatin features, as well RNA-seq profiling of HeLa cells after NF-Y inactivation. We double the number of sequence-specific TFs and co-factors reported. We catalogue them in 4 classes based on co-association criteria, infer target genes categorizations, identify positional bias of binding sites and gene expression changes. Larger and novel co-associations emerge, specifically concerning subunits of repressive complexes as well as RNA-binding proteins. On the one hand, these data better define NF-Y association with single members of major classes of TFs, on the other, they suggest that it might have a wider role in the control of mRNA production.

Language transfer and positional bias in English stress

This article shows that first language (L1) transfer may not be effectively maintained in the interlanguage due to confounding factors in the second language (L2). When two factors, [Formula: see text] and [Formula: see text], are correlated in the L2, second language learners may only acquire [Formula: see text], even if [Formula: see text] is present in the L1. Transfer may not be effective because [Formula: see text], being more robust in the input, conceals [Formula: see text]. Native speakers, on the other hand, generalize [Formula: see text] in spite of [Formula: see text]. The variables in question are weight-sensitivity ([Formula: see text]) and positional bias ([Formula: see text]) in English, both of which can predict the location of stress in the language. I show that two seemingly target-like groups of second language learners of English (speakers of Mandarin and speakers Portuguese) fail to accurately generalize weight-sensitivity in the language, and instead display response patterns which are predictable given the existing positional bias in English stress.

Positional specificity of different transcription factor classes within enhancers

Gene expression is controlled by sequence-specific transcription factors (TFs), which bind to regulatory sequences in DNA. TF binding occurs in nucleosome-depleted regions of DNA (NDRs), which generally encompass regions with lengths similar to those protected by nucleosomes. However, less is known about where within these regions specific TFs tend to be found. Here, we characterize the positional bias of inferred binding sites for 103 TFs within ∼500,000 NDRs across 47 cell types. We find that distinct classes of TFs display different binding preferences: Some tend to have binding sites toward the edges, some toward the center, and some at other positions within the NDR. These patterns are highly consistent across cell types, suggesting that they may reflect TF-specific intrinsic structural or functional characteristics. In particular, TF classes with binding sites at NDR edges are enriched for those known to interact with histones and chromatin remodelers, whereas TFs with central enrichment interact with other TFs and cofactors such as p300. Our results suggest distinct regiospecific binding patterns and functions of TF classes within enhancers.

Golgi stabilization, not its front-rear bias, is associated with EMT-enhanced fibrillar migration

ABSTRACTEpithelial-to-mesenchymal transition (EMT) and maturation of collagen fibrils in the tumor microenvironment play a significant role in cancer cell invasion and metastasis. Confinement along fiber-like tracks enhances cell migration. To what extent and in what manner EMT further promotes migration in a microenvironment already conducive to migration is poorly understood. Here, we show that TGFβ-mediated EMT significantly enhances migration on fiber-like micropatterned tracks of collagen, doubling migration speed and quadrupling persistence relative to untreated mammary epithelial cells. Thus, cell-intrinsic EMT and extrinsic fibrillar tracks have non-redundant effects on motility. To better understand EMT-enhanced fibrillar migration, we investigated the regulation of Golgi positioning, which is involved in front-rear polarization and persistent cell migration. Confinement along fiber-like tracks has been reported to favor posterior Golgi positioning, whereas anterior positioning is observed during 2d wound healing. While EMT also regulates cell polarity, little is known about its effect on Golgi positioning. Here, we show that EMT induces a 2:1 rearward bias in Golgi positioning; however, positional bias explains less than 5% of single-cell variability in migration speed and persistence. Meanwhile, EMT significantly stabilizes Golgi positioning. Cells that enhance migration in response to TGFβ maintain Golgi position for 3-4 fold longer than untreated counterparts, irrespective of whether the Golgi is ahead or behind the nucleus. In fact, 35% of cells that respond to TGFβ exhibit a fully-committed Golgi phenotype with the organelle either in the anterior or posterior position for over 90% of the time. Furthermore, single-cell differences in Golgi stability capture up to 30% of variations in migration speed and persistence. These results lead us to propose that the Golgi is part of a core physical scaffold that distributes cell-generated forces necessary for migration. A stable scaffold more consistently, and therefore more productively, distributes forces over time, leading to efficient migration.