Genome-wide Analysis Reflects Novel 5-Hydroxymethylcytosines Implicated in Diabetic Nephropathy

Long-term complications of type 2 diabetes (T2D) are the major causes for T2D-related disability and mortality. Notably, diabetic nephropathy (DN) has become the most frequent cause of end-stage renal disease (ESRD) in most countries. Understanding epigenetic contributors to DN can provide novel insights into this complex disorder and lay the foundation for more effective monitoring tools and preventive interventions, critical for achieving the ultimate goal of improving patient care and reducing healthcare burden. We have used a selective chemical labeling technique (5hmC-Seal) to profile genome-wide distributions of 5-hydroxymethylcytosines (5hmC), a gene activation mark, in patient-derived circulating cell-free DNA (cfDNA). Differentially modified 5hmC genes were identified across T2D patients with DN (n = 12), T2D patients with non-DN vascular complications (non-DN) (n = 29), and T2D patients with no complications (controls) (n = 14). Specifically, differential 5hmC markers between DN and controls revealed relevant pathways such as NOD-like receptor signaling pathway and tyrosine metabolism. A ten-gene panel was shown to provide differential 5hmC patterns between controls and DN, as well as between controls and non-DN patients using a machine learning approach. The 5hmC profiles in cfDNA reflected novel DN-associated epigenetic modifications relevant to the disease pathogenesis of DN. Importantly, these findings in cfDNA, a convenient liquid biopsy, have the potential to be exploited as a clinically useful tool for predicting DN in high risk T2D patients. Keywords: diabetes, nephropathy, epigenetics, 5-hydroxymethylcytosine, cfDNA

Replication of Dengue Virus in K562-Megakaryocytes Induces Suppression in the Accumulation of Reactive Oxygen Species

Dengue virus can infect human megakaryocytes leading to decreased platelet biogenesis. In this article, we report a study of Dengue replication in human K562 cells undergoing PMA-induced differentiation into megakaryocytes. PMA-induced differentiation in these cells recapitulates steps of megakaryopoiesis including gene activation, expression of CD41/61 and CD61 platelet surface markers and accumulation of intracellular reactive oxygen species (ROS). Our results show differentiating megakaryocyte cells to support higher viral replication without any apparent increase in virus entry. Further, Dengue replication suppresses the accumulation of ROS in differentiating cells, probably by only augmenting the activity of the transcription factor NFE2L2 without influencing the expression of the coding gene. Interestingly pharmacological modulation of NFE2L2 activity showed a simultaneous but opposite effect on intracellular ROS and virus replication suggesting the former to have an inhibitory effect on the later. Also cells that differentiated while supporting intracellular virus replication showed reduced level of surface markers compared to uninfected differentiated cells.

The pioneer factor hypothesis is not necessary to explain ectopic liver gene activation

The Pioneer Factor Hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (nonPFs) that activate batteries of silent genes. The PFH predicts that ectopic gene activation requires the sequential activity of qualitatively different TFs. We tested the PFH by expressing the endodermal PF FOXA1 and nonPF HNF4A in K562 lymphoblast cells. While co-expression of FOXA1 and HNF4A activated a burst of endoderm-specific gene expression, we found no evidence for a functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and 'pioneered' for each other, although FOXA1 required fewer copies of its motif for binding. A subset of targets required both TFs, but the predominant mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results we hypothesize an alternative to the PFH where 'pioneer activity' depends not on categorically different TFs but rather on the affinity of interaction between TF and DNA.

BRD9 regulates interferon-stimulated genes during macrophage activation via cooperation with BET protein BRD4

Macrophages induce a number of inflammatory response genes in response to stimulation with microbial ligands. In response to endotoxin Lipid A, a gene-activation cascade of primary followed by secondary-response genes is induced. Epigenetic state is an important regulator of the kinetics, specificity, and mechanism of gene activation of these two classes. In particular, SWI/SNF chromatin-remodeling complexes are required for the induction of secondary-response genes, but not primary-response genes, which generally exhibit open chromatin. Here, we show that a recently discovered variant of the SWI/SNF complex, the noncanonical BAF complex (ncBAF), regulates secondary-response genes in the interferon (IFN) response pathway. Inhibition of bromodomain-containing protein 9 (BRD9), a subunit of the ncBAF complex, with BRD9 bromodomain inhibitors (BRD9i) or a degrader (dBRD9) led to reduction in a number of interferon-stimulated genes (ISGs) following stimulation with endotoxin lipid A. BRD9-dependent genes overlapped highly with a subset of genes differentially regulated by BET protein inhibition with JQ1 following endotoxin stimulation. We find that the BET protein BRD4 is cobound with BRD9 in unstimulated macrophages and corecruited upon stimulation to ISG promoters along with STAT1, STAT2, and IRF9, components of the ISGF3 complex activated downstream of IFN-alpha receptor stimulation. In the presence of BRD9i or dBRD9, STAT1-, STAT2-, and IRF9-binding is reduced, in some cases with reduced binding of BRD4. These results demonstrate a specific role for BRD9 and the ncBAF complex in ISG activation and identify an activity for BRD9 inhibitors and degraders in dampening endotoxin- and IFN-dependent gene expression.

Chitinase Chi 2 Positively Regulates Cucumber Resistance against Fusarium oxysporum f. sp. cucumerinum

Cucumber (Cucumis sativus L.) is an important vegetable crop worldwide, and Fusarium wilt (FW), caused by Fusarium oxysporum f. sp. cucumerinum (Foc), severely restricts cucumber growth and yield. Accumulating lines of evidence indicate that chitinases play important roles in attacking the invading fungal pathogens through catalyzing their cell wall degradation. Here, we identified the chitinase (Chi) genes in cucumber and further screened the FW-responsive genes via a comparative transcriptome analysis and found that six common genes were predominantly expressed in roots but also significantly upregulated after Foc infection. Expression verification further conformed that Chi2 and Chi14 were obviously induced by Foc as well as by hormone treatments, compared with the controls. The purified Chi2 and Chi14 proteins significantly affected the growth of Foc in vitro, compared with the controls. Knockdown of Chi2 in cucumber by virus-induced gene silencing (VIGS) increased susceptibility to FW, compared with the Chi14-silenced and control plants, and silencing of Chi2 drastically impaired gene activation in the jasmonic acid pathway, suggesting that the Chi2 gene might play positive roles in cucumber FW defense and, therefore, can provide a gene resource for developing cucumber-FW-resistance breeding programs.

A Novel Approach for Calculating Exact Forms of mRNA Distribution in Single-Cell Measurements

Gene transcription is a stochastic process manifested by fluctuations in mRNA copy numbers in individual isogenic cells. Together with mathematical models of stochastic transcription, the massive mRNA distribution data that can be used to quantify fluctuations in mRNA levels can be fitted by Pm(t), which is the probability of producing m mRNA molecules at time t in a single cell. Tremendous efforts have been made to derive analytical forms of Pm(t), which rely on solving infinite arrays of the master equations of models. However, current approaches focus on the steady-state (t→∞) or require several parameters to be zero or infinity. Here, we present an approach for calculating Pm(t) with time, where all parameters are positive and finite. Our approach was successfully implemented for the classical two-state model and the widely used three-state model and may be further developed for different models with constant kinetic rates of transcription. Furthermore, the direct computations of Pm(t) for the two-state model and three-state model showed that the different regulations of gene activation can generate discriminated dynamical bimodal features of mRNA distribution under the same kinetic rates and similar steady-state mRNA distribution.

Reb1, Cbf1, and Pho4 bias histone sliding and deposition away from their binding sites

In transcriptionally active genes, nucleosome positions in promoters are regulated by nucleosome displacing factors (NDFs) and chromatin remodeling enzymes. Depletion of NDFs or the RSC chromatin remodeler shrinks or abolishes the nucleosome depleted regions (NDRs) in promoters, which can suppress gene activation and result in cryptic transcription. Despite their vital cellular functions, how the action of chromatin remodelers may be directly affected by site-specific binding factors like NDFs is poorly understood. Here we demonstrate that two NDFs, Reb1 and Cbf1, can direct both Chd1 and RSC chromatin remodeling enzymes in vitro , stimulating repositioning of the histone core away from their binding sites. Interestingly, although the Pho4 transcription factor had a much weaker effect on nucleosome positioning, both NDFs and Pho4 were able to similarly redirect positioning of hexasomes. In chaperone-mediated nucleosome assembly assays, Reb1 but not Pho4 showed an ability to block deposition of the histone H3/H4 tetramer, but Reb1 did not block addition of the H2A/H2B dimer to hexasomes. Our in vitro results show that NDFs bias the action of remodelers to increase the length of the free DNA in the vicinity of their binding sites. These results suggest that NDFs could directly affect NDR architecture through chromatin remodelers.