Conformational switching of Arp5 subunit differentially regulates INO80 chromatin remodeling.

The INO80 chromatin remodeler is a versatile enzyme capable of several functions, including spacing nucleosomes equal distances apart, precise positioning of nucleosomes based on DNA shape/sequence and exchanging histone dimers. Within INO80, the Arp5 subunit plays a central role in INO80 remodeling, evidenced by its interactions with the histone octamer, nucleosomal and extranucleosomal DNA, and its necessity in linking INO80's ATPase activity to nucleosome movement. Our investigation reveals that the grappler domain of Arp5 interacts with the acidic pocket of nucleosomes through two distinct mechanisms: an arginine anchor or a hydrophobic/acidic patch. These two modes of binding serve distinct functions within INO80 as shown in vivo by mutations in these regions resulting in varying phenotypes and in vitro by diverse effects on nucleosome mobilization. Our findings suggest that the hydrophobic/acidic patch of Arp5 is likely important for dimer exchange by INO80, while the arginine anchor is crucial for mobilizing nucleosomes.

Unravelling HDAC Selectivity for Erasing Acetyl Mark on Lys-5 of Histone H2B.

The reversible acetylation of specific Lysine residues of histones plays crucial role in the epigenetic regulation of chromatin activity. Importantly, perturbations of acetylation-deacetylation dynamics have important implications for cancer and neurological disorders. There are 18 human HDACs including sirtuins. The site-selective acetyl eraser specificity of HDACs is poorly defined. Deciphering the site specificity preference of HDACs from a gamut of lysine in histones may be critical for targeted inhibitor development and delineation of regulatory mechanisms associated with chromatin. Here, we have interrogated the propensity of HDACs to erase acetyl mark at Lys-5 of H2B namely, H2BK5Ac engineered by a peptide ligation reaction catalyzed by transpeptidase sortase. HDACs and Sirtuins were individually over-expressed in HEK293 cells and the deacetylation propensity of respective cell lysates was evaluated against H2BK5Ac for initial screening of potential acetyl erasers. This screen indicated HDAC1 as the prime eraser of acetyl mark in H2BK5Ac. The propensity of HDAC1 to erase acetyl mark of H2BK5Ac was further probed using semisynthetic designer nucleosomes with whole cell lysates, recombinant enzyme, and specific inhibitors. Consistent with the above data, siRNA knockdown of HDAC1 and closely related HDAC3 in HEK293 cells prevented the loss of H2BK5 acetylation.

New insights into the mechanism and DNA-sequence specificity of INO80 chromatin remodeling.

The INO80 complex stood out in a large family of ATP-dependent chromatin remodelers because of its ATPase domain binding and translocating on DNA at the edge of nucleosomes, rather than at two helical turns from the center of DNA that is wrapped around nucleosomes. This unique property of INO80 was thought to account for its singular role in nucleosome placement at gene promoters in a DNA-sequence dependent manner that is crucial for transcription regulation. Now, we uncover INO80 functions differently than previously thought with its ATPase domain translocating on DNA close to the center of nucleosomes, like other remodelers. Our discovery also reveals the physical properties of the first ~36 bp of DNA on the entry side of nucleosomes is the main determinant for the DNA specificity of INO80 rather than the properties of the extranucleosomal DNA. The DNA sequence sensitive step of INO80 is after DNA is displaced from the histone octamer on the entry side of nucleosomes and 20 bp of DNA are moved out the exit side. We find the ATPase domain and Arp5 subunit of INO80 are likely involved in INO80's DNA specificity and the mechanism of INO80 remodeling is substantially different than originally proposed.

Analysis of gut bacteriome of in utero arsenic-exposed mice using 16S rRNA-based metagenomic approach.

Introduction: Approximately 200 million people worldwide are affected by arsenic toxicity emanating from the consumption of drinking water containing inorganic arsenic above the prescribed maximum contaminant level. The current investigation deals with the role of prenatal arsenic exposure in modulating the gut microbial community and functional pathways of the host. Method: 16S rRNA-based next-generation sequencing was carried out to understand the effects of in utero 0.04 mg/kg (LD) and 0.4 mg/kg (HD) of arsenic exposure. This was carried out from gestational day 15 (GD-15) until the birth of pups to understand the alterations in bacterial diversity. Results: The study focused on gestational exposure to arsenic and the altered gut microbial community at phyla and genus levels, along with diversity indices. A significant decrease in firmicutes was observed in the gut microbiome of mice treated with arsenic. Functional analysis revealed that a shift in genes involved in crucial pathways such as insulin signaling and non-alcoholic fatty liver disease pathways may lead to metabolic diseases in the host. Discussion: The present investigation may hypothesize that in utero arsenic exposure can perturb the gut bacterial composition significantly as well as the functional pathways of the gestationally treated pups. This research paves the way to further investigate the probable mechanistic insights in the field of maternal exposure environments, which may play a key role in epigenetic modulations in developing various disease endpoints in the progeny.

A study on bisphenol S induced nephrotoxicity and assessment of altered downstream kidney metabolites using gas chromatography-mass spectrometry based metabolomics.

The global use of bisphenol S (BPS) has now been significantly increased for commensurate utilization as a substitute for BPA for its regulatory concerns. Though, previous reports indicated that BPS been also appeared as a toxic congener comparable to BPA. In the present study, we determined nephrotoxicity condition induced due to BPS exposure. Results indicated that BPS significantly promoted histopathological disturbance in the kidney, and altered the levels of biomarkers of kidney damage in serum and urine samples of Wistar rats. It is also indicated that BPS altered the expression of kidney damage biomarkers associated with glomerular and tubular injury. Additionally, we determined the perturbation of kidney metabolites in the underlying pathophysiological response of kidney injury due to BPS exposure. Gas chromatography-mass spectrometry based untargeted metabolomics exhibited 20 significantly perturbed metabolites. Moreover, metabolic pathway analysis revealed significant disturbance in the TCA cycle and pyruvate metabolism pathways.

Prenatal exposure to arsenic promotes sterile inflammation through the Polycomb repressive element EZH2 and accelerates skin tumorigenesis in mouse.

Prenatal and postnatal life stress could be a potent programmer of phenotype or disease state of an individual in the later life. Prenatal arsenic exposure has been shown to promote developmental defects, low birth weight, immunotoxicity and is associated with various cancers including skin cancer in adulthood. To investigate the association between prenatal arsenic exposure and adult life skin carcinogenesis, we used a two-stage cutaneous carcinogenesis model in which BALB/c mice were prenatally exposed to 0.04 mg/kg and 0.4 mg/kg arsenic (As). Exposure to arsenic was sufficient to shorten the tumor latency period and promote epidermal hyperplasia in the offspring upon challenge with dimethylbenz[a]/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA). The levels of inflammatory and tissue microenvironment remodeling factors such as IL-1α and TNF-α were persistently elevated in the skin, and their inhibition through diacerein led to a significant decrease in the tumor response, suggesting their role in tumorigenesis. While there was overexpression of multiple epigenetic regulators at tissue level, we found decreased enrichment of Polycomb repressive complex 2 (PRC2) member EZH2 and H3K27me3 mark at the upstream of the affected inflammatory genes. The higher expression of the inflammatory genes suggests the gene specific selective nature of EZH2 repression which was also associated with increased binding of the activator KDM6a (demethylase). Further, arsenic conditioned basal keratinocytes cells (BKCs) showed increased migration and proliferation along with higher expression of tumor associated cytokines. Inhibition of EZH2 in the BKCs lead to their further upregulation suggesting that BKCs might be the potential cell type for the interaction of EZH2 and inflammatory cytokines. The present study provides new evidence for the role of PRC2 group regulators in inflammatory conditioning and development of skin cancer in offspring prenatally exposed to arsenic.

Prenatal arsenic exposure induces immunometabolic alteration and renal injury in rats.

Arsenic (As) exposure is progressively associated with chronic kidney disease (CKD), a leading public health concern present worldwide. The adverse effect of As exposure on the kidneys of people living in As endemic areas have not been extensively studied. Furthermore, the impact of only prenatal exposure to As on the progression of CKD also has not been fully characterized. In the present study, we examined the effect of prenatal exposure to low doses of As 0.04 and 0.4 mg/kg body weight (0.04 and 0.4 ppm, respectively) on the progression of CKD in male offspring using a Wistar rat model. Interestingly, only prenatal As exposure was sufficient to elevate the expression of profibrotic (TGF-ß1) and proinflammatory (IL-1α, MIP-2α, RANTES, and TNF-α) cytokines at 2-day, 12- and 38-week time points in the exposed progeny. Further, alteration in adipogenic factors (ghrelin, leptin, and glucagon) was also observed in 12- and 38-week old male offspring prenatally exposed to As. An altered level of these factors coincides with impaired glucose metabolism and homeostasis accompanied by progressive kidney damage. We observed a significant increase in the deposition of extracellular matrix components and glomerular and tubular damage in the kidneys of 38-week-old male offspring prenatally exposed to As. Furthermore, the overexpression of TGF-ß1 in kidneys corresponds with hypermethylation of the TGF-ß1 gene-body, indicating a possible involvement of prenatal As exposure-driven epigenetic modulations of TGF-ß1 expression. Our study provides evidence that prenatal As exposure to males can adversely affect the immunometabolism of offspring which can promote kidney damage later in life.

Sortase-click strategy for defined protein conjugation on a heptavalent cyclodextrin scaffold.

Multivalent proteins or protein dendrimers are useful for clinical and biotechnological applications. However, assembly of chemically defined protein dendrimers is a challenging endeavor. In the past, majority of protein dendrimers have been developed on branched lysine scaffolds and are usually limited to a valency of two to four. The naturally occurring cyclodextrin (CD) scaffold composed of 6-8 glucose units offers the possibility of expanding the valency. Here we have adapted a chemoenzymatic-click strategy for displaying heptavalent peptides and large proteins on the ß-cyclodextrin (ß-CD) scaffold. We demonstrate that recombinant proteins (engineered with a LPXTG pentapeptide motif at the carboxy terminus), labeled with an alkyne moiety by sortase-mediated ligation, can be easily clicked on to the azide-derivatized ß-cyclodextrin through the Huisgen cycloaddition reaction yielding a well-defined heptavalent display of proteins.