Abstract P009: Differential Redox Signaling In Urine Derived Renal Proximal Tubule Cells Isolated From Inverse Salt Sensitive Vs Salt Resistant Clinical Study Participants

Increased morbidity and mortality occurs in some individuals consuming low sodium diets. We demonstrated that single nucleotide polymorphisms (SNPs) in the dopamine type 2 receptor (D2R) as well as lower expression of cellular and plasma membrane D2R are associated with inverse salt sensitivity (ISS, a paradoxical increase in blood pressure while on the low sodium diet). Urine derived human renal proximal tubule cells (RPTC) were cultured from 3 separate ISS and 3 salt resistant (SR, resists blood pressure change on both low and high sodium diets) participants. D2R reduces cellular redox signaling so we hypothesized that an aberrant redox state occurs in ISS and SR RPTC exposed to different NaCl concentrations. RPTC incubated in 90 mM NaCl (low salt - LS) for 2 hours in both ISS and SR RPTCs had a significant reduction of monochlorobimane (mBCl - reduced glutathione indicator) compared to 140 mM NaCl (normal salt -NS) (SR -7.1± 2.1% and ISS -9.7 ± 1.6% vs NS, n=12, p<0.05, t-test). Only ISS RPTC had a significant increase in mBCl fluorescence in 190 mM NaCl (ISS +7.0 ± 2.5% vs NS, n=12, p<0.05, t-test) and mBCl fluorescence was not affected by incubation with losartan (angiotensin type 1 receptor inhibitor). 3-Aminobenzamide (3AB, 1 mM) inhibits Parp1 (poly[ADP-ribose] synthase), a protein necessary for the transcriptional repressor CTCF insulator function. 3AB reversed the lower mBCl fluorescence only in the SR RPTC, suggesting that reducing CTCF insulator function only affects D2R wild type cells. mBCl fluorescence in ISS RPTC showed a greater reduction by LOS vs SR RPTC (ISS -49.3 ± 1.5% vs SR -37.3 ± 4.8%, n=12, p<0.05, t-test), which was blocked by nemonapride (NEM, 1 uM, D2R antagonist) and further reduced the mBCl fluorescence only in the SR RPTC. Whether these differences in salt sensitivities are due to differential effects of CTCF insulator function at the homozygous variant or wild type D2R warrants further studies.

Interaction between Two Transcriptional Factors CTCF and YB-1 – Truncated domains in Brain Cancer Cell line

CTCF is a protein involved in the regulation of transcription, insulator function, and the X-chromosome inactivation. It is an 11 ZF transcriptional factor which is highly conserved between the species. Identification of proteins interacting with CTCF can help to elucidate the function in the cell. Previously reported studies had identified numerous CTCF protein interacting partners, and one of the interacting partners chosen in this study is YB-1. Brain cancer cell –RGBM was selected as a model to study the interaction between CTCF and YB-1. Firstly, proteins were transformed and expressed in the bacterial expression system, and these proteins were chosen to further map the interaction via pull-down assay. Results showed CTCF-ZF was the only domain able to binds to YB-1 CSD. Other truncated areas did not show any interaction hence demonstrating the interaction between these two proteins took place at the ZF for CTCF and CSD for YB-1. Next, the significant of the interaction was further characterized using the mammalian two-hybrid system. Results show strong interaction when both we co-transfected into RGBM cells. Thus, this study shows a significant binding between CTCF/YB-1 interaction in the brain cell line.

BEN-solo factors partition active chromatin to ensure proper gene activation in Drosophila

The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in both transcriptional repression and chromatin insulation. These proteins all have a DNA binding BEN domain. A fourth protein Elba3 bridges Elba1 and Elba2 to form a heterotrimeric complex ELBA. Here we report comprehensive investigation on the in vivo functions of these proteins in Drosophila embryos. We generate ChIP-seq data of all these factors from all cognate and non-cognate mutants to assess common and distinct binding locations of Insv and ELBA, and genetic interdependencies. Notably, while most Elba1 and Elba2 binding requires the full ELBA complex, the adapter protein Elba3 can associate with chromatin and repress gene expression independently of Elba1 and Elba2. We also employ high-resolution ChIP-nexus mapping to show that Insv binds to DNA in a symmetric configuration while the ELBA complex binds asymmetrically in vivo. We observe that motifs of known insulator proteins are enriched in ELBA and Insv ChIP peaks and demonstrate that ELBA collaborates with other insulator factors to regulate developmental patterning in embryos. To differentiate the insulator function of ELBA and Insv from their repressor activity, we determined real-time transcription change in mutant embryos using precision nuclear run-on sequencing. ELBA factor mutants dampen expression differences between pairs of ELBA-bound neighboring genes. Finally, transgenic reporters confirm insulation activity of ELBA- and Insv-bound sites. Altogether, these findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators in partitioning transcription units.

Pharmacogenetic 3C interaction associated with the PDGFRA gene as a chromatin conformation marker for treatment with tyrosine kinase inhibitors.

e23054 Background: IDH1 mutations detected in glioma cells impair the insulator function between FIPL1L1 and PDGFRA at 4q12 ( Flavahan et al. 2016). We have used a high-resolution chromosome-conformation capture 3C analysis platform, EpiSwitch, and quantitative PCR, to map, evaluate, and quantify the TKI-sensitive conformational juxtaposition between FIP1L1 and PDGFRA. Loss of the insulator function in glioma prompted us to investigate the same interaction in the context of insulator loss with interstitial deletions at 4q12 in eosinophilic leukemias and AML. Methods: We tested a total of 72 primers in temperature gradient PCRs, with concentration matched negative controls, using the AML cell lines EOL-1 and HL-60. Products were sequenced in forward and reverse order. Dual label 5’FAM-BHQ1-3’hydrolysis probe assays, entirely specific for the PCR products, targeted the junction region of the 3C fragments. A reference 3C interaction was used as an internal copy number control for 3C library production. Results: EpiSwitch predicted and identified six 3C FIP1L1-PDGFRA interactions in different sequence orientations, within the 3D organization of the PDGFRA locus. The interaction D7 identified by the EpiSwitch qPCR assay was detected reproducibly in EOL-1 cells and glioblastoma tissue using both single step PCR and qPCR. An imatinib-sensitive AML cell line EOL-1 was used as a positive control for qPCR assays. Both AML and glioma cell lines tested positive using the assay as did glioma patient biopsies. The glioblastoma cell line DBTRG-05MG also tested positive for the D7 interaction at a maximum of 8.92 copies per 20 ng of the template. Conclusions: We confirmed and characterized, at high resolution, the conformational deregulation of FIP1L1 and PDGFRA in glioma. Additionally, our group detected the interaction in TKI-sensitive leukemia cell lines. The analysis of 3C microstructural alterations is consistent with latest insights into epigenetic regulation of PDGFRA. It provides a promising approach to the stratification of patients for tyrosine kinase inhibitor treatment, which could not be provided diagnostically with conventional sequencing approaches.

A chromatin extension model for insulator function based on comparison of high-resolution chromatin conformation capture and polytene banding maps

Insulator proteins bind to specific genomic loci and have been shown to play a role in partitioning genomes into independent domains of gene expression and chromatin structure. Despite decades of study, the mechanism by which insulators establish these domains remains elusive. Here, we use genome-wide chromatin conformation capture (Hi-C) to generate a high-resolution map of spatial interactions of chromatin from Drosophila melanogaster embryos. We show that from the earliest stages of development the genome is divided into distinct topologically associated domains (TADs), that we can map the boundaries between TADs to sub-kilobase resolution, and that these boundaries correspond to 500-2000 bp insulator elements. Comparing this map with a detailed assessment of the banding pattern of a region of a polytene chromosome, we show that these insulator elements correspond to low density polytene interbands that divide compacted bands, which correspond to TADs. It has been previously shown that polytene interbands have low packing ratios allowing the conversion of small genomic distances (in base pairs) into a large physical distances. We therefore suggest a simple mechanism for insulator function whereby insulators increase the physical space between adjacent domains via the unpacking and extension of intervening chromatin. This model provides an intuitive explanation for known features of insulators, including the ability to block enhancer-promoter interactions, limit the spread of heterochromatin, and organize the structural features of interphase chromosomes.

Targeted degradation of CTCF decouples local insulation of chromosome domains from higher-order genomic compartmentalization

The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, we show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and segmental organization into topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding, and CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Further, our data supports that CTCF mediates transcriptional insulator function through enhancer-blocking but not direct chromatin barrier activity. These results define the functions of CTCF in chromosome folding, and provide new fundamental insights into the rules governing mammalian genome organization.