TCL1A, a Novel Transcription Factor and a Coregulator of Nuclear Factor Kb P65: Single Nucleotide Polymorphism and Estrogen Dependence S

Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2018/03/28/jpet.118.247718.DC1 1521-0103/365/3/700–710$35.00 https://doi.org/10.1124/jpet.118.247718 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 365:700–710, June 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics TCL1A, a Novel Transcription Factor and a Coregulator of Nuclear Factor kB p65: Single Nucleotide Polymorphism and Estrogen Dependence s Ming-Fen Ho, Edroaldo Lummertz da Rocha, Cheng Zhang, James N. Ingle, Paul E. Goss, Lois E. Shepherd, Michiaki Kubo, Liewei Wang, Hu Li, and Richard M. Weinshilboum Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics (M.H., E.L.d.R., C.Z., L.W., H.L., R.M.W.), and Division of Medical Oncology, Department of Oncology (J.N.I.), Mayo Clinic, Rochester, Minnesota; Division of Hematology/Oncology, Department of Medicine, Massachusetts General Hospital Cancer Center, Boston, Massachusetts (P.E.G.); Canadian Cancer Trials Group, Kingston, Ontario, Canada (L.E.S.); and RIKEN Center for Integrative Downloaded from Medical Science, Yokohama, Japan (M.K.) Received January 10, 2018; accepted March 19, 2018 ABSTRACT T-cell leukemia 1A (TCL1A) single-nucleotide polymorphisms cell lines. RNA-seq identified 357 genes that were regulated jpet.aspetjournals.org (SNPs) have been associated with aromatase inhibitor-induced in a TCL1A SNP- and E2-dependent fashion with expression musculoskeletal adverse events. We previously demonstrated patterns that were 4OH-TAM reversible. ChIP-seq for the that TCL1A is inducible by estradiol (E2) and plays a critical role in same cells identified 57 TCL1A binding sites that could be the regulation of cytokines, chemokines, and Toll-like receptors regulated by E2 in a SNP-dependent fashion. Even more in a TCL1A SNP genotype and estrogen-dependent fashion. striking, nuclear factor-kB(NF-kB) p65 bound to those same Furthermore, TCLIA SNP-dependent expression phenotypes DNA regions. In summary, TCL1A is a novel transcription “ ” can be reversed by exposure to selective estrogen receptor factor with expression that is regulated in a SNP- and at ASPET Journals on September 27, 2021 modulators such as 4-hydroxytamoxifen (4OH-TAM). The pre- E2-dependent fashion—a pattern of expression that can be sent study was designed to comprehensively characterize the reversed by 4OH-TAM. Integrated RNA-seq and ChIP-seq role of TCL1A in transcriptional regulation across the genome by results suggest that TCL1A also acts as a transcriptional performing RNA sequencing (RNA-seq) and chromatin immuno- coregulator with NF-kB p65, an important immune system precipitation sequencing (ChIP-seq) assays with lymphoblastoid transcription factor. Introduction TCL1A expression was induced by estradiol (E2), but only in cell lines homozygous for variant genotypes for the TCL1A SNPs (Ho We previously performed a genome-wide association study et al., 2016a). These three SNPs appeared to act in concert to (GWAS) that identified three single-nucleotide polymorphisms influence estrogen-dependent TCL1A induction (Ho et al., 9 (SNPs) located 3 of the T-cell leukemia 1A (TCL1A)genethat 2016a). However, this expression pattern could be “reversed” were associated with musculoskeletal adverse events induced after estrogen receptor a blockadewiththeactivetamoxifen by aromatase inhibitors (AIs) (Ingle et al., 2010). The top hit SNP metabolite 4-hydroxytamoxifen (4OH-TAM). (rs11849538) from that GWAS was in tight linkage disequilib- We have also reported that mechanisms underlying this 2 5 rium with two other SNPs, rs7160302 and rs7359033 (R 0.92 drug-induced reversal of TCL1A expression are due, at least in and 0.98, respectively) (Ho et al., 2016a). Subsequently, we part, to altered estrogen receptor binding to estrogen response performed functional genomic studies using a human variation elements, two of which are at a distance from the SNPs panel of lymphoblastoid cell lines (LCLs), and we found that (Ho et al., 2016a). Even more striking, a series of downstream immune mediator genes, including those encoding cytokines This work was supported in part by the National Institutes of Health (Liu et al., 2012), chemokines (Ho et al., 2016a), and Toll-like National Institute of General Medical Sciences [U19 GM61388 and R01 receptors (TLRs) (Ho et al., 2017), responded in parallel with GM28157] and National Cancer Institute [P50 CA11620, R01 CA196648, and R01 CA138461]; the Avon Breast Cancer Crusade, the Breast Cancer Research TCL1A SNP- and estrogen-dependent transcription. These Foundation, and the Mayo Clinic Center for Individualized Medicine. R.M.W. observations have potential implications for the treatment of and L.W. are cofounders and stockholders in OneOme. https://doi.org/10.1124/jpet.118.247718. inflammatory diseases such as rheumatoid arthritis because s This article has supplemental material available at jpet.aspetjournals.org. their drug treatment targets these immune mediators. If the ABBREVIATIONS: AI, aromatase inhibitor; bp, base pair; ChIP, chromatin immunoprecipitation; ChIP-seq, chromatin immunoprecipitation sequencing; DAPI, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine; E2, estradiol; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; GWAS, genome-wide association study; IP, immunoprecipitation; kb; kilobase; LCL, lymphoblastoid cell line; NF-kB, nuclear factor kB; 4OH-TAM, 4-hydroxytamoxifen; RA, rheumatoid arthritis; PCR, polymerase chain reaction; RNA-seq, RNA sequencing; siRNA, small interfering RNA; SNP, single-nucleotide polymorphism; TCL1A, T-cell leukemia 1A; TLR, Toll-like receptor. 700 TCL1A, a Transcription Factor and Coregulator of NF-kB p65 701 TCL1A SNP genotypes were known, these observations raise data to identify genes that were up-regulated with log2FC .1 only in the possibility that immune mediator gene expression could be LCLs homozygous for wild-type sequences for the TCL1A SNPs in manipulated by the use of drugs such as 4OH-TAM. response to E2 treatment, and LCLs homozygous for variant genotypes The molecular mechanisms underlying the effect of the for the TCL1A SNPs with nonsignificant changes or down-regulated , TCL1A SNPs that result in changes in TCL1A expression in (log2FC 0.5) expression in response to E2 treatment. We also searched for genes that had “reversed” gene expression patterns in response to an estrogen- and 4OH-TAM-dependent fashion are not clear. E214OH-TAM treatment. Specifically, these were genes that were The molecular mechanisms by which TCL1A expression might up-regulated with log2FC .1 only in LCLs with homozygous variant be associated with AI-induced musculoskeletal adverse events sequences for the TCL1A SNPs in response to E214OH-TAM treat- also remain unclear. Our study was designed to explore those ment, and LCLs with homozygous wild-type genotypes for the TCL1A molecular mechanisms. Specifically, we set out to determine SNPs that displayed nonsignificant changes or down-regulation whether TCL1A might function as a transcription factor that acts (log2FC , 0.5) in response to E214OH-TAM treatment. Gene ontology broadly—genome-wide—in a SNP- and estrogen-dependent fash- terms for each data set were generated using Database for Annotation, ion, and to study underlying mechanisms responsible for this Visualization and Integrated Discovery (DAVID) v6.8 (https://david. genomic phenomenon using a human variation panel LCL model ncifcrf.gov/) (Huang et al., 2009). system. We should emphasize that this LCL panel represents a Chromatin Immunoprecipitation Sequencing and Data genomic data-rich cell line model system that has repeatedly Analysis Downloaded from demonstrated its utility in the generation and testing of The chromatin immunoprecipitation sequencing (ChIP-seq) experi- pharmacogenomic hypotheses (Ingle et al., 2010, 2013, 2016; ments were performed in duplicate using two LCLs with known TCL1A Liu et al., 2012; Ho et al., 2016a, 2017). Specifically, it allowed genotypes. Specifically, we selected one LCL from each genotype group us to select LCLs for study with any common genotype or from the five LCLs we used to perform RNA-seq. The ChIP-seq libraries combinations of genotypes, as demonstrated by the experi- were prepared using the Ovation ultralow DR Multiplex kit (NuGEN), ments described subsequently. and were subsequently sequenced to 51-bp paired ends using Illumina HiSeq 2000 at the Mayo Clinic Center for Individualized Medicine jpet.aspetjournals.org Medical Genomics Facility. The Fastq files were aligned against the Materials and Methods UCSC human reference genome (hg19) with Bowtie 1.1.0 using the following Bowtie parameters: 2sam 2chunkmbs 512 2p42k12m12e Human Variation Panel Lymphoblastoid Cell Lines 70 2l512best. We then used the HOMER function findPeaks with The human variation panel LCL model system consists of 300 LCLs default parameters to identify significantly enriched peaks in the from healthy subjects of three ethnicities (100 European American, experimental samples (Heinz et al., 2010). 100 African American, and 100 Han Chinese American). This LCL cell We used deepTools to visualize binding profiles for TCL1A around line model system was used to perform many of the experiments transcription start sites (22 kb, 12 kb) (Ramírez et al., 2016). Briefly, at ASPET Journals on September 27, 2021 described in this report. This cell line model system provides genome- sorted bam files

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