Global Chromatin Immunoprecipitation Research Articles

Abstract 846: Transcriptional regulation of Aurora-B kinase expression in B-cell acute lymphoblastic leukemia (B-ALL)

Abstract The Aurora kinases are serine/threonine kinases that are essential for mitosis. Aurora-B kinase is a component of the chromosomal passenger complex (CPC), chromosome condensation, the spindle-assembly checkpoint, and for cytokinesis. The activity of Aurora-B is essential for cellular division and proliferation. Inhibition of Aurora-B is cytotoxic, and inhibitor of Aurora-B - Barasertib (AZD1152) has been tested in clinical trials against different types of malignancies, including leukemia. However, regulation of Aurora-B expression is still largely unknown. Here, we present evidence that expression of Aurora-B in B-cell acute lymphoblastic leukemia (B-ALL) is regulated at the transcriptional level by Ikaros, a transcription factor and tumor suppressor protein. Analysis of global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) in several B-ALL primary human B-ALL cells and cell lines, showed a strong enrichment of Ikaros at the promoter of the Aurora-B gene. Ikaros functions as a tumor suppressor protein and deletion of Ikaros is associated with development of high-risk B-ALL. Ikaros binding to Aurora-B promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary B-ALL cells. The role of Ikaros in regulating Aurora-B transcription in B-ALL was tested using gain-of-function and loss-of-function experiments. Overexpression of Ikaros in human B-ALL was associated with strongly reduced transcription and overall expression of Aurora-B. Ikaros knock-down with shRNA results in increased expression of Aurora-B in B-ALL. Since Ikaros function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of Aurora-B in B-ALL. Overexpression of CK2 via retroviral transduction resulted in increased expression of the Aurora-B gene. Increased expression of CK2 was associated with a loss of Ikaros binding to the Aurora-B gene promoter. Molecular inhibition of CK2 using shRNA resulted in reduced expression of Aurora-B in human B-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the Aurora-B promoter. Ikaros knock-down abolished downregulation of Aurora-B expression in B-ALL cells following treatment with CK2 inhibitors. These data demonstrate that CK2 and Ikaros are critical regulators of Aurora-B expression in B-ALL. In conclusion, these results indicate that expression of the Aurora-B gene in B-ALL is regulated by the CK2-Ikaros signaling axis and provide a novel insight into mechanisms that regulate mitosis in leukemia. Citation Format: Yali Ding, Chunhua Song, Joseph Schramm, Soumya Maru, Daniel Bogus, Dhimant H. Desai, Arati K. Sharma, Chandrika Gowda, Sinisa Dovat. Transcriptional regulation of Aurora-B kinase expression in B-cell acute lymphoblastic leukemia (B-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 846.

Profiling of histone H3 trimethylation and distinct epigenetic pattern of chromosome Y in the transformed bronchial epithelial cells induced by consecutive arsenic treatment

Abstract Environmental arsenic is a known human carcinogen that is associated with cancers in lung, liver, skin, breast, bladder, kidney, prostate, etc. However, the underpinnings of malignant transformation of the normal cells in response to environmental arsenic exposure remain poorly understood, in part due to mixed signaling pathways elicited by arsenic, especially the inorganic trivalent arsenic (As 3+). In the present report we profiled the trimethylation status of histone H3 through global chromatin immunoprecipitation followed by sequencing (ChIP-seq) in the As 3+ -induced transformed cells and found an overall gain of active methylation of histone H3, the trimethylation of lysine 4 of histone H3 (H3K4me3), along with a diminished enrichment of the repressive marker H3K27me3 in the genome in the transformed cells relative to the control cells. Many oncogenic genes responsible for cell proliferation and selfrenewal of the cancer stem-like cells are enriched with H3K4me3 along with a reduced level of H3K27me3 and/or H3K9me3. In contrast, some genes encoding proteins with tumor suppressor-like activity showed reduced enrichment of the active marker, H3K4me3, but elevated levels of the repressive markers, H3K27me3 and H3K9me3. These data, thus, suggest that the carcinogenicity of As 3+ is achieved through regulating histone methylations that define the chromatin configuration and gene expression dynamics of the genome and the cell fate decision.

Dynamic association of the H3K64 trimethylation mark with genes encoding exported proteins in Plasmodium falciparum

Epigenetic modifications have emerged as critical regulators of virulence genes and stage-specific gene expression in Plasmodium falciparum. However, the specific roles of histone core epigenetic modifications in regulating the stage-specific gene expression are not well understood. In this study, we report an unconventional trimethylation at lysine 64 on histone 3 (H3K64me3) and characterize its functional relevance in P. falciparum. We show that PfSET4 and PfSET5 proteins of P. falciparum methylate H3K64 and that they prefer the nucleosome as a substrate over free histone 3 proteins. Structural analysis of PfSET5 revealed that it interacts with the nucleosome as a dimer. The H3K64me3 mark is dynamic, being enriched in the ring and trophozoite stages and drastically reduced in the schizont stages. Stage-specific global chromatin immunoprecipitation –sequencing analysis of the H3K64me3 mark revealed the selective enrichment of this methyl mark on the genes of exported family proteins in the ring and trophozoite stages and a significant reduction of the same in the schizont stages. Collectively, our data identify a novel epigenetic mark that is associated with the subset of genes encoding for exported proteins, which may regulate their expression in different stages of P. falciparum.

Ikaros‐Mediated Regulation of the Rab20 GTPase in B‐ALL is PP1 Dependent

Rab20 is a member of the Rab GTPase family, a group of over 60 small GTPases that play critical roles in membrane trafficking and vesicular transport in all eukaryotic cells. Rab20 has been found to play a critical role in the immune response and is highly expressed in macrophages where it plays a role in phagosome and endosome formation. RAB20 overexpression has been observed in various human malignancies. However, the role of RAB20 in leukemia is unknown. Here we present data that RAB20 expression is repressed by the Ikaros tumor suppressor protein in B‐cell acute lymphoblastic leukemia (B‐ALL). Additionally, we report that protein phosphatase 1 (PP1) regulates Ikaros’ function as a repressor of RAB20 transcription. Global chromatin immunoprecipitation coupled with next‐generation sequencing (ChIP‐Seq) demonstrated that Ikaros binds to the promoter of RAB20 in B‐ALL. Ikaros binding to the RAB20 promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in both B‐ALL cell lines and primary B‐ALL. The role of Ikaros in regulating RAB20 expression in B‐ALL was dissected using gain‐of‐function and loss‐of‐function experiments. Ikaros overexpression resulted in increased Ikaros binding to the RAB20 promoter and decreased RAB20 expression. shRNA‐mediated knockdown of Ikaros abolished Ikaros binding at the RAB20 promoter and resulted in increased RAB20 expression. A luciferase reporter assay demonstrated that Ikaros directly represses RAB20 promoter activity. The role of Ikaros in regulating the chromatin signature at the RAB20 promoter was examined by combining Ikaros overexpression and knockdown in B‐ALL with qChIP against markers of active chromatin (H3K9ac & H3K4me3) and repressive chromatin (H3K9me3). Ikaros overexpression results in an increase in H3K9me3 and a decrease in both H3K9ac and H3K4me3 at the RAB20 promoter—indicating formation of repressive chromatin. Ikaros knockdown resulted in an increase in H3K9ac and H3K4me3—indicating formation of active chromatin. Since our previous studies showed that protein phosphatase 1 (PP1) can regulate Ikaros function, we tested whether PP1 affects Ikaros‐mediated regulation of RAB20 expression. Treatment of B‐ALL cells with the calyculin and tautomycin PP1 inhibitors resulted in decreased Ikaros binding at the RAB20 promoter and increased RAB20 expression. qChIP demonstrated that mutating the PP1‐interacting site of Ikaros—IKZF1(465‐7A)—ablated Ikaros’ RAB20 promoter binding. Consequently, using an Ikaros mutant with both a mutated PP1‐interacting site and phosphoresistant mutants (simulating active PP1 dephosphorylation) restored Ikaros binding to the RAB20 promoter. Together, these data demonstrate that Ikaros transcriptionally represses RAB20 expression in B‐ALL in a PP1‐dependent manner. The results provide a novel insight into intersection of PP1 and Rab GTPase signaling pathways and suggest a potential mechanism by which PP1 regulates Rab20 activity in leukemia.Support or Funding InformationNIH R01 CA213912; NIH F30 CA221109; The Four Diamonds Fund; St. Baldrick’s Foundation; Hyundai Hope on Wheels; Alex’s Lemonade Stand; Bear Necessities Pediatric Cancer Foundation

Abstract 871: Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL)

Abstract CDC25A is a member of the CDC25 family of phosphatases that plays a major role in cell cycle progression. Here, we present evidence that expression of CDC25a in T-ALL is regulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a transcription factor and tumor suppressor protein. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both primary human acute lymphoblastic leukemia cells and cell lines, demonstrated that Ikaros binds to the promoter of the CDC25a gene. Ikaros functions as a tumor suppressor protein and deletion of which is associated with development of T-ALL. Ikaros binding to CDC25a promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. Ikaros knock-down with shRNA results in increased transcription of CDC25a in T-ALL. In mice, T-ALL cells that were derived from Ikaros-knockout mice express high levels of CDC25a. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC25a expression. Overexpression of CK2 via retroviral transduction resulted in increased transcription of the CDC25a gene, as measured by qRT-PCR, as well as increased overall expression of CDC25a, as measured by Western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the CDC25a gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor resulted in reduced expression of CDC25a in primary human T-ALL. CK2 inhibition was also associated with strong reduction in AKT phosphorylation, emphasizing that CK2 inhibition downregulates CDC25a and other cell cycle progression genes. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC25a. Ikaros knock-down restored high expression of CDC25a in T-ALL cells that were treated with CK2 inhibitors. These data showed that CK2 and Ikaros are major transcriptional regulators of CDC25a transcription in T-ALL and that CK2 inhibition represses CDC25a transcription via Ikaros-mediated repression. In conclusion, these results indicate that expression of the CDC25a oncogene in T-ALL is regulated by the CK2 which modulates Ikaros activity. Presented data revealed a novel mechanism of therapeutic action of CK2 inhibitors - repression of CDC25a expression via Ikaros. Results provide a rationale for the use of novel CK2 inhibitors in T-ALL. Citation Format: Soumya C. Iyer, Shriya Kane, Chandrika Gowda, Chunhua Song, Yali Ding, Jon Payne, Pavan Kumar Dhanyam Raju, Bihua Tan, Mary McGrath, Yevgeniya Bamme, Mario Solimon, Nathalia Moreno Cury, Dhimant Desai, Arati Sharma, Kimberly J. Payne, Sinisa Dovat. Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 871.

Abstract 4400: Epigenetic regulation of CDC16 expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL)

Abstract CDC16 is one of several subunits of the anaphase-promoting complex (APC) with crucial role in cell division. Here, we show evidence that expression of CDC16 in T-ALL is modulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a tumor suppressor protein and transcription factor. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both cell lines and primary human ALL, shows that Ikaros binds to the promoter of the CDC16 gene. Ikaros functions as a tumor suppressor protein and its deletion is associated with development of T-ALL. Ikaros binding to CDC16 promoter was confirmed via quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. The role of Ikaros in regulating CDC16 transcription in T-ALL was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with shRNA resulted in increased transcription of CDC16 in T-ALL. Overexpression of Ikaros in human T-ALL was associated with strong reduction in transcription of CDC16. In mice, T-ALL cells that are derived from Ikaros-knockout mice express high levels of CDC16. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC16 expression. Since Ikaros function in T-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of CDC16 in T-ALL. Overexpression of CK2 via retroviral transduction resulted in increased CDC16 gene transcription as measured by qRT-PCR, as well as increased overall expression of CDC16, as analyzed by Western blot. Increased expression of CK2 was associated with loss of Ikaros binding to the CDC16 gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor led to reduced expression of CDC16 in primary human T-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC16. Ikaros knock-down restored high expression of CDC16 in T-ALL cells that were treated with CK2 inhibitors. These data demonstrate that CK2 and Ikaros are major transcriptional regulators of CDC16 transcription in T-ALL and that inhibition of CK2 represses CDC16 transcription via Ikaros-mediated repression. In summary, these results provide evidence that expression of the CDC16 gene in T-ALL is regulated by the CK2 signaling pathway which modulates Ikaros funtion. Targeting CK2 with specific inhibitors has been used as a therapeutic strategy in preclinical models of T-ALL. Presented data reveals a novel mechanism of therapeutic action of CK2 inhibitors – repression of CDC16 expression via Ikaros. These results provide a rationale for the use of novel CK2 inhibitors in T-ALL to target CDC16. Citation Format: Pavan Kumar Dhanyam Raju, Shriya Kane, Chandrika Gowda, Chunhua Song, Yali Ding, Jonathan Payne, Soumya Iyer, Nathalia Moreno Curry, Mary McGrath, Yevgeniya Bamme, Bihua Tan, Mario Soliman, Dhimant Desai, Arati Sharma, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of CDC16 expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4400.

Regulation of mTOR expression by Casein Kinase II (CK2) signaling pathway in leukemia

The mammalian target of rapamycin(mTOR) is a kinase encoded by the MTOR gene. mTOR is a critical component of PI3K/mTOR/AKT signaling pathway that positively regulates proliferation of leukemia cells. Targeting mTOR and PI3K/mTOR/AKT pathway has been used to treat T‐cell acute lymphoblastic leukemia (T‐ALL). However, little is known about mechanisms that regulate transcription and expression of mTOR gene in T‐ALL. Here, we present the evidence that expression of mTOR in T‐ALL is regulated at transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of the Ikaros, a transcription factor and a tumor suppressor protein. Global chromatin immunoprecipitation coupled with the next‐generation sequencing (ChIP‐seq) studies in primary human T‐ALL, demonstrated that Ikaros binds of the promoter of mTOR gene. Ikaros binding to mTOR promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T‐ALL cells. The role of Ikaros in regulation of mTOR transcription in T‐ALL was tested using gain‐of‐function and loss‐of‐function experiments. Ikaros knock‐down with shRNA, results in increased transcription of mTOR in T‐ALL. Overexpression of Ikaros in human T‐ALL was associated with strongly reduced transcription of mTOR. In mice, T‐ALL cells that are derived from Ikaros‐knockout mice express high level of mTOR. Transduction of these cells with Ikaros‐containing retrovirus, results in sharp reduction of mTOR expression. Since Ikaros function in T‐ALL is negatively regulated by pro‐oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of mTOR in T‐ALL. Overexpression of CK2 via retroviral transduction resulted in increased transcription of the MTOR gene, as well as increased overall expression of mTOR, as measured by Western blot. Increased expression of CK2 was associated with loss of Ikaros binding to the promoter of the MTOR gene. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor resulted in reduced expression of mTOR in primary T‐ALL. Inhibition of CK2 was associated with increased Ikaros binding at promoter of mTOR. Ikaros knock‐down restored high expression of mTOR in T‐ALL cells that are treated with CK2 inhibitors. These data showed that CK2 and Ikaros are major transcriptional regulators of mTOR transcription in T‐ALL and that CK2 inhibition represses mTOR transcription via Ikaros‐mediated repression. CK2 inhibition, along with an increased Ikaros binding was associated with reduced histone H3K9ac and H3K4me3 marks at mTOR promoter suggesting that CK2 and Ikaros regulate mTOR transcription via chromatin remodeling. In conclusion, results presented the evidence that expression of mTOR oncogene is regulated by CK2 signaling pathway that involves Ikaros activity in T‐ALL. Results provide the rationale for the use of CK2 inhibitors in T‐ALL to target PI3K/mTOR/AKT pathway.Support or Funding InformationR01CA209829‐02 (SD and KP) and R01CA213912‐01A1 (SD)St. Baldrick's foundation ( SD and CG) and Hyundai Hope on wheels (SD and CG)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative Transcriptomics Highlights New Features of the Iron Starvation Response in the Human Pathogen Candida glabrata.

In this work, we used comparative transcriptomics to identify regulatory outliers (ROs) in the human pathogen Candida glabrata. ROs are genes that have very different expression patterns compared to their orthologs in other species. From comparative transcriptome analyses of the response of eight yeast species to toxic doses of selenite, a pleiotropic stress inducer, we identified 38 ROs in C. glabrata. Using transcriptome analyses of C. glabrata response to five different stresses, we pointed out five ROs which were more particularly responsive to iron starvation, a process which is very important for C. glabrata virulence. Global chromatin Immunoprecipitation and gene profiling analyses showed that four of these genes are actually new targets of the iron starvation responsive Aft2 transcription factor in C. glabrata. Two of them (HBS1 and DOM34b) are required for C. glabrata optimal growth in iron limited conditions. In S. cerevisiae, the orthologs of these two genes are involved in ribosome rescue by the NO GO decay (NGD) pathway. Hence, our results suggest a specific contribution of NGD co-factors to the C. glabrata adaptation to iron starvation.

Abstract 5426: Regulation of mTOR signaling pathway by Ikaros and CK2 in high risk leukemia

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common cancer in children. Alterations in IKZF1 have proven to be an indicator of inferior outcome in patients with high-risk ALL. Casein Kinase II (CK2) is a pro-oncogenic protein which is overexpressed in various cancers including leukemia. CK2 phosphorylates Ikaros and impairs its tumor suppressor functions. Targeted inhibition of CK2 restores Ikaros tumor suppressor function in high-risk B-ALL even in cases with single allele Ikaros deletion. Global chromatin immunoprecipitation (ChIP) coupled with the next-generation sequencing (ChIP-seq) studies in primary human B-ALL cells and in cell lines, demonstrated Ikaros occupancy of the promoter of genes involved in mTOR pathway. mTOR (mechanistic target of rapamycin) is the key component of the PI3K/AKT/mTOR pathway - a major signaling pathway that stimulates cellular proliferation in B-ALL. Several specific inhibitor of mTOR (rapamycin, Everolimus, sirolimus) are currently being used for the treatment of relapsed/refractory leukemia. Objective of this study is to understand the mechanisms of transcriptional regulation of mTOR in B- ALL. We hypothesize that Ikaros negatively regulates the mTOR pathway by repressing transcription of mTOR and PI3K genes. Ikaros binding to promoters of mTOR and genes that promote the PI3K pathway was confirmed using quantitative ChIP. Functional experiments such as overexpression of Ikaros in B-ALL cells results in transcriptional repression of mTOR and PI3K genes whereas Ikaros silencing using shRNA resulted in increased transcription. These results suggest that Ikaros negatively regulates the mTOR pathway by repressing transcription of mTOR and PI3K genes. Molecular inhibition of CK2 with shRNA targeting the CK2 catalytic subunit, as well as pharmacological targeting of CK2 with CX4945 resulted in transcriptional repression of mTOR and PI3K genes. CK2 inhibition was associated with increased Ikaros DNA-binding to the promoters of mTOR and PI3K genes. However, the ability of CX4945 to repress mTOR and PI3K genes is lost or severely reduced, in cells with shRNA silencing of Ikaros, as compared to cells with intact Ikaros. Moreover, similar results were noted following treatment with CX4945 in leukemia cells obtained from high risk B-ALL patients with deletion of one IKZF1 allele. Ikaros binds poorly to promoters of mTOR and PI3K genes in these cells. Treatment with CX4945 restores Ikaros DNA-binding to the promoters of both mTOR and PI3K genes, which is associated with strong repression of mTOR and PI3K genes. In conclusion, these results suggest that CK2 inhibition with CX4945 represses the mTOR pathway by enhancing Ikaros-mediated transcriptional repression of mTOR and PI3K pathway genes. Results provide the rationale for the use of CK2 inhibitors in combination with mTOR inhibitors in high risk leukemia with dysregulation of mTOR/PI3K pathway. Citation Format: Chandrika S. Gowda, Chunhua Song, Mary McGrath, Charyguly Annageldiyev, Kimberly payne, Sinisa Dovat. Regulation of mTOR signaling pathway by Ikaros and CK2 in high risk leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5426.

Abstract 2537: Regulation of LMO2 oncogene expression in high-risk B-cell acute lymphoblastic leukemia

Abstract LIM domain only protein 2 (LMO2) is an oncogene that is overexpressed in a subset of B-cell acute lymphoblastic leukemia (B-ALL). The mechanisms that regulate LMO2 expression in B-ALL are still unknown. Here, we show that transcription of LMO2 in B-ALL is regulated by Ikaros, a tumor suppressor protein, which is encoded by the IKZF1 gene, and whose deletion is associated with development of high-risk B-ALL. The use of global chromatin immunoprecipitation coupled with the next-generation sequencing (ChIP-seq) studies in primary human B-ALL cells and in cell lines revealed a strong Ikaros occupancy at the promoter of the LMO2 gene. Ikaros binding to the LMO2 promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP). We tested the role of Ikaros in regulating LMO2 transcription in B-ALL using gain-of-function and loss-of-function experiments. A luciferase reporter assay with the LMO2 promoter showed that Ikaros directly represses LMO2 transcription. Overexpression of Ikaros in B-ALL via retroviral transduction was associated with strongly reduced expression of LMO2. Furthermore, Ikaros knock-down with shRNA, resulted in increased transcription of LMO2 in B-ALL cells. These results suggest that Ikaros represses transcription of LMO2 in B-ALL. Since Ikaros' function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 inhibition affects Ikaros-mediated repression of LMO2. Both molecular inhibition with shRNA, and pharmacological inhibition of CK2 with a specific CK2 inhibitor, CX-4945, resulted in reduced expression of LMO2. Inhibition of CK2 was also associated with increased Ikaros occupancy at the LMO2 promoter. In high-risk B-ALL that have deletion of a single copy of the IKZF1 gene, Ikaros does not bind to the LMO2 promoter. Treatment of primary B-ALL cells that have IKZF1 haploinsufficiency restores Ikaros binding to the LMO2 promoter and results in reduced LMO2 expression. In conclusion, our data demonstrate that Ikaros and CK2 regulate transcription and overall expression of the LMO2 oncogene in B-ALL. Our results identify a novel mechanism of therapeutic action of CK2 inhibitors in high-risk B-ALL—repression of LMO2 expression via restoration of Ikaros' tumor suppressor function. Citation Format: Yali Ding, Jonathon L. Payne, Shriya Kane, Elanora Dovat, Mario Soliman, Chunhua Song, Sinisa Dovat. Regulation of LMO2 oncogene expression in high-risk B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2537.

PLAG1 and USF2 Co-regulate Expression of Musashi-2 in Human Hematopoietic Stem and Progenitor Cells.

SummaryMSI2, which is expressed predominantly in hematopoietic stem and progenitor cells (HSPCs), enforces HSPC expansion when overexpressed and is upregulated in myeloid leukemias, indicating its regulated transcription is critical to balanced self-renewal and leukemia restraint. Despite this, little is understood of the factors that enforce appropriate physiological levels of MSI2 in the blood system. Here, we define a promoter region that reports on endogenous expression of MSI2 and identify USF2 and PLAG1 as transcription factors whose promoter binding drives reporter activity. We show that these factors co-regulate, and are required for, efficient transactivation of endogenous MSI2. Coincident overexpression of USF2 and PLAG1 in primitive cord blood cells enhanced MSI2 transcription and yielded cellular phenotypes, including expansion of CD34+ cells in vitro, consistent with that achieved by direct MSI2 overexpression. Global chromatin immunoprecipitation sequencing analyses confirm a preferential co-binding of PLAG1 and USF2 at the promoter of MSI2, as well as regulatory regions corresponding to genes with roles in HSPC homeostasis. PLAG1 and USF2 cooperation is thus an important contributor to stem cell-specific expression of MSI2 and HSPC-specific transcriptional circuitry.

Abstract 5533: Signaling pathways that regulate LMO2 oncogene expression in pediatric high risk T cell acute lymphoblastic leukemia

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) accounts for about 15% of pediatric ALL and is characterized as a high-risk disease with frequent relapse, chemotherapy resistance, and a poorer prognosis. LIM domain only protein 2 (LMO2) is a regulator of hematopoiesis and an oncogene that is overexpressed in about 10% of T-ALL. The IKZF1 gene encodes a zinc finger protein called Ikaros which is a master regulator of lymphoid development and a tumor suppressor. In pediatric ALL, Ikaros alteration is considered independent prognostic marker for poor outcome. Pro oncogenic Casein Kinase II (CK2) is overexpressed in various malignancies including leukemia. CK2 directly phosphorylates Ikaros in vivo and inhibits its function as transcriptional regulator. Inhibition of CK2 restores Ikaros tumor suppressor function and results in anti-leukemic effect. Objective of this study is to understand the mechanisms of transcriptional regulation of LMO2 in T-cell ALL. Global chromatin immunoprecipitation (ChIP) coupled with the next-generation sequencing (ChIP-seq) studies in primary human ALL cells and in cell lines, demonstrated Ikaros occupancy of the promoter of LMO2 gene. We hypothesize that Ikaros negatively regulates expression of LMO2 at transcriptional level and CK2 impairs Ikaros mediated repression of LMO2 in T-cell ALL. Results: qChIP in primary leukemia cells confirmed that Ikaros binds to the promoter region of LMO2. Using gain-of-function and loss-of-function experiments we dissected the role of Ikaros in regulation of LMO2 transcription in T-ALL. Ikaros silencing using shRNA transfection revealed increase in LMO2 expression as measured by qRT PCR. Conversely, overexpression of Ikaros was associated with strongly reduced transcription of LMO2. T-ALL cells that are derived from Ikaros-knockout mouse models express high level of LMO2. Retroviral transduction of these cells with Ikaros, resulted in significant reduction of LMO2 expression. Next we investigated how CK2 affects the regulatory functions of Ikaros towards LMO2. Molecular and pharmacological inhibition of CK2 resulted in reduced expression of LMO2 in primary human T-ALL. Ikaros binding at promoter of LMO2 was noted to be significantly increased following CK2 inhibition. This effect was not seen when cell were subjected to CK2 inhibition after Ikaros silencing. Further, we analyzed changes in the histone markers at the heterochromatin at the LMO2 promoter following Increased Ikaros binding such as reduced histone H3K9ac and H3K4me3 markers. This suggests that Ikaros regulates LMO2 transcription via chromatin remodeling. Conclusion: This data reveals new regulatory mechanism for oncogene LMO2 in pediatric T-ALL. New evidence suggests that repression of LMO2 expression in T cell ALL via Ikaros can be potentiated using CK2 inhibitors. Findings provide the rationale for the use of CK2 inhibitors in T-ALL with LMO2 overexpression. Citation Format: Chandrika S. Gowda, Yali Ding, Chunhua Song, Malika Kapadia, Kimberly J. Payne, Sinisa Dovat. Signaling pathways that regulate LMO2 oncogene expression in pediatric high risk T cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5533. doi:10.1158/1538-7445.AM2017-5533

Transcriptional Repression of the LMO2 Oncogene By Ikaros in T-Cell Acute Lymphoblastic Leukemia

LIM domain only protein 2 (LMO2) is a regulator of hematopoiesis and an oncogene that is overexpressed in a subset of T-cell acute lymphoblastic leukemia (T-ALL). Overexpression of LMO2 in T-ALL is associated with a poor prognosis. The mechanisms that regulate LMO2 expression in T-ALL are still unknown. Here, we present evidence that expression of LMO2 in T-ALL is regulated at the transcriptional level by Ikaros, a tumor suppressor protein whose deletion is associated with the development T-ALL. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in primary human acute lymphoblastic leukemia cells and in cell lines demonstrated Ikaros occupancy of the LMO2 promoter. Ikaros binding at the LMO2 promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL and B-ALL cells. The role of Ikaros in the regulation of LMO2 transcription in T-ALL was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with siRNA resulted in increased transcription of LMO2 in T-ALL. Overexpression of Ikaros in human T-ALL was associated with strongly reduced transcription of LMO2. In mice, T-ALL cells that are derived from Ikaros-knockout mice express high levels of LMO2. Transduction of these cells with Ikaros-containing retrovirus, results in a sharp reduction of LMO2 expression. Since Ikaros function in T-ALL is negatively regulated by the pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 inhibition can enhance Ikaros-mediated transcriptional repression of LMO2. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor, resulted in reduced expression of LMO2 in primary human T-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the LMO2 promoter. Ikaros knock-down restored high expression of LMO2 in T-ALL cells that were treated with CK2 inhibitors. These data show that Ikaros is a major regulator of LMO2 transcription in T-ALL and that CK2 inhibition requires Ikaros activity to repress LMO2 transcription. Increased Ikaros binding was associated with reduced histone H3K9ac and H3K4me3 marks at the LMO2 promoter suggesting that Ikaros regulates LMO2 transcription via chromatin remodeling. In conclusion, these results provide evidence that expression of the LMO2 oncogene is regulated by Ikaros and CK2 in T-ALL. Targeting CK2 with specific inhibitors has been used as a therapeutic strategy in a preclinical model of T-ALL. The presented data reveal a novel mechanism of therapeutic action for CK2 inhibitors - repression of LMO2 expression via Ikaros. These results provide a rationale for the use of CK2 inhibitors in T-ALL with LMO2 overexpression. DisclosuresNo relevant conflicts of interest to declare.

Weighted enrichment method for prediction of transcription regulators from transcriptome and global chromatin immunoprecipitation data.

Predicting responsible transcription regulators on the basis of transcriptome data is one of the most promising computational approaches to understanding cellular processes and characteristics. Here, we present a novel method employing vast amounts of chromatin immunoprecipitation (ChIP) experimental data to address this issue. Global high-throughput ChIP data was collected to construct a comprehensive database, containing 8 578 738 binding interactions of 454 transcription regulators. To incorporate information about heterogeneous frequencies of transcription factor (TF)-binding events, we developed a flexible framework for gene set analysis employing the weighted t-test procedure, namely weighted parametric gene set analysis (wPGSA). Using transcriptome data as an input, wPGSA predicts the activities of transcription regulators responsible for observed gene expression. Validation of wPGSA with published transcriptome data, including that from over-expressed TFs, showed that the method can predict activities of various TFs, regardless of cell type and conditions, with results totally consistent with biological observations. We also applied wPGSA to other published transcriptome data and identified potential key regulators of cell reprogramming and influenza virus pathogenesis, generating compelling hypotheses regarding underlying regulatory mechanisms. This flexible framework will contribute to uncovering the dynamic and robust architectures of biological regulation, by incorporating high-throughput experimental data in the form of weights.

Canonical Wnt Signaling Negatively Modulates Regulatory T Cell Function

Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that Tcell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both invitro and invivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector Tcells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.

Non-canonical TAF complexes regulate active promoters in human embryonic stem cells

The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find, unexpectedly, that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2, 3, 5, 6, 7 and 11), whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP, whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results, hESCs contain a previously undescribed complex comprising TAFs 2, 6, 7, 11 and TBP. Altering the composition of hESC TAFs, either by depleting TAFs that are present or ectopically expressing TAFs that are absent, results in misregulated expression of pluripotency genes and induction of differentiation. Thus, the selective expression and use of TAFs underlies the ability of hESCs to self-renew.DOI:http://dx.doi.org/10.7554/eLife.00068.001.

CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells

Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer.

ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals.

Activation Domain-dependent Monoubiquitylation of Gal4 Protein Is Essential for Promoter Binding in Vivo

The Saccharomyces cerevisiae Gal4 protein is a paradigmatic transcriptional activator containing a C-terminal acidic activation domain (AD) of 34 amino acids. A mutation that results in the truncation of about two-thirds of the Gal4AD (gal4D) results in a crippled protein with only 3% the activity of the wild-type activator. We show here that although the Gal4D protein is not intrinsically deficient in DNA binding, it is nonetheless unable to stably occupy GAL promoters in vivo. This is because of the activity of the proteasomal ATPases, including Sug1/Rpt6, which bind to Gal4D via the remainder of the AD and strip it off of DNA. A mutation that suppressed the Gal4D "no growth on galactose" phenotype repressed the stripping activity of the ATPase complex but not other activities. We further demonstrate that Gal4D is hypersensitive to this stripping activity because of its failure to be monoubiquitylated efficiently in vivo and in vitro. Evidence is presented that the piece of the AD that is deleted in Gal4D protein is likely a recognition element for the E3 ubiquitin-protein ligase that modifies Gal4. These data argue that acidic ADs comprise at least two small peptide subdomains, one of which is responsible for activator monoubiquitylation and another that interacts with the proteasomal ATPases, coactivators and other transcription factors. This study validates the physiological importance of Gal4 monoubiquitylation and clarifies its major role as that of protecting the activator from being destabilized by the proteasomal ATPases.

Identification of Genes Directly Regulated by the Oncogene ZNF217 Using Chromatin Immunoprecipitation (ChIP)-Chip Assays

It has been proposed that ZNF217, which is amplified at 20q13 in various tumors, plays a key role during neoplastic transformation. ZNF217 has been purified in complexes that contain repressor proteins such as CtBP2, suggesting that it acts as a transcriptional repressor. However, the function of ZNF217 has not been well characterized due to a lack of known target genes. Using a global chromatin immunoprecipitation (ChIP)-chip approach, we identified thousands of ZNF217 binding sites in three tumor cell lines (MCF7, SW480, and Ntera2). Further analysis of ZNF217 in Ntera2 cells showed that many promoters are bound by ZNF217 and CtBP2 and that a subset of these promoters are activated upon removal of ZNF217. Thus, our in vivo studies corroborate the in vitro biochemical analyses of ZNF217-containing complexes and support the hypothesis that ZNF217 functions as a transcriptional repressor. Gene ontology analysis showed that ZNF217 targets in Ntera2 cells are involved in organ development, suggesting that one function of ZNF217 may be to repress differentiation. Accordingly we show that differentiation of Ntera2 cells with retinoic acid led to down-regulation of ZNF217. Our identification of thousands of ZNF217 target genes will enable further studies of the consequences of aberrant expression of ZNF217 during neoplastic transformation.