Global Chromatin Research Articles

MeCP2: A Critical Regulator of Chromatin in Neurodevelopment and Adult Brain Function.

Methyl CpG binding protein 2 (MeCP2) was first identified as a nuclear protein with a transcriptional repressor role that recognizes DNA methylation marks. MeCP2 has a well-established function in neurodevelopment, as evidenced by the severe neurological impairments characteristic of the Rett syndrome (RTT) pathology and the MeCP2 duplication syndrome (MDS), caused by loss or gain of MeCP2 function, respectively. Research aimed at the underlying pathophysiological mechanisms of RTT and MDS has significantly advanced our understanding of MeCP2 functions in the nervous system. It has revealed, however, that MeCP2 has more varied and complex roles than previously thought. Here we review recent insights into the functions of MeCP2 in neurodevelopment and the less explored requirement for MeCP2 in adult brain function. We focus on the emerging view that MeCP2 is a global chromatin organizer. Finally, we discuss how the individual functions of MeCP2 in neurodevelopment and adulthood are linked to its role as a chromatin regulator.

The BACH1–HMOX1 Regulatory Axis Is Indispensable for Proper Macrophage Subtype Specification and Skeletal Muscle Regeneration

The infiltration and subsequent in situ subtype specification of monocytes to effector/inflammatory and repair macrophages is indispensable for tissue repair upon acute sterile injury. However, the chromatin-level mediators and regulatory events controlling this highly dynamic macrophage phenotype switch are not known. In this study, we used a murine acute muscle injury model to assess global chromatin accessibility and gene expression dynamics in infiltrating macrophages during sterile physiological inflammation and tissue regeneration. We identified a heme-binding transcriptional repressor, BACH1, as a novel regulator of this process. Bach1 knockout mice displayed impaired muscle regeneration, altered dynamics of the macrophage phenotype transition, and transcriptional deregulation of key inflammatory and repair-related genes. We also found that BACH1 directly binds to and regulates distal regulatory elements of these genes, suggesting a novel role for BACH1 in controlling a broad spectrum of the repair response genes in macrophages upon injury. Inactivation of heme oxygenase-1 (Hmox1), one of the most stringently deregulated genes in the Bach1 knockout in macrophages, impairs muscle regeneration by changing the dynamics of the macrophage phenotype switch. Collectively, our data suggest the existence of a heme-BACH1--HMOX1 regulatory axis, that controls the phenotype and function of the infiltrating myeloid cells upon tissue damage, shaping the overall tissue repair kinetics.

CryoEM structures of Arabidopsis DDR complexes involved in RNA-directed DNA methylation

Transcription by RNA polymerase V (Pol V) in plants is required for RNA-directed DNA methylation, leading to transcriptional gene silencing. Global chromatin association of Pol V requires components of the DDR complex DRD1, DMS3 and RDM1, but the assembly process of this complex and the underlying mechanism for Pol V recruitment remain unknown. Here we show that all DDR complex components co-localize with Pol V, and we report the cryoEM structures of two complexes associated with Pol V recruitment—DR (DMS3-RDM1) and DDR′ (DMS3-RDM1-DRD1 peptide), at 3.6 Å and 3.5 Å resolution, respectively. RDM1 dimerization at the center frames the assembly of the entire complex and mediates interactions between DMS3 and DRD1 with a stoichiometry of 1 DRD1:4 DMS3:2 RDM1. DRD1 binding to the DR complex induces a drastic movement of a DMS3 coiled-coil helix bundle. We hypothesize that both complexes are functional intermediates that mediate Pol V recruitment.

BAT Hi-C maps global chromatin interactions in an efficient and economical way.

Chromosome Conformation Capture (3C)-based technologies, such as Hi-C, have represented a significant breakthrough in investigating the structure and function of higher-order genome architecture. However, the mapping of global chromatin interactions remains challenging across many biological conditions due to high background noise and financial constraints, especially for small laboratories. Here, we describe the Bridge linker-Alul-Tn5 Hi-C (BAT Hi-C) method, which is a simple and efficient method for delineating chromatin conformational features of mouse embryonic stem (mES) cells and uncover DNA loops. This protocol combines Alul fragmentation and biotinylated linker-mediated proximity ligation to obtain kilobase (kb) resolution with a marked increase in the amount of unique read pairs. The protocol also includes chromatin isolation to reduce background noise and Tn5 tagmentation to cut down on preparation time. Importantly, with only one-third sequencing depth, our method revealed the same spectrum of chromatin contacts as in situ Hi-C. BAT Hi-C is an economical (i.e., approximately $40 for library preparation) and straightforward (total hands-on time of 3 days) tool that is ideal for the in-depth analysis of long-range chromatin looping events in a genome-wide fashion.

3175 – HEMATOPOIETIC STEM CELL-DEPENDENT NOTCH TRANSCRIPTION IS MEDIATED BY P53 THROUGH THE HISTONE CHAPERONE SUPT16

Hematopoietic stem cells (HSCs) are rare cells that can self-renew and differentiate into all blood cell lineages for life. HSCs have long been the focus of developmental and regenerative studies, yet our understanding of the signaling events regulating their specification remains incomplete. In order to identify novel genes and transcription factors involved in hematopoietic specification, we performed a forward genetic screen to identify zebrafish mutants defective in HSC formation. Through large-scale whole mount in situ hybridization based screens followed by RNA-sequencing-based linkage mapping, we identified that supt16h, a component of the FAcilitates Chromatin Transcription (FACT) complex, is required for HSC formation. Zebrafish supt16h mutants express reduced levels of Notch signaling components, genes essential for HSC development, due to abrogated transcription. Although cellular functions of Supt16h is generally known for regulating transcription and reorganizing nucleosomes to alter chromatin accessibility, global chromatin accessibility in the zebrafish supt16h mutants is unaffected. However, we observe a specific increase in accessibility at the p53 locus leading to an accumulation of P53 protein in the supt16h mutants and abrogation of increased p53 levels in supt16h mutants rescues both loss of Notch and HSC phenotypes. We further demonstrate that P53 levels directly influence expression of the Polycomb Group protein, Phc1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues both loss of Notch and loss of HSC phenotypes in supt16h mutants. Taken together, our results highlight a previously uncharacterized relationship between supt16h, p53, and phc1 to specify HSCs via modulation of Notch signaling.

29 Introduction to functional annotation of animal genomes consortium

Abstract The annotation of the functional components of genomes is required for both basic and applied genomic research, yet farmed animal genomics are deficient in such annotation. This talk will introduce the Functional Annotation of ANimal Genomes (FAANG) consortium, which was initiated in 2014 to address this knowledge deficit. An overarching theme of early FAANG efforts is the collaborative approach required for such comprehensive research, thus another emphasis of this talk will be our community efforts to develop the environment for successful FAANG research. FAANG scientists have created a coordinated data collection and analysis enterprise crucial for success of this global effort, and funding of ~$20 million has been secured for current projects in several species, with an anticipated doubling of this support during 2019. Member FAANG consortium labs are producing genome-wide datasets on RNA expression, DNA methylation, chromatin modification, chromatin accessibility, and chromatin interactions for many agriculturally relevant animal species. The goal of a first-generation global chromatin state map for cattle, chicken, pig, and potentially other species is projected for completion in the next 3–5 years. These data will be used both to better understand animal genome function at the epigenetic level, as well as improve the precision and sensitivity of genomic selection for animal improvement. The functional annotation delivered by the FAANG initiative will add value and utility to the greatly improved genome sequences being established for domesticated animal species.

Ovine oocytes display a similar germinal vesicle configuration and global DNA methylation at prepubertal and adult ages

Epigenetic mechanisms are thought to be involved in the reduced developmental capacity of early prepubertal ewe oocytes compared to their adult counterparts. In this study, we have analyzed the global DNA methylation pattern and in vitro meiotic and developmental competence of oocytes at the germinal vesicle (GV) stage obtained from adult and 3-month-old donors. All oocytes were aspirated from antral follicles with a diameter ≥3 mm, and DNA methylation on 5-methylcytosine was detected by immunofluorescence using an anti-methyl cytosine antibody. The main global chromatin configuration pattern shown by both prepubertal and adult ovine oocytes corresponded to condensed chromatin localized close to the nuclear envelope (the SNE pattern). Immunofluorescence showed that a global bright nuclear staining of 5-methylcytosine (5-mC) occurred in all germinal vesicle stage oocytes and matched the propidium iodide staining pattern. The total fluorescence intensity values of lamb GVs were not lower than those observed in adult GVs. The meiotic competence and cleavage rates were similar in adult and prepubertal oocytes, however, the developmental competence of embryos to reach blastocysts was higher for adult oocytes than lamb oocytes (p<0.0001). In conclusion, our results indicate that adult-size oocytes derived from 3 to 4 month old prepubertal ewes show similar GV morphology and DNA methylation staining patterns to those obtained from adult animals, despite exhibiting a lower developmental competence.

Global DNA Methylation Patterns in Human Gliomas and Their Interplay with Other Epigenetic Modifications.

During the last two decades, several international consortia have been established to unveil the molecular background of human cancers including gliomas. As a result, a huge outbreak of new genetic and epigenetic data appeared. It was not only shown that gliomas share some specific DNA sequence aberrations, but they also present common alterations of chromatin. Many researchers have reported specific epigenetic features, such as DNA methylation and histone modifications being involved in tumor pathobiology. Unlike mutations in DNA, epigenetic changes are more global in nature. Moreover, many studies have shown an interplay between different types of epigenetic changes. Alterations in DNA methylation in gliomas are one of the best described epigenetic changes underlying human pathology. In the following work, we present the state of knowledge about global DNA methylation patterns in gliomas and their interplay with histone modifications that may affect transcription factor binding, global gene expression and chromatin conformation. Apart from summarizing the impact of global DNA methylation on glioma pathobiology, we provide an extract of key mechanisms of DNA methylation machinery.

Global Chromatin Profiling Fingerprints Reveal Therapeutic Efficacy in Breast Cancer

Regulatory abnormalities caused by epigenetic changes due to chromatin modifications are being increasingly recognized as contributors to cancer. While many molecularly targeted drugs have the potential to revert these modifications, their precise mechanism of action in cellular reprogramming is yet to be deciphered. We generated “network-based global chromatin profiling fingerprints” by integrating proteomic/phosphoproteomic, transcriptomic and regulatory genomic data to understand how unique chromatin alterations by post-translational histone modifications regulate cell state changes when treated with drugs in breast cancer. We find H3K27me3K36me3 as a key fingerprint, mediated by chromatin remodelers BRD4, NSD3, EZH2, and a proto-oncogene MYC. We show CDK inhibitors flavopiridol and dinaciclib display selective inhibitory potential toward BRD4/MYC, implicating them as potential therapeutic targets to restitute H3K27me3K36me3 status in breast cancer.

Abstract 5198: 5AZA and decitabine exhibit different molecular effects on non-Hodgkin lymphoma cells: Involvement of DNMT3a

Abstract Introductory sentence. Demethylating drugs represent a powerful tool for epigenetic modulation of chromatin structure in cancer cells. 5-azacytidine (5-AZA) and 5-aza-2’-deoxycytidine (decitabine) are promising therapeutic solutions also in mature B-cell neoplasms, although the molecular mechanisms of action have yet to be elucidated. Here we describe their effects on non-Hodgkin lymphoma cells and demonstrate how hypomethylation is not homogeneous across the genome. Experimental procedures. Toledo (GC-derived) and NU-DUL-1 (ABC-like) diffuse large-B cell lymphoma (DLBCL; non-Hodgkin) cell lines. Peripheral blood mononuclear cells (PBMCs) isolation. Epitect methyl II methylation qPCR and global chromatin methylation quantitative assays. Immunoblottings. Proliferation assays. CpG islands bioinformatics analysis. Comet assay and phosphorylation of gamma-H2AX histone assay. Gene silencing through siRNAs. New data. A panel of 9 CpG islands within a set of genes that we previously identified has been investigated as a potential target for hypomethylating activity of 5AZA and decitabine. KLF4, DAPK1 and SPG20 promoters (located on chr. n°9 the first two and n°3 the latter) are not affected by a single dose of 5AZA after 48h or 7 days of incubation (the CpG islands within these promoters are fully unmethylated in healthy PBMCs). However, global chromatin demethylation is clearly visible either after 48h or 7 days in both cell lines. By comparison, decitabine demethylating effects return to the initial values after 7 days. MZB1 promoter results demethylated by both treatments, while MGMT is demethylated only in NU-DUL-1 by decitabine but not 5AZA. These data suggest that hypomethylating agents act selectively on discrete regions of the genome. 5AZA and decitabine markedly down-regulate DNMT1 in a dose-dependent fashion and activate PARP. Despite DNMT1 downregulation, KLF4, DAPK1 and SPG20 promoters result unaffected. DNMT1 silencing in our cells do not cause any change in the promoter methylation of the selected targets. The de novo methyltransferase DNMT3a is expressed only by NU-DUL-1 and its silencing leads to a partial MZB1 demethylation and to the significant decrease in global chromatin methylation whereas DNMT1 silencing does not. In order to assess an involvement of the genotoxic damage, Comet assays were performed up to 24 hours of incubation with the drugs. We demonstrate that 5AZA does not induce any significant genotoxic damage while decitabine causes a tail formation starting at 6h after drug administration. Conclusions. Our data show that 5AZA and decitabine exhibit different mechanisms of action on lymphoma cells. Different DLBCL-derived cell lines display different changes when exposed to single doses of hypomethylating drugs, underlying the fact that cell of origin may play a role during the response. DNMT3a contributes to chromatin methylation in lymphoma cells. Citation Format: Raffaele Frazzi, Tonia De Simone, Olga Serra, Annamaria Buschini, Laura Canovi, Tiziana De Luca, Francesco Merli. 5AZA and decitabine exhibit different molecular effects on non-Hodgkin lymphoma cells: Involvement of DNMT3a [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 5198.

Abstract 4335: Epigenetic plasticity potentiates a rapid cyclical shift to and from an aggressive cancer phenotype

Abstract Introduction: Subpopulations of highly tumorigenic, drug resistant cancer stem-like cells (CSCs) play a key role in cancer recurrence and progression. Surprisingly, these aggressive cells can arise repeatedly de novo from bulk tumor cells independently of mutational events. We investigated whether transition to and from a cancer stem-like state is associated with epigenetic alterations, such as DNA methylation and chromatin accessibility. Materials and methods: Using FACS and Hoechst dye staining, side population (SP) cells marked by high tumorigenicity and drug resistance vs. non-side population (NSP) cells lacking these properties were isolated from bladder cancer cell lines. Global DNA methylation and chromatin accessibility were mapped for SP vs. NSP cells using AcceSssIble assay on the Infinium EPIC platform. Differential gene expression for SP vs. NSP cells was assayed by HuEx array. AcceSssIble and HuEx results were overlapped to generate a subset of genes with differential accessible promoter regions and expression. One such gene, E2F3, was validated by qRT-PCR and analyzed clinically using the Oncomine and Basespace online data mining engines, as well as functionally by lentiviral shRNA depletion in bladder cancer cells for cell migration, invasion and drug resistance. 5-AZA was used to modulate global DNA methylation in bladder cancer cells. Results: SP and NSP subpopulations exhibited differential DNA methylation and chromatin accessibility at thousands of gene loci by AcceSssIble assay, and a majority of differential chromatin accessibility was independent of DNA methylation. When overlapped with HuEx data, 38 genes exhibited differential expression associated with differential promoter chromatin accessibility. One such gene validated by qRT-PCR was E2F3, a cell cycle regulator that was found to be associated with bladder cancer in the Oncomine and Basespace databases. In vitro E2F3 depletion by lentiviral shRNA resulted in smaller SP subpopulations and reduced cell migration, invasiveness, and drug resistance. Epigenetic interference with 5-AZA blocked the phenotypic transition, reduced SP subpopulations, and lowered E2F3 expression. Conclusion: Rapid cyclical transition between the SP and NSP subpopulations was associated with dramatic shifts in chromatin accessibility with concomitant changes in gene expression of clinically relevant genes that drove the SP phenotype, cell migration, invasiveness, and drug resistance. These observations implicate epigenetic plasticity as a driver of the switch between cancer stem-like and non-cancer stem-like states, suggesting new potential therapeutic avenues to control cancer resistance and progression. Citation Format: Tong Xu, Hongtao Li, Rong Xu, Jenny Wei, Meng Li, Gangning Liang, Amir Goldkorn. Epigenetic plasticity potentiates a rapid cyclical shift to and from an aggressive cancer phenotype [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 4335.

Abstract 2883: Mithramycin inhibits KDM6A (UTX) and SWI/SNF to induce apoptosis in malignant rhabdoid tumor

Abstract BACKGROUND: Rhabdoid tumor (RT) is an aggressive pediatric malignancy defined by the genetic deletion of SMARCB1 (BAF47/SNF5/INI1), a subunit of the SWI/SNF chromatin remodeling complex. SMARCB1 loss redistributes the SWI/SNF complex to favor a proliferative non-differentiated cellular state. Nevertheless, while EZH2, the catalytic subunit of PRC2, is an established, synthetically lethal therapeutic target, other vulnerabilities that result from this imbalance between the SWI/SNF and PRC2 complexes are relatively unexplored. METHODS: In a screen of multiple pediatric cancer cell lines, we identified a heightened sensitivity of RT cells to mithramycin (MMA), a small molecule transcription inhibitor. We hypothesized the hypersensitivity of rhabdoid tumor to mithramycin was due to the disruption of SWI/SNF and PRC2 dynamics. Here, we describe an epigenetic mechanism of action for mithramycin in rhabdoid tumor. We characterize KDM6A, a H3K27me3 histone demethylase, as a novel therapeutic vulnerability in rhabdoid tumor with live cell imaging, western blot, small molecule inhibition, and chromatin immunoprecipitation (ChIP) sequencing. We identify SWI/SNF eviction from chromatin as a novel mechanism of action for mithramycin with biochemical fractionation, ChIP-seq, and ATAC-seq. Finally, we demonstrate these findings in vivo using RT xenograft models. RESULTS: Mithramycin induced apoptosis and a striking increase in H3K27me3, the catalytic mark of PRC2, in rhabdoid tumor cell lines in a dose and time-dependent manner. This amplification of H3K27me3 preceded suppression of cellular proliferation and induction of apoptosis suggesting H3K27me3 amplification may drive the apoptotic phenotype. In addition, knockdown of KDM6A, a H3K27me3 histone demethylase, potentiates the effects of MMA providing further evidence that KDM6A loss and H3K27me3 amplification lead to apoptosis. We have identified SP1 as the transcription factor driving KDM6A and mithramycin interferes with SP1-dependent activation of KDM6A by evicting SWI/SNF from chromatin. We are currently working on the mechanism of action for mithramycin in rhabdoid tumor cells, including H3K27me3 ChIP-seq and ATAC-seq to elucidate global transcription and chromatin changes. We have recapitulated these results in vivo with xenograft mouse models. CONCLUSIONS: Mithramycin inhibits KDM6A, a histone demethylase, and SWI/SNF leading to apoptosis through the amplification of H3K27me3. Eviction of SWI/SNF from chromatin is a novel mechanism of action for mithramycin. Overall, this study indicates KDM6A and SWI/SNF loss are novel therapeutic vulnerabilities in rhabdoid tumor and mithramycin may be a promising clinical candidate for the treatment of rhabdoid tumor. Citation Format: Maggie H. Chasse, Elissa Boguslawski, Katie Sorensen, Ian Beddows, Kristin Rybski, Zachary Madaj, Susan M. Kitchen-Goosen, Patrick J. Grohar. Mithramycin inhibits KDM6A (UTX) and SWI/SNF to induce apoptosis in malignant rhabdoid tumor [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 2883.

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.

Mitotic Chromosome Mechanics: How Cells Segregate Their Genome.

During mitosis, replicated chromosomes segregate such that each daughter cell receives one copy of the genome. Faithful mechanical transport during mitosis requires that chromosomes undergo extensive structural changes as the cell cycle progresses, resulting in the formation of compact, cylindrical bodies. Such structural changes encompass a range of different activities, including longitudinal condensation of the chromosome axis, global chromatin compaction, resolution of sister chromatids, and individualisation of chromosomes into separate bodies. After mitosis, chromosomes undergo further reorganisation to rebuild interphase cell nuclei. Here we review the requirements for mitotic chromosomes to successfully transmit genetic information to daughter cells and the biophysical principles that underpin such requirements.

Assessment of the global chromatin landscape and transcriptome of peripheral iNKT cell subsets

Abstract Vα14i invariant Natural Killer T (iNKT) cells are important modulators of immune responses. They differentiate into three effector cell subsets in the thymus, NKT1, NKT2, and NKT17, which resemble Th1, Th2, and Th17 CD4 T cells. After exiting the thymus, iNKT cells localize to tissues and do not recirculate, but the impact of tissue localization on the epigenetic landscape and transcriptome of each subset is not known. We assessed global chromatin accessibility and transcriptional activity using ATACseq and RNAseq in iNKT cell subsets isolated from the thymus, spleen, liver, and lung. By comparing iNKT cell subsets within each tissue, we found over 5,000 differentially accessible regions of chromatin. This is consistent with previous analyses showing great differences in the transcriptomes of the thymic iNKT cell subsets. Interestingly, relatively limited differences in the accessible chromatin or transcriptome were observed when comparing the same subset from different sites, e.g., NKT1 cells from different tissues. An exception is the unique set of accessible chromatin regions and transcripts identified in all iNKT cell subsets in the lung. Lung iNKT cells exhibited features of increased AP-1 transcription factor activity, which has been correlated with both activation and tissue residency, found in other lung lymphocyte populations. After antigen challenge, an iNKT cell subset with features similar to T follicular helper cells is abundant; these cells are highly different from the other subsets. Taken together, these data indicate that the epigenome and transcriptome of iNKT cells is highly impacted by subset decision, and much less by tissue localization, although dynamic changes occur after antigen exposure.

Su1833 – Elevated Cap-D3 Levels in Intestinal Epithelial Cells Cause Global Chromatin Disorganization and May Have Implications for Crohn’s Disease

Su1833 – Elevated Cap-D3 Levels in Intestinal Epithelial Cells Cause Global Chromatin Disorganization and May Have Implications for Crohn’s Disease

Angiotensin II and TGF-β1 Induce Alterations in Human Amniotic Fluid-Derived Mesenchymal Stem Cells Leading to Cardiomyogenic Differentiation Initiation.

Background and ObjectivesHuman amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may be a valuable source for cardiovascular tissue engineering and cell therapy. The aim of this study is to verify angiotensin II and transforming growth factor-beta 1 (TGF-β1) as potential cardiomyogenic differentiation inducers of AF-MSCs.Methods and ResultsAF-MSCs were obtained from amniocentesis samples from second-trimester pregnant women, isolated and characterized by the expression of cell surface markers (CD44, CD90, CD105 positive; CD34 negative) and pluripotency genes (OCT4, SOX2, NANOG, REX1). Cardiomyogenic differentiation was induced using different concentrations of angiotensin II and TGF-β1. Successful initiation of differentiation was confirmed by alterations in cell morphology, upregulation of cardiac genes-markers NKX2-5, TBX5, GATA4, MYH6, TNNT2, DES and main cardiac ion channels genes (sodium, calcium, potassium) as determined by RT-qPCR. Western blot and immunofluorescence analysis revealed the increased expression of Connexin43, the main component of gap junctions, and Nkx2.5, the early cardiac transcription factor. Induced AF-MSCs switched their phenotype towards more energetic and started utilizing oxidative phosphorylation more than glycolysis for energy production as assessed using Agilent Seahorse XF analyzer. The immune analysis of chromatin-modifying enzymes DNMT1, HDAC1/2 and Polycomb repressive complex 1 and 2 (PRC1/2) proteins BMI1, EZH2 and SUZ12 as well as of modified histones H3 and H4 indicated global chromatin remodeling during the induced differentiation.ConclusionsAngiotensin II and TGF-β1 are efficient cardiomyogenic inducers of human AF-MSCs; they initiate alterations at the gene and protein expression, metabolic and epigenetic levels in stem cells leading towards cardiomyocyte-like phenotype formation.

Robust CTCF-Based Chromatin Architecture Underpins Epigenetic Changes in the Heart Failure Stress-Gene Response.

The human genome folds in 3 dimensions to form thousands of chromatin loops inside the nucleus, encasing genes and cis-regulatory elements for accurate gene expression control. Physical tethers of loops are anchored by the DNA-binding protein CTCF and the cohesin ring complex. Because heart failure is characterized by hallmark gene expression changes, it was recently reported that substantial CTCF-related chromatin reorganization underpins the myocardial stress-gene response, paralleled by chromatin domain boundary changes observed in CTCF knockout. We undertook an independent and orthogonal analysis of chromatin organization with mouse pressure-overload model of myocardial stress (transverse aortic constriction) and cardiomyocyte-specific knockout of Ctcf. We also downloaded published data sets of similar cardiac mouse models and subjected them to independent reanalysis. We found that the cardiomyocyte chromatin architecture remains broadly stable in transverse aortic constriction hearts, whereas Ctcf knockout resulted in ≈99% abolition of global chromatin loops. Disease gene expression changes correlated instead with differential histone H3K27-acetylation enrichment at their respective proximal and distal interacting genomic enhancers confined within these static chromatin structures. Moreover, coregulated genes were mapped out as interconnected gene sets on the basis of their multigene 3D interactions. This work reveals a more stable genome-wide chromatin framework than previously described. Myocardial stress-gene transcription responds instead through H3K27-acetylation enhancer enrichment dynamics and gene networks of coregulation. Robust and intact CTCF looping is required for the induction of a rapid and accurate stress response.

MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation

Actin cytoskeleton is well-known for providing structural/mechanical support, but whether and how it regulates chromatin and cell fate reprogramming is far less clear. Here, we report that MKL1, the key transcriptional co-activator of many actin cytoskeletal genes, regulates genomic accessibility and cell fate reprogramming. The MKL1-actin pathway weakens during somatic cell reprogramming by pluripotency transcription factors. Cells that reprogram efficiently display low endogenous MKL1 and inhibition of actin polymerization promotes mature pluripotency activation. Sustained MKL1 expression at a level seen in typical fibroblasts yields excessive actin cytoskeleton, decreases nuclear volume and reduces global chromatin accessibility, stalling cells on their trajectory toward mature pluripotency. In addition, the MKL1-actin imposed block of pluripotency can be bypassed, at least partially, when the Sun2-containing linker of the nucleoskeleton and cytoskeleton (LINC) complex is inhibited. Thus, we unveil a previously unappreciated aspect of control on chromatin and cell fate reprogramming exerted by the MKL1-actin pathway.