Combined Chromatin Immunoprecipitation and Bisulfite Methylation Sequencing Analysis

Abstract Aim Reduced calcium sensing receptor (CaSR) expression has been implicated in parathyroid tumorigenesis, but the underlying mechanism remains elusive. Accordingly, we aimed to explore the epigenetic changes (DNA methylation and histone modifications) involved in CaSR regulation in sporadic parathyroid adenomas and correlate epigenetic state with disease indices. Experimental Design Forty sporadic parathyroid adenomas and 10 control parathyroid tissues were studied. Real-time quantitative PCR (qPCR) for mRNA and immunohistochemistry for protein expression of CaSR were performed. The methylation status of the CaSR promoter 2 was determined by bisulphite sequencing analysis of sodium bisulphite-converted DNA. To determine the role of histone modifications in the CaSR regulation, chromatin immunoprecipitation-qPCR assay was performed. Results Real-time qPCR revealed reduced CaSR mRNA expression with a fold reduction of 0.12 (P < 0.0001) in parathyroid adenomas. Immunohistochemistry revealed reduced protein expression of CaSR in 90% (36/40) of adenomas. The promoter 2 region of CaSR displayed significant hypermethylation in 45% (18/40) of the adenomas compared with the controls (6.7%; 1 of 10) (P < 0.002). Bisulphite sequencing analysis revealed maximum methylated CpG at glial cell missing 2 binding site on the CaSR promoter 2 compared to other CpG sites. The methylation status of CaSR correlated directly with plasma intact parathyroid hormone levels in patients with parathyroid adenoma. With chromatin immunoprecipitation-qPCR analysis, H3K9me3 levels showed increased enrichment by 10-fold in adenomas and correlated with CaSR-mRNA expression (r = 0.61; P < 0.003). Treatment with 5-aza-2′deoxycytidine restored the expression of CaSR in a parathyroid cell line. Conclusion Our data suggest that hypermethylation and increased H3K9me3 of the CaSR promoter 2 are involved in silencing CaSR expression in sporadic parathyroid adenoma.

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Negative MAPK-ERK regulation sustains CIC-DUX4 oncoprotein expression in undifferentiated sarcoma

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2009137117 ◽

2020 ◽

Vol 117 (34) ◽

pp. 20776-20784 ◽

Cited By ~ 2

Author(s):

Yone Kawe Lin ◽

Wei Wu ◽

Rovingaile Kriska Ponce ◽

Ji Won Kim ◽

Ross A. Okimoto

Keyword(s):

Chromatin Immunoprecipitation ◽

Soft Tissue Sarcomas ◽

Erk Signaling ◽

Sequencing Analysis ◽

Round Cell ◽

Undifferentiated Sarcoma ◽

Erk Activation ◽

Chromatin Immunoprecipitation Sequencing ◽

Erk Activity ◽

Apoptotic Induction

Transcription factor fusions (TFFs) are present in ∼30% of soft-tissue sarcomas. TFFs are not readily “druggable” in a direct pharmacologic manner and thus have proven difficult to target in the clinic. A prime example is the CIC-DUX4 oncoprotein, which fuses Capicua (CIC) to the double homeobox 4 gene, DUX4. CIC-DUX4 sarcoma is a highly aggressive and lethal subtype of small round cell sarcoma found predominantly in adolescents and young adults. To identify new therapeutic targets in CIC-DUX4 sarcoma, we performed chromatin immunoprecipitation sequencing analysis using patient-derived CIC-DUX4 cells. We uncovered multiple CIC-DUX4 targets that negatively regulate MAPK-ERK signaling. Mechanistically, CIC-DUX4 transcriptionally up-regulates these negative regulators of MAPK to dampen ERK activity, leading to sustained CIC-DUX4 expression. Genetic and pharmacologic MAPK-ERK activation through DUSP6 inhibition leads to CIC-DUX4 degradation and apoptotic induction. Collectively, we reveal a mechanism-based approach to therapeutically degrade the CIC-DUX4 oncoprotein and provide a precision-based strategy to combat this lethal cancer.

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Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division inStreptomyces

mBio ◽

10.1128/mbio.00523-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 43

Author(s):

Matthew J. Bush ◽

Govind Chandra ◽

Maureen J. Bibb ◽

Kim C. Findlay ◽

Mark J. Buttner

Keyword(s):

Transcription Factor ◽

Cell Division ◽

Dna Binding ◽

Chromatin Immunoprecipitation ◽

Sequencing Analysis ◽

Founding Member ◽

Genome Wide ◽

Biochemical Function ◽

Chromatin Immunoprecipitation Sequencing

ABSTRACTWhiB is the founding member of a family of proteins (theWhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. InStreptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) inStreptomyces venezuelae. In the first demonstration ofin vivogenome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes ofwhiAandwhiBmutants. Using ChIP-seq, we demonstrated thatin vivoDNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiAin vivo.IMPORTANCEDespite the central importance ofWhiB-like (Wbl) proteins in actinomycete biology, a conclusive demonstration of their biochemical function has been elusive, and they have been difficult to study, particularlyin vitro, largely because they carry an oxygen-sensitive [4Fe-4S] cluster. Here we used genome-wide ChIP-seq to investigate the function ofStreptomycesWhiB, the founding member of the Wbl family. The advantage of this approach is that the oxygen sensitivity of the [4Fe-4S] cluster becomes irrelevant once the protein has been cross-linked to DNAin vivo. Our data provide the most compellingin vivoevidence to date that WhiB, and, by extension, probably all Wbl proteins, function as transcription factors. Further, we show that WhiB does not act independently but rather coregulates its regulon of sporulation genes with a partner transcription factor, WhiA.

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Stemness, Pluripotentiality, and Wnt Antagonism: sFRP4, a Wnt antagonist Mediates Pluripotency and Stemness in Glioblastoma

Cancers ◽

10.3390/cancers11010025 ◽

2018 ◽

Vol 11 (1) ◽

pp. 25 ◽

Cited By ~ 15

Author(s):

Gurubharathi Bhuvanalakshmi ◽

Naisarg Gamit ◽

Manasi Patil ◽

Frank Arfuso ◽

Gautam Sethi ◽

...

Keyword(s):

Stem Cells ◽

Rna Sequencing ◽

Chromatin Immunoprecipitation ◽

Sequencing Analysis ◽

Oxygen Species ◽

Related Protein ◽

Chip Sequencing ◽

Apoptosis Inducer ◽

Multipotent Mesenchymal Stem Cells ◽

U87 Cells

Background: Chemotherapeutic resistance of glioblastoma has been attributed to a self-renewing subpopulation, the glioma stem cells (GSCs), which is known to be maintained by the Wnt β−catenin pathway. Our previous findings demonstrated that exogeneous addition of the Wnt antagonist, secreted fizzled-related protein 4 (sFRP4) hampered stem cell properties in GSCs. Methods: To understand the molecular mechanism of sFRP4, we overexpressed sFRP4 (sFRP4 OE) in three human glioblastoma cell lines U87MG, U138MG, and U373MG. We also performed chromatin immunoprecipitation (ChIP) sequencing of sFRP4 OE and RNA sequencing of sFRP4 OE and sFRP4 knocked down U87 cells. Results: We observed nuclear localization of sFRP4, suggesting an unknown nuclear role. ChIP-sequencing of sFRP4 pulldown DNA revealed a homeobox Cphx1, related to the senescence regulator ETS proto-oncogene 2 (ETS2). Furthermore, miRNA885, a p53-mediated apoptosis inducer, was upregulated in sFRP4 OE cells. RNA sequencing analysis suggested that sFRP4-mediated apoptosis is via the Fas-p53 pathway by activating the Wnt calcium and reactive oxygen species pathways. Interestingly, sFRP4 OE cells had decreased stemness, but when knocked down in multipotent mesenchymal stem cells, pluripotentiality was induced and the Wnt β-catenin pathway was upregulated. Conclusions: This study unveils a novel nuclear role for sFRP4 to promote apoptosis by a possible activation of DNA damage machinery in glioblastoma.

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ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B–H4K8ac–BRD4 axis

Nucleic Acids Research ◽

10.1093/nar/gkaa815 ◽

2020 ◽

Vol 48 (19) ◽

pp. 10848-10866

Author(s):

Shuhei Ishikura ◽

Kazuhiko Nakabayashi ◽

Masayoshi Nagai ◽

Toshiyuki Tsunoda ◽

Senji Shirasawa

Keyword(s):

Rna Polymerase Ii ◽

Chromatin Immunoprecipitation ◽

Noncoding Rna ◽

High Throughput Sequencing ◽

Binding Motif ◽

Sequencing Analysis ◽

Histone H4 ◽

Mouse Cells ◽

Genomic Regions ◽

Human And Mouse

Abstract Centromeres are genomic regions essential for faithful chromosome segregation. Transcription of noncoding RNA (ncRNA) at centromeres is important for their formation and functions. Here, we report the molecular mechanism by which the transcriptional regulator ZFAT controls the centromeric ncRNA transcription in human and mouse cells. Chromatin immunoprecipitation with high-throughput sequencing analysis shows that ZFAT binds to centromere regions at every chromosome. We find a specific 8-bp DNA sequence for the ZFAT-binding motif that is highly conserved and widely distributed at whole centromere regions of every chromosome. Overexpression of ZFAT increases the centromeric ncRNA levels at specific chromosomes, whereas its silencing reduces them, indicating crucial roles of ZFAT in centromeric transcription. Overexpression of ZFAT increases the centromeric levels of both the histone acetyltransferase KAT2B and the acetylation at the lysine 8 in histone H4 (H4K8ac). siRNA-mediated knockdown of KAT2B inhibits the overexpressed ZFAT-induced increase in centromeric H4K8ac levels, suggesting that ZFAT recruits KAT2B to centromeres to induce H4K8ac. Furthermore, overexpressed ZFAT recruits the bromodomain-containing protein BRD4 to centromeres through KAT2B-mediated H4K8ac, leading to RNA polymerase II-dependent ncRNA transcription. Thus, ZFAT binds to centromeres to control ncRNA transcription through the KAT2B–H4K8ac–BRD4 axis.

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Constitutive centromere-associated network controls centromere drift in vertebrate cells

The Journal of Cell Biology ◽

10.1083/jcb.201605001 ◽

2016 ◽

Vol 216 (1) ◽

pp. 101-113 ◽

Cited By ~ 14

Author(s):

Tetsuya Hori ◽

Naoko Kagawa ◽

Atsushi Toyoda ◽

Asao Fujiyama ◽

Sadahiko Misu ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Chromatin Immunoprecipitation ◽

Sequencing Analysis ◽

Epigenetic Mechanisms ◽

Short Term ◽

Parallel Sequencing ◽

Cell Divisions ◽

Centromere Position ◽

Dt40 Cells

Centromeres are specified by sequence-independent epigenetic mechanisms, and the centromere position may drift at each cell cycle, but once this position is specified, it may not be frequently moved. Currently, it is unclear whether the centromere position is stable. To address this question, we systematically analyzed the position of nonrepetitive centromeres in 21 independent clones isolated from a laboratory stock of chicken DT40 cells using chromatin immunoprecipitation combined with massive parallel sequencing analysis with anti–CENP-A antibody. We demonstrated that the centromere position varies among the clones, suggesting that centromere drift occurs during cell proliferation. However, when we analyzed this position in the subclones obtained from one isolated clone, the position was found to be relatively stable. Interestingly, the centromere drift was shown to occur frequently in CENP-U– and CENP-S–deficient cells. Based on these results, we suggest that the centromere position can change after many cell divisions, but this drift is suppressed in short-term cultures, and the complete centromere structure contributes to the suppression of the centromere drift.

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Replication stress induces 53BP1-containing OPT domains in G1 cells

The Journal of Cell Biology ◽

10.1083/jcb.201011083 ◽

2011 ◽

Vol 193 (1) ◽

pp. 97-108 ◽

Cited By ~ 204

Author(s):

Jeanine A. Harrigan ◽

Rimma Belotserkovskaya ◽

Julia Coates ◽

Daniela S. Dimitrova ◽

Sophie E. Polo ◽

...

Keyword(s):

Dna Damage ◽

Chromatin Immunoprecipitation ◽

Massively Parallel Sequencing ◽

Fragile Sites ◽

Nuclear Bodies ◽

Sequencing Analysis ◽

Metaphase Chromosomes ◽

Common Fragile Sites ◽

Damage Response ◽

G1 Cells

Chromosomal deletions and rearrangements in tumors are often associated with common fragile sites, which are specific genomic loci prone to gaps and breaks in metaphase chromosomes. Common fragile sites appear to arise through incomplete DNA replication because they are induced after partial replication inhibition by agents such as aphidicolin. Here, we show that in G1 cells, large nuclear bodies arise that contain p53 binding protein 1 (53BP1), phosphorylated H2AX (γH2AX), and mediator of DNA damage checkpoint 1 (MDC1), as well as components of previously characterized OPT (Oct-1, PTF, transcription) domains. Notably, we find that incubating cells with low aphidicolin doses increases the incidence and number of 53BP1-OPT domains in G1 cells, and by chromatin immunoprecipitation and massively parallel sequencing analysis of γH2AX, we demonstrate that OPT domains are enriched at common fragile sites. These findings invoke a model wherein incomplete DNA synthesis during S phase leads to a DNA damage response and formation of 53BP1-OPT domains in the subsequent G1.

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