bims-crepig 2021-06-20 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2021–06–20
thirty-one papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit

3D genome alterations associated with dysregulated HOXA13 expression in high-risk T-lineage acute lymphoblastic leukemia.
Arginine methylation of METTL14 promotes RNA N6-methyladenosine modification and endoderm differentiation of mouse embryonic stem cells.
Chromatin-directed proteomics-identified network of endogenous androgen receptor in prostate cancer cells.
INO80 promotes H2A.Z occupancy to regulate cell fate transition in pluripotent stem cells.
BET family members Bdf1/2 modulate global transcription initiation and elongation in Saccharomyces cerevisiae.
Subtype-specific collaborative transcription factor networks are promoted by OCT4 in the progression of prostate cancer.
Methylscaper: an R/shiny app for joint visualization of DNA methylation and nucleosome occupancy in single-molecule and single-cell data.
Functional coordination of BET family proteins underlies altered transcription associated with memory impairment in fragile X syndrome.
Acute depletion of METTL3 implicates N6-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome.
Dnmt1 has de novo activity targeted to transposable elements.
NUCOME: A comprehensive database of nucleosome organization referenced landscapes in mammalian genomes.
Cell segmentation-free inference of cell types from in situ transcriptomics data.
Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening.
RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays.
Mapping chromatin accessibility and active regulatory elements reveals pathological mechanisms in human gliomas.
Increased peak detection accuracy in over-dispersed ChIP-seq data with supervised segmentation models.
TIMEOR: a web-based tool to uncover temporal regulatory mechanisms from multi-omics data.
DGLinker: flexible knowledge-graph prediction of disease-gene associations.
The epigenetic regulator LSH maintains fork protection and genomic stability via MacroH2A deposition and RAD51 filament formation.
Defective folate metabolism causes germline epigenetic instability and distinguishes Hira as a phenotype inheritance biomarker.
The loss of heterochromatin is associated with multiscale three-dimensional genome reorganization and aberrant transcription during cellular senescence.
Spatial and cell type transcriptional landscape of human cerebellar development.
NF-κB dynamics determine the stimulus specificity of epigenomic reprogramming in macrophages.
Hakai is required for stabilization of core components of the m6A mRNA methylation machinery.
SOX9 inactivation affects the proliferation and differentiation of human lung organoids.
Pancreas morphogenesis and homeostasis depends on tightly regulated Zeb1 levels in epithelial cells.
Epigenetics Identifier screens reveal regulators of chromatin acylation and limited specificity of acylation antibodies.
Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer.
SMARCA4 oncogenic potential via IRAK1 enhancer to activate Gankyrin and AKR1B10 in liver cancer.
RUNX2 Phosphorylation by Tyrosine Kinase ABL Promotes Breast Cancer Invasion.
Metabolic alterations mediated by STAT3 promotes drug persistence in CML.

Nat Commun. 2021 06 17. 12(1): 3708

3D genome alterations associated with dysregulated HOXA13 expression in high-risk T-lineage acute lymphoblastic leukemia.

Lu Yang, Fengling Chen, Haichuan Zhu, Yang Chen, Bingjie Dong, Minglei Shi, Weitao Wang, Qian Jiang, Leping Zhang, Xiaojun Huang, Michael Q Zhang, Hong Wu.

3D genome alternations can dysregulate gene expression by rewiring enhancer-promoter interactions and lead to diseases. We report integrated analyses of 3D genome alterations and differential gene expressions in 18 newly diagnosed T-lineage acute lymphoblastic leukemia (T-ALL) patients and 4 healthy controls. 3D genome organizations at the levels of compartment, topologically associated domains and loop could hierarchically classify different subtypes of T-ALL according to T cell differentiation trajectory, similar to gene expressions-based classification. Thirty-four previously unrecognized translocations and 44 translocation-mediated neo-loops are mapped by Hi-C analysis. We find that neo-loops formed in the non-coding region of the genome could potentially regulate ectopic expressions of TLX3, TAL2 and HOXA transcription factors via enhancer hijacking. Importantly, both translocation-mediated neo-loops and NUP98-related fusions are associated with HOXA13 ectopic expressions. Patients with HOXA11-A13 expressions, but not other genes in the HOXA cluster, have immature immunophenotype and poor outcomes. Here, we highlight the potentially important roles of 3D genome alterations in the etiology and prognosis of T-ALL.

DOI: https://doi.org/10.1038/s41467-021-24044-5
Nat Commun. 2021 Jun 18. 12(1): 3780

Arginine methylation of METTL14 promotes RNA N6-methyladenosine modification and endoderm differentiation of mouse embryonic stem cells.

Xiaona Liu, Hailong Wang, Xueya Zhao, Qizhi Luo, Qingwen Wang, Kaifen Tan, Zihan Wang, Jia Jiang, Jinru Cui, Enhui Du, Linjian Xia, Wenyi Du, Dahua Chen, Laixin Xia, Shan Xiao.

RNA N6-methyladenosine (m6A), the most abundant internal modification of mRNAs, plays key roles in human development and health. Post-translational methylation of proteins is often critical for the dynamic regulation of enzymatic activity. However, the role of methylation of the core methyltransferase METTL3/METTL14 in m6A regulation remains elusive. We find by mass spectrometry that METTL14 arginine 255 (R255) is methylated (R255me). Global mRNA m6A levels are greatly decreased in METTL14 R255K mutant mouse embryonic stem cells (mESCs). We further find that R255me greatly enhances the interaction of METTL3/METTL14 with WTAP and promotes the binding of the complex to substrate RNA. We show that protein arginine N-methyltransferases 1 (PRMT1) interacts with and methylates METTL14 at R255, and consistent with this, loss of PRMT1 reduces mRNA m6A modification globally. Lastly, we find that loss of R255me preferentially affects endoderm differentiation in mESCs. Collectively, our findings show that arginine methylation of METTL14 stabilizes the binding of the m6A methyltransferase complex to its substrate RNA, thereby promoting global m6A modification and mESC endoderm differentiation. This work highlights the crosstalk between protein methylation and RNA methylation in gene expression.

DOI: https://doi.org/10.1038/s41467-021-24035-6
Oncogene. 2021 Jun 14.

Chromatin-directed proteomics-identified network of endogenous androgen receptor in prostate cancer cells.

Kaisa-Mari Launonen, Ville Paakinaho, Gianluca Sigismondo, Marjo Malinen, Reijo Sironen, Jaana M Hartikainen, Hanna Laakso, Tapio Visakorpi, Jeroen Krijgsveld, Einari A Niskanen, Jorma J Palvimo.

Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating SMARCA4's functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target.

DOI: https://doi.org/10.1038/s41388-021-01887-2
Nucleic Acids Res. 2021 Jun 17. pii: gkab476. [Epub ahead of print]

INO80 promotes H2A.Z occupancy to regulate cell fate transition in pluripotent stem cells.

Hongyao Yu, Jiajia Wang, Brad Lackford, Brian Bennett, Jian-Liang Li, Guang Hu.

The INO80 chromatin remodeler is involved in many chromatin-dependent cellular functions. However, its role in pluripotency and cell fate transition is not fully defined. We examined the impact of Ino80 deletion in the naïve and primed pluripotent stem cells. We found that Ino80 deletion had minimal effect on self-renewal and gene expression in the naïve state, but led to cellular differentiation and de-repression of developmental genes in the transition toward and maintenance of the primed state. In the naïve state, INO80 pre-marked gene promoters that would adopt bivalent histone modifications by H3K4me3 and H3K27me3 upon transition into the primed state. In the primed state, in contrast to its known role in H2A.Z exchange, INO80 promoted H2A.Z occupancy at these bivalent promoters and facilitated H3K27me3 installation and maintenance as well as downstream gene repression. Together, our results identified an unexpected function of INO80 in H2A.Z deposition and gene regulation. We showed that INO80-dependent H2A.Z occupancy is a critical licensing step for the bivalent domains, and thereby uncovered an epigenetic mechanism by which chromatin remodeling, histone variant deposition and histone modification coordinately control cell fate.

DOI: https://doi.org/10.1093/nar/gkab476
Elife. 2021 Jun 17. pii: e69619. [Epub ahead of print]10

BET family members Bdf1/2 modulate global transcription initiation and elongation in Saccharomyces cerevisiae.

Rafal Donczew, Steven Hahn.

  Human bromodomain-containing BET family members are promising targets for therapy of cancer and immunoinflammatory diseases, but their mechanisms of action and functional redundancies are poorly understood. Bdf1/2, yeast homologues of the human BET factors, were previously proposed to target transcription factor TFIID to acetylated histone H4, analogous to bromodomains that are present within the largest subunit of metazoan TFIID. We investigated the genome-wide roles of Bdf1/2 and found that their important contributions to transcription extend beyond TFIID function, as transcription of many genes is more sensitive to Bdf1/2 than to TFIID depletion. Bdf1/2 co-occupy the majority of yeast promoters and affect preinitiation complex formation through recruitment of TFIID, Mediator and basal transcription factors to chromatin. Surprisingly, we discovered that hypersensitivity of genes to Bdf1/2 depletion results from combined defects in transcription initiation and early elongation, a striking functional similarity to human BET proteins, most notably Brd4. Our results establish Bdf1/2 as critical for yeast transcription and provide important mechanistic insights into the function of BET proteins in all eukaryotes.

Keywords:  S. cerevisiae; chromosomes; gene expression; genetics; genomics

DOI:  https://doi.org/10.7554/eLife.69619
Nat Commun. 2021 Jun 18. 12(1): 3766

Subtype-specific collaborative transcription factor networks are promoted by OCT4 in the progression of prostate cancer.

Ken-Ichi Takayama, Takeo Kosaka, Takashi Suzuki, Hiroshi Hongo, Mototsugu Oya, Tetsuya Fujimura, Yutaka Suzuki, Satoshi Inoue.

Interactive networks of transcription factors (TFs) have critical roles in epigenetic and gene regulation for cancer progression. It is required to clarify underlying mechanisms for transcriptional activation through concerted efforts of TFs. Here, we show the essential role of disease phase-specific TF collaboration changes in advanced prostate cancer (PC). Investigation of the transcriptome in castration-resistant PC (CRPC) revealed OCT4 as a key TF in the disease pathology. OCT4 confers epigenetic changes by promoting complex formation with FOXA1 and androgen receptor (AR), the central signals for the progression to CRPC. Meanwhile, OCT4 facilitates a distinctive complex formation with nuclear respiratory factor 1 (NRF1) to gain chemo-resistance in the absence of AR. Mechanistically, we reveal that OCT4 increases large droplet formations with AR/FOXA1 as well as NRF1 in vitro. Disruption of TF collaborations using a nucleoside analogue, ribavirin, inhibited treatment-resistant PC tumor growth. Thus, our findings highlight the formation of TF collaborations as a potent therapeutic target in advanced cancer.

DOI: https://doi.org/10.1038/s41467-021-23974-4
Bioinformatics. 2021 Jun 14. pii: btab438. [Epub ahead of print]

Methylscaper: an R/shiny app for joint visualization of DNA methylation and nucleosome occupancy in single-molecule and single-cell data.

Parker Knight, Marie-Pierre L Gauthier, Carolina E Pardo, Russell P Darst, Kevin Kapadia, Hadley Browder, Eliza Morton, Alberto Riva, Michael P Kladde, Rhonda Bacher.

SUMMARY: Differential DNA methylation and chromatin accessibility are associated with disease development, particularly cancer. Methods that allow profiling of these epigenetic mechanisms in the same reaction and at the single-molecule or single-cell level continue to emerge. However, a challenge lies in jointly visualizing and analyzing the heterogeneous nature of the data and extracting regulatory insight. Here, we present methylscaper, a visualization framework for simultaneous analysis of DNA methylation and chromatin accessibility landscapes. Methylscaper implements a weighted principal component analysis that orders DNA molecules, each providing a record of the chromatin state of one epiallele, and reveals patterns of nucleosome positioning, transcription factor occupancy, and DNA methylation. We demonstrate methylscaper's utility on a long-read, single-molecule methyltransferase accessibility protocol for individual templates (MAPit-BGS) dataset and a single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) dataset. In comparison to other procedures, methylscaper is able to readily identify chromatin features that are biologically relevant to transcriptional status while scaling to larger datasets.
AVAILABILITY AND IMPLEMENTATION: Methylscaper, is implemented in R (version > 4.1) and available on Bioconductor: https://bioconductor.org/packages/methylscaper/, GitHub: https://github.com/rhondabacher/methylscaper/, and Web: https://methylscaper.com.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btab438
Sci Adv. 2021 May;pii: eabf7346. [Epub ahead of print]7(21):

Functional coordination of BET family proteins underlies altered transcription associated with memory impairment in fragile X syndrome.

Seung-Kyoon Kim, Xihui Liu, Jongmin Park, Dahun Um, Gokhul Kilaru, Cheng-Ming Chiang, Mingon Kang, Kimberly M Huber, Keunsoo Kang, Tae-Kyung Kim.

Bromodomain and extraterminal proteins (BET) are epigenetic readers that play critical roles in gene regulation. Pharmacologic inhibition of the bromodomain present in all BET family members is a promising therapeutic strategy for various diseases, but its impact on individual family members has not been well understood. Using a transcriptional induction paradigm in neurons, we have systematically demonstrated that three major BET family proteins (BRD2/3/4) participated in transcription with different recruitment kinetics, interdependency, and sensitivity to a bromodomain inhibitor, JQ1. In a mouse model of fragile X syndrome (FXS), BRD2/3 and BRD4 showed oppositely altered expression and chromatin binding, correlating with transcriptional dysregulation. Acute inhibition of CBP/p300 histone acetyltransferase (HAT) activity restored the altered binding patterns of BRD2 and BRD4 and rescued memory impairment in FXS. Our study emphasizes the importance of understanding the BET coordination controlled by a balanced action between HATs with different substrate specificity.

DOI: https://doi.org/10.1126/sciadv.abf7346
Genome Res. 2021 Jun 15. pii: gr.271635.120. [Epub ahead of print]

Acute depletion of METTL3 implicates N6-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome.

Guifeng Wei, Mafalda Almeida, Greta Pintacuda, Heather Coker, Joseph S Bowness, Jernej Ule, Neil Brockdorff.

RNA N6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed cotranscriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m6A peaks are located in alternative introns/exons, often close to 5' splice sites. m6A peaks significantly overlap with RBM15 RNA-binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5' splice sites that are proximal to m6A peaks. For terminal or variable-length exons, m6A peaks are generally located on or immediately downstream of a 5' splice site that is suppressed in the presence of m6A, and upstream of a 5' splice site that is promoted in the presence of m6A. Genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m6A machinery and the regulation of RNA splicing.

DOI: https://doi.org/10.1101/gr.271635.120
Nat Struct Mol Biol. 2021 Jun 17.

Dnmt1 has de novo activity targeted to transposable elements.

Chuck Haggerty, Helene Kretzmer, Christina Riemenschneider, Abhishek Sampath Kumar, Alexandra L Mattei, Nina Bailly, Judith Gottfreund, Pay Giesselmann, Raha Weigert, Björn Brändl, Pascal Giehr, René Buschow, Christina Galonska, Ferdinand von Meyenn, Melissa B Pappalardi, Michael T McCabe, Lars Wittler, Claudia Giesecke-Thiel, Thorsten Mielke, David Meierhofer, Bernd Timmermann, Franz-Josef Müller, Jörn Walter, Alexander Meissner.

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.

DOI: https://doi.org/10.1038/s41594-021-00603-8
BMC Bioinformatics. 2021 Jun 13. 22(1): 321

NUCOME: A comprehensive database of nucleosome organization referenced landscapes in mammalian genomes.

Xiaolan Chen, Hui Yang, Guifen Liu, Yong Zhang.

   BACKGROUND: Nucleosome organization is involved in many regulatory activities in various organisms. However, studies integrating nucleosome organization in mammalian genomes are very limited mainly due to the lack of comprehensive data quality control (QC) assessment and uneven data quality of public data sets.
RESULTS: The NUCOME is a database focused on filtering qualified nucleosome organization referenced landscapes covering various cell types in human and mouse based on QC metrics. The filtering strategy guarantees the quality of nucleosome organization referenced landscapes and exempts users from redundant data set selection and processing. The NUCOME database provides standardized, qualified data source and informative nucleosome organization features at a whole-genome scale and on the level of individual loci.
CONCLUSIONS: The NUCOME provides valuable data resources for integrative analyses focus on nucleosome organization. The NUCOME is freely available at http://compbio-zhanglab.org/NUCOME .

Keywords:  Database; MNase; Nucleosome; Transcriptional regulation

DOI:  https://doi.org/10.1186/s12859-021-04239-9
Nat Commun. 2021 06 10. 12(1): 3545

Cell segmentation-free inference of cell types from in situ transcriptomics data.

Jeongbin Park, Wonyl Choi, Sebastian Tiesmeyer, Brian Long, Lars E Borm, Emma Garren, Thuc Nghi Nguyen, Bosiljka Tasic, Simone Codeluppi, Tobias Graf, Matthias Schlesner, Oliver Stegle, Roland Eils, Naveed Ishaque.

Multiplexed fluorescence in situ hybridization techniques have enabled cell-type identification, linking transcriptional heterogeneity with spatial heterogeneity of cells. However, inaccurate cell segmentation reduces the efficacy of cell-type identification and tissue characterization. Here, we present a method called Spot-based Spatial cell-type Analysis by Multidimensional mRNA density estimation (SSAM), a robust cell segmentation-free computational framework for identifying cell-types and tissue domains in 2D and 3D. SSAM is applicable to a variety of in situ transcriptomics techniques and capable of integrating prior knowledge of cell types. We apply SSAM to three mouse brain tissue images: the somatosensory cortex imaged by osmFISH, the hypothalamic preoptic region by MERFISH, and the visual cortex by multiplexed smFISH. Here, we show that SSAM detects regions occupied by known cell types that were previously missed and discovers new cell types.

DOI: https://doi.org/10.1038/s41467-021-23807-4
Cell. 2021 Jun 10. pii: S0092-8674(21)00629-2. [Epub ahead of print]

Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening.

Sandra Schilbach, Shintaro Aibara, Christian Dienemann, Frauke Grabbe, Patrick Cramer.

  Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.

Keywords:  RNA polymerase II; cryo-electron microscopy; in vitro transcription; pre-initiation complex; promoter DNA melting; promoter DNA opening; structure; transcription bubble; transcription factors; transcription initiation

DOI:  https://doi.org/10.1016/j.cell.2021.05.012
PLoS Genet. 2021 Jun 14. 17(6): e1009326

RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays.

Wei Guo, Dafang Wang, Damon Lisch.

In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on Mediator of Paramutation 1 (Mop1), a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via histone H3 lysine 9 dimethylation (H3K9me2), and histone H3 lysine27 dimethylation (H3K27me2), even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by histone H3 lysine 27 trimethylation (H3K27me3), a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild-type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.

DOI: https://doi.org/10.1371/journal.pgen.1009326
Nat Commun. 2021 06 15. 12(1): 3621

Mapping chromatin accessibility and active regulatory elements reveals pathological mechanisms in human gliomas.

Karolina Stępniak, Magdalena A Machnicka, Jakub Mieczkowski, Anna Macioszek, Bartosz Wojtaś, Bartłomiej Gielniewski, Katarzyna Poleszak, Malgorzata Perycz, Sylwia K Król, Rafał Guzik, Michał J Dąbrowski, Michał Dramiński, Marta Jardanowska, Ilona Grabowicz, Agata Dziedzic, Hanna Kranas, Karolina Sienkiewicz, Klev Diamanti, Katarzyna Kotulska, Wiesława Grajkowska, Marcin Roszkowski, Tomasz Czernicki, Andrzej Marchel, Jan Komorowski, Bozena Kaminska, Bartek Wilczyński.

Chromatin structure and accessibility, and combinatorial binding of transcription factors to regulatory elements in genomic DNA control transcription. Genetic variations in genes encoding histones, epigenetics-related enzymes or modifiers affect chromatin structure/dynamics and result in alterations in gene expression contributing to cancer development or progression. Gliomas are brain tumors frequently associated with epigenetics-related gene deregulation. We perform whole-genome mapping of chromatin accessibility, histone modifications, DNA methylation patterns and transcriptome analysis simultaneously in multiple tumor samples to unravel epigenetic dysfunctions driving gliomagenesis. Based on the results of the integrative analysis of the acquired profiles, we create an atlas of active enhancers and promoters in benign and malignant gliomas. We explore these elements and intersect with Hi-C data to uncover molecular mechanisms instructing gene expression in gliomas.

DOI: https://doi.org/10.1038/s41467-021-23922-2
BMC Bioinformatics. 2021 Jun 14. 22(1): 323

Increased peak detection accuracy in over-dispersed ChIP-seq data with supervised segmentation models.

Arnaud Liehrmann, Guillem Rigaill, Toby Dylan Hocking.

   BACKGROUND: Histone modification constitutes a basic mechanism for the genetic regulation of gene expression. In early 2000s, a powerful technique has emerged that couples chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq). This technique provides a direct survey of the DNA regions associated to these modifications. In order to realize the full potential of this technique, increasingly sophisticated statistical algorithms have been developed or adapted to analyze the massive amount of data it generates. Many of these algorithms were built around natural assumptions such as the Poisson distribution to model the noise in the count data. In this work we start from these natural assumptions and show that it is possible to improve upon them.
RESULTS: Our comparisons on seven reference datasets of histone modifications (H3K36me3 & H3K4me3) suggest that natural assumptions are not always realistic under application conditions. We show that the unconstrained multiple changepoint detection model with alternative noise assumptions and supervised learning of the penalty parameter reduces the over-dispersion exhibited by count data. These models, implemented in the R package CROCS ( https://github.com/aLiehrmann/CROCS ), detect the peaks more accurately than algorithms which rely on natural assumptions.
CONCLUSION: The segmentation models we propose can benefit researchers in the field of epigenetics by providing new high-quality peak prediction tracks for H3K36me3 and H3K4me3 histone modifications.

Keywords:  ChIP-seq; Histone modifications; Likelihood inference; Multiple changepoint detection; Over-dispersion; Peak calling; Supervised learning

DOI:  https://doi.org/10.1186/s12859-021-04221-5
Nucleic Acids Res. 2021 Jun 14. pii: gkab384. [Epub ahead of print]

TIMEOR: a web-based tool to uncover temporal regulatory mechanisms from multi-omics data.

Ashley Mae Conard, Nathaniel Goodman, Yanhui Hu, Norbert Perrimon, Ritambhara Singh, Charles Lawrence, Erica Larschan.

Uncovering how transcription factors regulate their targets at DNA, RNA and protein levels over time is critical to define gene regulatory networks (GRNs) and assign mechanisms in normal and diseased states. RNA-seq is a standard method measuring gene regulation using an established set of analysis stages. However, none of the currently available pipeline methods for interpreting ordered genomic data (in time or space) use time-series models to assign cause and effect relationships within GRNs, are adaptive to diverse experimental designs, or enable user interpretation through a web-based platform. Furthermore, methods integrating ordered RNA-seq data with protein-DNA binding data to distinguish direct from indirect interactions are urgently needed. We present TIMEOR (Trajectory Inference and Mechanism Exploration with Omics data in R), the first web-based and adaptive time-series multi-omics pipeline method which infers the relationship between gene regulatory events across time. TIMEOR addresses the critical need for methods to determine causal regulatory mechanism networks by leveraging time-series RNA-seq, motif analysis, protein-DNA binding data, and protein-protein interaction networks. TIMEOR's user-catered approach helps non-coders generate new hypotheses and validate known mechanisms. We used TIMEOR to identify a novel link between insulin stimulation and the circadian rhythm cycle. TIMEOR is available at https://github.com/ashleymaeconard/TIMEOR.git and http://timeor.brown.edu.

DOI: https://doi.org/10.1093/nar/gkab384
Nucleic Acids Res. 2021 Jun 14. pii: gkab449. [Epub ahead of print]

DGLinker: flexible knowledge-graph prediction of disease-gene associations.

Jiajing Hu, Rosalba Lepore, Richard J B Dobson, Ammar Al-Chalabi, Daniel M Bean, Alfredo Iacoangeli.

As a result of the advent of high-throughput technologies, there has been rapid progress in our understanding of the genetics underlying biological processes. However, despite such advances, the genetic landscape of human diseases has only marginally been disclosed. Exploiting the present availability of large amounts of biological and phenotypic data, we can use our current understanding of disease genetics to train machine learning models to predict novel genetic factors associated with the disease. To this end, we developed DGLinker, a webserver for the prediction of novel candidate genes for human diseases given a set of known disease genes. DGLinker has a user-friendly interface that allows non-expert users to exploit biomedical information from a wide range of biological and phenotypic databases, and/or to upload their own data, to generate a knowledge-graph and use machine learning to predict new disease-associated genes. The webserver includes tools to explore and interpret the results and generates publication-ready figures. DGLinker is available at https://dglinker.rosalind.kcl.ac.uk. The webserver is free and open to all users without the need for registration.

DOI: https://doi.org/10.1093/nar/gkab449
Nat Commun. 2021 06 10. 12(1): 3520

The epigenetic regulator LSH maintains fork protection and genomic stability via MacroH2A deposition and RAD51 filament formation.

Xiaoping Xu, Kai Ni, Yafeng He, Jianke Ren, Chongkui Sun, Yie Liu, Mirit I Aladjem, Sandra Burkett, Richard Finney, Xia Ding, Shyam K Sharan, Kathrin Muegge.

The Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is caused by mutations in LSH/HELLS, a chromatin remodeler promoting incorporation of histone variant macroH2A. Here, we demonstrate that LSH depletion results in degradation of nascent DNA at stalled replication forks and the generation of genomic instability. The protection of stalled forks is mediated by macroH2A, whose knockdown mimics LSH depletion and whose overexpression rescues nascent DNA degradation. LSH or macroH2A deficiency leads to an impairment of RAD51 loading, a factor that prevents MRE11 and EXO1 mediated nascent DNA degradation. The defect in RAD51 loading is linked to a disbalance of BRCA1 and 53BP1 accumulation at stalled forks. This is associated with perturbed histone modifications, including abnormal H4K20 methylation that is critical for BRCA1 enrichment and 53BP1 exclusion. Altogether, our results illuminate the mechanism underlying a human syndrome and reveal a critical role of LSH mediated chromatin remodeling in genomic stability.

DOI: https://doi.org/10.1038/s41467-021-23809-2
Nat Commun. 2021 06 17. 12(1): 3714

Defective folate metabolism causes germline epigenetic instability and distinguishes Hira as a phenotype inheritance biomarker.

Georgina E T Blake, Xiaohui Zhao, Hong Wa Yung, Graham J Burton, Anne C Ferguson-Smith, Russell S Hamilton, Erica D Watson.

The mechanism behind transgenerational epigenetic inheritance is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrr hypomorphic mutation results in transgenerational epigenetic inheritance of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. Here, we use genome-wide approaches to reveal genetic stability in the Mtrr model and genome-wide differential DNA methylation in the germline of Mtrr mutant maternal grandfathers. We observe that, while epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional changes that correlate with germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos for at least three wildtype generations in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance.

DOI: https://doi.org/10.1038/s41467-021-24036-5
Genome Res. 2021 Jun 17.

The loss of heterochromatin is associated with multiscale three-dimensional genome reorganization and aberrant transcription during cellular senescence.

Xianglin Zhang, Xuehui Liu, Zhenhai Du, Lei Wei, Huan Fang, Qiongye Dong, Jing Niu, Yanda Li, Juntao Gao, Michael Q Zhang, Wei Xie, Xiaowo Wang.

Heterochromatin remodeling is critical for various cell processes. In particular, the "loss of heterochromatin" phenotype in cellular senescence is associated with the process of aging and age-related disorders. Although biological processes of senescent cells, including senescence-associated heterochromatin foci (SAHF) formation, chromosome compaction, and redistribution of key proteins, have been closely associated with high-order chromatin structure, the relationship between the high-order chromatin reorganization and the loss of heterochromatin phenotype during senescence has not been fully understood. By using senescent and deep senescent fibroblasts induced by DNA damage harboring the "loss of heterochromatin" phenotype, we observed progressive 3D reorganization of heterochromatin during senescence. Facultative and constitutive heterochromatin marked by H3K27me3 and H3K9me3, respectively, show different alterations. Facultative heterochromatin tends to switch from the repressive B-compartment to the active A-compartment, whereas constitutive heterochromatin shows no significant changes at the compartment level but enhanced interactions between themselves. Both types of heterochromatin show increased chromatin accessibility and gene expression leakage during senescence. Furthermore, increased chromatin accessibility in potential CTCF binding sites accompanies the establishment of novel loops in constitutive heterochromatin. Finally, we also observed aberrant expression of repetitive elements, including LTR (long terminal repeat) and satellite classes. Overall, facultative and constitutive heterochromatin show both similar and distinct multiscale alterations in the 3D map, chromatin accessibility, and gene expression leakage. This study provides an epigenomic map of heterochromatin reorganization during senescence.

DOI: https://doi.org/10.1101/gr.275235.121
Nat Neurosci. 2021 Jun 17.

Spatial and cell type transcriptional landscape of human cerebellar development.

Kimberly A Aldinger, Zachary Thomson, Ian G Phelps, Parthiv Haldipur, Mei Deng, Andrew E Timms, Matthew Hirano, Gabriel Santpere, Charles Roco, Alexander B Rosenberg, Belen Lorente-Galdos, Forrest O Gulden, Diana O'Day, Lynne M Overman, Steven N Lisgo, Paula Alexandre, Nenad Sestan, Dan Doherty, William B Dobyns, Georg Seelig, Ian A Glass, Kathleen J Millen.

The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.

DOI: https://doi.org/10.1038/s41593-021-00872-y
Science. 2021 Jun 18. 372(6548): 1349-1353

NF-κB dynamics determine the stimulus specificity of epigenomic reprogramming in macrophages.

Quen J Cheng, Sho Ohta, Katherine M Sheu, Roberto Spreafico, Adewunmi Adelaja, Brooks Taylor, Alexander Hoffmann.

The epigenome of macrophages can be reprogrammed by extracellular cues, but the extent to which different stimuli achieve this is unclear. Nuclear factor κB (NF-κB) is a transcription factor that is activated by all pathogen-associated stimuli and can reprogram the epigenome by activating latent enhancers. However, we show that NF-κB does so only in response to a subset of stimuli. This stimulus specificity depends on the temporal dynamics of NF-κB activity, in particular whether it is oscillatory or non-oscillatory. Non-oscillatory NF-κB opens chromatin by sustained disruption of nucleosomal histone-DNA interactions, enabling activation of latent enhancers that modulate expression of immune response genes. Thus, temporal dynamics can determine a transcription factor's capacity to reprogram the epigenome in a stimulus-specific manner.

DOI: https://doi.org/10.1126/science.abc0269
Nat Commun. 2021 Jun 18. 12(1): 3778

Hakai is required for stabilization of core components of the m6A mRNA methylation machinery.

Praveen Bawankar, Tina Lence, Chiara Paolantoni, Irmgard U Haussmann, Migle Kazlauskiene, Dominik Jacob, Jan B Heidelberger, Florian M Richter, Mohanakarthik P Nallasivan, Violeta Morin, Nastasja Kreim, Petra Beli, Mark Helm, Martin Jinek, Matthias Soller, Jean-Yves Roignant.

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.

DOI: https://doi.org/10.1038/s41467-021-23892-5
Stem Cell Res Ther. 2021 Jun 10. 12(1): 343

SOX9 inactivation affects the proliferation and differentiation of human lung organoids.

Lian Li, Jianqi Feng, Shanshan Zhao, Zhili Rong, Ying Lin.

   BACKGROUND: The regulation of the transcription factor sex-determining region Y-box transcription factor 9 (SOX9) in lung development has been described in mouse, but the same principles apply to human lung development is unknown due to a lack of appropriate experimental approaches and models.
METHODS: Here, we used gene editing technology to inactivate SOX9 in human embryonic stem cells that were then induced to differentiate into lung organoids to investigate the role of SOX9 in human lung epithelium development.
RESULTS: Complete knockout of the transactivation domain of SOX9 by gene editing resulted in indels in both alleles of SOX9. SOX9-/- hESCs could be induced to differentiate into lung progenitor organoids. In vitro long-term expansion showed that SOX9 inactivation did not affect the differentiation of pulmonary epithelial cells, but promoted apoptosis and reduced proliferative capacity in the organoids. When lung progenitor organoids were transplanted under the kidney capsule of immunodeficient mice, expression of the club cell marker secretoglobin family 1A member 1 (SCGB1A1) was detected in SOX9-/- transplants but was absent in wild-type (WT) transplants. The maturation of goblet cells was also affected by SOX9 inactivation, as evidenced by the presence of mucin 5 AC (MUC5AC) in the cytoplasm of SOX9-/- grafts as compared to WT grafts in which most MUC5AC was secreted into the lumen. In vivo lung orthotopic transplantations showed that SOX9 inactivation had a limited effect on the differentiation of alveolar cells and lung regeneration in injured mice.
CONCLUSIONS: SOX9 modulates the proliferative capacity of lung epithelium but is not an indispensable transcription factor in the regulation of human lung epithelium development.

Keywords:  CRISPR/Cas9; Differentiation; Lung organoids; Proliferation; SOX9

DOI:  https://doi.org/10.1186/s13287-021-02422-6
Cell Death Discov. 2021 Jun 11. 7(1): 138

Pancreas morphogenesis and homeostasis depends on tightly regulated Zeb1 levels in epithelial cells.

María Lasierra Losada, Melissa Pauler, Niels Vandamme, Steven Goossens, Geert Berx, Moritz Leppkes, Harald Schuhwerk, Simone Brabletz, Thomas Brabletz, Marc P Stemmler.

The pancreas is comprised of exocrine and endocrine compartments releasing digestive enzymes into the duodenum and regulating blood glucose levels by insulin and glucagon release. Tissue homeostasis is depending on transcription factor networks, involving Ptf1α, Ngn3, Nkx6.1, and Sox9, which are already activated during organogenesis. However, proper organ function is challenged by diets of high sugar and fat content, increasing the risk of type 2 diabetes and other disorders. A detailed understanding of processes that are important for homeostasis and are impaired during type 2 diabetes is lacking. Here, we show that Zeb1-a transcription factor known for its pivotal role in epithelial-mesenchymal transition, cell plasticity, and metastasis in cancer-is expressed at low levels in epithelial cells of the pancreas and is crucial for organogenesis and pancreas function. Loss of Zeb1 in these cells result in an increase of islet mass, impaired glucose tolerance, and sensitizes to develop liver and pancreas steatosis during diabetes and obesity. Interestingly, moderate overexpression of Zeb1 results in severe pancreas agenesis and lethality after birth, due to islet insufficiency and lack of acinar structures. We show that Zeb1 induction interferes with proper differentiation, cell survival, and proliferation during pancreas formation, due to deregulated expression of endocrine-specific transcription factors. In summary, our analysis suggests a novel role of Zeb1 for homeostasis in epithelial cells that is indispensable for pancreas morphogenesis and proper organ function involving a tight regulation of Zeb1 expression.

DOI: https://doi.org/10.1038/s41420-021-00522-z
Sci Rep. 2021 Jun 17. 11(1): 12795

Epigenetics Identifier screens reveal regulators of chromatin acylation and limited specificity of acylation antibodies.

Leonie Kollenstart, Sophie C van der Horst, Kees Vreeken, George M C Janssen, Fabrizio Martino, Hanneke Vlaming, Peter A van Veelen, Fred van Leeuwen, Haico van Attikum.

The collection of known posttranslational modifications (PTMs) has expanded rapidly with the identification of various non-acetyl histone lysine acylations, such as crotonylation, succinylation and butyrylation, yet their regulation is still not fully understood. Through an unbiased chromatin immunoprecipitation (ChIP)-based approach called Epigenetics-IDentifier (Epi-ID), we aimed to identify regulators of crotonylation, succinylation and butyrylation in thousands of yeast mutants simultaneously. However, highly correlative results led us to further investigate the specificity of the pan-K-acyl antibodies used in our Epi-ID studies. This revealed cross-reactivity and lack of specificity of pan-K-acyl antibodies in various assays. Our findings suggest that the antibodies might recognize histone acetylation in vivo, in addition to histone acylation, due to the vast overabundance of acetylation compared to other acylation modifications in cells. Consequently, our Epi-ID screen mostly identified factors affecting histone acetylation, including known (e.g. GCN5, HDA1, and HDA2) and unanticipated (MET7, MTF1, CLB3, and RAD26) factors, expanding the repertoire of acetylation regulators. Antibody-independent follow-up experiments on the Gcn5-Ada2-Ada3 (ADA) complex revealed that, in addition to acetylation and crotonylation, ADA has the ability to butyrylate histones. Thus, our Epi-ID screens revealed limits of using pan-K-acyl antibodies in epigenetics research, expanded the repertoire of regulators of histone acetylation, and attributed butyrylation activity to the ADA complex.

DOI: https://doi.org/10.1038/s41598-021-91359-0
Elife. 2021 Jun 14. pii: e66524. [Epub ahead of print]10

Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer.

Yusuke Inoue, Ana Nikolic, Dylan Farnsworth, Rocky Shi, Fraser D Johnson, Alvin Liu, Marc Ladanyi, Romel Somwar, Marco Gallo, William W Lockwood.

  Lineage transformation between lung cancer subtypes is a poorly understood phenomenon associated with resistance to treatment and poor patient outcomes. Here, we aimed to model this transition to define underlying biological mechanisms and identify potential avenues for therapeutic intervention. Small cell lung cancer (SCLC) is neuroendocrine in identity and, in contrast to non-SCLC (NSCLC), rarely contains mutations that drive the MAPK pathway. Likewise, NSCLCs that transform to SCLC concomitantly with development of therapy resistance downregulate MAPK signaling, suggesting an inverse relationship between pathway activation and lineage state. To test this, we activated MAPK in SCLC through conditional expression of mutant KRAS or EGFR, which revealed suppression of the neuroendocrine differentiation program via ERK. We found that ERK induces the expression of ETS factors that mediate transformation into a NSCLC-like state. ATAC-seq demonstrated ERK-driven changes in chromatin accessibility at putative regulatory regions and global chromatin rewiring at neuroendocrine and ETS transcriptional targets. Further, ERK-mediated induction of ETS factors as well as suppression of neuroendocrine differentiation were dependent on histone acetyltransferase activities of CBP/p300. Overall, we describe how the ERK-CBP/p300-ETS axis promotes a lineage shift between neuroendocrine and non-neuroendocrine lung cancer phenotypes and provide rationale for the disruption of this program during transformation-driven resistance to targeted therapy.

Keywords:  cancer biology; human

DOI:  https://doi.org/10.7554/eLife.66524
Oncogene. 2021 Jun 17.

SMARCA4 oncogenic potential via IRAK1 enhancer to activate Gankyrin and AKR1B10 in liver cancer.

Sang Yean Kim, Qingyu Shen, Keunhong Son, Hyung Seok Kim, Hee Doo Yang, Min Jeong Na, Eunbi Shin, Suji Yu, Keunsoo Kang, Jueng Soo You, Kyung-Rok Yu, Seung Min Jeong, Eun Kyung Lee, Young Min Ahn, Won Sang Park, Suk Woo Nam.

SWItch/Sucrose Non-Fermentable (SWI/SNF) is a multiprotein complex essential for the regulation of eukaryotic gene expression. SWI/SNF complex genes are genetically altered in over 20% of human malignancies, but the aberrant regulation of the SWI/SNF subunit genes and subsequent dysfunction caused by abnormal expression of subunit gene in cancer, remain poorly understood. Among the SWI/SNF subunit genes, SMARCA4, SMARCC1, and SMARCA2 were identified to be overexpressed in human hepatocellular carcinoma (HCC). Modulation of SMARCA4, SMARCC1, and SMARCA2 inhibited in vitro tumorigenesis of HCC cells. However, SMARCA4-targeting elicited remarkable inhibition in an in vivo Ras-transgenic mouse HCC model (Ras-Tg), and high expression levels of SMARCA4 significantly associated with poor prognosis in HCC patients. Furthermore, most HCC patients (72-86%) showed SMARCA4 overexpression compared to healthy controls. To identify SMARCA4-specific active enhancers, mapping, and analysis of chromatin state in liver cancer cells were performed. Integrative analysis of SMARCA4-regulated genes and active chromatin enhancers suggested 37 genes that are strongly activated by SMARCA4 in HCC. Through chromatin immunoprecipitation-qPCR and luciferase assays, we demonstrated that SMARCA4 activates Interleukin-1 receptor-associated kinase 1 (IRAK1) expression through IRAK1 active enhancer in HCC. We then showed that transcriptional activation of IRAK1 induces oncoprotein Gankyrin and aldo-keto reductase family 1 member B10 (AKR1B10) in HCC. The regulatory mechanism of the SMARCA4-IRAK1-Gankyrin, AKR1B10 axis was further demonstrated in HCC cells and in vivo Ras-Tg mice. Our results suggest that aberrant overexpression of SMARCA4 causes SWI/SNF to promote IRAK1 enhancer to activate oncoprotein Gankyrin and AKR1B10, thereby contributing to hepatocarcinogenesis.

DOI: https://doi.org/10.1038/s41388-021-01875-6
Front Oncol. 2021 ;11 665273

RUNX2 Phosphorylation by Tyrosine Kinase ABL Promotes Breast Cancer Invasion.

Fang He, Yoshinori Matsumoto, Yosuke Asano, Yuriko Yamamura, Takayuki Katsuyama, Jose La Rose, Nahoko Tomonobu, Ni Luh Gede Yoni Komalasari, Masakiyo Sakaguchi, Robert Rottapel, Jun Wada.

  Activity of transcription factors is normally regulated through interaction with other transcription factors, chromatin remodeling proteins and transcriptional co-activators. In distinction to these well-established transcriptional controls of gene expression, we have uncovered a unique activation model of transcription factors between tyrosine kinase ABL and RUNX2, an osteoblastic master transcription factor, for cancer invasion. We show that ABL directly binds to, phosphorylates, and activates RUNX2 through its SH2 domain in a kinase activity-dependent manner and that the complex formation of these proteins is required for expression of its target gene MMP13. Additionally, we show that the RUNX2 transcriptional activity is dependent on the number of its tyrosine residues that are phosphorylated by ABL. In addition to regulation of RUNX2 activity, we show that ABL transcriptionally enhances RUNX2 expression through activation of the bone morphogenetic protein (BMP)-SMAD pathway. Lastly, we show that ABL expression in highly metastatic breast cancer MDA-MB231 cells is associated with their invasive capacity and that ABL-mediated invasion is abolished by depletion of endogenous RUNX2 or MMP13. Our genetic and biochemical evidence obtained in this study contributes to a mechanistic insight linking ABL-mediated phosphorylation and activation of RUNX2 to induction of MMP13, which underlies a fundamental invasive capacity in cancer and is different from the previously described model of transcriptional activation.

Keywords:  ABL - Abelson murine leukemia viral oncogene homolog; Runx2 (runt-related transcription factor 2); invasion; phosphorylation; tyrosine

DOI:  https://doi.org/10.3389/fonc.2021.665273
Leukemia. 2021 Jun 12.

Metabolic alterations mediated by STAT3 promotes drug persistence in CML.

Sweta B Patel, Travis Nemkov, Davide Stefanoni, Gloria A Benavides, Mahmoud A Bassal, Brittany L Crown, Victoria R Matkins, Virginia Camacho, Valeriya Kuznetsova, Ashley T Hoang, Danielle E Tenen, Samuel L Wolock, Jihye Park, Li Ying, Zongliang Yue, Jake Y Chen, Henry Yang, Daniel G Tenen, Paul Brent Ferrell, Rui Lu, Victor Darley-Usmar, Angelo D'Alessandro, Ravi Bhatia, Robert S Welner.

Leukemic stem cells (LSCs) can acquire non-mutational resistance following drug treatment leading to therapeutic failure and relapse. However, oncogene-independent mechanisms of drug persistence in LSCs are incompletely understood, which is the primary focus of this study. We integrated proteomics, transcriptomics, and metabolomics to determine the contribution of STAT3 in promoting metabolic changes in tyrosine kinase inhibitor (TKI) persistent chronic myeloid leukemia (CML) cells. Proteomic and transcriptional differences in TKI persistent CML cells revealed BCR-ABL-independent STAT3 activation in these cells. While knockout of STAT3 inhibited the CML cells from developing drug-persistence, inhibition of STAT3 using a small molecule inhibitor sensitized the persistent CML cells to TKI treatment. Interestingly, given the role of phosphorylated STAT3 as a transcription factor, it localized uniquely to genes regulating metabolic pathways in the TKI-persistent CML stem and progenitor cells. Subsequently, we observed that STAT3 dysregulated mitochondrial metabolism forcing the TKI-persistent CML cells to depend on glycolysis, unlike TKI-sensitive CML cells, which are more reliant on oxidative phosphorylation. Finally, targeting pyruvate kinase M2, a rate-limiting glycolytic enzyme, specifically eradicated the TKI-persistent CML cells. By exploring the role of STAT3 in altering metabolism, we provide critical insight into identifying potential therapeutic targets for eliminating TKI-persistent LSCs.

DOI: https://doi.org/10.1038/s41375-021-01315-0