bims-crepig 2021-10-24 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2021‒10‒24
twenty-nine papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

Decoding the function of bivalent chromatin in development and cancer.
Changing and stable chromatin accessibility supports transcriptional overhaul during neural stem cell activation and is altered with age.
Identification and prediction of developmental enhancers in sea urchin embryos.
Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia.
TET2 mutations are associated with hypermethylation at key regulatory enhancers in normal and malignant hematopoiesis.
The chromatin remodeler Ino80 mediates RNAPII pausing site determination.
Dual detection of chromatin accessibility and DNA methylation using ATAC-Me.
Simple oligonucleotide-based multiplexing of single-cell chromatin accessibility.
The glucose-sensing transcription factor MLX balances metabolism and stress to suppress apoptosis and maintain spermatogenesis.
Differential m6A RNA landscapes across hematopoiesis reveal a role for IGF2BP2 in preserving hematopoietic stem cell function.
A balancing act in transcription regulation by response regulators: titration of transcription factor activity by decoy DNA binding sites.
KLF17 promotes human naïve pluripotency but is not required for its establishment.
The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation.
Efficient hemogenic endothelial cell specification by RUNX1 is dependent on baseline chromatin accessibility of RUNX1-regulated TGFβ target genes.
Characterizing batch effects and binding site-specific variability in ChIP-seq data.
YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis.
MINTIE: identifying novel structural and splice variants in transcriptomes using RNA-seq data.
KLF4 Induces Mesenchymal-Epithelial Transition (MET) by Suppressing Multiple EMT-Inducing Transcription Factors.
Loss of the wild-type KRAS allele promotes pancreatic cancer progression through functional activation of YAP1.
Activation of Notch and Myc signaling via B cell-restricted depletion of Dnmt3a generates a consistent murine model of chronic lymphocytic leukemia.
The Hda1 histone deacetylase limits divergent non-coding transcription and restricts transcription initiation frequency.
Short-chain fatty acids activate acetyltransferase p300.
Drosophila Keap1 xenobiotic response factor regulates developmental transcription through binding to chromatin.
Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.
Comprehensive multi-omics integration identifies differentially active enhancers during human brain development with clinical relevance.
Osteoclasts adapt to physioxia perturbation through DNA demethylation.
Single-cell profiling of proteins and chromatin accessibility using PHAGE-ATAC.
A GO catalogue of human DNA-binding transcription factors.
Identification of histone methyltransferase NSD2 as an important oncogenic gene in colorectal cancer.

Genome Res. 2021 Oct 19. pii: gr.275736.121. [Epub ahead of print]

Decoding the function of bivalent chromatin in development and cancer.

Dhirendra Kumar, Senthilkumar Cinghu, Andrew J Oldfield, Pengyi Yang, Raja Jothi.

Bivalent chromatin is characterized by the simultaneous presence of H3K4me3 and H3K27me3, histone modifications generally associated with transcriptionally active and repressed chromatin, respectively. Prevalent in embryonic stem cells (ESCs), bivalency is postulated to poise/prime lineage-controlling developmental genes for rapid activation during embryogenesis while maintaining a transcriptionally repressed state in the absence of activation cues; however, this hypothesis remains to be directly tested. Most gene promoters DNA-hypermethylated in adult human cancers are bivalently marked in ESCs, and it was speculated that bivalency predisposes them for aberrant de novo DNA methylation and irreversible silencing in cancer, but evidence supporting this model is largely lacking. Here we show that bivalent chromatin does not poise genes for rapid activation but protects promoters from de novo DNA methylation. Genome-wide studies in differentiating ESCs reveal that activation of bivalent genes is no more rapid than that of other transcriptionally silent genes, challenging the premise that H3K4me3 is instructive for transcription. H3K4me3 at bivalent promoters, a product of the underlying DNA sequence, persists in nearly all cell types irrespective of gene expression and confers protection from de novo DNA methylation. Bivalent genes in ESCs that are frequent targets of aberrant hypermethylation in cancer are particularly strongly associated with loss of H3K4me3/bivalency in cancer. Altogether, our findings suggest that bivalency protects reversibly repressed genes from irreversible silencing and that loss of H3K4me3 may make them more susceptible to aberrant DNA methylation in diseases such as cancer. Bivalency may thus represent a distinct regulatory mechanism for maintaining epigenetic plasticity.

DOI: https://doi.org/10.1101/gr.275736.121
Aging Cell. 2021 Oct 23. e13499

Changing and stable chromatin accessibility supports transcriptional overhaul during neural stem cell activation and is altered with age.

Sun Y Maybury-Lewis, Abigail K Brown, Mitchell Yeary, Anna Sloutskin, Shleshma Dhakal, Tamar Juven-Gershon, Ashley E Webb.

  Neural stem cells (NSCs) in the adult and aged brain are largely quiescent, and require transcriptional reprogramming to re-enter the cell cycle. However, the mechanisms underlying these changes and how they are altered with age remain undefined. Here, we identify the chromatin accessibility differences between primary neural stem/progenitor cells in quiescent and activated states. These distinct cellular states exhibit shared and unique chromatin profiles, both associated with gene regulation. Accessible chromatin states specific to activation or quiescence are active enhancers bound by key pro-neurogenic and quiescence factors. In contrast, shared sites are enriched for core promoter elements associated with translation and metabolism. Unexpectedly, through integrated analysis, we find that many sites that become accessible during NSC activation are linked to gene repression and associated with pro-quiescence factors, revealing a novel mechanism that may preserve quiescence re-entry. Furthermore, we report that in aged NSCs, chromatin regions associated with metabolic and transcriptional functions bound by key pro-quiescence transcription factors lose accessibility, suggesting a novel mechanism of age-associated NSC dysfunction. Together, our findings reveal how accessible chromatin states regulate the transcriptional switch between NSC quiescence and activation, and how this switch is affected with age.

Keywords:  aging; chromatin accessibility; neural stem cells; stem cell activation

DOI:  https://doi.org/10.1111/acel.13499
BMC Genomics. 2021 Oct 19. 22(1): 751

Identification and prediction of developmental enhancers in sea urchin embryos.

César Arenas-Mena, Sofija Miljovska, Edward J Rice, Justin Gurges, Tanvi Shashikant, Zihe Wang, Sevinç Ercan, Charles G Danko.

  BACKGROUND: The transcription of developmental regulatory genes is often controlled by multiple cis-regulatory elements. The identification and functional characterization of distal regulatory elements remains challenging, even in tractable model organisms like sea urchins.RESULTS: We evaluate the use of chromatin accessibility, transcription and RNA Polymerase II for their ability to predict enhancer activity of genomic regions in sea urchin embryos. ATAC-seq, PRO-seq, and Pol II ChIP-seq from early and late blastula embryos are manually contrasted with experimental cis-regulatory analyses available in sea urchin embryos, with particular attention to common developmental regulatory elements known to have enhancer and silencer functions differentially deployed among embryonic territories. Using the three functional genomic data types, machine learning models are trained and tested to classify and quantitatively predict the enhancer activity of several hundred genomic regions previously validated with reporter constructs in vivo.
CONCLUSIONS: Overall, chromatin accessibility and transcription have substantial power for predicting enhancer activity. For promoter-overlapping cis-regulatory elements in particular, the distribution of Pol II is the best predictor of enhancer activity in blastula embryos. Furthermore, ATAC- and PRO-seq predictive value is stage dependent for the promoter-overlapping subset. This suggests that the sequence of regulatory mechanisms leading to transcriptional activation have distinct relevance at different levels of the developmental gene regulatory hierarchy deployed during embryogenesis.

Keywords:  Cis-regulatory element; Developmental gene regulation; Enhancer prediction

DOI:  https://doi.org/10.1186/s12864-021-07936-0
Nat Genet. 2021 Oct 18.

Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia.

Derek H Janssens, Michael P Meers, Steven J Wu, Ekaterina Babaeva, Soheil Meshinchi, Jay F Sarthy, Kami Ahmad, Steven Henikoff.

Acute myeloid and lymphoid leukemias often harbor chromosomal translocations involving the KMT2A gene, encoding the KMT2A lysine methyltransferase (also known as mixed-lineage leukemia-1), and produce in-frame fusions of KMT2A to other chromatin-regulatory proteins. Here we map fusion-specific targets across the genome for diverse KMT2A oncofusion proteins in cell lines and patient samples. By modifying CUT&Tag chromatin profiling for full automation, we identify common and tumor-subtype-specific sites of aberrant chromatin regulation induced by KMT2A oncofusion proteins. A subset of KMT2A oncofusion-binding sites are marked by bivalent (H3K4me3 and H3K27me3) chromatin signatures, and single-cell CUT&Tag profiling reveals that these sites display cell-to-cell heterogeneity suggestive of lineage plasticity. In addition, we find that aberrant enrichment of H3K4me3 in gene bodies is sensitive to Menin inhibitors, demonstrating the utility of automated chromatin profiling for identifying therapeutic vulnerabilities. Thus, integration of automated and single-cell CUT&Tag can uncover epigenomic heterogeneity within patient samples and predict sensitivity to therapeutic agents.

DOI: https://doi.org/10.1038/s41588-021-00941-9
Nat Commun. 2021 Oct 18. 12(1): 6061

TET2 mutations are associated with hypermethylation at key regulatory enhancers in normal and malignant hematopoiesis.

Morten Tulstrup, Mette Soerensen, Jakob Werner Hansen, Linn Gillberg, Maria Needhamsen, Katja Kaastrup, Kristian Helin, Kaare Christensen, Joachim Weischenfeldt, Kirsten Grønbæk.

Mutations in the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). Here, we investigate associations between TET2 mutations and DNA methylation in whole blood in 305 elderly twins, 15 patients with CCUS and 18 healthy controls. We find that TET2 mutations are associated with DNA hypermethylation at enhancer sites in whole blood in CHIP and in both granulocytes and mononuclear cells in CCUS. These hypermethylated sites are associated with leukocyte function and immune response and ETS-related and C/EBP-related transcription factor motifs. While the majority of TET2-associated hypermethylation sites are shared between CHIP and in AML, we find a set of AML-specific hypermethylated loci at active enhancer elements in hematopoietic stem cells. In summary, we show that TET2 mutations is associated with hypermethylated enhancers involved in myeloid differentiation in both CHIP, CCUS and AML patients.

DOI: https://doi.org/10.1038/s41467-021-26093-2
Genome Biol. 2021 Oct 18. 22(1): 294

The chromatin remodeler Ino80 mediates RNAPII pausing site determination.

Youngseo Cheon, Sungwook Han, Taemook Kim, Daehee Hwang, Daeyoup Lee.

  BACKGROUND: Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step for the precise regulation of gene expression. Despite the apparent close relationship between promoter-proximal pausing and nucleosome, the role of chromatin remodeler governing this step has mainly remained elusive.RESULTS: Here, we report highly confined RNAPII enrichments downstream of the transcriptional start site in Saccharomyces cerevisiae using PRO-seq experiments. This non-uniform distribution of RNAPII exhibits both similar and different characteristics with promoter-proximal pausing in Schizosaccharomyces pombe and metazoans. Interestingly, we find that Ino80p knockdown causes a significant upstream transition of promoter-proximal RNAPII for a subset of genes, relocating RNAPII from the main pausing site to the alternative pausing site. The proper positioning of RNAPII is largely dependent on nucleosome context. We reveal that the alternative pausing site is closely associated with the + 1 nucleosome, and nucleosome architecture around the main pausing site of these genes is highly phased. In addition, Ino80p knockdown results in an increase in fuzziness and a decrease in stability of the + 1 nucleosome. Furthermore, the loss of INO80 also leads to the shift of promoter-proximal RNAPII toward the alternative pausing site in mouse embryonic stem cells.
CONCLUSIONS: Based on our collective results, we hypothesize that the highly conserved chromatin remodeler Ino80p is essential in establishing intact RNAPII pausing during early transcription elongation in various organisms, from budding yeast to mouse.

Keywords:  + 1 nucleosome; AID system; Alternative pausing site; PRO-seq; Promoter-proximal RNAPII pausing; The chromatin remodeler Ino80p

DOI:  https://doi.org/10.1186/s13059-021-02500-1
Nat Protoc. 2021 Oct 18.

Dual detection of chromatin accessibility and DNA methylation using ATAC-Me.

Lindsey N Guerin, Kelly R Barnett, Emily Hodges.

The epigenome is multidimensional, with individual molecular components operating on different levels to control transcriptional output. Techniques that combine measurements of these features can reveal their precise correspondence in genomic space, or temporal connectivity, to better understand how they jointly regulate genes. ATAC-Me is an integrated method to probe DNA methylation and chromatin accessibility from a single DNA fragment library. Intact nuclei undergo Tn5 transposition to isolate DNA fragments within nucleosome-free regions. Isolated fragments are exposed to sodium bisulfite before library amplification and sequencing. A typical ATAC-Me experiment detects ~60,000-75,000 peak regions (P < 0.05), covering ~3-4 million CpG sites with at least 5× coverage. These sites display a range of methylation values depending on the cellular and genomic context. The approach is well suited for time course studies that aim to capture chromatin and DNA methylation dynamics in tandem during cellular differentiation. The protocol is completed in 2 d with standard molecular biology equipment and expertise. Analysis of resulting data uses publicly available software requiring basic bioinformatics skills to interpret results.

DOI: https://doi.org/10.1038/s41596-021-00608-z
Mol Cell. 2021 Oct 21. pii: S1097-2765(21)00795-4. [Epub ahead of print]81(20): 4319-4332.e10

Simple oligonucleotide-based multiplexing of single-cell chromatin accessibility.

Kaile Wang, Zhenna Xiao, Yun Yan, Rui Ye, Min Hu, Shanshan Bai, Emi Sei, Yawei Qiao, Hui Chen, Bora Lim, Steven H Lin, Nicholas E Navin.

  Microdroplet single-cell ATAC-seq is widely used to measure chromatin accessibility, however, highly scalable and simple sample multiplexing procedures are not available. Here, we present a transposome-assisted single nucleus barcoding approach for ATAC-seq (SNuBar-ATAC) that utilizes a single oligonucleotide adaptor for multiplexing samples during the existing tagmentation step and does not require a pre-labeling procedure. The accuracy and scalability of SNuBar-ATAC was evaluated using cell line mixture experiments. We applied SNuBar-ATAC to investigate treatment-induced chromatin accessibility dynamics by multiplexing 28 mice with lung tumors that received different combinations of chemo, radiation, and targeted immunotherapy. We also applied SNuBar-ATAC to study spatial epigenetic heterogeneity by multiplexing 32 regions from a human breast tissue. Additionally, we show that SNuBar can multiplex single cell ATAC and RNA multiomic assays in cell lines and human breast tissue samples. Our data show that SNuBar is a highly accurate, easy-to-use, and scalable system for multiplexing scATAC-seq and scATAC and RNA co-assay experiments.

Keywords:  SNuBar; cell-type-specific transcription factors; multiplexing therapy combinations; single-cell ATAC sequencing; single-cell multiomics multiplexing; single-cell multiomics sequencing; single-cell multiplexing; spatial epigenetic heterogeneity

DOI:  https://doi.org/10.1016/j.molcel.2021.09.026
PLoS Biol. 2021 Oct;19(10): e3001085

The glucose-sensing transcription factor MLX balances metabolism and stress to suppress apoptosis and maintain spermatogenesis.

Patrick A Carroll, Brian W Freie, Pei Feng Cheng, Sivakanthan Kasinathan, Haiwei Gu, Theresa Hedrich, James A Dowdle, Vivek Venkataramani, Vijay Ramani, Xiaoying Wu, Daniel Raftery, Jay Shendure, Donald E Ayer, Charles H Muller, Robert N Eisenman.

Male germ cell (GC) production is a metabolically driven and apoptosis-prone process. Here, we show that the glucose-sensing transcription factor (TF) MAX-Like protein X (MLX) and its binding partner MondoA are both required for male fertility in the mouse, as well as survival of human tumor cells derived from the male germ line. Loss of Mlx results in altered metabolism as well as activation of multiple stress pathways and GC apoptosis in the testes. This is concomitant with dysregulation of the expression of male-specific GC transcripts and proteins. Our genomic and functional analyses identify loci directly bound by MLX involved in these processes, including metabolic targets, obligate components of male-specific GC development, and apoptotic effectors. These in vivo and in vitro studies implicate MLX and other members of the proximal MYC network, such as MNT, in regulation of metabolism and differentiation, as well as in suppression of intrinsic and extrinsic death signaling pathways in both spermatogenesis and male germ cell tumors (MGCTs).

DOI: https://doi.org/10.1371/journal.pbio.3001085
Cell Stem Cell. 2021 Oct 13. pii: S1934-5909(21)00390-8. [Epub ahead of print]

Differential m6A RNA landscapes across hematopoiesis reveal a role for IGF2BP2 in preserving hematopoietic stem cell function.

Rong Yin, Jiwei Chang, Yashu Li, Zhuying Gao, Qiang Qiu, Qifan Wang, Guoqiang Han, Jihua Chai, Mengdie Feng, Peipei Wang, Tiantian Zhang, Xueqin Xie, Jin Hu, Ying Cheng, Chengli Guo, Jing Wang, Kexin Gao, Manman Cui, Shaoguang Li, Yuhuan Zheng, Wei Jiang, Yiguo Hu, Qing-Yong Yang, Haojian Zhang.

  N6-methyladenosine (m6A) on mRNA plays critical roles in various cellular processes. However, the landscape and dynamics of m6A modification in hematopoietic system remain unknown. Here, we delineate a comprehensive m6A landscape across hematopoietic hierarchy and uncover that IGF2BP2 is required for preserving the function of hematopoietic stem cells (HSCs). Our data reveal a cell-type-specific m6A landscape in hematopoiesis. m6A modifications arise mostly in the early stage of hematopoiesis and prefer to play distinct roles for determining mRNA fates in HSCs and committed progenitors. Mechanistically, increased m6A-IGF2BP2 expression controls transcriptional state and maintenance of HSCs. IGF2BP2 deficiency induces quiescence loss and impairs HSC function. Moreover, IGF2BP2 loss increases mitochondrial activity of HSCs by accelerating Bmi1 mRNA decay, leading to de-repression of mitochondria-related genes. Collectively, our results present a fascinating portrait of m6A modification of hematopoietic hierarchy and reveal a key role of IGF2BP2 in maintaining HSC function by restraining mitochondrial activity.

Keywords:  IGF2BP2; N6-methyladenosine; hematopoiesis; hematopoietic stem cells

DOI:  https://doi.org/10.1016/j.stem.2021.09.014
Nucleic Acids Res. 2021 Oct 20. pii: gkab935. [Epub ahead of print]

A balancing act in transcription regulation by response regulators: titration of transcription factor activity by decoy DNA binding sites.

Rong Gao, Libby J Helfant, Ti Wu, Zeyue Li, Samantha E Brokaw, Ann M Stock.

Studies of transcription regulation are often focused on binding of transcription factors (TFs) to a small number of promoters of interest. It is often assumed that TFs are in great excess to their binding sites (TFBSs) and competition for TFs between DNA sites is seldom considered. With increasing evidence that TFBSs are exceedingly abundant for many TFs and significant variations in TF and TFBS numbers occur during growth, the interplay between a TF and all TFBSs should not be ignored. Here, we use additional decoy DNA sites to quantitatively analyze how the relative abundance of a TF to its TFBSs impacts the steady-state level and onset time of gene expression for the auto-activated Escherichia coli PhoB response regulator. We show that increasing numbers of decoy sites progressively delayed transcription activation and lowered promoter activities. Perturbation of transcription regulation by additional TFBSs did not require extreme numbers of decoys, suggesting that PhoB is approximately at capacity for its DNA sites. Addition of decoys also converted a graded response to a bi-modal response. We developed a binding competition model that captures the major features of experimental observations, providing a quantitative framework to assess how variations in TFs and TFBSs influence transcriptional responses.

DOI: https://doi.org/10.1093/nar/gkab935
Development. 2021 Oct 18. pii: dev.199378. [Epub ahead of print]

KLF17 promotes human naïve pluripotency but is not required for its establishment.

Rebecca A Lea, Afshan McCarthy, Stefan Boeing, Todd Fallesen, Kay Elder, Phil Snell, Leila Christie, Sarah Adkins, Valerie Shaikly, Mohamed Taranissi, Kathy K Niakan.

  Current knowledge of the transcriptional regulation of human pluripotency is incomplete, with lack of inter-species conservation observed. Single-cell transcriptomics analysis of human embryos previously enabled us to identify transcription factors, including the zinc-finger protein KLF17, that are enriched in the human epiblast and naïve hESCs. Here we show that KLF17 is expressed coincident with the known pluripotency-associated factors NANOG and SOX2 across human blastocyst development. We investigate the function of KLF17 using primed and naïve hESCs for gain- and loss-of-function analyses. We find that ectopic expression of KLF17 in primed hESCs is sufficient to induce a naïve-like transcriptome and that KLF17 can drive transgene-mediated resetting to naïve pluripotency. This implies a role for KLF17 in establishing naïve pluripotency. However, CRISPR-Cas9-mediated knockout studies reveal that KLF17 is not required for naïve pluripotency acquisition in vitro. Transcriptome analysis of naïve hESCs identifies subtle effects on metabolism and signalling pathways following KLF17 loss-of-function, and possible redundancy with other KLF paralogues. Overall, we show that KLF17 is sufficient, but not necessary, for naïve pluripotency under the given in vitro conditions.

Keywords:  Epiblast; Human embryonic stem cells; KLF factors; KLF17; Naïve pluripotency

DOI:  https://doi.org/10.1242/dev.199378
EMBO Rep. 2021 Oct 18. e52764

The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation.

Sarah El Kharraz, Vanessa Dubois, Martin E van Royen, Adriaan B Houtsmuller, Ekatarina Pavlova, Nina Atanassova, Tien Nguyen, Arnout Voet, Roy Eerlings, Florian Handle, Stefan Prekovic, Elien Smeets, Lisa Moris, Wout Devlies, Claes Ohlsson, Matti Poutanen, Kevin J Verstrepen, Geert Carmeliet, Kaisa-Mari Launonen, Laura Helminen, Jorma J Palvimo, Claude Libert, Dirk Vanderschueren, Christine Helsen, Frank Claessens.

  Whereas dimerization of the DNA-binding domain of the androgen receptor (AR) plays an evident role in recognizing bipartite response elements, the contribution of the dimerization of the ligand-binding domain (LBD) to the correct functioning of the AR remains unclear. Here, we describe a mouse model with disrupted dimerization of the AR LBD (ARLmon/Y ). The disruptive effect of the mutation is demonstrated by the feminized phenotype, absence of male accessory sex glands, and strongly affected spermatogenesis, despite high circulating levels of testosterone. Testosterone replacement studies in orchidectomized mice demonstrate that androgen-regulated transcriptomes in ARLmon/Y mice are completely lost. The mutated AR still translocates to the nucleus and binds chromatin, but does not bind to specific AR binding sites. In vitro studies reveal that the mutation in the LBD dimer interface also affects other AR functions such as DNA binding, ligand binding, and co-regulator binding. In conclusion, LBD dimerization is crucial for the development of AR-dependent tissues through its role in transcriptional regulation in vivo. Our findings identify AR LBD dimerization as a possible target for AR inhibition.

Keywords:  androgen receptor; chromatin binding; dimerization; ligand-binding domain; transcriptional activation

DOI:  https://doi.org/10.15252/embr.202152764
Genes Dev. 2021 Oct 21.

Efficient hemogenic endothelial cell specification by RUNX1 is dependent on baseline chromatin accessibility of RUNX1-regulated TGFβ target genes.

Elizabeth D Howell, Amanda D Yzaguirre, Peng Gao, Raphael Lis, Bing He, Melike Lakadamyali, Shahin Rafii, Kai Tan, Nancy A Speck.

  Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo from hemogenic endothelial cells (HECs) via an endothelial-to-hematopoietic transition (EHT) that requires the transcription factor RUNX1. Ectopic expression of RUNX1 alone can efficiently promote EHT and HSPC formation from embryonic endothelial cells (ECs), but less efficiently from fetal or adult ECs. Efficiency correlated with baseline accessibility of TGFβ-related genes associated with endothelial-to-mesenchymal transition (EndoMT) and participation of AP-1 and SMAD2/3 to initiate further chromatin remodeling along with RUNX1 at these sites. Activation of TGFβ signaling improved the efficiency with which RUNX1 specified fetal ECs as HECs. Thus, the ability of RUNX1 to promote EHT depends on its ability to recruit the TGFβ signaling effectors AP-1 and SMAD2/3, which in turn is determined by the changing chromatin landscape in embryonic versus fetal ECs. This work provides insight into regulation of EndoMT and EHT that will guide reprogramming efforts for clinical applications.

Keywords:  RUNX1; TGFβ; endothelial-to-hematopoietic transition; endothelial-to-mesenchymal transition; hematopoiesis; hemogenic endothelium

DOI:  https://doi.org/10.1101/gad.348738.121
NAR Genom Bioinform. 2021 Dec;3(4): lqab098

Characterizing batch effects and binding site-specific variability in ChIP-seq data.

Mingxiang Teng, Dongliang Du, Danfeng Chen, Rafael A Irizarry.

Multiple sources of variability can bias ChIP-seq data toward inferring transcription factor (TF) binding profiles. As ChIP-seq datasets increase in public repositories, it is now possible and necessary to account for complex sources of variability in ChIP-seq data analysis. We find that two types of variability, the batch effects by sequencing laboratories and differences between biological replicates, not associated with changes in condition or state, vary across genomic sites. This implies that observed differences between samples from different conditions or states, such as cell-type, must be assessed statistically, with an understanding of the distribution of obscuring noise. We present a statistical approach that characterizes both differences of interests and these source of variability through the parameters of a mixed effects model. We demonstrate the utility of our approach on a CTCF binding dataset composed of 211 samples representing 90 different cell-types measured across three different laboratories. The results revealed that sites exhibiting large variability were associated with sequence characteristics such as GC-content and low complexity. Finally, we identified TFs associated with high-variance CTCF sites using TF motifs documented in public databases, pointing the possibility of these being false positives if the sources of variability are not properly accounted for.

DOI: https://doi.org/10.1093/nargab/lqab098
EMBO Mol Med. 2021 Oct 19. e14351

YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis.

Ruize Gao, Ravi K R Kalathur, Mairene Coto-Llerena, Caner Ercan, David Buechel, Song Shuang, Salvatore Piscuoglio, Michael T Dill, Fernando D Camargo, Gerhard Christofori, Fengyuan Tang.

  Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In this study, a combination of shRNA-mediated synthetic lethality screening and transcriptomic analysis revealed the transcription factors YAP/TAZ as key drivers of Sorafenib resistance in hepatocellular carcinoma (HCC) by repressing Sorafenib-induced ferroptosis. Mechanistically, in a TEAD-dependent manner, YAP/TAZ induce the expression of SLC7A11, a key transporter maintaining intracellular glutathione homeostasis, thus enabling HCC cells to overcome Sorafenib-induced ferroptosis. At the same time, YAP/TAZ sustain the protein stability, nuclear localization, and transcriptional activity of ATF4 which in turn cooperates to induce SLC7A11 expression. Our study uncovers a critical role of YAP/TAZ in the repression of ferroptosis and thus in the establishment of Sorafenib resistance in HCC, highlighting YAP/TAZ-based rewiring strategies as potential approaches to overcome HCC therapy resistance.

Keywords:  ATF4; Hippo signaling; YAP/TAZ; ferroptosis; liver cancer

DOI:  https://doi.org/10.15252/emmm.202114351
Genome Biol. 2021 Oct 22. 22(1): 296

MINTIE: identifying novel structural and splice variants in transcriptomes using RNA-seq data.

Marek Cmero, Breon Schmidt, Ian J Majewski, Paul G Ekert, Alicia Oshlack, Nadia M Davidson.

Calling fusion genes from RNA-seq data is well established, but other transcriptional variants are difficult to detect using existing approaches. To identify all types of variants in transcriptomes we developed MINTIE, an integrated pipeline for RNA-seq data. We take a reference-free approach, combining de novo assembly of transcripts with differential expression analysis to identify up-regulated novel variants in a case sample. We compare MINTIE with eight other approaches, detecting > 85% of variants while no other method is able to achieve this. We posit that MINTIE will be able to identify new disease variants across a range of disease types.

DOI: https://doi.org/10.1186/s13059-021-02507-8
Cancers (Basel). 2021 Oct 13. pii: 5135. [Epub ahead of print]13(20):

KLF4 Induces Mesenchymal-Epithelial Transition (MET) by Suppressing Multiple EMT-Inducing Transcription Factors.

Ayalur Raghu Subbalakshmi, Sarthak Sahoo, Isabelle McMullen, Aaditya Narayan Saxena, Sudhanva Kalasapura Venugopal, Jason A Somarelli, Mohit Kumar Jolly.

  Epithelial-Mesenchymal Plasticity (EMP) refers to reversible dynamic processes where cells can transition from epithelial to mesenchymal (EMT) or from mesenchymal to epithelial (MET) phenotypes. Both these processes are modulated by multiple transcription factors acting in concert. While EMT-inducing transcription factors (TFs)-TWIST1/2, ZEB1/2, SNAIL1/2/3, GSC, and FOXC2-are well-characterized, the MET-inducing TFs are relatively poorly understood (OVOL1/2 and GRHL1/2). Here, using mechanism-based mathematical modeling, we show that transcription factor KLF4 can delay the onset of EMT by suppressing multiple EMT-TFs. Our simulations suggest that KLF4 overexpression can promote a phenotypic shift toward a more epithelial state, an observation suggested by the negative correlation of KLF4 with EMT-TFs and with transcriptomic-based EMT scoring metrics in cancer cell lines. We also show that the influence of KLF4 in modulating the EMT dynamics can be strengthened by its ability to inhibit cell-state transitions at the epigenetic level. Thus, KLF4 can inhibit EMT through multiple parallel paths and can act as a putative MET-TF. KLF4 associates with the patient survival metrics across multiple cancers in a context-specific manner, highlighting the complex association of EMP with patient survival.

Keywords:  Epithelial–Mesenchymal Plasticity (EMP); KLF4; Mesenchymal–Epithelial Transition (MET); epigenetics; mathematical modeling

DOI:  https://doi.org/10.3390/cancers13205135
Oncogene. 2021 Oct 18.

Loss of the wild-type KRAS allele promotes pancreatic cancer progression through functional activation of YAP1.

Han Yan, Chih-Chieh Yu, Stuart A Fine, Ayman Lee Youssof, Ye-Ran Yang, Jun Yan, Dillon C Karg, Edwin C Cheung, Richard A Friedman, Haoqiang Ying, Emily I Chen, Ji Luo, Yi Miao, Wanglong Qiu, Gloria H Su.

Human pancreatic ductal adenocarcinoma (PDAC) harboring one KRAS mutant allele often displays increasing genomic loss of the remaining wild-type (WT) allele (known as LOH at KRAS) as tumors progress to metastasis, yet the molecular ramification of this WT allelic loss is unknown. In this study, we showed that the restoration of WT KRAS expression in human PDAC cell lines with LOH at KRAS significantly attenuated the malignancy of PDAC cells both in vitro and in vivo, demonstrating a tumor-suppressive role of the WT KRAS allele. Through RNA-Seq, we identified the HIPPO signaling pathway to be positively regulated by WT KRAS in PDAC cells. In accordance with this observation, PDAC cells with LOH at KRAS exhibited increased nuclear localization and activation of transcriptional co-activator YAP1. Mechanistically, we discovered that WT KRAS expression sequestered YAP1 from the nucleus, through enhanced 14-3-3zeta interaction with phosphorylated YAP1 at S127. Consistently, expression of a constitutively-active YAP1 mutant in PDAC cells bypassed the growth inhibitory effects of WT KRAS. In patient samples, we found that the YAP1-activation genes were significantly upregulated in tumors with LOH at KRAS, and YAP1 nuclear localization predicted poor survival for PDAC patients. Collectively, our results reveal that the WT allelic loss leads to functional activation of YAP1 and enhanced tumor malignancy, which explains the selection advantage of the tumor cells with LOH at KRAS during pancreatic cancer clonal evolution and progression to metastasis, and should be taken into consideration in future therapeutic strategies targeting KRAS.

DOI: https://doi.org/10.1038/s41388-021-02040-9
Cancer Res. 2021 Oct 22. pii: canres.CAN-21-1273-A.2021. [Epub ahead of print]

Activation of Notch and Myc signaling via B cell-restricted depletion of Dnmt3a generates a consistent murine model of chronic lymphocytic leukemia.

Anat Biran, Shanye Yin, Helene Kretzmer, Elisa Ten Hacken, Salma Parvin, Fabienne Lucas, Mohamed Uduman, Catherine Gutierrez, Nathan Dangle, Leah Billington, Fara Faye Regis, Laura Z Rassenti, Arman Mohammad, Gabriela Brunsting Hoffmann, Kristen Stevenson, Mei Zheng, Elizabeth Witten, Stacey Fernandes, Eugen Tausch, Clare Sun, Stephan Stilgenbauer, Jennifer R Brown, Thomas J Kipps, Jon C Aster, Andreas Gnirke, Donna S Neuberg, Anthony Letai, Lili Wang, Ruben D Carrasco, Alexander Meissner, Catherine J Wu.

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell-of-origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc-expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in 3 other CLL mouse models tested (Sf3b1-Atm, Ikzf3 and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-1273
EMBO J. 2021 Oct 18. e108903

The Hda1 histone deacetylase limits divergent non-coding transcription and restricts transcription initiation frequency.

Uthra Gowthaman, Maxim Ivanov, Isabel Schwarz, Heta P Patel, Niels A Müller, Desiré García-Pichardo, Tineke L Lenstra, Sebastian Marquardt.

  Nucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA and a divergent non-coding (DNC) transcript of unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast. Using high-throughput reverse genetic screens based on quantitative single-cell fluorescence measurements, we identified the Hda1 histone deacetylase complex (Hda1C) as a repressor of DNC transcription. Nascent transcription profiling showed a genome-wide role of Hda1C in repression of DNC transcription. Live-cell imaging of transcription revealed that mutations in the Hda3 subunit increased the frequency of DNC transcription. Hda1C contributed to decreased acetylation of histone H3 in DNC transcription regions, supporting DNC transcription repression by histone deacetylation. Our data support the interpretation that DNC transcription results as a consequence of the NDR-based architecture of eukaryotic promoters, but that it is governed by locus-specific repression to maintain genome fidelity.

Keywords:  RNA polymerase II transcription; divergent non-coding (DNC) transcription; live-cell imaging; non-coding RNA (ncRNA)

DOI:  https://doi.org/10.15252/embj.2021108903
Elife. 2021 Oct 22. pii: e72171. [Epub ahead of print]10

Short-chain fatty acids activate acetyltransferase p300.

Sydney P Thomas, John M Denu.

  Short-chain fatty acids (SCFAs) acetate, propionate, and butyrate are produced in large quantities by the gut microbiome and contribute to a wide array of physiological processes. While the underlying mechanisms are largely unknown, many effects of SCFAs have been traced to changes in the cell's epigenetic state. Here, we systematically investigate how SCFAs alter the epigenome. Using quantitative proteomics of histone modification states, we identified rapid and sustained increases in histone acetylation after addition of butyrate or propionate, but not acetate. While decades of prior observations would have suggested that hyperacetylation induced by SCFAs are attributed to inhibition of histone deacetylases (HDACs), we found that propionate and butyrate instead activate the acetyltransferase p300. Propionate and butyrate are rapidly converted to the corresponding acyl-CoAs which are then used by p300 to catalyze auto-acylation of the autoinhibitory loop, activating the enzyme for histone/protein acetylation. This data challenges the long-held belief that SCFAs mainly regulate chromatin by inhibiting HDACs, and instead reveals a previously unknown mechanism of HAT activation that can explain how an influx of low levels of SCFAs alters global chromatin states.

Keywords:  biochemistry; chemical biology; human; infectious disease; microbiology

DOI:  https://doi.org/10.7554/eLife.72171
Dev Biol. 2021 Oct 15. pii: S0012-1606(21)00225-6. [Epub ahead of print]

Drosophila Keap1 xenobiotic response factor regulates developmental transcription through binding to chromatin.

Jennifer Carlson, Lindsey Price, Isabel Cook, Huai Deng.

  The Keap1-Nrf2 complex is a central regulator that mediates transcriptional responses to xenobiotic stimuli and is highly related with multiple human diseases. The molecular mechanisms and biological functions of Keap1 and Nrf2 are not fully understood. The Drosophila Keap1 homolog (dKeap1) is conserved with mammalian Keap1 except that dKeap1 contains a 156 aa C-terminal tail (CTD). A dKeap1 truncation with the CTD removed (dKeap1-ΔCTD) shows abolished nuclear localization and chromatin-binding. Expression of dKeap1-ΔCTD in the dKeap1 null background significantly rescues this mutant to the adult stage, but the files showed partial lethality, sterility and defects in adipose tissue. In the rescued flies, expression levels of ecdysone-response genes, ecdysone-synthetic genes and adipogenesis genes were down-regulated in specific tissues, indicating that the chromatin-binding of dKeap1 mediates the activation of these developmental genes. As the same time, dKeap1-ΔCTD can still suppress the basal expression of detoxifying genes and mediate the activation of these genes in response to xenobiotic stimuli, suggesting that the chromatin-binding of dKeap1 is not required for the regulation of detoxifying genes. These results support a model in which dKeap1 on one hand functions as an inhibitor for the Nrf2-mediated transcription in the xenobiotic response pathway and on the other hand functions as a chromatin-binding transcription activator in the developmental pathway. Our study reveals a novel mechanism whereby Keap1-Nrf2 xenobiotic response signaling regulates development using a mechanism independent of redox signaling.

Keywords:  Adipogenesis; Chromatin binding; Development; Ecdysone; Keap1-Nrf2; Oogenesis; Oxidative and xenobiotic responses; Transcription activator; dKeap1-CncC

DOI:  https://doi.org/10.1016/j.ydbio.2021.10.003
Cell Rep. 2021 Oct 19. pii: S2211-1247(21)01300-0. [Epub ahead of print]37(3): 109836

Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.

Mario de la Fuente Revenga, Bohan Zhu, Christopher A Guevara, Lynette B Naler, Justin M Saunders, Zirui Zhou, Rudy Toneatti, Salvador Sierra, Jennifer T Wolstenholme, Patrick M Beardsley, George W Huntley, Chang Lu, Javier González-Maeso.

  Clinical evidence suggests that rapid and sustained antidepressant action can be attained with a single exposure to psychedelics. However, the biological substrates and key mediators of psychedelics' enduring action remain unknown. Here, we show that a single administration of the psychedelic DOI produces fast-acting effects on frontal cortex dendritic spine structure and acceleration of fear extinction via the 5-HT2A receptor. Additionally, a single dose of DOI leads to changes in chromatin organization, particularly at enhancer regions of genes involved in synaptic assembly that stretch for days after the psychedelic exposure. These DOI-induced alterations in the neuronal epigenome overlap with genetic loci associated with schizophrenia, depression, and attention deficit hyperactivity disorder. Together, these data support that epigenomic-driven changes in synaptic plasticity sustain psychedelics' long-lasting antidepressant action but also warn about potential substrate overlap with genetic risks for certain psychiatric conditions.

Keywords:  5-HT2A receptor; GPCR; depression; epigenomics; hallucinogens; psychedelics; psychosis; schizophrenia; serotonin (5-HT); synaptic plasticity

DOI:  https://doi.org/10.1016/j.celrep.2021.109836
Genome Med. 2021 Oct 19. 13(1): 162

Comprehensive multi-omics integration identifies differentially active enhancers during human brain development with clinical relevance.

Soheil Yousefi, Ruizhi Deng, Kristina Lanko, Eva Medico Salsench, Anita Nikoncuk, Herma C van der Linde, Elena Perenthaler, Tjakko J van Ham, Eskeatnaf Mulugeta, Tahsin Stefan Barakat.

  BACKGROUND: Non-coding regulatory elements (NCREs), such as enhancers, play a crucial role in gene regulation, and genetic aberrations in NCREs can lead to human disease, including brain disorders. The human brain is a complex organ that is susceptible to numerous disorders; many of these are caused by genetic changes, but a multitude remain currently unexplained. Understanding NCREs acting during brain development has the potential to shed light on previously unrecognized genetic causes of human brain disease. Despite immense community-wide efforts to understand the role of the non-coding genome and NCREs, annotating functional NCREs remains challenging.METHODS: Here we performed an integrative computational analysis of virtually all currently available epigenome data sets related to human fetal brain.
RESULTS: Our in-depth analysis unravels 39,709 differentially active enhancers (DAEs) that show dynamic epigenomic rearrangement during early stages of human brain development, indicating likely biological function. Many of these DAEs are linked to clinically relevant genes, and functional validation of selected DAEs in cell models and zebrafish confirms their role in gene regulation. Compared to enhancers without dynamic epigenomic rearrangement, DAEs are subjected to higher sequence constraints in humans, have distinct sequence characteristics and are bound by a distinct transcription factor landscape. DAEs are enriched for GWAS loci for brain-related traits and for genetic variation found in individuals with neurodevelopmental disorders, including autism.
CONCLUSION: This compendium of high-confidence enhancers will assist in deciphering the mechanism behind developmental genetics of human brain and will be relevant to uncover missing heritability in human genetic brain disorders.

Keywords:  Clinical genetics; Computational analysis; Data integration; Enhancer; Epigenome; Gene regulatory elements; Human brain development; Mendelian disorders; Meta-analysis; Non-coding genome

DOI:  https://doi.org/10.1186/s13073-021-00980-1
EMBO Rep. 2021 Oct 18. e53035

Osteoclasts adapt to physioxia perturbation through DNA demethylation.

Keizo Nishikawa, Shigeto Seno, Toshitada Yoshihara, Ayako Narazaki, Yuki Sugiura, Reito Shimizu, Junichi Kikuta, Reiko Sakaguchi, Norio Suzuki, Norihiko Takeda, Hiroaki Semba, Masamichi Yamamoto, Daisuke Okuzaki, Daisuke Motooka, Yasuhiro Kobayashi, Makoto Suematsu, Haruhiko Koseki, Hideo Matsuda, Masayuki Yamamoto, Seiji Tobita, Yasuo Mori, Masaru Ishii.

  Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two-photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia-inducible factor activity. We observe that hypoxia decreases ten-eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen-dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation.

Keywords:  bone metabolism; epigenetic regulation; intravital imaging; osteoclast; oxygen

DOI:  https://doi.org/10.15252/embr.202153035
Nat Biotechnol. 2021 Oct 21.

Single-cell profiling of proteins and chromatin accessibility using PHAGE-ATAC.

Evgenij Fiskin, Caleb A Lareau, Leif S Ludwig, Gökcen Eraslan, Feimei Liu, Aaron M Ring, Ramnik J Xavier, Aviv Regev.

Multimodal measurements of single-cell profiles are proving increasingly useful for characterizing cell states and regulatory mechanisms. In the present study, we developed PHAGE-ATAC (Assay for Transposase-Accessible Chromatin), a massively parallel droplet-based method that uses phage displaying, engineered, camelid single-domain antibodies ('nanobodies') for simultaneous single-cell measurements of protein levels and chromatin accessibility profiles, and mitochondrial DNA-based clonal tracing. We use PHAGE-ATAC for multimodal analysis in primary human immune cells, sample multiplexing, intracellular protein analysis and the detection of SARS-CoV-2 spike protein in human cell populations. Finally, we construct a synthetic high-complexity phage library for selection of antigen-specific nanobodies that bind cells of particular molecular profiles, opening an avenue for protein detection, cell characterization and screening with single-cell genomics.

DOI: https://doi.org/10.1038/s41587-021-01065-5
Biochim Biophys Acta Gene Regul Mech. 2021 Oct 18. pii: S1874-9399(21)00083-3. [Epub ahead of print] 194765

A GO catalogue of human DNA-binding transcription factors.

Ruth C Lovering, Pascale Gaudet, Marcio L Acencio, Alex Ignatchenko, Arttu Jolma, Oriol Fornes, Martin Kuiper, Ivan V Kulakovskiy, Astrid Lægreid, Maria J Martin, Colin Logie.

  To control gene transcription, DNA-binding transcription factors recognise specific sequence motifs in gene regulatory regions. A complete and reliable GO annotation of all DNA-binding transcription factors is key to investigating the delicate balance of gene regulation in response to environmental and developmental stimuli. The need for such information is demonstrated by the many lists of transcription factors that have been produced over the past decade. The COST Action Gene Regulation Ensemble Effort for the Knowledge Commons (GREEKC) Consortium brought together experts in the field of transcription with the aim of providing high quality and interoperable gene regulatory data. The Gene Ontology (GO) Consortium provides strict definitions for gene product function, including factors that regulate transcription. The collaboration between the GREEKC and GO Consortia has enabled the application of those definitions to produce a new curated catalogue of over 1400 human DNA-binding transcription factors, that can be accessed at https://www.ebi.ac.uk/QuickGO/targetset/dbTF. This catalogue has facilitated an improvement in the GO annotation of human DNA-binding transcription factors and led to the GO annotation of almost sixty thousand DNA-binding transcription factors in over a hundred species. Thus, this work will aid researchers investigating the regulation of transcription in both biomedical and basic science.

Keywords:  Biocuration; DNA-binding transcription factor; Gene ontology

DOI:  https://doi.org/10.1016/j.bbagrm.2021.194765
Cell Death Dis. 2021 Oct 20. 12(11): 974

Identification of histone methyltransferase NSD2 as an important oncogenic gene in colorectal cancer.

Li-Hao Zhao, Quan Li, Zhi-Jun Huang, Mi-Xue Sun, Jing-Jing Lu, Xiao-Hua Zhang, Gang Li, Fang Wu.

Colorectal cancer (CRC) is the second common cause of cancer-related human mortalities. Dysregulation of histone 3 (H3) methylation could lead to transcriptional activation of multiple oncogenes, which is closely associated with CRC tumorigenesis and progression. Nuclear receptor-binding SET Domain protein 2 (NSD2) is a key histone methyltransferase catalyzing histone H3 lysine 36 dimethylation (H3K36me2). Its expression, the potential functions, and molecular mechanisms in CRC are studied here. Gene Expression Profiling Interactive Analysis (GEPIA) bioinformatics results showed that the NSD2 mRNA expression is elevated in both colon cancers and rectal cancers. Furthermore, NSD2 mRNA and protein expression levels in local colon cancer tissues are significantly higher than those in matched surrounding normal tissues. In primary human colon cancer cells and established CRC cell lines, shRNA-induced silencing or CRISPR/Cas9-induced knockout of NSD2 inhibited cell viability, proliferation, cell cycle progression, migration, and invasion. Furthermore, NSD2 shRNA or knockout induced mitochondrial depolarization, DNA damage, and apoptosis in the primary and established CRC cells. Contrarily, ectopic NSD2 overexpression in primary colon cancer cells further enhanced cell proliferation, migration, and invasion. H3K36me2, expressions of multiple oncogenes (ADAM9, EGFR, Sox2, Bcl-2, SYK, and MET) and Akt activation were significantly decreased after NSD2 silencing or knockout in primary colon cancer cells. Their levels were however increased after ectopic NSD2 overexpression. A catalytic inactive NSD2 (Y1179A) also inhibited H3K36me2, multiple oncogenes expression, and Akt activation, as well as cell proliferation and migration in primary colon cancer cells. In vivo, intratumoral injection of adeno-associated virus (AAV)-packed NSD2 shRNA largely inhibited primary colon cancer cell xenograft growth in nude mice. Together, NSD2 exerted oncogenic functions in CRC and could be a promising therapeutic target.

DOI: https://doi.org/10.1038/s41419-021-04267-6