bims-crepig 2020-10-18 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2020‒10‒18
twenty-nine papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

DOT1L-mediated murine neuronal differentiation associates with H3K79me2 accumulation and preserves SOX2-enhancer accessibility.
LncRNA Platr22 promotes super-enhancer activity and stem cell pluripotency.
Dephosphorylation of the proneural transcription factor ASCL1 re-engages a latent post-mitotic differentiation programme in neuroblastoma.
DeepC: predicting 3D genome folding using megabase-scale transfer learning.
The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization.
Arid4b is critical for mouse embryonic stem cell differentiation towards mesoderm and endoderm, linking epigenetics to pluripotency exit.
Histone H3.3 G34 mutations promote aberrant PRC2 activity and drive tumor progression.
GuidingNet: revealing transcriptional cofactor and predicting binding for DNA methyltransferase by network regularization.
MYC as a driver of stochastic chromatin networks: implications for the fitness of cancer cells.
Establishment and function of chromatin modification at enhancers.
TET1 interacts directly with NANOG via independent domains containing hydrophobic and aromatic residues.
EpiMogrify Models H3K4me3 Data to Identify Signaling Molecules that Improve Cell Fate Control and Maintenance.
Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry.
Multivalent interactions drive nucleosome binding and efficient chromatin deacetylation by SIRT6.
A Structural Model of the Endogenous Human BAF Complex Informs Disease Mechanisms.
Aberrant methylation underlies insulin gene expression in human insulinoma.
GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm.
Cancer-Associated Gain-of-Function Mutations Activate a SWI/SNF-Family Regulatory Hub.
Single-cell RNA cap and tail sequencing (scRCAT-seq) reveals subtype-specific isoforms differing in transcript demarcation.
SOX2 regulates homeostasis of taste bud cells and lingual epithelial cells in posterior tongue.
ATAC-seq Reveals an Isl1 Enhancer that Regulates Sinoatrial Node Development and Function.
Whole transcriptomic network analysis using Co-expression Differential Network Analysis (CoDiNA).
Cell-type-specific 3D epigenomes in the developing human cortex.
Phase-Separated Transcriptional Condensates Accelerate Target-Search Process Revealed by Live-Cell Single-Molecule Imaging.
Predicting 3D genome folding from DNA sequence with Akita.
Nuclear metabolism and the regulation of the epigenome.
H3 K27M and EZHIP Impede H3K27-Methylation Spreading by Inhibiting Allosterically Stimulated PRC2.
Senescence-activated enhancer landscape orchestrates the senescence-associated secretory phenotype in murine fibroblasts.
SilencerDB: a comprehensive database of silencers.

Nat Commun. 2020 10 15. 11(1): 5200

DOT1L-mediated murine neuronal differentiation associates with H3K79me2 accumulation and preserves SOX2-enhancer accessibility.

Francesco Ferrari, Laura Arrigoni, Henriette Franz, Annalisa Izzo, Ludmila Butenko, Eirini Trompouki, Tanja Vogel, Thomas Manke.

During neuronal differentiation, the transcriptional profile and the epigenetic context of neural committed cells is subject to significant rearrangements, but a systematic quantification of global histone modification changes is still missing. Here, we show that H3K79me2 increases and H3K27ac decreases globally during in-vitro neuronal differentiation of murine embryonic stem cells. DOT1L mediates all three degrees of methylation of H3K79 and its enzymatic activity is critical to modulate cellular differentiation and reprogramming. In this context, we find that inhibition of DOT1L in neural progenitor cells biases the transcriptional state towards neuronal differentiation, resulting in transcriptional upregulation of genes marked with H3K27me3 on the promoter region. We further show that DOT1L inhibition affects accessibility of SOX2-bound enhancers and impairs SOX2 binding in neural progenitors. Our work provides evidence that DOT1L activity gates differentiation of progenitors by allowing SOX2-dependent transcription of stemness programs.

DOI: https://doi.org/10.1038/s41467-020-19001-7
J Mol Cell Biol. 2020 Oct 13. pii: mjaa056. [Epub ahead of print]

LncRNA Platr22 promotes super-enhancer activity and stem cell pluripotency.

Pixi Yan, J Yuyang Lu, Jing Niu, Juntao Gao, Michael Q Zhang, Yafei Yin, Xiaohua Shen.

  Super-enhancers (SEs) comprise large clusters of enhancers, which are co-occupied by multiple lineage-specific and master transcription factors, and play pivotal roles in regulating gene expression and cell fate determination. However, it is still largely unknown whether and how SEs are regulated by the non-coding portion of the genome. Here, through genome-wide analysis, we found that long noncoding RNA (lncRNA) genes preferentially lie next to SEs. In mouse embryonic stem cells (mESCs), depletion of SE-associated lncRNA transcripts dysregulated the activity of their nearby SEs. Specifically, we revealed a critical regulatory role of the lncRNA gene Platr22 in modulating the activity of a nearby SE and the expression of the nearby pluripotency regulator ZFP281. Through these regulatory events, Platr22 contributes to pluripotency maintenance and proper differentiation of mESCs. Mechanistically, Platr22 transcripts coat chromatin near the SE region and interact with DDX5 and hnRNP-L. DDX5 further recruits p300 and other factors related to active transcription. We propose that these factors assemble into a transcription hub, thus promoting an open and active epigenetic chromatin state. Our study highlights an unanticipated role for a class of lncRNAs in epigenetically controlling the activity and vulnerability to perturbation of nearby SEs for cell fate determination.

Keywords:   Platr22 ; lncRNA; pluripotency; super-enhancer

DOI:  https://doi.org/10.1093/jmcb/mjaa056
Mol Cancer Res. 2020 Oct 12. pii: molcanres.0693.2020. [Epub ahead of print]

Dephosphorylation of the proneural transcription factor ASCL1 re-engages a latent post-mitotic differentiation programme in neuroblastoma.

Fahad R Ali, Daniel Marcos, Igor Chernukhin, Laura M Woods, Lydia M Parkinson, Luke A Wylie, Tatiana D Papkovskaia, John D Davies, Jason S Carroll, Anna Philpott.

Paediatric cancers often resemble trapped developmental intermediate states that fail to engage the normal differentiation programme, typified by high-risk neuroblastoma arising from the developing sympathetic nervous system. Neuroblastoma cells resemble arrested neuroblasts trapped by a stable but aberrant epigenetic programme controlled by sustained expression of a core transcriptional circuit of developmental regulators in conjunction with elevated MYCN or MYC (MYC). The transcription factor ASCL1 is a key master regulator in neuroblastoma and has oncogenic and tumour suppressive activities in several other tumour types. Using functional mutational approaches, we find that preventing CDK-dependent phosphorylation of ASCL1 in neuroblastoma cells drives co-ordinated suppression of the MYC-driven core circuit supporting neuroblast identity and proliferation, while simultaneously activating an enduring gene programme driving mitotic exit and neuronal differentiation. Implications: These findings indicate that targeting phosphorylation of ASCL1 may offer a new approach to development of differentiation therapies in neuroblastoma.

DOI: https://doi.org/10.1158/1541-7786.MCR-20-0693
Nat Methods. 2020 Oct 12.

DeepC: predicting 3D genome folding using megabase-scale transfer learning.

Ron Schwessinger, Matthew Gosden, Damien Downes, Richard C Brown, A Marieke Oudelaar, Jelena Telenius, Yee Whye Teh, Gerton Lunter, Jim R Hughes.

Predicting the impact of noncoding genetic variation requires interpreting it in the context of three-dimensional genome architecture. We have developed deepC, a transfer-learning-based deep neural network that accurately predicts genome folding from megabase-scale DNA sequence. DeepC predicts domain boundaries at high resolution, learns the sequence determinants of genome folding and predicts the impact of both large-scale structural and single base-pair variations.

DOI: https://doi.org/10.1038/s41592-020-0960-3
Genes Dev. 2020 Oct 15.

The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization.

Bence Daniel, Zsolt Czimmerer, Laszlo Halasz, Pal Boto, Zsuzsanna Kolostyak, Szilard Poliska, Wilhelm K Berger, Petros Tzerpos, Gergely Nagy, Attila Horvath, György Hajas, Timea Cseh, Aniko Nagy, Sascha Sauer, Jean Francois-Deleuze, Istvan Szatmari, Attila Bacsi, Laszlo Nagy.

  Macrophages polarize into functionally distinct subtypes while responding to microenvironmental cues. The identity of proximal transcription factors (TFs) downstream from the polarization signals are known, but their activity is typically transient, failing to explain the long-term, stable epigenomic programs developed. Here, we mapped the early and late epigenomic changes of interleukin-4 (IL-4)-induced alternative macrophage polarization. We identified the TF, early growth response 2 (EGR2), bridging the early transient and late stable gene expression program of polarization. EGR2 is a direct target of IL-4-activated STAT6, having broad action indispensable for 77% of the induced gene signature of alternative polarization, including its autoregulation and a robust, downstream TF cascade involving PPARG. Mechanistically, EGR2 binding results in chromatin opening and the recruitment of chromatin remodelers and RNA polymerase II. Egr2 induction is evolutionarily conserved during alternative polarization of mouse and human macrophages. In the context of tissue resident macrophages, Egr2 expression is most prominent in the lung of a variety of species. Thus, EGR2 is an example of an essential and evolutionarily conserved broad acting factor, linking transient polarization signals to stable epigenomic and transcriptional changes in macrophages.

Keywords:  EGR2; IL-4; epigenomic regulation; macrophage polarization; transcription factor network

DOI:  https://doi.org/10.1101/gad.343038.120
J Biol Chem. 2020 Oct 15. pii: jbc.RA120.015534. [Epub ahead of print]

Arid4b is critical for mouse embryonic stem cell differentiation towards mesoderm and endoderm, linking epigenetics to pluripotency exit.

Nihal Terzi Cizmecioglu, Jialiang Huang, Ezgi G Keskin, Xiaofeng Wang, Idil Esen, Fei Chen, Stuart H Orkin.

  Distinct cell types emerge from embryonic stem cells through a precise and coordinated execution of gene expression programs during lineage commitment. This is established by the action of lineage specific transcription factors along with chromatin complexes. Numerous studies have focused on epigenetic factors that affect ESC self-renewal and pluripotency. However, the contribution of chromatin to lineage decisions at the exit from pluripotency has not been as extensively studied. Using a pooled epigenetic shRNA screen strategy, we identified chromatin related factors critical for differentiation towards mesodermal and endodermal lineages. Here we reveal a critical role for the chromatin protein, Arid4b. Arid4b-deficient mESCs are similar to wild-type mESCs in the expression of pluripotency factors and their self-renewal. However, Arid4b loss results in defects in upregulation of the meso/endodermal gene expression program. It was previously shown that Arid4b resides in a complex with Sin3a and Hdacs 1 and 2. We identified a physical and functional interaction of Arid4b with Hdac1 rather than Hdac2, suggesting functionally distinct Sin3a sub-complexes might regulate cell fate decisions Finally, we observed that Arid4b deficiency leads to increased H3K27me3 and reduced H3K27Ac level in key developmental gene loci, while a subset of genomic regions gain H3K27Ac marks. Our results demonstrate that epigenetic control through Arid4b plays a key role in the execution of lineage-specific gene expression programs at pluripotency exit.

Keywords:  cell differentiation; chromatin modification; embryonic stem cell; epigenetics; gene expression

DOI:  https://doi.org/10.1074/jbc.RA120.015534
Proc Natl Acad Sci U S A. 2020 Oct 16. pii: 202006076. [Epub ahead of print]

Histone H3.3 G34 mutations promote aberrant PRC2 activity and drive tumor progression.

Siddhant U Jain, Sima Khazaei, Dylan M Marchione, Stefan M Lundgren, Xiaoshi Wang, Daniel N Weinberg, Shriya Deshmukh, Nikoleta Juretic, Chao Lu, C David Allis, Benjamin A Garcia, Nada Jabado, Peter W Lewis.

  A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

Keywords:  H3.3 G34 mutations; NSD1/2; PRC2; SETD2; oncohistones

DOI:  https://doi.org/10.1073/pnas.2006076117
Brief Bioinform. 2020 Oct 13. pii: bbaa245. [Epub ahead of print]

GuidingNet: revealing transcriptional cofactor and predicting binding for DNA methyltransferase by network regularization.

Lixin Ren, Caixia Gao, Zhana Duren, Yong Wang.

  The DNA methyltransferases (DNMTs) (DNMT3A, DNMT3B and DNMT3L) are primarily responsible for the establishment of genomic locus-specific DNA methylation patterns, which play an important role in gene regulation and animal development. However, this important protein family's binding mechanism, i.e. how and where the DNMTs bind to genome, is still missing in most tissues and cell lines. This motivates us to explore DNMTs and TF's cooperation and develop a network regularized logistic regression model, GuidingNet, to predict DNMTs' genome-wide binding by integrating gene expression, chromatin accessibility, sequence and protein-protein interaction data. GuidingNet accurately predicted methylation experimental data validated DNMTs' binding, outperformed single data source based and sparsity regularized methods and performed well in within and across tissue prediction for several DNMTs in human and mouse. Importantly, GuidingNet can reveal transcription cofactors assisting DNMTs for methylation establishment. This provides biological understanding in the DNMTs' binding specificity in different tissues and demonstrate the advantage of network regularization. In addition to DNMTs, GuidingNet achieves good performance for other chromatin regulators' binding. GuidingNet is freely available at https://github.com/AMSSwanglab/GuidingNet.

Keywords:  DNA methyltransferase; data integration; network regularization; transcription cofactor

DOI:  https://doi.org/10.1093/bib/bbaa245
Nucleic Acids Res. 2020 Oct 13. pii: gkaa817. [Epub ahead of print]

MYC as a driver of stochastic chromatin networks: implications for the fitness of cancer cells.

Noriyuki Sumida, Emmanouil G Sifakis, Narsis A Kiani, Anna Lewandowska Ronnegren, Barbara A Scholz, Johanna Vestlund, David Gomez-Cabrero, Jesper Tegner, Anita Göndör, Rolf Ohlsson.

The relationship between stochastic transcriptional bursts and dynamic 3D chromatin states is not well understood. Using an innovated, ultra-sensitive technique, we address here enigmatic features underlying the communications between MYC and its enhancers in relation to the transcriptional process. MYC thus interacts with its flanking enhancers in a mutually exclusive manner documenting that enhancer hubs impinging on MYC detected in large cell populations likely do not exist in single cells. Dynamic encounters with pathologically activated enhancers responsive to a range of environmental cues, involved <10% of active MYC alleles at any given time in colon cancer cells. Being the most central node of the chromatin network, MYC itself likely drives its communications with flanking enhancers, rather than vice versa. We submit that these features underlie an acquired ability of MYC to become dynamically activated in response to a diverse range of environmental cues encountered by the cell during the neoplastic process.

DOI: https://doi.org/10.1093/nar/gkaa817
Open Biol. 2020 Oct;10(10): 200255

Establishment and function of chromatin modification at enhancers.

Amanuel Tafessu, Laura A Banaszynski.

  How a single genome can give rise to distinct cell types remains a fundamental question in biology. Mammals are able to specify and maintain hundreds of cell fates by selectively activating unique subsets of their genome. This is achieved, in part, by enhancers-genetic elements that can increase transcription of both nearby and distal genes. Enhancers can be identified by their unique chromatin signature, including transcription factor binding and the enrichment of specific histone post-translational modifications, histone variants, and chromatin-associated cofactors. How each of these chromatin features contributes to enhancer function remains an area of intense study. In this review, we provide an overview of enhancer-associated chromatin states, and the proteins and enzymes involved in their establishment. We discuss recent insights into the effects of the enhancer chromatin state on ongoing transcription versus their role in the establishment of new transcription programmes, such as those that occur developmentally. Finally, we highlight the role of enhancer chromatin in new conceptual advances in gene regulation such as condensate formation.

Keywords:  chromatin; enhancer; histone variants; phase separation; post-translational modification; transcription

DOI:  https://doi.org/10.1098/rsob.200255
J Mol Biol. 2020 Oct 12. pii: S0022-2836(20)30584-2. [Epub ahead of print]

TET1 interacts directly with NANOG via independent domains containing hydrophobic and aromatic residues.

Raphaël Pantier, Nicholas Mullin, Elisa Hall-Ponsele, Ian Chambers.

  The DNA demethylase TET1 is highly expressed in embryonic stem cells and is important both for lineage commitment, and reprogramming to naïve pluripotency. TET1 interacts with the pluripotency transcription factor NANOG which may contribute to its biological activity in pluripotent cells. However, how TET1 interacts with other proteins is largely unknown. Here, we characterise the physical interaction between TET1 and NANOG using embryonic stem cells and bacterial expression systems. TET1 and NANOG interact through multiple binding sites that act independently. Critically, mutating conserved hydrophobic and aromatic residues within TET1 and NANOG abolishes the interaction. On chromatin, NANOG is predominantly localised at ESC enhancers. While TET1 binds to CpG dinucleotides in promoters using its CXXC domain, TET1 also binds to enhancers, though the mechanism involved is unknown. Comparative ChIP-seq analysis identifies genomic loci bound by both TET1 and NANOG, that correspond predominantly to pluripotency enhancers. Importantly, around half of NANOG transcriptional target genes are associated with TET1-NANOG co-bound sites. These results indicate a mechanism by which TET1 protein may be targeted to specific sites of action at enhancers by direct interaction with a transcription factor.

Keywords:  chromatin; embryonic stem cells; enhancers; pluripotency; protein-protein interactions

DOI:  https://doi.org/10.1016/j.jmb.2020.10.008
Cell Syst. 2020 Oct 06. pii: S2405-4712(20)30334-3. [Epub ahead of print]

EpiMogrify Models H3K4me3 Data to Identify Signaling Molecules that Improve Cell Fate Control and Maintenance.

Uma S Kamaraj, Joseph Chen, Khairunnisa Katwadi, John F Ouyang, Yu Bo Yang Sun, Yu Ming Lim, Xiaodong Liu, Lusy Handoko, Jose M Polo, Enrico Petretto, Owen J L Rackham.

  The need to derive and culture diverse cell or tissue types in vitro has prompted investigations on how changes in culture conditions affect cell states. However, the identification of the optimal conditions (e.g., signaling molecules and growth factors) required to maintain cell types or convert between cell types remains a time-consuming task. Here, we developed EpiMogrify, an approach that leverages data from ∼100 human cell/tissue types available from ENCODE and Roadmap Epigenomics consortia to predict signaling molecules and factors that can either maintain cell identity or enhance directed differentiation (or cell conversion). EpiMogrify integrates protein-protein interaction network information with a model of the cell's epigenetic landscape based on H3K4me3 histone modifications. Using EpiMogrify-predicted factors for maintenance conditions, we were able to better potentiate the maintenance of astrocytes and cardiomyocytes in vitro. We report a significant increase in the efficiency of astrocyte and cardiomyocyte differentiation using EpiMogrify-predicted factors for conversion conditions.

Keywords:  cell conversion; cell maintenance; computational method; data driven; predictions

DOI:  https://doi.org/10.1016/j.cels.2020.09.004
Nat Commun. 2020 Oct 16. 11(1): 5250

Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry.

Alexandra Stützer, Luisa M Welp, Monika Raabe, Timo Sachsenberg, Christin Kappert, Alexander Wulf, Andy M Lau, Stefan-Sebastian David, Aleksandar Chernev, Katharina Kramer, Argyris Politis, Oliver Kohlbacher, Wolfgang Fischle, Henning Urlaub.

Protein-DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein-DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein-RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.

DOI: https://doi.org/10.1038/s41467-020-19047-7
Nat Commun. 2020 Oct 16. 11(1): 5244

Multivalent interactions drive nucleosome binding and efficient chromatin deacetylation by SIRT6.

Wallace H Liu, Jie Zheng, Jessica L Feldman, Mark A Klein, Vyacheslav I Kuznetsov, Craig L Peterson, Patrick R Griffin, John M Denu.

The protein deacetylase SIRT6 maintains cellular homeostasis through multiple pathways that include the deacetylation of histone H3 and repression of transcription. Prior work suggests that SIRT6 is associated with chromatin and can substantially reduce global levels of H3 acetylation, but how SIRT6 is able to accomplish this feat is unknown. Here, we describe an exquisitely tight interaction between SIRT6 and nucleosome core particles, in which a 2:1 enzyme:nucleosome complex assembles via asymmetric binding with distinct affinities. While both SIRT6 molecules associate with the acidic patch on the nucleosome, we find that the intrinsically disordered SIRT6 C-terminus promotes binding at the higher affinity site through recognition of nucleosomal DNA. Together, multivalent interactions couple productive binding to efficient deacetylation of histones on endogenous chromatin. Unique among histone deacetylases, SIRT6 possesses the intrinsic capacity to tightly interact with nucleosomes for efficient activity.

DOI: https://doi.org/10.1038/s41467-020-19018-y
Cell. 2020 Oct 09. pii: S0092-8674(20)31248-4. [Epub ahead of print]

A Structural Model of the Endogenous Human BAF Complex Informs Disease Mechanisms.

Nazar Mashtalir, Hiroshi Suzuki, Daniel P Farrell, Akshay Sankar, Jie Luo, Martin Filipovski, Andrew R D'Avino, Roodolph St Pierre, Alfredo M Valencia, Takashi Onikubo, Robert G Roeder, Yan Han, Yuan He, Jeffrey A Ranish, Frank DiMaio, Thomas Walz, Cigall Kadoch.

  Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.

Keywords:  ATP-dependent chromatin remodeling; BAF complex; cancer; cross-linking mass spectrometry; cryoelectron microscopy; homology modeling; mammalian SWI/SNF complexes; mutations

DOI:  https://doi.org/10.1016/j.cell.2020.09.051
Nat Commun. 2020 10 15. 11(1): 5210

Aberrant methylation underlies insulin gene expression in human insulinoma.

Esra Karakose, Huan Wang, William Inabnet, Rajesh V Thakker, Steven Libutti, Gustavo Fernandez-Ranvier, Hyunsuk Suh, Mark Stevenson, Yayoi Kinoshita, Michael Donovan, Yevgeniy Antipin, Yan Li, Xiaoxiao Liu, Fulai Jin, Peng Wang, Andrew Uzilov, Carmen Argmann, Eric E Schadt, Andrew F Stewart, Donald K Scott, Luca Lambertini.

Human insulinomas are rare, benign, slowly proliferating, insulin-producing beta cell tumors that provide a molecular "recipe" or "roadmap" for pathways that control human beta cell regeneration. An earlier study revealed abnormal methylation in the imprinted p15.5-p15.4 region of chromosome 11, known to be abnormally methylated in another disorder of expanded beta cell mass and function: the focal variant of congenital hyperinsulinism. Here, we compare deep DNA methylome sequencing on 19 human insulinomas, and five sets of normal beta cells. We find a remarkably consistent, abnormal methylation pattern in insulinomas. The findings suggest that abnormal insulin (INS) promoter methylation and altered transcription factor expression create alternative drivers of INS expression, replacing canonical PDX1-driven beta cell specification with a pathological, looping, distal enhancer-based form of transcriptional regulation. Finally, NFaT transcription factors, rather than the canonical PDX1 enhancer complex, are predicted to drive INS transactivation.

DOI: https://doi.org/10.1038/s41467-020-18839-1
Elife. 2020 Oct 15. pii: e53278. [Epub ahead of print]9

GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm.

Arun Sharma, Lauren K Wasson, Jon Al Willcox, Sarah U Morton, Joshua M Gorham, Daniel M DeLaughter, Meraj Neyazi, Manuel Schmid, Radhika Agarwal, Min Young Jang, Christopher N Toepfer, Tarsha Ward, Yuri Kim, Alexandre C Pereira, Steven R DePalma, Angela Tai, Seongwon Kim, David Conner, Daniel Bernstein, Bruce D Gelb, Wendy K Chung, Elizabeth Goldmuntz, George Porter, Martin Tristani-Firouzi, Deepak Srivastava, Jonathan G Seidman, Christine E Seidman.

  Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. Loss of function variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2 and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.

Keywords:  developmental biology; human; regenerative medicine; stem cells

DOI:  https://doi.org/10.7554/eLife.53278
Mol Cell. 2020 Oct 06. pii: S1097-2765(20)30654-7. [Epub ahead of print]

Cancer-Associated Gain-of-Function Mutations Activate a SWI/SNF-Family Regulatory Hub.

Cedric R Clapier, Naveen Verma, Timothy J Parnell, Bradley R Cairns.

  SWI/SNF-family remodelers (BAF/PBAF in mammals) are essential chromatin regulators, and mutations in human BAF/PBAF components are associated with ∼20% of cancers. Cancer-associated missense mutations in human BRG1 (encoding the catalytic ATPase) have been characterized previously as conferring loss-of-function. Here, we show that cancer-associated missense mutations in BRG1, when placed into the orthologous Sth1 ATPase of the yeast RSC remodeler, separate into two categories: loss-of-function enzymes, or instead, gain-of-function enzymes that greatly improve DNA translocation efficiency and nucleosome remodeling in vitro. Our work identifies a structural "hub," formed by the association of several Sth1 domains, that regulates ATPase activity and DNA translocation efficiency. Remarkably, all gain-of-function cancer-associated mutations and all loss-of-function mutations physically localize to distinct adjacent regions in the hub, which specifically regulate and implement DNA translocation, respectively. In vivo, only gain-of-function cancer-associated mutations conferred precocious chromatin accessibility. Taken together, we provide a structure-function mechanistic basis for cancer-associated hyperactivity.

Keywords:  BAF; BRG1; DNA accessibility; RSC; STH1; SWI/SNF; cancer; chromatin remodeling; nucleosome

DOI:  https://doi.org/10.1016/j.molcel.2020.09.024
Nat Commun. 2020 10 13. 11(1): 5148

Single-cell RNA cap and tail sequencing (scRCAT-seq) reveals subtype-specific isoforms differing in transcript demarcation.

Youjin Hu, Jiawei Zhong, Yuhua Xiao, Zheng Xing, Katherine Sheu, Shuxin Fan, Qin An, Yuanhui Qiu, Yingfeng Zheng, Xialin Liu, Guoping Fan, Yizhi Liu.

The differences in transcription start sites (TSS) and transcription end sites (TES) among gene isoforms can affect the stability, localization, and translation efficiency of mRNA. Gene isoforms allow a single gene diverse functions across different cell types, and isoform dynamics allow different functions over time. However, methods to efficiently identify and quantify RNA isoforms genome-wide in single cells are still lacking. Here, we introduce single cell RNA Cap And Tail sequencing (scRCAT-seq), a method to demarcate the boundaries of isoforms based on short-read sequencing, with higher efficiency and lower cost than existing long-read sequencing methods. In conjunction with machine learning algorithms, scRCAT-seq demarcates RNA transcripts with unprecedented accuracy. We identified hundreds of previously uncharacterized transcripts and thousands of alternative transcripts for known genes, revealed cell-type specific isoforms for various cell types across different species, and generated a cell atlas of isoform dynamics during the development of retinal cones.

DOI: https://doi.org/10.1038/s41467-020-18976-7
PLoS One. 2020 ;15(10): e0240848

SOX2 regulates homeostasis of taste bud cells and lingual epithelial cells in posterior tongue.

Makoto Ohmoto, Weiwei Lei, Junpei Yamashita, Junji Hirota, Peihua Jiang, Ichiro Matsumoto.

Taste bud cells arise from local epithelial stem cells in the oral cavity and are continuously replaced by newborn cells throughout an animal's life. However, little is known about the molecular and cellular mechanisms of taste cell turnover. Recently, it has been demonstrated that SOX2, a transcription factor expressed in epithelial stem/progenitor cells of the oral cavity, regulates turnover of anterior tongue epithelium including gustatory and non-gustatory papillae. Yet, the role of SOX2 in regulating taste cell turnover in the posterior tongue is unclear. Prompted by the fact that there are regional differences in the cellular and molecular composition of taste buds and stem/progenitor cells in the anterior and posterior portions of tongue, which are derived from distinct embryonic origins, we set out to determine the role of SOX2 in epithelial tissue homeostasis in the posterior tongue. Here we report the differential requirement of SOX2 in the stem/progenitor cells for the normal turnover of lingual epithelial cells in the posterior tongue. Sox2 deletion in the stem/progenitor cells neither induced active caspase 3-mediated apoptotic cell death nor altered stem/progenitor cell population in the posterior tongue. Nevertheless, morphology and molecular feature of non-gustatory epithelial cells were impaired in the circumvallate papilla but not in the filiform papillae. Remarkably, taste buds became thinner, collapsed, and undetectable over time. Lineage tracing of Sox2-deleted stem/progenitor cells demonstrated an almost complete lack of newly generated basal precursor cells in the taste buds, suggesting mechanistically that Sox2 is involved in determining stem/progenitor cells to differentiate to gustatory lineage cells. Together, these results demonstrate that SOX2 plays key roles in regulating epithelial tissue homeostasis in the posterior tongue, similar but not identical to its function in the anterior tongue.

DOI: https://doi.org/10.1371/journal.pone.0240848
Circ Res. 2020 Oct 12.

ATAC-seq Reveals an Isl1 Enhancer that Regulates Sinoatrial Node Development and Function.

Giselle Galang, Ravi Mandla, Hongmei Ruan, Catherine Jung, Tanvi Sinha, Nicole R Stone, Roland S Wu, Brandon J Mannion, Prasanna Kr Allu, Kevin Chang, Ashwin Rammohan, Marie B Shi, Len A Pennacchio, Brian L Black, Vasanth Vedantham.

Rationale: Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1, Tbx3, and Shox2, have been identified, the cis-regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified. Objective: To define the epigenetic profile of PCs using comparative ATAC-seq and to identify novel enhancers involved in SAN gene regulation, development and function. Methods and Results: We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched ATAC-seq peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at E8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations. Conclusions: (1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) Cis-regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human ISL1 enhancer may regulate human SAN function.

DOI: https://doi.org/10.1161/CIRCRESAHA.120.317145
PLoS One. 2020 ;15(10): e0240523

Whole transcriptomic network analysis using Co-expression Differential Network Analysis (CoDiNA).

Deisy Morselli Gysi, Tiago de Miranda Fragoso, Fatemeh Zebardast, Wesley Bertoli, Volker Busskamp, Eivind Almaas, Katja Nowick.

Biological and medical sciences are increasingly acknowledging the significance of gene co-expression-networks for investigating complex-systems, phenotypes or diseases. Typically, complex phenotypes are investigated under varying conditions. While approaches for comparing nodes and links in two networks exist, almost no methods for the comparison of multiple networks are available and-to best of our knowledge-no comparative method allows for whole transcriptomic network analysis. However, it is the aim of many studies to compare networks of different conditions, for example, tissues, diseases, treatments, time points, or species. Here we present a method for the systematic comparison of an unlimited number of networks, with unlimited number of transcripts: Co-expression Differential Network Analysis (CoDiNA). In particular, CoDiNA detects links and nodes that are common, specific or different among the networks. We developed a statistical framework to normalize between these different categories of common or changed network links and nodes, resulting in a comprehensive network analysis method, more sophisticated than simply comparing the presence or absence of network nodes. Applying CoDiNA to a neurogenesis study we identified candidate genes involved in neuronal differentiation. We experimentally validated one candidate, demonstrating that its overexpression resulted in a significant disturbance in the underlying gene regulatory network of neurogenesis. Using clinical studies, we compared whole transcriptome co-expression networks from individuals with or without HIV and active tuberculosis (TB) and detected signature genes specific to HIV. Furthermore, analyzing multiple cancer transcription factor (TF) networks, we identified common and distinct features for particular cancer types. These CoDiNA applications demonstrate the successful detection of genes associated with specific phenotypes. Moreover, CoDiNA can also be used for comparing other types of undirected networks, for example, metabolic, protein-protein interaction, ecological and psychometric networks. CoDiNA is publicly available as an R package in CRAN (https://CRAN.R-project.org/package=CoDiNA).

DOI: https://doi.org/10.1371/journal.pone.0240523
Nature. 2020 Oct 14.

Cell-type-specific 3D epigenomes in the developing human cortex.

Michael Song, Mark-Phillip Pebworth, Xiaoyu Yang, Armen Abnousi, Changxu Fan, Jia Wen, Jonathan D Rosen, Mayank N K Choudhary, Xiekui Cui, Ian R Jones, Seth Bergenholtz, Ugomma C Eze, Ivan Juric, Bingkun Li, Lenka Maliskova, Jerry Lee, Weifang Liu, Alex A Pollen, Yun Li, Ting Wang, Ming Hu, Arnold R Kriegstein, Yin Shen.

Lineage-specific epigenomic changes during human corticogenesis have been difficult to study owing to challenges with sample availability and tissue heterogeneity. For example, previous studies using single-cell RNA sequencing identified at least 9 major cell types and up to 26 distinct subtypes in the dorsal cortex alone1,2. Here we characterize cell-type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational samples of the human cortex. We show that chromatin interactions underlie several aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell-type-specific manner. In addition, promoters with increased levels of chromatin interactivity-termed super-interactive promoters-are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a technique that integrates immunostaining, CRISPR interference, RNAscope, and image analysis to validate cell-type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our findings provide insights into cell-type-specific gene expression patterns in the developing human cortex and advance our understanding of gene regulation and lineage specification during this crucial developmental window.

DOI: https://doi.org/10.1038/s41586-020-2825-4
Cell Rep. 2020 Oct 13. pii: S2211-1247(20)31237-7. [Epub ahead of print]33(2): 108248

Phase-Separated Transcriptional Condensates Accelerate Target-Search Process Revealed by Live-Cell Single-Molecule Imaging.

Samantha Kent, Kyle Brown, Chou-Hsun Yang, Njood Alsaihati, Christina Tian, Haobin Wang, Xiaojun Ren.

  Compartmentalization by liquid-liquid phase separation is implicated in transcription. It remains unclear whether and how transcriptional condensates accelerate the search of transcriptional regulatory factors for their target sites. Furthermore, the molecular mechanisms by which regulatory factors nucleate on chromatin to assemble transcriptional condensates remain incompletely understood. The CBX-PRC1 complexes compartmentalize key developmental regulators for repression through phase-separated condensates driven by the chromobox 2 (CBX2) protein. Here, by using live-cell single-molecule imaging, we show that CBX2 nucleates on chromatin independently of H3K27me3 and CBX-PRC1. The interactions between CBX2 and DNA are essential for nucleating CBX-PRC1 on chromatin to assemble condensates. The assembled condensates shorten 3D diffusion time and reduce trials for finding specific sites through revisiting the same or adjacent sites repetitively, thereby accelerating CBX2 in searching for target sites. Overall, our data suggest a generic mechanism by which transcriptional regulatory factors nucleate to assemble condensates that accelerate their target-search process.

Keywords:  CBX2; PRC1; PcG; chromatin; compartmentalization; epigenetics; liquid-liquid phase separation; nucleation; single-molecule imaging; target-search kinetics

DOI:  https://doi.org/10.1016/j.celrep.2020.108248
Nat Methods. 2020 Oct 12.

Predicting 3D genome folding from DNA sequence with Akita.

Geoff Fudenberg, David R Kelley, Katherine S Pollard.

In interphase, the human genome sequence folds in three dimensions into a rich variety of locus-specific contact patterns. Cohesin and CTCF (CCCTC-binding factor) are key regulators; perturbing the levels of either greatly disrupts genome-wide folding as assayed by chromosome conformation capture methods. Still, how a given DNA sequence encodes a particular locus-specific folding pattern remains unknown. Here we present a convolutional neural network, Akita, that accurately predicts genome folding from DNA sequence alone. Representations learned by Akita underscore the importance of an orientation-specific grammar for CTCF binding sites. Akita learns predictive nucleotide-level features of genome folding, revealing effects of nucleotides beyond the core CTCF motif. Once trained, Akita enables rapid in silico predictions. Accounting for this, we demonstrate how Akita can be used to perform in silico saturation mutagenesis, interpret eQTLs, make predictions for structural variants and probe species-specific genome folding. Collectively, these results enable decoding genome function from sequence through structure.

DOI: https://doi.org/10.1038/s41592-020-0958-x
Nat Metab. 2020 Oct 12.

Nuclear metabolism and the regulation of the epigenome.

Ruben Boon, Giorgia G Silveira, Raul Mostoslavsky.

Cellular metabolism has emerged as a major biological node governing cellular behaviour. Metabolic pathways fuel cellular energy needs, providing basic chemical molecules to sustain cellular homeostasis, proliferation and function. Changes in nutrient consumption or availability therefore can result in complete reprogramming of cellular metabolism towards stabilizing core metabolite pools, such as ATP, S-adenosyl methionine, acetyl-CoA, NAD/NADP and α-ketoglutarate. Because these metabolites underlie a variety of essential metabolic reactions, metabolism has evolved to operate in separate subcellular compartments through diversification of metabolic enzyme complexes, oscillating metabolic activity and physical separation of metabolite pools. Given that these same core metabolites are also consumed by chromatin modifiers in the establishment of epigenetic signatures, metabolite consumption on and release from chromatin directly influence cellular metabolism and gene expression. In this Review, we highlight recent studies describing the mechanisms determining nuclear metabolism and governing the redistribution of metabolites between the nuclear and non-nuclear compartments.

DOI: https://doi.org/10.1038/s42255-020-00285-4
Mol Cell. 2020 Oct 07. pii: S1097-2765(20)30680-8. [Epub ahead of print]

H3 K27M and EZHIP Impede H3K27-Methylation Spreading by Inhibiting Allosterically Stimulated PRC2.

Siddhant U Jain, Andrew Q Rashoff, Samuel D Krabbenhoft, Dominik Hoelper, Truman J Do, Tyler J Gibson, Stefan M Lundgren, Eliana R Bondra, Shriya Deshmukh, Ashot S Harutyunyan, Nikoleta Juretic, Nada Jabado, Melissa M Harrison, Peter W Lewis.

  Diffuse midline gliomas and posterior fossa type A ependymomas contain the recurrent histone H3 lysine 27 (H3 K27M) mutation and express the H3 K27M-mimic EZHIP (CXorf67), respectively. H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3 lysine 27 trimethylation (H3K27me3) levels; however, residual peaks of H3K27me3 remain at CpG islands (CGIs) through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with PRC2 that is allosterically activated by H3K27me3 at CGIs and impede its spreading. Moreover, H3 K27M oncohistones reduce H3K27me3 in trans, independent of their incorporation into the chromatin. Although EZHIP is not found outside placental mammals, expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved mechanism. Our results provide mechanistic insights for the retention of residual H3K27me3 in tumors driven by H3 K27M and EZHIP.

Keywords:  Drosophila PREs; EZHIP/CXorf67; H3.1 K27M; H3.3 K27M; PRC2; Weaver syndrome EED R302S mutation; diffuse midline gliomas; posterior fossa type-A ependymoma

DOI:  https://doi.org/10.1016/j.molcel.2020.09.028
Nucleic Acids Res. 2020 Oct 12. pii: gkaa858. [Epub ahead of print]

Senescence-activated enhancer landscape orchestrates the senescence-associated secretory phenotype in murine fibroblasts.

Yiting Guan, Chao Zhang, Guoliang Lyu, Xiaoke Huang, Xuebin Zhang, Tenghan Zhuang, Lumeng Jia, Lijun Zhang, Chen Zhang, Cheng Li, Wei Tao.

The three-dimensional configuration of the chromatin architecture is known to be crucial for alterations in the transcriptional network; however, the underlying mechanisms of epigenetic control of senescence-related gene expression by modulating the chromatin architecture remain unknown. Here, we demonstrate frequent chromosomal compartment switching during mouse embryonic fibroblasts (MEFs) replicative senescence as characterized by senescence-inactivated (SIAEs) and -activated enhancers (SAEs) in topologically associated domains (TADs). Mechanistically, SAEs are closely correlated with senescence-associated secretory phenotype (SASP) genes, which are a key transcriptional feature of an aging microenvironment that contributes to tumor progression, aging acceleration, and immunoinflammatory responses. Moreover, SAEs can positively regulate robust changes in SASP expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is capable of enhancing SAE activity, which accelerates the emergence of SAEs flanking SASPs and the secretion of downstream factors, contributing to the progression of senescence. Our results provide novel insight into the TAD-related control of SASP gene expression by revealing hierarchical roles of the chromatin architecture, transcription factors, and enhancer activity in the regulation of cellular senescence.

DOI: https://doi.org/10.1093/nar/gkaa858
Nucleic Acids Res. 2020 Oct 12. pii: gkaa839. [Epub ahead of print]

SilencerDB: a comprehensive database of silencers.

Wanwen Zeng, Shengquan Chen, Xuejian Cui, Xiaoyang Chen, Zijing Gao, Rui Jiang.

Gene regulatory elements, including promoters, enhancers, silencers, etc., control transcriptional programs in a spatiotemporal manner. Though these elements are known to be able to induce either positive or negative transcriptional control, the community has been mostly studying enhancers which amplify transcription initiation, with less emphasis given to silencers which repress gene expression. To facilitate the study of silencers and the investigation of their potential roles in transcriptional control, we developed SilencerDB (http://health.tsinghua.edu.cn/silencerdb/), a comprehensive database of silencers by manually curating silencers from 2300 published articles. The current version, SilencerDB 1.0, contains (1) 33 060 validated silencers from experimental methods, and (ii) 5 045 547 predicted silencers from state-of-the-art machine learning methods. The functionality of SilencerDB includes (a) standardized categorization of silencers in a tree-structured class hierarchy based on species, organ, tissue and cell line and (b) comprehensive annotations of silencers with the nearest gene and potential regulatory genes. SilencerDB, to the best of our knowledge, is the first comprehensive database at this scale dedicated to silencers, with reliable annotations and user-friendly interactive database features. We believe this database has the potential to enable advanced understanding of silencers in regulatory mechanisms and to empower researchers to devise diverse applications of silencers in disease development.

DOI: https://doi.org/10.1093/nar/gkaa839