bims-crepig 2021-10-17 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2021‒10‒17
thirty papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

A Polycomb domain found in committed cells impairs differentiation when introduced into PRC1 in pluripotent cells.
Integrative epigenomic and high-throughput functional enhancer profiling reveals determinants of enhancer heterogeneity in gastric cancer.
KrasG12D induces changes in chromatin territories that differentially impact early nuclear reprogramming in pancreatic cells.
Simultaneous profiling of multiple chromatin proteins in the same cells.
STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells.
Morc3 silences endogenous retroviruses by enabling Daxx-mediated histone H3.3 incorporation.
NRF1 association with AUTS2-Polycomb mediates specific gene activation in the brain.
Multiscale and integrative single-cell Hi-C analysis with Higashi.
Pioneer factors in development and cancer.
Reorganization of the 3D genome pinpoints non-coding drivers of primary prostate tumors.
Ssl2/TFIIH function in transcription start site scanning by RNA Polymerase II in Saccharomyces cerevisiae.
Chromatin Velocity reveals epigenetic dynamics by single-cell profiling of heterochromatin and euchromatin.
Structural basis of chromatin regulation by histone variant H2A.Z.
greenCUT&RUN: Efficient Genomic Profiling of GFP-Tagged Transcription Factors and Chromatin Regulators.
The dynamic, combinatorial cis-regulatory lexicon of epidermal differentiation.
The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle.
Global profiling of RNA-chromatin interactions reveals co-regulatory gene expression networks in Arabidopsis.
Scalable dual-omics profiling with single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2).
A global screening identifies chromatin-enriched RNA-binding proteins and the transcriptional regulatory activity of QKI5 during monocytic differentiation.
Metabolic gene regulation by Drosophila GATA transcription factor Grain.
SUMOylation Potentiates ZIC Protein Activity to Influence Murine Neural Crest Cell Specification.
Targeting KDM6A suppresses SREBP1c-dependent lipid metabolism and prostate tumorigenesis.
Coupling high-throughput mapping with proteomics analysis delineates cis-regulatory elements at high resolution.
Therapeutically expanded human regulatory T-cells are super-suppressive due to HIF1A induced expression of CD73.
Segmentation and genome annotation algorithms for identifying chromatin state and other genomic patterns.
Inhibition of GATA2 in prostate cancer by a clinically available small molecule.
Gene bookmarking by the heat shock transcription factor programs the insulin-like signaling pathway.
Single-cell atlas of lineage states, tumor microenvironment and subtype-specific expression programs in gastric cancer.
The comprehensive interactomes of human adenosine RNA methyltransferases and demethylases reveal distinct functional and regulatory features.
Pathway dynamics can delineate the sources of transcriptional noise in gene expression.

Mol Cell. 2021 Oct 01. pii: S1097-2765(21)00752-8. [Epub ahead of print]

A Polycomb domain found in committed cells impairs differentiation when introduced into PRC1 in pluripotent cells.

Elizabeth S Jaensch, Jinjin Zhu, Jesse C Cochrane, Sharon K Marr, Theresa A Oei, Manashree Damle, Ethan Z McCaslin, Robert E Kingston.

  The CBX family of proteins is central to proper mammalian development via key roles in Polycomb-mediated maintenance of repression. CBX proteins in differentiated lineages have chromatin compaction and phase separation activities that might contribute to maintaining repressed chromatin. The predominant CBX protein in pluripotent cells, CBX7, lacks the domain required for these activities. We inserted this functional domain into CBX7 in embryonic stem cells (ESCs) to test the hypothesis that it contributes a key epigenetic function. ESCs expressing this chimeric CBX7 were impaired in their ability to properly form embryoid bodies and neural progenitor cells and showed reduced activation of lineage-specific genes across differentiation. Neural progenitors exhibited a corresponding inappropriate maintenance of Polycomb binding at neural-specific loci over the course of differentiation. We propose that a switch in the ability to compact and phase separate is a central aspect of Polycomb group function during the transition from pluripotency to differentiated lineages.

Keywords:  PRC1; Polycomb repressive complex; chromatin; development; gene repression; nucleosome compaction; phase separation; stem cells

DOI:  https://doi.org/10.1016/j.molcel.2021.09.018
Genome Med. 2021 Oct 11. 13(1): 158

Integrative epigenomic and high-throughput functional enhancer profiling reveals determinants of enhancer heterogeneity in gastric cancer.

Taotao Sheng, Shamaine Wei Ting Ho, Wen Fong Ooi, Chang Xu, Manjie Xing, Nisha Padmanabhan, Kie Kyon Huang, Lijia Ma, Mohana Ray, Yu Amanda Guo, Ngak Leng Sim, Chukwuemeka George Anene-Nzelu, Mei Mei Chang, Milad Razavi-Mohseni, Michael A Beer, Roger Sik Yin Foo, Raghav Sundar, Yiong Huak Chan, Angie Lay Keng Tan, Xuewen Ong, Anders Jacobsen Skanderup, Kevin P White, Sudhakar Jha, Patrick Tan.

  BACKGROUND: Enhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity-however, most predicted enhancer regions remain to be functionally tested.METHODS: We analyzed 132 epigenomic histone modification profiles of 18 primary gastric cancer (GC) samples, 18 normal gastric tissues, and 28 GC cell lines using Nano-ChIP-seq technology. We applied Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to assess functional enhancer activity. An Activity-by-contact (ABC) model was employed to explore the effects of histone acetylation and CapSTARR-seq levels on enhancer-promoter interactions.
RESULTS: We report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are GC-associated in vivo (> 50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying CapSTARR-seq to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers, even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. We identified cancer-relevant genes (ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans-acting factor associated with GC enhancer heterogeneity.
CONCLUSIONS: Our results indicate that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, providing insights into the relative contribution of genetic (cis) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity.

Keywords:  CapSTARR-seq; Enhancer heterogeneity; Enhancer landscape; Enhancer-promoter interactions; Gastric cancer

DOI:  https://doi.org/10.1186/s13073-021-00970-3
Genome Biol. 2021 Oct 14. 22(1): 289

KrasG12D induces changes in chromatin territories that differentially impact early nuclear reprogramming in pancreatic cells.

Angela J Mathison, Romica Kerketta, Thiago Milech de Assuncao, Elise Leverence, Atefeh Zeighami, Guillermo Urrutia, Timothy J Stodola, Marina Pasca di Magliano, Juan L Iovanna, Michael T Zimmermann, Gwen Lomberk, Raul Urrutia.

  BACKGROUND: Pancreatic ductal adenocarcinoma initiation is most frequently caused by Kras mutations.RESULTS: Here, we apply biological, biochemical, and network biology methods to validate GEMM-derived cell models using inducible KrasG12D expression. We describe the time-dependent, chromatin remodeling program that impacts function during early oncogenic signaling. We find that the KrasG12D-induced transcriptional response is dominated by downregulated expression concordant with layers of epigenetic events. More open chromatin characterizes the ATAC-seq profile associated with a smaller group of upregulated genes and epigenetic marks. RRBS demonstrates that promoter hypermethylation does not account for the silencing of the extensive gene promoter network. Moreover, ChIP-Seq reveals that heterochromatin reorganization plays little role in this early transcriptional program. Notably, both gene activation and silencing primarily depend on the marking of genes with a combination of H3K27ac, H3K4me3, and H3K36me3. Indeed, integrated modeling of all these datasets shows that KrasG12D regulates its transcriptional program primarily through unique super-enhancers and enhancers, and marking specific gene promoters and bodies. We also report chromatin remodeling across genomic areas that, although not contributing directly to cis-gene transcription, are likely important for KrasG12D functions.
CONCLUSIONS: In summary, we report a comprehensive, time-dependent, and coordinated early epigenomic program for KrasG12D in pancreatic cells, which is mechanistically relevant to understanding chromatin remodeling events underlying transcriptional outcomes needed for the function of this oncogene.

Keywords:  Epigenomics; KRAS; Pancreatic cancer

DOI:  https://doi.org/10.1186/s13059-021-02498-6
Mol Cell. 2021 Oct 06. pii: S1097-2765(21)00753-X. [Epub ahead of print]

Simultaneous profiling of multiple chromatin proteins in the same cells.

Sneha Gopalan, Yuqing Wang, Nicholas W Harper, Manuel Garber, Thomas G Fazzio.

  Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of co-localization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here, we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and characterization of cell-type-specific chromatin architecture. In sum, multi-CUT&Tag increases the information content per cell of epigenomic maps, facilitating direct analysis of the interplay of different chromatin proteins.

Keywords:  H3K27ac; H3K27me3; RNA polymerase; chromatin; histones; profiling; single-cell; transcription

DOI:  https://doi.org/10.1016/j.molcel.2021.09.019
Nucleic Acids Res. 2021 Oct 14. pii: gkab864. [Epub ahead of print]

STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells.

Laia Richart, Eleonora Lapi, Vera Pancaldi, Mirabai Cuenca-Ardura, Enrique Carrillo-de-Santa Pau, Miguel Madrid-Mencía, Hélène Neyret-Kahn, François Radvanyi, Juan Antonio Rodríguez, Yasmina Cuartero, François Serra, François Le Dily, Alfonso Valencia, Marc A Marti-Renom, Francisco X Real.

Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium.

DOI: https://doi.org/10.1093/nar/gkab864
Nat Commun. 2021 Oct 14. 12(1): 5996

Morc3 silences endogenous retroviruses by enabling Daxx-mediated histone H3.3 incorporation.

Sophia Groh, Anna Viktoria Milton, Lisa Katherina Marinelli, Cara V Sickinger, Angela Russo, Heike Bollig, Gustavo Pereira de Almeida, Andreas Schmidt, Ignasi Forné, Axel Imhof, Gunnar Schotta.

Endogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1-mediated H3K9me3 and DNA methylation. However, the protein network which regulates the deposition of these chromatin modifications is still incompletely understood. Here, we perform a genome-wide single guide RNA (sgRNA) screen for genes involved in ERV silencing and identify the GHKL ATPase protein Morc3 as a top-scoring hit. Morc3 knock-out (ko) cells display de-repression, reduced H3K9me3, and increased chromatin accessibility of distinct ERV families. We find that the Morc3 ATPase cycle and Morc3 SUMOylation are important for ERV chromatin regulation. Proteomic analyses reveal that Morc3 mutant proteins fail to interact with the histone H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation and Daxx SUMO binding. Notably, in Morc3 ko cells, we observe strongly reduced histone H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as a critical regulator of Daxx-mediated histone H3.3 incorporation to ERV regions.

DOI: https://doi.org/10.1038/s41467-021-26288-7
Mol Cell. 2021 Oct 06. pii: S1097-2765(21)00754-1. [Epub ahead of print]

NRF1 association with AUTS2-Polycomb mediates specific gene activation in the brain.

Sanxiong Liu, Kimberly A Aldinger, Chi Vicky Cheng, Takae Kiyama, Mitali Dave, Hanna K McNamara, Wukui Zhao, Stefano G Caraffi, Ivan Ivanovski, Edoardo Errichiello, Christiane Zweier, Orsetta Zuffardi, Michael Schneider, Antigone S Papavasiliou, M Scott Perry, Jennifer Humberson, Megan T Cho, Astrid Weber, Andrew Swale, Tudor C Badea, Chai-An Mao, Livia Garavelli, William B Dobyns, Danny Reinberg.

  The heterogeneous family of complexes comprising Polycomb repressive complex 1 (PRC1) is instrumental for establishing facultative heterochromatin that is repressive to transcription. However, two PRC1 species, ncPRC1.3 and ncPRC1.5, are known to comprise novel components, AUTS2, P300, and CK2, that convert this repressive function to that of transcription activation. Here, we report that individuals harboring mutations in the HX repeat domain of AUTS2 exhibit defects in AUTS2 and P300 interaction as well as a developmental disorder reflective of Rubinstein-Taybi syndrome, which is mainly associated with a heterozygous pathogenic variant in CREBBP/EP300. Moreover, the absence of AUTS2 or mutation in its HX repeat domain gives rise to misregulation of a subset of developmental genes and curtails motor neuron differentiation of mouse embryonic stem cells. The transcription factor nuclear respiratory factor 1 (NRF1) has a novel and integral role in this neurodevelopmental process, being required for ncPRC1.3 recruitment to chromatin.

Keywords:  AUTS2; NRF1; P300; RSTS; active transcription; brain development; ncPRC1.3; polycomb

DOI:  https://doi.org/10.1016/j.molcel.2021.09.020
Nat Biotechnol. 2021 Oct 11.

Multiscale and integrative single-cell Hi-C analysis with Higashi.

Ruochi Zhang, Tianming Zhou, Jian Ma.

Single-cell Hi-C (scHi-C) can identify cell-to-cell variability of three-dimensional (3D) chromatin organization, but the sparseness of measured interactions poses an analysis challenge. Here we report Higashi, an algorithm based on hypergraph representation learning that can incorporate the latent correlations among single cells to enhance overall imputation of contact maps. Higashi outperforms existing methods for embedding and imputation of scHi-C data and is able to identify multiscale 3D genome features in single cells, such as compartmentalization and TAD-like domain boundaries, allowing refined delineation of their cell-to-cell variability. Moreover, Higashi can incorporate epigenomic signals jointly profiled in the same cell into the hypergraph representation learning framework, as compared to separate analysis of two modalities, leading to improved embeddings for single-nucleus methyl-3C data. In an scHi-C dataset from human prefrontal cortex, Higashi identifies connections between 3D genome features and cell-type-specific gene regulation. Higashi can also potentially be extended to analyze single-cell multiway chromatin interactions and other multimodal single-cell omics data.

DOI: https://doi.org/10.1038/s41587-021-01034-y
iScience. 2021 Oct 22. 24(10): 103132

Pioneer factors in development and cancer.

Benjamin D Sunkel, Benjamin Z Stanton.

  Transcription factors (TFs) are essential mediators of epigenetic regulation and modifiers of penetrance. Studies from the past decades have revealed a sub-class of TF that is capable of remodeling closed chromatin states through targeting nucleosomal motifs. This pioneer factor (PF) class of chromatin remodeler is ATP independent in its roles in epigenetic initiation, with nucleosome-motif recognition and association with repressive chromatin regions. Increasing evidence suggests that the fundamental properties of PFs can be coopted in human cancers. We explore the role of PFs in the larger context of tissue-specific epigenetic regulation. Moreover, we highlight an emerging class of chimeric PF derived from translocation partners in human disease and PFs associated with rare tumors. In the age of site-directed genome editing and targeted protein degradation, increasing our understanding of PFs will provide access to next-generation therapy for human disease driven from altered transcriptional circuitry.

Keywords:  Cancer systems biology; Epigenetics; Molecular biology; Systems biology

DOI:  https://doi.org/10.1016/j.isci.2021.103132
Cancer Res. 2021 Oct 12. pii: canres.CAN-21-2056-E.2021. [Epub ahead of print]

Reorganization of the 3D genome pinpoints non-coding drivers of primary prostate tumors.

James R Hawley, Stanley Zhou, Christopher Arlidge, Giacomo Grillo, Ken J Kron, Rupert Hugh-White, Theodorus H van der Kwast, Michael Fraser, Paul C Boutros, Robert G Bristow, Mathieu Lupien.

Prostate cancer is a heterogeneous disease whose progression is linked to genome instability. However, the impact of this instability on the non-coding genome and its three-dimensional organization to aid progression is unclear. Using primary benign and tumor tissue, we find a high concordance in higher order three-dimensional genome organization. This concordance argues for constraints to the topology of prostate tumor genomes. Nonetheless, we identified changes in focal chromatin interactions, typical of loops bridging non-coding cis-regulatory elements, and showed how structural variants can induce these changes to guide cis-regulatory element hijacking. Such events resulted in opposing differential expression of genes found at antipodes of rearrangements. Collectively, these results argue that changes to focal chromatin interactions, as opposed to higher order genome organization, allow for aberrant gene regulation and are repeatedly mediated by structural variants in primary prostate cancer.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-2056
Elife. 2021 Oct 15. pii: e71013. [Epub ahead of print]10

Ssl2/TFIIH function in transcription start site scanning by RNA Polymerase II in Saccharomyces cerevisiae.

Tingting Zhao, Irina O Vvedenskaya, William Km Lai, Shrabani Basu, B Franklin Pugh, Bryce E Nickels, Craig D Kaplan.

  In Saccharomyces cerevisiae, RNA Polymerase II (Pol II) selects transcription start sites (TSS) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window ~40-120 basepairs downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.

Keywords:  S. cerevisiae; genetics; genomics

DOI:  https://doi.org/10.7554/eLife.71013
Nat Biotechnol. 2021 Oct 11.

Chromatin Velocity reveals epigenetic dynamics by single-cell profiling of heterochromatin and euchromatin.

Martina Tedesco, Francesca Giannese, Dejan Lazarević, Valentina Giansanti, Dalia Rosano, Silvia Monzani, Irene Catalano, Elena Grassi, Eugenia R Zanella, Oronza A Botrugno, Leonardo Morelli, Paola Panina Bordignon, Giulio Caravagna, Andrea Bertotti, Gianvito Martino, Luca Aldrighetti, Sebastiano Pasqualato, Livio Trusolino, Davide Cittaro, Giovanni Tonon.

Recent efforts have succeeded in surveying open chromatin at the single-cell level, but high-throughput, single-cell assessment of heterochromatin and its underlying genomic determinants remains challenging. We engineered a hybrid transposase including the chromodomain (CD) of the heterochromatin protein-1α (HP-1α), which is involved in heterochromatin assembly and maintenance through its binding to trimethylation of the lysine 9 on histone 3 (H3K9me3), and developed a single-cell method, single-cell genome and epigenome by transposases sequencing (scGET-seq), that, unlike single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), comprehensively probes both open and closed chromatin and concomitantly records the underlying genomic sequences. We tested scGET-seq in cancer-derived organoids and human-derived xenograft (PDX) models and identified genetic events and plasticity-driven mechanisms contributing to cancer drug resistance. Next, building upon the differential enrichment of closed and open chromatin, we devised a method, Chromatin Velocity, that identifies the trajectories of epigenetic modifications at the single-cell level. Chromatin Velocity uncovered paths of epigenetic reorganization during stem cell reprogramming and identified key transcription factors driving these developmental processes. scGET-seq reveals the dynamics of genomic and epigenetic landscapes underlying any cellular processes.

DOI: https://doi.org/10.1038/s41587-021-01031-1
Nucleic Acids Res. 2021 Oct 13. pii: gkab907. [Epub ahead of print]

Structural basis of chromatin regulation by histone variant H2A.Z.

Tyler S Lewis, Vladyslava Sokolova, Harry Jung, Honkit Ng, Dongyan Tan.

The importance of histone variant H2A.Z in transcription regulation has been well established, yet its mechanism-of-action remains enigmatic. Conflicting evidence exists in support of both an activating and a repressive role of H2A.Z in transcription. Here we report cryo-electron microscopy (cryo-EM) structures of nucleosomes and chromatin fibers containing H2A.Z and those containing canonical H2A. The structures show that H2A.Z incorporation results in substantial structural changes in both nucleosome and chromatin fiber. While H2A.Z increases the mobility of DNA terminus in nucleosomes, it simultaneously enables nucleosome arrays to form a more regular and condensed chromatin fiber. We also demonstrated that H2A.Z's ability to enhance nucleosomal DNA mobility is largely attributed to its characteristic shorter C-terminus. Our study provides the structural basis for H2A.Z-mediated chromatin regulation, showing that the increase flexibility of the DNA termini in H2A.Z nucleosomes is central to its dual-functions in chromatin regulation and in transcription.

DOI: https://doi.org/10.1093/nar/gkab907
Curr Protoc. 2021 Oct;1(10): e266

greenCUT&RUN: Efficient Genomic Profiling of GFP-Tagged Transcription Factors and Chromatin Regulators.

Stefanie Koidl, H T Marc Timmers.

  Genome-wide mapping of transcription factors and chromatin regulators is important to distinguish their direct from indirect effects on gene transcription or chromatin function. Novel approaches for studying their genomic localization under native conditions, such us cleavage under target and release using nuclease (CUT&RUN), offer higher resolution and lower sequencing costs than classical chromatin immunoprecipitation (ChIP) assays, and require fewer cells but they still depend on the availability of high-quality antibodies. Here, we describe detailed and robust protocols for greenCUT&RUN, which is a generic CUT&RUN-based approach for mapping the genome-wide localization of green fluorescent protein (GFP)-tagged factors in intact mammalian cells. The greenCUT&RUN method makes use of a micrococcal nuclease (MNase) coupled to a high affinity nanobody against GFP, which exploits the accessibility of multiple surfaces of the GFP tag, thus eliminating issues of antibody variability and availability. We also provide efficient protocols for the expression and purification of two different GFP nanobodies, which recognize non-overlapping GFP epitopes and can be combined for a further gain in sensitivity and accuracy. Compared to traditional CUT&RUN, genomic localization by greenCUT&RUN reduces handling time and experimental variability. GreenCUT&RUN is a versatile, robust, and universal procedure for surveying the genome-wide localization of GFP-tagged versions of proteins that drive key transcriptional programs and regulate chromatin function. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Standard greenCUT&RUN for GFP-tagged proteins in mammalian cells Alternate Protocol: High-Ca++ /low-salt greenCUT&RUN for GFP-tagged histone proteins in mammalian cells Support Protocol: Expression and purification of GFP nanobody-MNase fusion proteins for greenCUT&RUN.

Keywords:  CUT&RUN; GFP-fusion proteins; genome localization; genomic profiling; greenCUT&RUN; micrococcal nuclease mapping; transcription factor binding

DOI:  https://doi.org/10.1002/cpz1.266
Nat Genet. 2021 Oct 14.

The dynamic, combinatorial cis-regulatory lexicon of epidermal differentiation.

Daniel S Kim, Viviana I Risca, David L Reynolds, James Chappell, Adam J Rubin, Namyoung Jung, Laura K H Donohue, Vanessa Lopez-Pajares, Arwa Kathiria, Minyi Shi, Zhixin Zhao, Harsh Deep, Mahfuza Sharmin, Deepti Rao, Shin Lin, Howard Y Chang, Michael P Snyder, William J Greenleaf, Anshul Kundaje, Paul A Khavari.

Transcription factors bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multiomic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep-learning framework to model the cis-regulatory logic of chromatin accessibility. This analysis framework identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter assay analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach shows the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code of dynamic gene regulation.

DOI: https://doi.org/10.1038/s41588-021-00947-3
Nat Commun. 2021 Oct 14. 12(1): 6013

The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle.

Giovanna Rigillo, Valentina Basile, Silvia Belluti, Mirko Ronzio, Elisabetta Sauta, Alessia Ciarrocchi, Lucia Latella, Marielle Saclier, Susanna Molinari, Antonio Vallarola, Graziella Messina, Roberto Mantovani, Diletta Dolfini, Carol Imbriano.

The transcription factor NF-Y promotes cell proliferation and its activity often declines during differentiation through the regulation of NF-YA, the DNA binding subunit of the complex. In stem cell compartments, the shorter NF-YA splice variant is abundantly expressed and sustains their expansion. Here, we report that satellite cells, the stem cell population of adult skeletal muscle necessary for its growth and regeneration, express uniquely the longer NF-YA isoform, majorly associated with cell differentiation. Through the generation of a conditional knock out mouse model that selectively deletes the NF-YA gene in satellite cells, we demonstrate that NF-YA expression is fundamental to preserve the pool of muscle stem cells and ensures robust regenerative response to muscle injury. In vivo and ex vivo, satellite cells that survive to NF-YA loss exit the quiescence and are rapidly committed to early differentiation, despite delayed in the progression towards later states. In vitro results demonstrate that NF-YA-depleted muscle stem cells accumulate DNA damage and cannot properly differentiate. These data highlight a new scenario in stem cell biology for NF-Y activity, which is required for efficient myogenic differentiation.

DOI: https://doi.org/10.1038/s41467-021-26293-w
Nat Plants. 2021 Oct;7(10): 1364-1378

Global profiling of RNA-chromatin interactions reveals co-regulatory gene expression networks in Arabidopsis.

Lanxia Li, Haofei Luo, Do-Hwan Lim, Lu Han, Yan Li, Xiang-Dong Fu, Yijun Qi.

It is increasingly evident that various RNAs can bind chromatin to regulate gene expression and genome organization. Here we adapted a sequencing-based technique to profile RNA-chromatin interactions at a genome-wide scale in Arabidopsis seedlings. We identified more than 10,000 RNA-chromatin interactions mediated by protein-coding RNAs and non-coding RNAs. Cis and intra-chromosomal interactions are mainly mediated by protein-coding RNAs, whereas inter-chromosomal interactions are primarily mediated by non-coding RNAs. Many RNA-chromatin interactions tend to positively correlate with DNA-DNA interactions, suggesting their mutual influence and reinforcement. We further show that some RNA-chromatin interactions undergo alterations in response to biotic and abiotic stresses and that altered RNA-chromatin interactions form co-regulatory networks. Our study provides a global view on RNA-chromatin interactions in Arabidopsis and a rich resource for future investigations of regulatory roles of RNAs in gene expression and genome organization.

DOI: https://doi.org/10.1038/s41477-021-01004-x
Nat Protoc. 2021 Oct 14.

Scalable dual-omics profiling with single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2).

Nongluk Plongthongkum, Dinh Diep, Song Chen, Blue B Lake, Kun Zhang.

Comprehensive characterization of cellular heterogeneity and the underlying regulatory landscapes of tissues and organs requires a highly robust and scalable method to acquire matched RNA and chromatin accessibility profiles on the same cells. Here, we describe a single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2) assay, implemented with cellular combinatorial indexing. This method involves tagmentation within permeabilized and fixed single-nucleus isolates to capture accessible chromatin (AC) regions, followed by the capture and reverse transcription of RNA transcripts. Through combinatorial split pool ligations, cDNA and AC within each single nucleus become appended with a common cell barcode combination. The captured cDNA and AC are then co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. This protocol is compatible with both nuclei and whole cells and can be completed in 3.5 d. SNARE-seq2 permits robust generation of high-quality, joint single-cell RNA and AC sequencing libraries from hundreds of thousands of single cells per experiment.

DOI: https://doi.org/10.1038/s41596-021-00507-3
Genome Biol. 2021 Oct 14. 22(1): 290

A global screening identifies chromatin-enriched RNA-binding proteins and the transcriptional regulatory activity of QKI5 during monocytic differentiation.

Yue Ren, Yue Huo, Weiqian Li, Manman He, Siqi Liu, Jiabin Yang, Hongmei Zhao, Lingjie Xu, Yuehong Guo, Yanmin Si, Hualu Zhao, Shuan Rao, Jing Wang, Yanni Ma, Xiaoshuang Wang, Jia Yu, Fang Wang.

  BACKGROUND: Cellular RNA-binding proteins (RBPs) have multiple roles in post-transcriptional control, and some are shown to bind DNA. However, the global localization and the general chromatin-binding ability of RBPs are not well-characterized and remain undefined in hematopoietic cells.RESULTS: We first provide a full view of RBPs' distribution pattern in the nucleus and screen for chromatin-enriched RBPs (Che-RBPs) in different human cells. Subsequently, by generating ChIP-seq, CLIP-seq, and RNA-seq datasets and conducting combined analysis, the transcriptional regulatory potentials of certain hematopoietic Che-RBPs are predicted. From this analysis, quaking (QKI5) emerges as a potential transcriptional activator during monocytic differentiation. QKI5 is over-represented in gene promoter regions, independent of RNA or transcription factors. Furthermore, DNA-bound QKI5 activates the transcription of several critical monocytic differentiation-associated genes, including CXCL2, IL16, and PTPN6. Finally, we show that the differentiation-promoting activity of QKI5 is largely dependent on CXCL2, irrespective of its RNA-binding capacity.
CONCLUSIONS: Our study indicates that Che-RBPs are versatile factors that orchestrate gene expression in different cellular contexts, and identifies QKI5, a classic RBP regulating RNA processing, as a novel transcriptional activator during monocytic differentiation.

Keywords:  Monocytic differentiation; QKI5; RNA-binding proteins (RBPs); Transcriptional regulation

DOI:  https://doi.org/10.1186/s13059-021-02508-7
PLoS Genet. 2021 Oct 11. 17(10): e1009855

Metabolic gene regulation by Drosophila GATA transcription factor Grain.

Krista Kokki, Nicole Lamichane, Anni I Nieminen, Hanna Ruhanen, Jack Morikka, Marius Robciuc, Bohdana M Rovenko, Essi Havula, Reijo Käkelä, Ville Hietakangas.

Nutrient-dependent gene regulation critically contributes to homeostatic control of animal physiology in changing nutrient landscape. In Drosophila, dietary sugars activate transcription factors (TFs), such as Mondo-Mlx, Sugarbabe and Cabut, which control metabolic gene expression to mediate physiological adaptation to high sugar diet. TFs that correspondingly control sugar responsive metabolic genes under conditions of low dietary sugar remain, however, poorly understood. Here we identify a role for Drosophila GATA TF Grain in metabolic gene regulation under both low and high sugar conditions. De novo motif prediction uncovered a significant over-representation of GATA-like motifs on the promoters of sugar-activated genes in Drosophila larvae, which are regulated by Grain, the fly ortholog of GATA1/2/3 subfamily. grain expression is activated by sugar in Mondo-Mlx-dependent manner and it contributes to sugar-responsive gene expression in the fat body. On the other hand, grain displays strong constitutive expression in the anterior midgut, where it drives lipogenic gene expression also under low sugar conditions. Consistently with these differential tissue-specific roles, Grain deficient larvae display delayed development on high sugar diet, while showing deregulated central carbon and lipid metabolism primarily on low sugar diet. Collectively, our study provides evidence for the role of a metazoan GATA transcription factor in nutrient-responsive metabolic gene regulation in vivo.

DOI: https://doi.org/10.1371/journal.pgen.1009855
Int J Mol Sci. 2021 Sep 28. pii: 10437. [Epub ahead of print]22(19):

SUMOylation Potentiates ZIC Protein Activity to Influence Murine Neural Crest Cell Specification.

Helen M Bellchambers, Kristen S Barratt, Koula E M Diamand, Ruth M Arkell.

  The mechanisms of neural crest cell induction and specification are highly conserved among vertebrate model organisms, but how similar these mechanisms are in mammalian neural crest cell formation remains open to question. The zinc finger of the cerebellum 1 (ZIC1) transcription factor is considered a core component of the vertebrate gene regulatory network that specifies neural crest fate at the neural plate border. In mouse embryos, however, Zic1 mutation does not cause neural crest defects. Instead, we and others have shown that murine Zic2 and Zic5 mutate to give a neural crest phenotype. Here, we extend this knowledge by demonstrating that murine Zic3 is also required for, and co-operates with, Zic2 and Zic5 during mammalian neural crest specification. At the murine neural plate border (a region of high canonical WNT activity) ZIC2, ZIC3, and ZIC5 function as transcription factors to jointly activate the Foxd3 specifier gene. This function is promoted by SUMOylation of the ZIC proteins at a conserved lysine immediately N-terminal of the ZIC zinc finger domain. In contrast, in the lateral regions of the neurectoderm (a region of low canonical WNT activity) basal ZIC proteins act as co-repressors of WNT/TCF-mediated transcription. Our work provides a mechanism by which mammalian neural crest specification is restricted to the neural plate border. Furthermore, given that WNT signaling and SUMOylation are also features of non-mammalian neural crest specification, it suggests that mammalian neural crest induction shares broad conservation, but altered molecular detail, with chicken, zebrafish, and Xenopus neural crest induction.

Keywords:  Foxd3; TCF; ZIC; co-factor; mouse neural crest cell; post-translational modification; transcription factor

DOI:  https://doi.org/10.3390/ijms221910437
Cancer Res. 2021 Oct 13. pii: canres.1825.2021. [Epub ahead of print]

Targeting KDM6A suppresses SREBP1c-dependent lipid metabolism and prostate tumorigenesis.

Dong-E Tang, Jia-Xi He, Yong Dai, Hui-Fen Zhou, Cancan Zhang, Qi-Xin Leng, Xinyan Geng, Dexue Fu, Hao-Wu Jiang, Rui Sun, Song-Hui Xu.

The histone demethylase KDM6A controls gene expression by the epigenetic regulation of H3K27 methylation and functions in diverse processes, including differentiation, development, and cancer. Here, we investigated the role of KDM6A in prostate cancer (PCa). Specific homozygous deletion of KDM6A in the adult mouse prostate epithelium strongly inhibited tumor progression initiated by the homozygous loss of PTEN. Mechanistically, KDM6A promoted prostate tumorigenesis and lipid metabolism by binding to the SREBP1c promoter to increase SREBP1c transcription. USP7 deubiquitinated KDM6A to increase its expression. KDM6A was significantly up-regulated in PCa and positively associated with USP7 expression. Furthermore, targeting KDM6A stability by inhibiting USP7 in conditional knockout mice and xenograft models markedly suppressed PCa growth and significantly enhanced KDM6A inhibitor efficacy. Collectively, these findings indicate that KDM6A regulates prostate lipid metabolism and is essential for prostate tumorigenesis initiated by PTEN loss. Targeting USP7/KDM6A could be a valuable strategy to ameliorate prostate cancer progression and therapeutic resistance.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-1825
Nucleic Acids Res. 2021 Oct 11. pii: gkab890. [Epub ahead of print]

Coupling high-throughput mapping with proteomics analysis delineates cis-regulatory elements at high resolution.

Ting Wu, Danli Jiang, Meijuan Zou, Wei Sun, Di Wu, Jing Cui, Ian Huntress, Xinxia Peng, Gang Li.

Growing evidence suggests that functional cis-regulatory elements (cis-REs) not only exist in epigenetically marked but also in unmarked sites of the human genome. While it is already difficult to identify cis-REs in the epigenetically marked sites, interrogating cis-REs residing within the unmarked sites is even more challenging. Here, we report adapting Reel-seq, an in vitro high-throughput (HTP) technique, to fine-map cis-REs at high resolution over a large region of the human genome in a systematic and continuous manner. Using Reel-seq, as a proof-of-principle, we identified 408 candidate cis-REs by mapping a 58 kb core region on the aging-related CDKN2A/B locus that harbors p16INK4a. By coupling Reel-seq with FREP-MS, a proteomics analysis technique, we characterized two cis-REs, one in an epigenetically marked site and the other in an epigenetically unmarked site. These elements are shown to regulate the p16INK4a expression over an ∼100 kb distance by recruiting the poly(A) binding protein PABPC1 and the transcription factor FOXC2. Downregulation of either PABPC1 or FOXC2 in human endothelial cells (ECs) can induce the p16INK4a-dependent cellular senescence. Thus, we confirmed the utility of Reel-seq and FREP-MS analyses for the systematic identification of cis-REs at high resolution over a large region of the human genome.

DOI: https://doi.org/10.1093/nar/gkab890
Commun Biol. 2021 Oct 14. 4(1): 1186

Therapeutically expanded human regulatory T-cells are super-suppressive due to HIF1A induced expression of CD73.

Lorna B Jarvis, Daniel B Rainbow, Valerie Coppard, Sarah K Howlett, Zoya Georgieva, Jessica L Davies, Harpreet Kaur Mullay, Joanna Hester, Tom Ashmore, Aletta Van Den Bosch, James T Grist, Alasdair J Coles, Hani S Mousa, Stefano Pluchino, Krishnaa T Mahbubani, Julian L Griffin, Kourosh Saeb-Parsy, Fadi Issa, Luca Peruzzotti-Jametti, Linda S Wicker, Joanne L Jones.

The adoptive transfer of regulatory T-cells (Tregs) is a promising therapeutic approach in transplantation and autoimmunity. However, because large cell numbers are needed to achieve a therapeutic effect, in vitro expansion is required. By comparing their function, phenotype and transcriptomic profile against ex vivo Tregs, we demonstrate that expanded human Tregs switch their metabolism to aerobic glycolysis and show enhanced suppressive function through hypoxia-inducible factor 1-alpha (HIF1A) driven acquisition of CD73 expression. In conjunction with CD39, CD73 expression enables expanded Tregs to convert ATP to immunosuppressive adenosine. We conclude that for maximum therapeutic benefit, Treg expansion protocols should be optimised for CD39/CD73 co-expression.

DOI: https://doi.org/10.1038/s42003-021-02721-x
PLoS Comput Biol. 2021 Oct;17(10): e1009423

Segmentation and genome annotation algorithms for identifying chromatin state and other genomic patterns.

Maxwell W Libbrecht, Rachel C W Chan, Michael M Hoffman.

Segmentation and genome annotation (SAGA) algorithms are widely used to understand genome activity and gene regulation. These algorithms take as input epigenomic datasets, such as chromatin immunoprecipitation-sequencing (ChIP-seq) measurements of histone modifications or transcription factor binding. They partition the genome and assign a label to each segment such that positions with the same label exhibit similar patterns of input data. SAGA algorithms discover categories of activity such as promoters, enhancers, or parts of genes without prior knowledge of known genomic elements. In this sense, they generally act in an unsupervised fashion like clustering algorithms, but with the additional simultaneous function of segmenting the genome. Here, we review the common methodological framework that underlies these methods, review variants of and improvements upon this basic framework, and discuss the outlook for future work. This review is intended for those interested in applying SAGA methods and for computational researchers interested in improving upon them.

DOI: https://doi.org/10.1371/journal.pcbi.1009423
Endocr Relat Cancer. 2021 Oct 01. pii: ERC-21-0085.R1. [Epub ahead of print]

Inhibition of GATA2 in prostate cancer by a clinically available small molecule.

Salma Kaochar, Aleksandra Rusin, Christopher Foley, Kimal Rajapakshe, Matthew Robertson, Darlene Skapura, Cammy Mason, Karen BermandeRuiz, Alexey Mikhailovich Tyryshkin, Jenny Deng, Jin Na Shin, Warren Fiskus, Jianrong Dong, Shixia Huang, Nora M Navone, Christel M Davis, Erik A Ehli, Cristian Coarfa, Nicholas Mitsiades.

Castration-resistant prostate cancer (CRPC) remains highly lethal and in need of novel, actionable therapeutic targets. The pioneer factor GATA2 is a significant prostate cancer (PC) driver and linked to poor prognosis. GATA2 directly promotes androgen receptor (AR) gene expression (both full-length and splice-variant) and facilitates AR binding to chromatin, recruitment of coregulators, and target gene transcription. Unfortunately, there is no clinically applicable GATA2 inhibitor available at the moment. Using a bioinformatics algorithm, we screened in silico 2,650 clinically relevant drugs for a potential GATA2 inhibitor. Validation studies used cytotoxicity assays (MTT), global gene expression analysis, reporter assay, reverse phase protein array analysis (RPPA), and immunoblotting. We examined target engagement via cellular thermal shift assay (CETSA), ChIP-qPCR, and GATA2 DNA-binding assay. We identified the vasodilator dilazep as a potential GATA2 inhibitor and confirmed on-target activity via CETSA. Dilazep exerted anticancer activity across a broad panel of GATA2-dependent PC cell lines in vitro and in a PDX model in vivo. Dilazep inhibited GATA2 recruitment to chromatin and suppressed the cell cycle program, transcriptional programs driven by GATA2, AR, and c-MYC, and the expression of several oncogenic drivers, including AR, c-MYC, FOXM1, CENPF, EZH2, UBE2C, and RRM2, as well as of several mediators of metastasis, DNA damage repair and stemness. In conclusion, we provide, via an extensive compendium of methodologies, proof-of-principle that a small molecule can inhibit GATA2 function and suppress its downstream AR, c-MYC, and other PC-driving effectors. We propose GATA2 as a therapeutic target in CRPC.

DOI: https://doi.org/10.1530/ERC-21-0085
Mol Cell. 2021 Oct 05. pii: S1097-2765(21)00775-9. [Epub ahead of print]

Gene bookmarking by the heat shock transcription factor programs the insulin-like signaling pathway.

Srijit Das, Sehee Min, Veena Prahlad.

  Maternal stress can have long-lasting epigenetic effects on offspring. To examine how epigenetic changes are triggered by stress, we examined the effects of activating the universal stress-responsive heat shock transcription factor HSF-1 in the germline of Caenorhabditis elegans. We show that, when activated in germ cells, HSF-1 recruits MET-2, the putative histone 3 lysine 9 (H3K9) methyltransferase responsible for repressive H3K9me2 (H3K9 dimethyl) marks in chromatin, and negatively bookmarks the insulin receptor daf-2 and other HSF-1 target genes. Increased H3K9me2 at these genes persists in adult progeny and shifts their stress response strategy away from inducible chaperone expression as a mechanism to survive stress and instead rely on decreased insulin/insulin growth factor (IGF-1)-like signaling (IIS). Depending on the duration of maternal heat stress exposure, this epigenetic memory is inherited by the next generation. Thus, paradoxically, HSF-1 recruits the germline machinery normally responsible for erasing transcriptional memory but, instead, establishes a heritable epigenetic memory of prior stress exposure.

Keywords:  C. elegans; H3K9 methyltransferase; HSF1; MET-2; daf-2; epigenetic; heat shock; stress; transcriptional memory; transgenerational

DOI:  https://doi.org/10.1016/j.molcel.2021.09.022
Cancer Discov. 2021 Oct 12. pii: candisc.0683.2021. [Epub ahead of print]

Single-cell atlas of lineage states, tumor microenvironment and subtype-specific expression programs in gastric cancer.

Vikrant Kumar, Kalpana Ramnarayanan, Raghav Sundar, Nisha Padmanbahan, Supriya Srivastava, Mayu Koiwa, Tadahito Yasuda, Vivien Koh, Kie Kyon Huang, Su Ting Tay, Shamaine Wei Ting Ho, Angie Lay Keng Tan, Takatsugu Ishimoto, Guowei Kim, Asim Shabbir, Qingfeng Chen, Zhang Biyan, Shengli Xu, Kong-Peng Lam, Huey Yew Jeffrey Lum, Ming Teh, Wei Peng Yong, Jimmy Bok-Yan So, Patrick Tan.

Gastric cancer (GC) heterogeneity represents a barrier to disease management. We generated a comprehensive single-cell atlas of GC (>200,000 cells) comprising 48 samples from 31 patients across clinical stages and histological subtypes. We identified 34 distinct cell-lineage states including novel rare cell populations. Many lineage states exhibited distinct cancer-associated expression profiles, individually contributing to a combined tumor-wide molecular collage. We observed increased plasma cell proportions in diffuse-type tumors associated with epithelial-resident KLF2, and stage-wise accrual of cancer-associated fibroblast sub-populations marked by high INHBA and FAP co-expression. Single-cell comparisons between patient-derived organoids (PDOs) and primary tumors highlighted inter- and intra-lineage similarities and differences, demarcating molecular boundaries of PDOs as experimental models. We complemented these findings by spatial transcriptomics, orthogonal validation in independent bulk RNA-seq cohorts, and functional demonstration using in vitro and in vivo models. Our results provide a high-resolution molecular resource of intra- and inter-patient lineage-states across distinct GC subtypes.

DOI: https://doi.org/10.1158/2159-8290.CD-21-0683
Nucleic Acids Res. 2021 Oct 11. pii: gkab900. [Epub ahead of print]

The comprehensive interactomes of human adenosine RNA methyltransferases and demethylases reveal distinct functional and regulatory features.

Helena Covelo-Molares, Ales Obrdlik, Ivana Poštulková, Michaela Dohnálková, Pavlína Gregorová, Ranjani Ganji, David Potěšil, Lisa Gawriyski, Markku Varjosalo, Štěpánka Vaňáčová.

N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology. They function mainly by regulating interactions with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date, three methyltransferases and two demethylases have been identified that modify adenosines in mammalian mRNAs. Here, we present a comprehensive analysis of the interactomes of these enzymes. PCIF1 protein network comprises mostly factors involved in nascent RNA synthesis by RNA polymerase II, whereas ALKBH5 is closely linked with most aspects of pre-mRNA processing and mRNA export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome positions this demethylase at a crossroad between RNA transcription, RNA processing and DNA replication and repair. Altogether, these enzymes share limited spatial interactomes, pointing to specific molecular mechanisms of their regulation.

DOI: https://doi.org/10.1093/nar/gkab900
Elife. 2021 Oct 12. pii: e69324. [Epub ahead of print]10

Pathway dynamics can delineate the sources of transcriptional noise in gene expression.

Lucy Ham, Marcel Jackson, Michael Stumpf.

  Single-cell expression profiling opens up new vistas on cellular processes. Extensive cell-to-cell variability at the transcriptomic and proteomic level has been one of the stand-out observations. Because most experimental analyses are destructive we only have access to snapshot data of cellular states. This loss of temporal information presents significant challenges for inferring dynamics, as well as causes of cell-to-cell variability. In particular, we typically cannot separate dynamic variability from within cells ('intrinsic noise') from variability across the population ('extrinsic noise'). Here we make this non-identifiability mathematically precise, allowing us to identify new experimental set-ups that can assist in resolving this non-identifiability. We show that multiple generic reporters from the same biochemical pathways (e.g. mRNA and protein) can infer magnitudes of intrinsic and extrinsic transcriptional noise, identifying sources of heterogeneity. Stochastic simulations support our theory, and demonstrate that 'pathway-reporters' compare favourably to the well-known, but often difficult to implement, dual-reporter method.

Keywords:  chromosomes; computational biology; gene expression; none; systems biology

DOI:  https://doi.org/10.7554/eLife.69324